WO2014152282A1

WO2014152282A1 - Allogeneic stromal vascular fraction transplantation by blood type matching

Info

Publication number: WO2014152282A1
Application number: PCT/US2014/027160
Authority: WO
Inventors: Steven VICTOR
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-03-14
Filing date: 2014-03-14
Publication date: 2014-09-25
Anticipated expiration: 2015-09-14

Abstract

Allogeneic transplantation of stromal vascular fraction for use in treating a disease, injury, or condition comprising matching the donor and recipient by blood type. The stromal vascular fraction may be derived from a vascularized tissue obtained from living and non-living sources (e.g., post-mortem) using mechanical (e.g., ultrasonic cavitation), enzymatic, and/or chemical treatment, wherein the tissue cells and blood vessels are lysed thereby releasing intact stromal vascular fraction cells while substantially maintaining the viability of the stromal vascular fraction.

Description

ALLOGENEIC STROMAL VASCULAR FRACTION TRANSPLANTATION BY BLOOD TYPE MATCHING

FIELD OF THE INVENTION

[0001] The present invention relates to allogeneic transplantation of stromal vascular fraction for use in treating a disease, injury, or condition comprising matching the donor and recipient by blood type.

BACKGROUND OF THE INVENTION

Stromal Vascular Fraction

[0002] Within the last 10 years, adipose tissues have attracted attention as a resource of multipotent stem cells. See Zuk, et al. (2002) Mol Biol Cell. 13: 4279-4295. Adipose tissue comprises two fractions— the adipocyte fraction and the stromal-vascular fraction (SVF).

Adipose tissue may be found in the visceral adipose tissue (VAT) and subcutaneous adipose tissue deposits (SAT). See Peinado, et al. (2010) Proteomics 10(18): 3356-66; Novakofski & Hu (1987) J Anim Sci 65: 12-24. The stromal vascular fraction (SVF) comprises preadipocytes, mesenchymal stem cells (MSC), endothelial progenitor cells, blood endothelial cells, fibroblasts, pericytes, T regulatory cells, and macrophages. Koh, et al. (2011) Arterioscler Thromb Vase Biol. 31(5): 1141-50. The mesenchymanl stem cells (MSC) can be differentiated into a variety of cell lineages including adipogenic, chondrogenic, myogenic, and osteogeneic lineages.

Rodriguez, et al. (2012) International Archives of Medicine 5: 1-9; Zuk, et al. (2001) Tissue Eng. 7: 211-228; Hicok, et al. (2004) Tissue Eng. 10: 371-380; Erickson, et al. (2002) Biochem Biophys Res Commun. 290: 763-769; Cousin, et al. (2003) Biochem Biophys Res Commun. 301: 1016-1022; Safford, et al. (2002) Biochem Biophys Res Commun. 294: 371-379;

Miranville, et al. (2004) Circulation. 110: 349-355; Planat-Benard, et al. (2004) Circ Res. 94: 223-229; and Planat-Benard, et al. (2004) Circulation. 109: 656-663. The stromal vascular fraction, including the mesenchymal stem cells contained therein, are useful in research and therapeutics.

[0003] A common source of adipose tissue, including the stromal vascular fraction, is

"lipoaspirate," the adipose tissue harvested from patients undergoing liposuction. The lipoasperiate comprises adipocytes, fat, connective tissue, blood vessels, and stromal vascular fraction. The adipose tissue is usually separated from non-adipose tissue using a tissue collection container that utilizes decantation, sedimentation, and/or centrifugation techniques to separate the materials. The adipose tissue is then disaggregated using methods such as mechanical force (mincing or shear forces), enzymatic digestion with single or combinatorial proteolytic enzymes, such as collagenase, trypsin, lipase, liberase HI, pepsin, or a combination of mechanical and enzymatic methods. U.S. Patent No. 7,429,488. For example, a lipoaspirate may be obtained. The fatty portion diluted with PBS and centrifuged to exclude all

hematopoietic cells. The sample is then incubated with collagenase followed by centrifugation and filtration to isolate a stromal vascular fraction. Godthardt, et al. (2008) MACS Miltenyi Biotec Information Pamphlet. Other methods involve centrifugation and culturing the lipoaspirate. See Francis, et al. (2010) Organogenesis 6(1): 11-14. U.S. Patent Application Publication No. 2006/0051865 describes the use of ultrasonic methods to release adult stem cells from adipose tissue. U.S. Patent Application Publication 2007/0148766 also discloses the isolation of stem cells from liposuction-derived aspirates.

Allogeneic Transplantation

[0004] SVF cells may be used in transplantation therapy, but are limited to autologous transplantation methods. Ichim "Cell Therapy for Transplant Rejection." Tom Ichim, Cell Therapy for Transplant Rejection. ACVS Veterinary Symposium, & American College of Veterinary Surgeons. (2010). This is due to concerns about transplant rejection. The immune system plays an important role in the success of any allogeneic stem cell transplant. The immune system acts to destroy anything in the body it sees as foreign, such as bacteria or viruses. A working immune system recognizes cells from other people as foreign, too. If the tissue type match between donor and recipient is not close, the patient's immune system may see the new stem cells as foreign and destroy them. This is called graft rejection, and it can lead to graft failure. This is rare, because the pre-transplant treatment (chemo and/or radiation) mostly destroys the recipient's immune system, and donor matching is carefully done. Another problem that can happen is that when the donor stem cells make their own immune cells, the new cells may see the patient's cells as foreign and turn against their new home. This type of attack is called grafl-versus-host disease. The grafted stem cells attack the body of the person who got the transplant. This is a common problem, and it's the main reason every effort is made to find the closest match possible. HLA matching

[0005] Many factors play a role in how the immune system knows the difference between self and non-self, but the most important for transplants is the human leukocyte antigen (HLA) system. Each person has a number of pairs of HLA antigens. We inherit one of each of these pairs from each of our parents (and pass one of each pair on to each of our children). Human leukocyte antigens are proteins found on the surface of most cells. They make up a person's tissue type, which is different from a person's blood type.

[0006] How well the donor's and recipient's HLA tissue types match plays a large part in whether the transplant will work. A match is better when all 6 of the known major HLA antigens are the same— a 6 out of 6 match. People with these matches have a lower chance of graft- versus-host disease, graft rejection, having a weak immune system, and getting serious infections. There are thousands of different combinations of possible HLA tissue types. This can make it hard to find an exact match.

Blood Type

[0007] In 1901, Karl Landsteiner, an Austrian pathologist, randomly combined the serum and red blood cells of his colleagues. From the reactions he observed in test tubes, he discovered the ABO blood group system. This discovery earned him the 1930 Nobel Prize in Medicine.

[0008] A person's ABO blood type— A, B, AB, or O— is based on the presence or absence of the A and B antigens on his red blood cells. The A blood type has only the A antigen and the B blood type has only the B antigen. The AB blood type has both A and B antigens, and the O blood type has neither A nor B antigen.

[0009] By the time a person is six months old, he naturally will have developed antibodies against the antigens his red blood cells lack. That is, a person with A blood type will have anti-B antibodies, and a person with B blood type will have anti-A antibodies. A person with AB blood type will have neither antibody, but a person with O blood type will have both anti-A and anti-B antibodies. Although the distribution of each of the four ABO blood types varies, O is the most common and AB is the least common.

[0010] ABO typing is the first test done on blood when it is tested for transfusion. A person must receive ABO-matched blood. ABO incompatibilities are the major cause of fatal transfusion reactions. [0011] The Rh, or Rhesus, system was first detected in 1940 by Landsteiner and Wiener when they injected blood from rhesus monkeys into guinea pigs and rabbits. More than 50 antigens have since been discovered belonging to this system, making it the most complex red blood cell antigen system.

[0012] Currently, SVF transplantation therapy relies on autologous transplantation (e.g., the patient donates adipose tissue that is processed to yield SVF cells which are then transplanted into the same patient). However, there exists a need in the art for a broader base of donors and matching of donors potential recipients for stromal vascular fraction cell transplantation therapy. SUMMARY OF THE INVENTION

[0013] In one embodiment, a method for matching a donor with a recipient for a SVF transplantation may comprise (a) selecting SVF donor; (b) determining the SVF donor's blood type; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's blood type; and (e) matching the donor with a compatible recipient based on blood type.

[0014] In one embodiment, a method for stromal vascular fraction transplantation therapy may comprise (a) selecting SVF donor; (b) determining the SVF donor's blood type; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's blood type; (e) matching the donor with a compatible recipient based on blood type; (f) isolating the SVF from the SVF donor; and (h) administering the SVF composition to the SVF recipient.

[0015] In one embodiment, a method for matching a donor with a recipient for a SVF transplantation may comprise (a) selecting a SVF recipient in need of SVF therapy; (b) determining the SVF recipient's blood type; and (c) matching the SVF recipient with a SVF composition from a donor with a compatible blood type.

[0016] In one embodiment, a method for stromal vascular fraction transplantation therapy may comprise (a) selecting SVF donor; (b) determining the SVF donor's blood type; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's blood type; (e) matching the donor with a compatible recipient; (f) isolating the SVF from the SVF donor; and (h) administering the SVF composition to the SVF recipient.

[0017] In one embodiment, a method for matching a donor with a recipient for a SVF transplantation may comprise (a) selecting SVF donor; (b) determining the SVF donor's HLA; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's HLA and; (e) matching the donor with a compatible recipient. [0018] In one embodiment, a method for stromal vascular fraction transplantation may comprise (a) selecting SVF donor; (b) determining the SVF donor's HLA type; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's HLA type; (e) matching the donor with a compatible recipient; (f) isolating the SVF from the SVF donor; (g) admixing the SVF to form a SVF composition; and (h) administering the SVF composition to the SVF recipient.

[0019] In one embodiment, an allogeneic SVF composition for use in the manufacture of cosmetic surgery products, wherein the donor of the SVF may be matched to the recipient by blood type.

[0020] In one embodiment, an allogeneic SVF composition for use in the manufacture of cosmetic surgery products, wherein the donor of the SVF may be matched to the recipient by HLA.

[0021] In one embodiment, a method for cosmetic surgery may comprise (a) selecting SVF donor; (b) determining the SVF donor's blood type; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's blood type; (e) matching the donor with a compatible recipient; (f) isolating the SVF from the SVF donor; (g) admixing the SVF to form a SVF composition; and (h) administering the SVF composition to the SVF recipient.

[0022] In one embodiment, a method for cosmetic surgery may comprise (a) selecting SVF donor; (b) determining the SVF donor's HLA type; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's HLA type; (e) matching the donor with a compatible recipient; (f) isolating the SVF from the SVF donor; (g) admixing the SVF to form a SVF composition; and (h) administering the SVF composition to the SVF recipient.

[0023] In one embodiment, a method for allogeneic transplantation of SVF may comprise matching the SVF donor and SVF recipient by blood type, isolating SVF from said donor, and administering said isolated allogeneic SVF to said recipient.

[0024] In one embodiment, a method for allogeneic transplantation of SVF may comprise matching the SVF donor and SVF recipient by HLA type, isolating SVF from said donor, and administering said isolated allogeneic SVF to said recipient.

[0025] In one embodiment, an allogeneic composition for cosmetic surgery may comprise an effective amount of an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by blood type. [0026] In one embodiment, an allogeneic composition for cosmetic surgery may comprise an effective amount of an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by HLA type.

[0027] In one embodiment, an allogeneic composition for cosmetic surgery may comprise an effective amount of an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by blood type.

[0028] In one embodiment, an allogeneic composition for cosmetic uses may comprise an effective amount of an allogeneic SVF, wherein the donor of the SVF may be matched to the recipient by blood type.

[0029] In one embodiment, an allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally may comprise dermal fillers, optionally for the reduction of a skin defect, wherein the donor of the SVF may be matched to the recipient by blood typeA

[0030] In one embodiment, a method for reducing a skin defect may comprise administering an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by blood type.

[0031] In one embodiment, an allogeneic composition for reduction of a skin defect may comprise an effective amount of an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by blood type.

[0032] In one embodiment, an allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally may comprise dermal fillers, for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin, wherein the donor of the SVF may be matched to the recipient by blood type.

[0033] In one embodiment, a method for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin may comprise administering an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by blood type.

[0034] In one embodiment, an allogeneic composition for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improve skin texture, and tighten skin may comprise an effective amount of the SVF composition, wherein the donor of the SVF may be matched to the recipient by blood type. [0035] In one embodiment, an allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally may comprise dermal fillers, optionally for the reduction of a skin defect, wherein the donor of the SVF may be matched to the recipient by HLA type.

[0036] In one embodiment, a method for reducing a skin defect may comprise administering an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by HLA type.

[0037] In one embodiment, an allogeneic composition for reduction of a skin defect may comprise an effective amount of an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by HLA type.

[0038] In one embodiment, an allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally may comprise dermal fillers, for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin, wherein the donor of the SVF may be matched to the recipient by HLA type.

[0039] In one embodiment, a method for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin may comprise administering an allogeneic SVF composition, wherein the donor of the SVF may be matched to the recipient by HLA type.

[0040] In one embodiment, an allogeneic composition for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improve skin texture, and tighten skin may comprise an effective amount of the SVF composition, wherein the donor of the SVF may be matched to the recipient by HLA type.

[0041] In one embodiment, the use of the stromal vascular fraction in the manufacture of allogeneic cosmetic surgery products.

[0042] In one embodiment, a method for cosmetic surgery may comprise administering an allogeneic stromal vascular fraction composition.

[0043] In one embodiment, a composition for cosmetic surgery may comprise an effective amount of an allogeneic stromal vascular fraction composition.

[0044] In one embodiment, the use of an allogeneic stromal vascular fraction composition in the manufacture of a medicament for the treatment of a disease.

[0045] In one embodiment, a cosmetic composition may comprise an effective amount of an allogeneic stromal vascular fraction. In another embodiment, the composition may be in a form selected from the group consisting of a balm, solution, suspension, emulsion, ointment, foam, paste, gel, cream, lotion, powder, salve, soap, surfactant-containing cleansing, oil, serum, drops, liposomes, nanoparticles, nanoboots, and spray. In another embodiment, the composition may be formulated for topical administration.

[0046] In one embodiment, a method of treating a disease may comprise administering an allogeneic stromal vascular fraction composition.

[0047] In one embodiment, a composition for treating a disease may comprise an effective amount of an allogeneic stromal vascular fraction composition.

[0048] In one embodiment, the use of an allogeneic stromal vascular fraction composition in the manufacture of medicament for allogeneic transplantation to treat a disease.

[0049] In one embodiment, a method for treating a disease may comprise administering an allogeneic transplant may comprise an allogeneic stromal vascular fraction composition.

[0050] In one embodiment, a composition for allogeneic transplantation may comprise an effective amount of an allogeneic stromal vascular fraction composition.

[0051] In one embodiment, a method for treating a disease may comprise obtaining stromal vascular fraction from a patient and administering the stromal vascular fraction to the same patient to treat said disease.

[0052] In one embodiment, a method for allogeneic transplantation may comprise transplanting an allogeneic stromal vascular fraction composition.

[0053] In one embodiment, a pharmaceutical composition for the treatment of a disease may comprise an allogeneic stromal vascular fraction composition.

[0054] In one embodiment, a pharmaceutical composition for the treatment of a disease may comprise an allogeneic stromal vascular fraction composition.

[0055] In one embodiment, a method for treating rosacea, psoriasis, acne, eczema, and atopic dermatitis may comprise administering an allogeneic stromal vascular fraction composition, optionally the SVF composition may be applied topically.

[0056] In one embodiment, a cosmetic method for treating wrinkles, tone, text, large pores, dullness, or loose skin, may comprise administering an effective amount of an allogeneic stromal vascular fraction composition, optionally the allogeneic SVF composition may be applied topically. [0057] In one embodiment, a method for augmenting soft tissue to provide relief of a skin defect may comprise administering an effective amount of an allogeneic stromal vascular fraction composition.

[0058] In one embodiment, a method for augmenting soft tissue to provide reduction of a skin defect may comprise topically applying to the skin defect an allogeneic stromal vascular fraction composition.

[0059] In one embodiment, a method for augmenting soft tissue to provide reduction of a skin defect, optionally wherein the skin defect may be a dynamic wrinkle, a fine wrinkles or a static wrinkle, may comprise administering an effective amount of an allogeneic stromal vascular fraction composition.

[0060] In one embodiment, a method of augmenting soft tissue to provide long-term reduction of a skin defect may comprise administering an effective amount of an allogeneic stromal vascular fraction composition. In another embodiment, the skin defect may be the result of loss of collagen and hyaluronic acid in the skin during the aging process. In another embodiment, the skin defect may be the result of premature aging, said premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders. In another embodiment, the stromal vascular fraction are used in a cosmetic surgery application, to promote wound healing, are used in a tissue filler or in association with breast augmentation, breast reconstruction, tissue engineering, or burn treatment.

[0061] In another embodiment, the SVF may be allogeneic and matched with the donor by blood type. In another embodiment, the SVF may be allogeneic and matched with the donor by HLA.

[0062] In another embodiment, the SVF composition does not include the addition of an endopeptidase, optionally collagenase.

[0063] In another embodiment, the animal may be a mammal, optionally a human.

[0064] In another embodiment, the SVF may be obtained from the stromal or mesenchymal compartment of a human cadaver, tissue bank, organ donation, solid fat obtained from a human cadaver, or a liposuction derived aspirate. In another embodiment, the animal may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post-mortem. In another embodiment, the animal may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-mortem. [0065] In another embodiment, the stromal vascular fraction comprises mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts,

megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, CD29+ cells, CD166+ cells, Thy-1+ stem cells, CD90+ stem cells, CD44+ cells, monocytes, leukocytes, lymphocytes, B cells, T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, and neutrophil granulocytes.

[0066] In another embodiment, the allogeneic hematopoietic stem cells are isolated from the allogeneic stromal vascular fraction.

[0067] In another embodiment, the allogeneic hematopoietic stem cells are used in lieu of bone marrow for therapeutic uses.

[0068] In another embodiment, the SVF may be isolated by mechanical, enzymatic, and/or chemical treatment.

[0069] In another embodiment, the SVF composition comprises at least about l lO⁶ to lxlO⁷ stromal vascular cells per mL.

[0070] In another embodiment, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of said stromal vascular fraction cells are viable.

[0071] In another embodiment, the method may further comprise administering stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells.

[0072] In another embodiment, the isolated stromal vascular fraction does not comprise any exogenous collagenase.

[0073] In another embodiment, the stromal vascular fraction comprises hematopoietic elements.

[0074] In another embodiment, the stromal vascular fraction comprises extracellular matrix (ECM) materials.

[0075] In another embodiment, the white blood cells have been removed from the stromal vascular fraction. In another embodiment, the stromal vascular fraction may be essentially free of white blood cells.

[0076] In another embodiment, the disease may be gum recession, loss of bone, including the jaw, amyotrophic lateral sclerosis (ALS), arthritis, optionally rheumatoid arthritis, autism, diabetes, optionally Type I diabetes, bone fractures, chronic obstructive pulmonary disease (COPD), dermal treatment for burns and non-healing wounds, enterocutaneous fistula

(HULPUTC), gingival gum regeneration, hair loss (in both men and women), gum recession, ischemic heart failure, microvascular protection treatment in a myocardial infarction, migraine, multiple sclerosis, orthopedic problems, osteoarthritis, plantar fascitis, recto-vaginal fistula, rheumatoid arthritis, rotator cuff injuries, sports medicine injuries, optionally tears and sprains of the ligaments and tendons, tennis elbow, tinnitus, ulcers, and non-healing wounds.

[0077] In another embodiment, the composition may further comprise tissue fillers. In another embodiment, the use, method, or composition comprises treatment alone or in combination with tissue fillers.

[0078] In one embodiment, the use, method, or composition may be for the reduction of a skin defect. In another embodiment, the skin defect may be a dynamic wrinkle, a fine wrinkles or a static wrinkle. In another embodiment, the dynamic wrinkle may be a forehead crease, a brow burrow or an eye line (crow's feet). In another embodiment, the static wrinkle may be a skin fold wrinkle resulting from sagging skin. In another embodiment, the skin defect may be a medical condition selected from the group consisting of an acne scar, optionally a "rolling" scar, a "boxcar" scar or an "ice pick" scar, a surgical scar, trauma scar, a large pore and a soft tissue contour defect. In another embodiment, the wrinkle or scar may be the result of loss of collagen and hyaluronic acid in the skin during the aging process. In another embodiment, the wrinkle or scar may be the result of premature aging, optionally premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

[0079] In one embodiment, the medical condition may be a deformity that requires re- contouring, such as a small tissue defect, optionally after animal bite(s)) or a deformity related to trauma wherein the deformity may be cosmetically unappealing. In another embodiment, the augmentation may be done after plastic surgery to achieve symmetry or a desired result. In another embodiment, the long-term reduction of a skin defect may be of a duration of at least one year, one year to about five years, five years to about ten years, or ten years or longer.

[0080] In another embodiment, the stromal vascular faction may be isolated from a tissue or organ may comprise subjecting said tissue to mechanical, chemical, and or enzymatic treatment to release the stromal vascular fraction.

[0081] In one embodiment, the method comprises subjecting said tissue to ultrasonic cavitation, wherein the cells in the tissue and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction. In another embodiment, the method comprises subjecting a tissue to ultrasonic cavitation may comprise bringing said tissue into contact with a powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate. In another embodiment, the method may be performed in situ in a patient.

[0082] In another embodiment, the method may further comprise removing the lysed tissue may comprise the lysed blood vessels and the stromal vascular fraction, optionally adipose tissue. In another embodiment, the method may further comprise identifying and targeting the tissue using ultrasound.

[0083] In one embodiment, the ultrasonic cavitation may be applied directly externally to the tissue, optionally applied to the patient's skin and the blood vessels are lysed to release the stromal vascular fraction. In another embodiment, the ultrasonic cavitation rod may be inserted through a puncture or incision in the skin into the patient's tissue and lyse the blood vessels to release stromal vascular fraction. In another embodiment, the tissue may be located using ultrasound device and then removed using indirect cavitation device and subject to ultrasonic cavitation to release the stromal vascular fraction. In another embodiment, the tissue is, optionally located using ultrasound device, removed subject to ultrasonic cavitation by an ultrasonic flow cell device, optionally, an Ultrasonic Mini Row Cell, to release the stromal vascular fraction, optionally, wherein the stromal vascular fraction may be further isolated.

[0084] In another embodiment, the stromal vascular fraction may be isolated by a method may comprise providing about 40-60 mL of adipose tissue obtained from a nonliving animal; treating said adipose tissue with ultrasonic cavitation using an about 13-14 mm probe for about 10 minutes at about 24 kHz, wherein the adipose cells and blood vessels in the adipose tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated adipose tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction. In another embodiment, the ultrasonic cavitation may be effected for about 5 minutes, paused, and then continued for another 5 minutes for a total of 10 minutes. In another embodiment, the probe may be placed towards the bottom of the tissue sample for a first 5 minute period, paused, and then the probe may be moved upwards to about half-way in the tissue sample and continued for the second 5 minute period. In another embodiment, the ultrasonic cavitation may be at a frequency of about 20-30 kHz, optionally about 20, 21, 22, 23, 24, 24, or 25 kHz, or optionally about 20-23 kHz or 23-25 kHz. In another embodiment, the ultrasonic cavitation may be performed using an ultrasonic probe of about 10-15 mm, about 13 or 14 mm probe, optionally about 14 mm. In another embodiment, the probe may be 14 mm. In another embodiment, the ultrasonic cavitation device may have a 200W-500W generator, optionally a 200W generator.

[0085] In another embodiment, the stromal vascular fraction may be isolated by a method may comprise providing a tissue obtained from an animal; mixing said a tissue with tumescent fluid; and homogenizing said tissue with beads for about 30 seconds to 6 minutes, wherein the tissue cells and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction. In another embodiment, the method may further comprise isolating the stromal vascular fraction (SVF), optionally removing the beads. In another embodiment, the adipose tissue may be comminuted prior to homogenization with beads. In another embodiment, the beads are stainless steel, zirconium oxide, tungsten carbide, ceramic, zirconium silicate, or glass beads, optionally zirconium oxide beads. In another embodiment, the beads are about 0.01-2 mm beads, optionally 2 mm beads. In another embodiment, the beads are added at a bead-to- sample ratio of about 1:1-1:4, optionally 1:4 (25% beads by volume). In another embodiment, the sample comprises about 7 mL of tissue, optionally adipose tissue. In another embodiment, the sample may be mixed with about 3 mL of tumescent fluid.

[0086] In another embodiment, the method may comprise providing about 7 mL of tissue obtained from an animal; mixing said tissue with about 3 mL of tumescent fluid; homogenizing said tissue with about 5 mL of 2 mm zirconium oxide beads for about 3 minutes at a bead-to- sample ratio of 1:3, wherein the tissue cells and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction; and isolating the stromal vascular fraction (SVF) cells, optionally removing the beads.

[0087] In another embodiment, the sample may be homogenized with beads for about 3 minutes. In another embodiment, the tissue may be mechanically treated using a homogenizer, optionally a rotor stator homogenizer; a dounce; mortar and pestle; tissue mill, mixer-mill, or bead-beater assembly; blender; spin column homogenizer; or a sonicator. In another embodiment, the tissue may be mechanically comminuted, optionally by grinding, dicing, slicing, chopping up, comminuting, grinding with mortar and pestal, granulating, pressing, cubing, mincing, milling, grating, grading, crushing, rolling, shearing, dividing, or hewing.

[0088] In another embodiment, after mechanical comminuting, the tissue may be further treated with enzymes, chemicals, or ultrasonic cavitation.

[0089] In another embodiment, the amount of tissue may be about 50 mL. In another embodiment, the sample comprises about 40, 45, 50, 55, or 60 cc of tissue.

[0090] In another embodiment, the method may further comprise isolating the stromal vascular fraction (SVF). In another embodiment, the method does not include the addition of an endopeptidase, optionally collagenase.

[0091] In another embodiment, the animal may be a mammal, optionally a human. In another embodiment, the tissue may be obtained from the stromal or mesenchymal compartment of a human cadaver, tissue bank, organ donation, solid fat obtained from a human cadaver, or a liposuction derived aspirate. In another embodiment, the animal may be at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post-mortem. In another embodiment, the animal may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days postmortem.

[0092] In another embodiment, the tissue may be comprised in phosphate buffered saline, normal saline, or another biologically acceptable liquid.

[0093] In another embodiment, the tissue comprises blood vessels. In another embodiment, the tissue may be selected from the group consisting of connective tissue, muscle tissue, adipose tissue, nervous tissue, and epithelial tissue. In another embodiment, the connective tissue may be selected from the group consisting of blood, bone, and extracellular matrix. In another embodiment, the nervous tissue may be selected from the group consisting of neural tissue selected from the group consisting of central nervous system may comprise the brain and spinal cord and peripheral nervous system may comprise cranial nerves and spinal nerves may comprise motor neurons. In another embodiment, the muscle tissue may be selected from the group consisting of skeletal (striated) muscle, cardiac muscle, and smooth muscle. In another embodiment, the epithelial tissue may be selected from the group consisting of squamous epithelium, cuboidal epithelium, columnar epithelium, glandular epithelium, and ciliated epithelium.

[0094] In another embodiment, the organ comprises blood vessels. In another embodiment, the organ may be heart, lung, liver, bladder, kidney, pancreas, or stomach.

[0095] In another embodiment, the method may further comprise allowing the treated tissue, optionally adipose tissue, to settle or may be centrifuged, optionally for about 3 minutes at 500

RCF (relative centrifugal force), resulting in the fat rising to the top of the sample.

[0096] In another embodiment, the stromal vascular fraction comprises mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts,

megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, CD29+ cells, CD 166+ cells, Thy-1+ stem cells, CD90+ stem cells,

CD44+ cells, monocytes, leukocytes, lymphocytes, B cells, T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, and neutrophil granulocytes.

[0097] In another embodiment, after mechanical, enzymatic, and/or chemical treatment, the sample may be assayed, optionally by flow cytometry, for the presence of adipose-derived stem cells including CD34 and/or Thy-1 or CD90 expressing stem cells.

[0098] In another embodiment, after mechanical, enzymatic, and/or chemical treatment, the sample may be fractionated using fluorescence activated call sorting (FACS) based on cell surface antigens which are specific to stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells.

[0099] In another embodiment, the method may further comprise isolating the stromal vascular fraction and cryopreserving said stromal vascular fraction. In another embodiment, the method results in a yield of at least about lxlO⁶ to lxlO⁷ stromal vascular cells per mL of adipose tissue. In another embodiment, at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of said stromal vascular fraction cells isolated are viable.

[0100] In another embodiment, the method may further comprise isolating stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, from said stromal vascular fraction.

[0101] In another embodiment, the stromal vascular fraction are isolated from a tissue may comprise (a) subjecting said tissue to ultrasonic cavitation may comprise bringing said tissue into contact with a powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate, optionally wherein said method may be performed in situ in a patient, wherein the cells in the tissue and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction, and (b) removing the lysed tissue may comprise the lysed blood vessels and the stromal vascular fraction. In another embodiment, the said tissue may be adipose tissue.

[0102] In a further embodiment, the stromal vascular fraction are isolated from a tissue may comprise (a) identifying the tissue using ultrasound; (b) subjecting said tissue to ultrasonic cavitation may comprise bringing said tissue into contact with a powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate, wherein said ultrasonic cavitation may be applied directly externally to the tissue, optionally applied to the patient's skin, and wherein the blood vessels are lysed, dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction, and (c) removing the lysed tissue may comprise the lysed blood vessels and the stromal vascular fraction. In another embodiment, the method may be performed in situ in a patient. In another embodiment, the tissue may be adipose tissue.

[0103] In a further embodiment, the stromal vascular fraction are isolated from a tissue may comprise (a) identifying the tissue using ultrasound; (b) subjecting said tissue to ultrasonic cavitation may comprise inserting a cavitation rod through a puncture or incision in the skin into the patient's tissue and lyse the blood vessels to release stromal vascular fraction while substantially maintaining the viability of the cells constituting the stromal vascular fraction, and (c) removing the lysed tissue may comprise the lysed blood vessels and the stromal vascular fraction.

[0104] In a further embodiment, the stromal vascular fraction are isolated from a tissue may comprise (a) subjecting said tissue to ultrasonic cavitation may comprise inserting a cannulae through a puncture or incision in the skin into the patient's tissue, and (c) extracting the tissue may comprise blood vessels and the stromal vascular fraction. In another embodiment, the tissue may be subject to indirect ultrasonic cavitation to release the stromal vascular fraction. In another embodiment, the ultrasonic cavitation may be by an ultrasonic flow cell device, optionally an Ultrasonic Mini Flow Cell, powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate.

[0105] In one embodiment, a composition may comprise the stromal vascular fraction.In another embodiment, the composition may be in a form selected from the group consisting of a balm, solution, suspension, emulsion, ointment, foam, paste, gel, cream, lotion, powder, salve, soap, surfactant-containing cleansing, oil, serum, drops, liposomes, nanoparticles, nanoboots, and spray. In another embodiment, the composition may be a cream, lotion, or solution.

[0106] In one embodiment, a method for formulating an autologous skin cream may comprise isolating SVF from a patient and compounding to form a skin cream may comprise autologous

SVF for said patient. In another embodiment, the SVF composition may be administered topically. In one embodiment, a method for formulating an allogeneic SVF topical skin cream may comprise matching a SVF composition with a patient by blood type and compounding to form a topical skin cream may comprise allogeneic SVF.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0107] In order that the invention herein described may be fully understood, the following detailed description is set forth. Various embodiments of the invention are described in detail and may be further illustrated by the provided examples. Additional viable variations of the embodiments can easily be envisioned.

Definitions

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs.

[0109] As used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the" includes plural reference unless the context clearly dictates otherwise.

[0110] "About," will be understood by persons of ordinary skill in the art and will vary to some extent based on the context in which it is used.

[0111] "Adipose tissue-derived cell," as used herein, refers broadly to a cell that originates in adipose tissue, from the blood vessels contained therein. The initial cell population isolated from adipose tissue is a heterogeneous cell population including, but not limited to, stromal or mesenchymal vascular fraction (SVF). [0112] "Adipose tissue," as used herein, refers broadly to any fat tissue. The adipose tissue may be brown or white adipose tissue. The adipose may be mesenchymal or stromal. Preferably, the adipose tissue is subcutaneous white adipose tissue. The adipose tissue may be from any organism having fat tissue. Preferably the adipose tissue is mammalian, most preferably the adipose tissue is human. A convenient source of human adipose tissue is that derived from liposuction surgery or other surgery. Adipose tissue may be obtained from non-living donors, including animals, including mammals, post-mortem.

[0113] "Adipose-derived stem cell (ADSC or ASC) ," as used herein, refers broadly to mesenchymal stem cells that originate from blood vessels found in adipose tissue which can serve as stem cell-like precursors to a variety of different cell types including but not limited to adipocytes, osteocytes, chondrocytes, muscle and neuronal/glial cell lineages. Adipose-derived stem cells make up a subset population derived from adipose tissue which can be separated from other components of the adipose tissue using standard culturing procedures or other methods disclosed herein. In addition, adipose-derived adult stem cells can be isolated from a mixture of cells using the cell surface markers disclosed herein. Also, adipose-derived stem cells are known as mesenchymal stem cells in the art.

[0114] "Adipose cell," as used herein, refers broadly to any type of adipose tissue, including an undifferentiated adipose-derived adult stem cell and a differentiated adipose-derived adult stem cell.

[0115] "Allogeneic," as used herein, refers broadly to any material derived from a different mammal of the same species.

[0116] "Allograft," as used herein, refers broadly to a tissue graft from a donor genetically unrelated to the recipient.

[0117] "Allotransplantation," as used herein, refers broadly to the transplantation of cells, tissues, or organs, to a recipient from a (genetically non-identical) donor from the same species. Allotransplants may be referred to an allograft, allogeneic transplant, or homograft in the art.

[0118] "Applicator," as used herein, refers broadly to any device including, but not limited to, a hypodermic syringe, a pipette, for administering the compounds and compositions of the invention.

[0119] "Autograft," as used herein, refers broadly to a tissue transplanted from one site to another on the same patient (e.g., removal of SVF cells and transplant to another site). [0120] "Autologous," as used herein, refers broadly to any material derived from the same individual to which it is later to be re-introduced.

[00100] "Blood type," as used herein, refers broadly to the blood type based on the presence or absence of the A and B antigens on the red blood cells (e.g.. A, B, O, and AB blood types). "ABO Blood Type," may be used interchangeably with blood type. "Rh Blood Type," as used herein, refers broadly to the presence (+) or absence (-) of D antigen on red blood cells. Further, "Blood type" and "ABO blood type" may be used interchangeably to refer to the blood type of an individual according to the presence of A, B, O, and D antigens (e.g., A+, A-, AB-). Finding a compatible match between a donor and recipient based on blood type is well known in the art.

[00101] "Crossmatch," as used herein, refers broadly to a laboratory test done to confirm that blood from a donor and blood from the recipient are compatible.

[0121] "Central nervous system," as used herein, refers broadly to include brain and/or the spinal cord of a mammal. The term may also include the eye and optic nerve in some instances.

[0122] "Cosmetically or aesthetically effective amount," as used herein, refers broadly to a compound or cells is that amount of compound or cells which is sufficient to provide a cosmetically or aesthetically beneficial effect to the subject to which the compound or cells are administered such as skin rejuvenation, enhancement in plumpness or volume or appearance of treated tissue such as the cheeks, lips, buttocks, or breast tissue. Also, as used herein, a

"cosmetically effective amount" is the amount of cells which is sufficient to provide a beneficial effect to the subject to which the cells are administered.

[0123] "Differentiated," as used herein, refers broadly to a cell that has achieved a terminal state of maturation such that the cell has developed fully and demonstrates biological specialization and/or adaptation to a specific environment and/or function. Typically, a differentiated cell is characterized by expression of genes that encode differentiation-associated proteins in that cell. For example expression of GALC in a leukocyte is a typical example of a terminally

differentiated leukocyte.

[0124] "Differentiation medium," as used herein, refers broadly to a cell growth medium comprising an additive or a lack of an additive such that a stem cell, adipose tissue derived stromal cell, embryonic stem cell, ES-like cell, MSCs, neurosphere, NSC or other such progenitor cell, that is not fully differentiated when incubated in the medium, develops into a cell with some or all of the characteristics of a differentiated cell.

[0125] "Differentiating," as used herein, refers broadly to a cell that is in the process of being differentiated.

[0126] "Differentiated adipose-derived adult stem cell" refers broadly to an adipose-derived adult stem cell isolated from any adipose tissue that has differentiated as defined herein.

[0127] "Undifferentiated adipose-derived adult stem cell," as used herein, refers broadly to a cell isolated from adipose tissue and cultured to promote proliferation, but has no detectably expressed proteins or other phenotypic characteristics indicative of biological specialization and/or adaptation.

[0128] "Effective amount," as used herein, refers broadly to a compound is that amount of compound or cells which is sufficient to provide a beneficial effect to the subject to which the compound is administered. Also, as used herein, an "effective amount" is the amount of cells which is sufficient to provide a beneficial effect to the subject to which the cells are

administered.

[0129] "Endogenous," as used herein, refers broadly to any material from or produced inside an organism, cell or system.

[0130] "Exogenous," as used herein, refers broadly to any material introduced from or produced outside an organism, cell, or system. In particular exogenous may refer to a material that is not present in the treated adipose tissue.

[0131] "Isolated," as used herein, refers broadly to material removed from its original environment in which it naturally occurs, and thus is altered by the hand of man from its natural environment. Isolated material may be, for example, exogenous nucleic acid included in a vector system, exogenous nucleic acid contained within a host cell, or any material which has been removed from its original environment and thus altered by the hand of man (e.g., "isolated cell"). For example, "isolated" or "purified," as used herein, refers broadly to a protein, cell, DNA, antibody, RNA, or biologically active portion thereof, that is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the biological substance is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. For example, "isolated cell" refers broadly to a cell which has been separated from other components and/or cells which naturally accompany the isolated cell in a tissue or mammal.

[0132] "Isograft," as used herein, refers broadly to a transplanted organ or tissue from a genetically identical donor (i.e., identical twin).

[0133] "Graft," as used herein, refers broadly to a cell, tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect. A graft may further comprise a scaffold. The tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: "autograft", "autologous transplant", "autologous implant" and "autologous graft". A graft comprising cells from a genetically different individual of the same species is referred to herein by the following interchangeable terms: "allograft", "allogeneic transplant", "allogeneic implant" and "allogeneic graft". A graft from an individual to his identical twin is referred to herein as an "isograft", a "syngeneic transplant", a "syngeneic implant" or a "syngeneic graft". A "xenograft",

"xenogeneic transplant" or "xenogeneic implant" refers to a graft from one individual to another of a different species.

[0134] "Immunophenotype," as used herein, refers broadly to cell is used herein to refer to the phenotype of a cell in terms of the surface protein profile of a cell.

[0135] "Late passaged adipose tissue-derived stromal cell," as used herein, refers broadly to a cell exhibiting a less immunogenic characteristic when compared to an earlier passaged cell. The immunogenicity of an adipose tissue-derived stromal cell corresponds to the number of passages. Preferably, the cell has been passaged up to at least the second passage, more preferably, the cell has been passaged up to at least the third passage, and most preferably, the cell has been passaged up to at least the fourth passage.

[0136] "Mammal," as used herein, refers broadly to any and all warm-blooded vertebrate animals of the class Mammalia, including humans, characterized by a covering of hair on the skin and, in the female, milk-producing mammary glands for nourishing the young. Examples of mammals include but are not limited to alpacas, armadillos, capybaras, cats, camels,

chimpanzees, chinchillas, cattle, dogs, elephants, goats, gorillas, hamsters, horses, humans, lemurs, llamas, mice, non-human primates, pigs, rats, sheep, shrews, squirrels, tapirs, and voles. Mammals include but are not limited to bovine, canine, equine, feline, murine, ovine, porcine, primate, and rodent species. Mammal also includes any and all those listed on the Mammal Species of the World maintained by the National Museum of Natural History, Smithsonian Institution in Washington DC.

[0137] "Mesenchymal stromal cells" or "Mesenchymal stem cells," as used herein, refers broadly to cells derived from a stromal vascular fraction that have a multipotent differentiation potential (e.g., adipogenic, chondrogenic, and osteogenic) and may show CD73⁺, CD90⁺, CD105⁺, CD1 lb/14^", CD19/CD73b^~, CD34^", CD45^", and HLA-DR^". See, e.g., Gimble, et al. (2011) Stem Cells 29: 749-754 and Alexander (2012) Journal of Prolotherapy [online].

[0138] "Multipotential" or "multipotentiality," as used herein, refers broadly to the capability of a stem cell to differentiate into more than one type of cell.

[0139] "Phenotypic characteristics," as used herein, refers broadly to mean at least one of the following characteristics: morphological appearance, the expression of a specific protein, a staining pattern or the ability to be stained with a substance.

[0140] "Precursor cell," "progenitor cell," and "stem cell" are used interchangeably in the art and herein and refer broadly either to a pluripotent, or lineage-uncommitted, progenitor cell, which is potentially capable of an unlimited number of mitotic divisions to either renew itself or to produce progeny cells which will differentiate into the desired cell type. In contrast to pluripotent stem cells, lineage-committed progenitor cells are generally considered to be incapable of giving rise to numerous cell types that phenotypically differ from each other.

Instead, progenitor cells give rise to one or possibly two lineage-committed cell types.

[0141] "Rh blood type," as used herein refers broadly to the blood type based on the presence or absence of the Rh factor antigen (e.g., D antigen) on the red blood cells (e.g., A- indicates an A type blood type lacking the Rh factor).

[0142] "Stromal vascular fraction," as used herein, refers broadly to a cell fraction derived from blood vessels found in adipose tissue that comprises different cell types including by way of example mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts, megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells, CD29+ cells, CD166+ cells, Thy-1+ or CD90+ stem cells, CD44+ cells, immune cells such as monocytes, leukocytes, lymphocytes, B and T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, neutrophil granulocytes, and the like including immune and other cells that express one or more of the following markers: CD3, CD 14

(macrophage marker), CD19, CD20 (B cell marker), CD29 (integrin unit), CD31 (endothelial, platelet, macrophage, Kupffer cell, dendritic cell, granulocyte, T/NK cells, lymphocytes, megakaryocytes, osteoclasts, neutrophils, et al.), CD44 (Hyaluronic acid receptor), CD45 (B and T cell marker), C56, CD73 (lymphocyte differentiation marker), CD105. Also, it includes cells expressing any of the markers or any combination thereof disclosed in this application.

[0143] "Treat," as used herein, refers broadly to reduce the frequency of the disease or disorder reducing the frequency with which a symptom of the one or more symptoms disease or disorder is experienced by an animal.

[0144] "Vascularized" or "vascular," as used herein, refers broadly to of, relating to, affecting, or consisting of a vessel or vessels, especially, those that carry blood. Vascularized refers to any tissue that comprises a blood vessel.

[0145] "Xenogeneic," as used herein, refers broadly to any material derived from a mammal of a different species.

[0146] "Xenograft," as used herein, refers broadly to a transplant from another species (e.g., transplant from a donor of one species to recipient of another different species).

Allogeneic Transplantation of SVF Based on Blood Type and HLA

[0147] Blood banking has been a staple of medical practice for decades. Allogeneic blood for transfusion is normally separated by blood type and removal of the leukocytes and white blood cells to reduce the immune response and limit rejection by the recipient. Stromal vascular fraction cells are vascular cells because these cells reside in the blood vessel walls of every blood vessel in the animal organism. As blood transfusion is generally considered safe when separated by blood typing ensuring that donor and recipient have a compatible blood type, the same method may apply for the cells residing in the blood vessel walls of donor and recipient. Thus, blood type compatibility may be used as a proxy for determining the compatibility of SVF donors and recipients. For example, if a donor and recipient have a compatible blood type for purposes of blood transfusion, these same rules of determining compatibility of blood transfusion may be used for determining compatibility of SVF transplantation. For example, generally a person with an "A" blood type may accept blood from an A donor. A person with a "B" blood type may accept blood from an B donor. A person with an "AB" blood type may accept blood from an AB donor. A person with an "O" blood type may accept blood from an O donor. With the addition of the Rh factor (+ or -) a person may make a determination of whether a donor and recipient have compatible blood types. For example, a person with a "Rh positive" ("+") blood type may accept either + or - blood type. Therefore, a person with an "A+" blood type may accept blood from an A+ or A- donor.

[0148] As the SVF is vascular in origin, SVF derived from whole blood and blood types and Rh factors are used to find matches for blood transfusion, the same practice may work for SVF administration. For example, if the recipient has a compatible blood type and Rh factor with the SVF donor, this may be used as a means for determining an allogeneic match. The method may comprise determining a patient's blood type, matching the patient's blood type to the blood type of the SVF donor, and administering SVF composition to said patient if the blood type of the patient matches the SVF donor. The determination of allogeneicy for the SVF transplant may be the same as the criteria for standard whole blood transfusion (e.g., match the blood type with compatible blood types and Rh factor status with compatible Rh factor status, e.g., Rh negative can be donated to Rh negative and positive patients, but Rh positive SVF may only be donated to Rh positive recipients). Methods for determining a compatible match between a donor and recipient based on blood type is well known in the art. See American Red Cross website "Blood Types" (2013). Methods for determining a person's blood type are also routine and well-known in the art. The inventor surprisingly discovered that the SVF may be determined to be allogeneic based on the blood type of the donor and recipient.

TABLE 1: BLOOD TYPE COMPATIBILITY CHART AS PROXY FOR SVF

C

[0149] Therefore, an allogeneic product may be developed, from living and non-living donors, by processing the SVF, removing the leukocytes (white blood cells), cryopreserving the processed cells, and manufacturing them to cGMP and/or cGTP standards for in-human and research use.

[0150] The donor may be the same patient who is to be treated with the stromal or mesenchymal stem cells derived therefrom (e.g., autograft). Additionally, the donor may be an allogeneic donor that is immune compatible with the treated individual. For example, a patient may undergo liposuction to obtain the tissue. The tissue may be subjected to the methods described herein to isolate the stromal vascular fraction (SVF), optionally mesenchymal stem cells derived therefrom. The stromal vascular fraction (SVF) may be isolated by ultrasonic cavitation as described in U.S. Patent Application Publication No. 2012/0164113 or the methods described in U.S. Provisional Patent Application Nos. 61/674,116; 61/721,917; 61/693,982; or 61/773,482. The stromal vascular fraction (SVF), optionally mesenchymal stem cells derived therefrom, may then be used in therapeutic methods, uses, or compositions.

[0151] Cells may be derived from the individual to be treated or a matched donor. Those having ordinary skill in the art can readily identify matched donors using standard techniques and criteria. For example, the stromal vascular fraction cells are vascular and reside in the walls of all blood vessels in the body and now that the method described herein can be used to harvest these cells from non-living tissue. The harvested cells can be stored in cryostorage based on their donor's blood type and be used allogenically with culturing. For example, the SVF may be stored and be readily available to treat patients for a multitude of injuries (e.g., orthopedic, post myocardial infarction). The stromal vascular fraction cells stored may be matched to the patient by blood type and/or tissue type to ensure an allogeneic match. The stromal vascular fraction cells may be stored at +4°C, -20°C, or -70°C.

[0152] Cells may be derived from the individual to be treated or a matched donor. Those having ordinary skill in the art can readily identify matched donors using standard techniques and criteria. For example, the stromal vascular fraction cells are vascular and reside in the walls of all blood vessels in the body and now that the method described herein can be used to harvest these cells from non-living tissue. The harvested cells can be stored in cryostorage based on their donor's blood type and be used allogenically with culturing. For example, the SVF may be stored and be readily available to treat patients for a multitude of injuries (e.g., orthopedic, post myocardiacal infarction). The stromal vascular fraction cells stored may be matched to the patient by blood type and/or tissue type to ensure an allogeneic match. The stromal vascular fraction cells may be stored at +4°C, -20°C, or -70°C.

[0153] The allogeneic SVF compositions described herein may be used in autograft methods. For example, a patient may have tissue removed, optionally adipose tissue (e.g., by liposuction), the SVF cells may be isolated from the tissue, optionally adipose tissue, using the methods described herein, and then transplanted to another area of the same patient.

[0154] The allogeneic SVF compositions described herein may be used in allogeneic transplant methods. For example, a first patient may have tissue removed, optionally adipose tissue (e.g., by liposuction), the SVF cells may be isolated from the tissue, optionally adipose tissue, using the methods described herein, and then transplanted to a second allogeneic patient.

[0155] The allogeneic SVF compositions described herein may be used in autograft methods. For example, a patient may have tissue removed, optionally adipose tissue (e.g., by liposuction), the SVF cells may be isolated from the tissue, optionally adipose tissue, using the methods described herein, and then transplanted to another area of the same patient.

[0156] The allogeneic SVF compositions described herein may be used in allogeneic transplant methods. For example, a first patient may have tissue removed, optionally adipose tissue (e.g., by liposuction), the SVF cells may be isolated from the tissue, optionally adipose tissue, using the methods described herein, and then transplanted to a second allogeneic patient.

[0157] The SVF may be autologous, i.e., derived from the same donor or the SVF may be derived from a compatible donor. Methods of HLA tissue matching are well known in the art. Accordingly, a recipient patient may be matched using HLA tissue matching with a donor patient (e.g., the whole blood may be harvested, the SVF isolated therefrom, and then administered to an HLA-matched recipient.)

[0158] Major histocompatibility complex or HLA antigens may be used for matching between a SVF donor and a SVF recipient. In this process, it is analyzed which six of the HLA antigens both individuals have and a determination of the closeness of tissue matching is made. A six- antigen match (both people have the same set of six antigens) is the best compatibility between a donor recipient pair who is not identical twins. This match occurs 25 percent of the time between siblings having the same mother and father and also occurs from time-to-time in the general population. Thus, The allogeneic SVF compositions described herein may be used in allogeneic matches for SVF composition therapies based on HLA antigen matching.

[0159] The donor may be the same patient who is to be treated with the SVF composition (e.g., autograft). Additionally, the donor may be an allogeneic donor that is immune compatible with the treated individual. The SVF may be derived from the individual to be treated or a matched donor. Those having ordinary skill in the art can readily identify matched donors using standard techniques and criteria.

[0160] The harvested cells can be stored in cryostorage based on their donor's blood type and be used allogenically. For example, the SVF may be stored and be readily available to treat patients for a multitude of injuries (e.g., orthopedic, post myocardiacal infarction). The SVF composition may be matched to the patient by blood type and/or tissue type to ensure an allogeneic match. The SVF may be tested to match common blood types and Rh factors to recipients and then allogeneic and frozen to be stored for use by patients requiring cellular therapies in physician's offices and hospitals at future dates. The inventor surprisingly discovered that as SVF is derived from whole blood, and the patient's blood type may be used to find matches for blood transfusion, the same practice unexpectedly applies to SVF administration.

[0161] The allogeneic SVF compositions described herein may be used in autograft methods. For example, a patient may have whole blood removed, the SVF may be isolated from the blood, using methods known in the art, admixed to form a SVF composition as described herein and then administered to the same patient. The SVF may be stored at 4°C, 25°C, -17°C, -20°C, or - 70°C prior to administration.

[0162] The allogeneic SVF compositions described herein may be used in allogeneic transplant methods. For example, a first patient may have whole blood removed, the blood type determined, the SVF may be isolated from the blood, using methods known in the art, admixed to form a SVF composition as described herein and then administered to a second patient whose blood type is compatible with the blood type of the first patient. The rules for determining compatible blood types for donors and recipients is well known in the art. For example, a recipient who is A+, may accept A+, A-, 0+, O- blood, or combinations thereof. These same rules apply to determining compatibility for matching SVF donor and recipient using the blood type as a proxy for compatibility.

Stromal Vascular Fraction [0163] The inventor surprisingly discovered that tissues, optionally connective, muscle, nervous, adipose, and/or epithelial tissue, e.g., derived from surgical excision or aspirated via liposuction may be treated by ultrasonic cavitation for a sufficient amount of time to explode or lyse the fat cells and the blood vessels contained therein and thereby release stromal vascular fraction cells contained within the blood vessels in the tissue including stromal and mesenchymal stem cells, endothelial precursor cells, and other cell types which constitute the stromal vascular fraction. The stromal vascular fraction (SVF) may be isolated by ultrasonic cavitation as described in U.S. Patent Application Publication No. 2012/0164113 or the methods described in U.S. Provisional Patent Application Nos. 61/674,116; 61/721,917; 61/693,982; or 61/773,482.

[0164] The invention provides a novel method of obtaining a stromal vascular fraction from tissue, optionally connective, muscle, nervous, adipose, and/or epithelial tissue, that does not include the use of collagenase or other enzymes to digest the collagen bonds that hold together the tissue. While collagenase works well for this purpose, and indeed is conventionally used by those skilled in the art to degrade collagen and separate the tissue into discrete cells, the use of this enzyme may be disadvantageous for cellular products that are to be used in humans, e.g., cells or cell fractions which are to be used in tissue reconstruction or regeneration, e.g., breast reconstruction procedures, cosmetic skin rejuvenation or usage in cosmetic tissue fillers that are used during plastic surgery. Particularly the FDA may consider that the use of this enzyme (to derive desired cells) results in a "maximally manipulated" cellular product. This is

disadvantageous as the use of collagenase would potentially place stromal or mesenchymal vascular cells derived from tissue in a category that requires drug approval, even if the cell fraction is to be used cosmetically and not clinically.

[0165] Also, the use of enzymes such as collagenase is further disfavored as these enzymes result in more cell death, thereby reducing the number of the desired cells which are isolatable, and further this results in more cellular debris, thereby resulting in a less useful cell product, especially if the cells are to be used therapeutically.

[0166] In collagenase protocol requires washing out blood. The blood contains hematopoietic stem cells, blood is retained, red blood cells are destroyed but hematopoietic stem cells are surprisingly retained. This has the unexpected result of producing a stromal vascular faction product with better therapeutic properties than prior stromal vascular fraction products. Further, the stromal vascular fraction products isolated by the methods described herein do not have any enzymes which must be remove prior to clinical use.

[0167] The SVF cells obtained by the ultrasonic cavitation methods described in U.S. Patent Application Publication No. 2012/0164113 are different than the SVF cell product produced using enzymes, e.g., collagenase, in part because the methods described herein do not wash out the blood. Thus, all of the hematopoietic elements are present in the stromal vascular fraction cells prepared by the ultrasonic cavitation methods described in U.S. Patent Application Publication No. 2012/0164113 whereas they are not in SVF cells produced by prior art methods which use enzymes. Further, stromal vascular cells (SVF) isolated by these ultrasonic cavitation methods retain hematopoietic factors. Also, in samples digested by enzymes it is necessary to wash out the blood components before processing (e.g.. hematopoietic stem cells, glucose). The stromal vascular fraction cells prepared by the ultrasonic cavitation methods described in U.S. Patent Application Publication No. 2012/0164113 retain these important components. The presence of the blood components, specifically glucose, gives the stromal vascular fraction cells prepared by the ultrasonic cavitation methods described in U.S. Patent Application Publication No. 2012/0164113 a longer viability when stored at room temperature.

[0168] Additionally, the stromal vascular fraction cells prepared by the ultrasonic cavitation methods described in U.S. Patent Application Publication No. 2012/0164113 are different than the SVF cell product produced using enzymes, e.g., collagenase, in part because the methods described herein do not wash out the extracellular matrix (ECM). The extracellular

matrix (ECM) is the extracellular part of animal tissue that usually provides structural support to the animal cells in addition to performing various other important functions. The extracellular matrix is the defining feature of connective tissue in animals. Extracellular matrix includes the interstitial matrix and the basement membrane. Interstitial matrix is present between various animal cells (i.e., in the intercellular spaces). Gels of polysaccharides and fibrous proteins fill the interstitial space and act as a compression buffer against the stress placed on the

ECM. Basement membranes are sheet-like depositions of ECM on which various epithelial cells rest. Due to its diverse nature and composition, the ECM can serve many functions, such as providing support, segregating tissues from one another, and regulating intercellular communication. The extracellular matrix regulates a cell's dynamic behavior. In addition, it sequesters a wide range of cellular growth factors, and acts as a local depot for them. Changes in physiological conditions can trigger protease activities that cause local release of such depots. This allows the rapid and local growth factor-mediated activation of cellular functions, without de novo synthesis. Formation of the extracellular matrix is essential for processes like growth, wound healing and fibrosis. Therefore, the stromal vascular fraction cells prepared by the ultrasonic cavitation methods described in U.S. Patent Application Publication No.

2012/0164113 comprise extracellular matrix components, unlike SVF prepared by enzymatic treatment, and thus may have better healing properties.

Table 2: Comparison of SVF prepared by ultrasonic cavitation methods described herein and collagenase methods.

[0169] Accordingly it is desirable to provide alternative methods, e.g., mechanical methods, that produce a stromal vascular fraction (containing mesenchymal stem cells, endothelial cells, and other cells found in adipose tissues) which is suitable for administration to patients via local or systemic administration such as via injection, infusion, topical administration, or which is administered in association with implants, matrices, tissue fillers, wherein the adipose tissue derived composition does not include collagenase and is not "maximally manipulated" according to the FDA. Further, the stromal vascular fraction cells prepared by the ultrasonic cavitation methods described in U.S. Patent Application Publication No. 2012/0164113 comprise both the hematopoietic factors and extracellular matrix components.

[0170] The inventor has surprisingly discovered that tissues, optionally connective, muscle, nervous, adipose, and/or epithelial tissue {e.g., derived from surgical excision or aspirated via liposuction), may be treated ex vivo or in situ by ultrasonic cavitation for a sufficient amount of time to explode or lyse the tissue cells and the blood vessels contained therein thereby releasing the stromal vascular fraction cells contained within the outer layer of blood vessel walls contained in the tissue including mesenchymal stem cells, endothelial precursor cells, and other cell types which constitute the "stromal vascular fraction." The inventor has unexpectedly found that the treatment of tissue by use of ultrasonic cavitation under appropriate conditions such as exemplified in the working examples, not only explodes or lyses the tissue cells, but further explodes or lyses the blood vessels contained therein, without adversely affecting the viability of stromal and mesenchymal stem cells, thereby releasing high numbers of viable stromal and mesenchymal stem cells, endothelial precursor cells, and other cell types which constitute the "stromal vascular fraction" which stromal and mesenchymal stem cells may be recovered and used in desired cosmetic or therapeutic methods wherein these cells are of beneficial value. This was unexpectedly expanded to cover any vascularized tissue, e.g., any tissue that comprises blood vessels may be subjected to ultrasonic cavitation or other means to isolate a stromal vascular fraction. See U.S. Provisional Patent Application No. 61/773,482.

[0171] The stromal vascular fraction may comprise stem and other cells that express at least one protein selected from the group consisting of CD13, CD14, CD29, CD31, CD34, CD36, CD44, CD45. CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3, or CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3 and/or CD31, CD45, CD117 and CD146. Further, the stromal vascular fraction may comprise stem and other cells that do not express CD56.

[0172] The stromal vascular fraction may comprise stem and other cells that express at least one protein selected from the group consisting of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135 and CD144, and does not express CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD135 and CD144. Also, the stromal vascular fraction may comprise stem and other cells that express CD49d but do not express CD56.

[0173] Cells contained therein and markers isolatable from the stromal vascular fraction of tissue according to the methods described herein include by way of example mesenchymal stem cells, hematopoietic cells, endothelial precursor cells (EPC), hematopoietic stem cells, platelets, Kupffer cells, osteoclasts, megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells, CD29+ cells, CD166+ cells, Thy-1+ or CD90+ stem cells, CD44+ cells, immune cells such as monocytes, leukocytes, lymphocytes, B and T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, neutrophil granulocytes, and the like including immune and other cells that express one or more of the following markers: CD3, CD14

(macrophage marker), CD19, CD20 (B cell marker),CD29 (integrin unit) CD31 (endothelial, platelet, macrophage, Kupffer cell, granulocyte, T NK cells, lymphocytes, megakaryocytes, osteoclasts, neutrophils, et al.), CD44 (Hyaluronic acid receptor) CD45 (B and T cell marker), C56, CD73 (lymphocyte differentiation marker), CD105 et al. Also, it includes cells expressing any of the markers disclosed in this application or any combination of these markers.

[0174] The stromal vascular fraction may comprise adipose- derived stem cells that express at least one protein selected from the group consisting of CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3, or CD13, CD29, CD34, CD36, CD44, CD49d, CD54, CD58, CD71, CD73, CD90, CD105, CD106, CD151 and SH3 and/or CD31, CD45, CD 117 and CD 146 and will not express CD56.

[0175] The stromal vascular fraction may comprise stem cells that express at least one protein selected from the group consisting of CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD 104, CD 135 and CD 144, and does not express CD3, CD4, CD14, CD15, CD16, CD19, CD33, CD38, CD56, CD61, CD62e, CD62p, CD69, CD104, CD 135 and CD 144 or expresses CD49d and does not express CD56.

[0176] While the stromal vascular fraction (SVF) or isolated cells derived from tissue may be used directly for treatment, alternatively the cells may be expanded in culture such that a single milliliter of tissue yields over 400,000 cells. Aust, et al. (2004) Cvtotherapy 6: 1-8.

Undifferentiated human adipocyte cells express a distinct immunophenotype based on flow cytometric analyses and, following induction, produce additional adipocyte specific proteins. Aust, et al. (2004) Cvtotherapy 6: 1-8; Gronthos, et al. (2001) J. Cell Physiol. 189: 54-63; Halvorsen, et al. (2001) Metabolism 50: 407-413; Sen, (2001) J. Cell. Biochem. 81: 312-319; Zuk, et al. (2002) Mol. Biol. Cell. 13: 4279^1295. Human adipose-derived adult stem cells (huASCs) display multipotentiality, with the capability of differentiating along the adipocyte, chondrocyte, myogenic, neuronal, and osteoblast lineages. Aust, et al. (2004) Cvtotherapy 6: 1- 8; Gronthos, et al. (2001) J. Cell Physiol. 189: 54-63; Halvorsen, et al, (2001) Metabolism 50: 407-413; Sen (2001) J. Cell. Biochem. 81: 312-319; Zuk, et al. (2002) Mol. Biol. Cell. 13: 4279^295; Ashjian, et al. (2003) Plast. Reconstr. Surg. 111: 1922-19231; Awad, et al. (2003) Tissue Engineering 9: 1301-1312; Awad, et al. (2004) Biomaterials 25: 3211-3222; Halvorsen, et al. (2001) Tissue Eng. 7: 729-741; Hicok, et al. (2004) Tissue Engineering 10: 371-380; Mizuno, et al. (2002) Plast. Reconstr. Surg. 109: 199-209; Safford, et al. (2002) Biochem.

Biophvs. Res. Commun. 294: 371-379; Safford, et al. (2004) Experimental Neurology 187: 319- 328; Wickham, et al. (2003) Clin. Orthop. 412: 196-212; Winter, et al. (2003) Arthritis Rheum. 48: 418^429; Zuk, et al. (2001) Tissue Eng. 7: 211-28. In the presence of dexamethasone, insulin, isobutylmethylxanthine and a thiazolidinedione, the undifferentiated human adipocyte cells undergo adipogenesis as evidenced by the fact that between 30% to 80% of the cells, based on flow cytometric methods, accumulate lipid vacuoles, which can be stained for neutral lipid with Oil Red O dye. Halvorsen, et al. (2001) Metabolism 50: 407-^13; Sen, et al. (2001) J. Cell. Biochem. 81: 312-319. Further, the stromal vascular fraction isolated by the methods described herein may be cultured.

[0177] Methods to isolate and expand SVF cells are known in the art and described, for example in U.S. Patent Nos. 6,391 ,297 Bl; 6,777,231B1; Burris, et al (1999) Mol Endocrinol 13:410-7; Erickson, et al. (2002) Biochem Biophvs Res Commun. 290(2):763-9; Gronthos et al. (2001) Journal of Cellular Physiology. 189:54-63; Halvorsen, et al. (2001) Metabolism 50:407-413: Halvorsen, et al. (2001) Tissue Eng. 7(6):729-41; Harp, et al. (2001) Biochem Biophvs Res Commun 281:907-912; Saladin, et al. (1999) Cell Growth & Diff 10:43^8; Sen et al. (2001) Journal of Cellular Biochemistry 81:312-319; Zhou, et al. (1999) Biotechnol. Techniques 13: 513-517; Zuk et al. (2001) Tissue Eng. 7: 211-228.

[0178] Methods to isolate and expand SVF cells are also described in U.S. Patent Application Publication No. 2012/0164113 or the methods described in U.S. Provisional Patent Application Nos. 61/674,116; 61/721,917; 61/693,982; or 61/773,482.

[0179] Mesenchymal stem cells isolated from the SVF obtained using the methods described in U.S. Patent Application Publication No. 2012/0164113 or the methods described in U.S.

Provisional Patent Application Nos. 61/674,116; 61/721,917; 61/693,982; or 61/773,482 may be cultured without differentiation using standard cell culture media, referred to herein as control medium {e.g., DMEM, typically supplemented with 5-15% serum {e.g., fetal bovine serum, horse serum). The stem cells can be passaged at least five times or even more than twenty times in this or similar medium without differentiating to obtain a substantially homogeneous population of SVF cells. The SVF cells can be identified by phenotypic identification. To phenotypically separate the SVF cells, the cells are plated at any suitable density which may be anywhere from between about 100 cells/cm² to about 100,000 cells/cm² (e.g., about 500 cells/cm² to about 50,000 cells/cm², between about 1,000 cells/cm² to about 20,000 cells/cm²).

[0180] After passaging for several days, SVF cells initially plated at lower densities (at less than 500 cells/cm², or alternatively, less than about 300 cells/cm² or alternatively, at less than 100 cells/cm ) can be used to obtain a clonal population of SVF cells by any suitable method such as by physically picking and seeding cells into separate plates (such as the well of a multi-well plate). Alternatively, the stem cells can be subcloned into a multi-well plate at a statistical ratio for facilitating placing a single cell into each well (e.g., from about 0.1 to about 1 cell/well or even about 0.25 to about 0.5 cells/well, such as 0.5 cells/well). Cloning can be facilitated by the use of cloning rings. See MacFarland (2000) Methods in Cell Sci. 22:63-66. Alternatively, where an irradiation source is available, clones can be obtained by permitting the cells to grow into a monolayer and then shielding one and irradiating the rest of the cells within the monolayer. The surviving cell then will grow into a clonal population. Alternatively, plated cells can be diluted to a density of 10 cells/ml and plated on Nunclon 96-well plates (Nalge Nunc International). Only wells that contain a single cell at the outset of the culture period are assayed for colony formation. Clones are detectable by microscopy after 4 to 5 days.

[0181] An exemplary culture condition for cloning stem cells comprises about 213 F12 medium+20% serum (preferably fetal bovine serum) and about 113 standard medium that has been conditioned with stromal cells or 15% FBS, 1% antibiotic/antimycotic in F-12/DMEM [1:1]) (e.g., cells from the stromal vascular fraction of liposuction aspirate, the relative proportions can be determined volumetrically).

[0182] The white blood cells (WBCs), as known as leukocytes, may be removed from the allogeneic SVF compositions described herein. The concentration of white blood cells in human whole blood is about 4,000-11 ,000 white blood cells per microliter. The WBC count in a microliter of the allogeneic SVF composition may be between about 1,000-4,000; about 1,000- 4,000; about 500-2,000; about 1,500-2,000; about 200-1,000; about 200-500; about 500-750; or 75-100 white blood cells per microliter. The SVF composition may be essentially free of white blood cells. The SVF composition may comprise less than 1,000 WBCs.

Methods of Isolating

[0183] To isolate the stromal vascular fraction, the tissue may be subject to mechanical, chemical, and/or enzymatic treatment to release the stromal vascular fraction. The stromal vascular fraction (SVF) may be isolated by ultrasonic cavitation as described in U.S. Patent Application Publication No. 2012/0164113 or the methods described in U.S. Provisional Patent Application Nos. 61/674,116; 61/721,917; 61/693,982; or 61/773,482. Further, U.S. Provisional Patent Application No. 61/773,482, filed March 6, 2013, describes several methods for isolating SVF from a variety of vascular tissues.

[0184] For example, the ultrasonic cavitation method described in U.S. Patent Application Publication No. 2012/0164113 may result in a yield of about 2,000,000 up to about 22,000,000 stromal vascular cells per mL of tissue. The ultrasonic cavitation method may result in a yield of about lxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, lOxlO⁶, l lxlO⁶, 12xl0⁶, 13xl0⁶, 14xl0⁶, 15xl0⁶, 16xl0⁶, 17xl0⁶, 18xl0⁶, 19xl0⁶, 20xl0⁶, 21xl0⁶, or 22xl0⁶ stromal vascular cells per mL of tissue. The ultrasonic cavitation method may result in a yield of about lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, or lOxlO⁷ stromal vascular cells per mL of tissue. The ultrasonic cavitation method may result in a yield of at least about 1x10 stromal vascular cells per mL of tissue.

[0185] The ultrasonic cavitation method may result in a cell yield with stromal vascular cells with at least about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% viability. The ultrasonic cavitation method may result in a cell yield with stromal vascular cells with at least about 80%, 85%, 90%, or 95% viability.

[0186] The tissue, optionally connective, muscle, nervous, adipose, and/or epithelial tissue, may be obtained from a non-living animal, including mammals. The tissue may be obtained from a mammal at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 hours post-mortem. The tissue may be obtained from a mammal at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 days post-mortem.

Tissue Sources [0187] The inventor surprisingly discovered that the methods described herein may be used to isolate stromal vascular fraction (SVF) including stromal vascular fraction cells from a vascularized tissue source. This was unexpected because stromal vascular fraction has only been isolated from adipose tissue. The inventor surprisingly discovered that stromal vascular fraction cells can be isolated from any tissue source comprising blood vessels including but not limited to adipose tissue, optionally subcutaneous fat. U.S. Provisional Patent Application No. 61/773,482, filed March 6, 2013, describes several methods for isolating SVF from a variety of vascular tissues.

[0188] The inventors also surprisingly discovered that stromal vascular cells (SVF) may be isolated from blood vessels in any tissue. Thus, the inventors surprisingly discovered that stromal vascular fraction cells may be isolated from any tissue including but not limited to connective, muscle, nervous, adipose, and epithelial tissue. Stromal vascular fraction cells may be isolated from connective tissue including but not limited to blood, bone, and extracellular matrix. Further, stromal vascular fraction cells may be isolated from nervous tissue including but not limited to neural tissue including but not limited to central nervous system comprising the brain and spinal cord, or peripheral nervous system comprise cranial nerves and spinal nerves including motor neurons. Stromal vascular fraction cells may be isolated from muscle tissue including but not limited to skeletal (striated) muscle, cardiac muscle, or smooth muscle.

Stromal vascular fraction cells may be isolated from adipose tissue. Stromal vascular fraction cells may be isolated from epithelial tissue including but not limited to squamous epithelium, cuboidal epithelium, columnar epithelium, glandular epithelium, and ciliated epithelium.

[0189] The adipose tissue treated by the claimed method described herein may be obtained from a variety of living and non-living sources including but not limited to animals (e.g., cows, chickens, sheep, goats, pigs) and humans (e.g., lipoaspirate, removed during surgery, or from cadavers).

Dosages of Stromal Vascular Fraction

[0190] The allogeneic SVF cell compositions may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, or 100,000 SVF cells. The allogeneic SVF cell compositions may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 SVF cells. The allogeneic SVF cell compositions may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, lxlO⁴, 2xl0⁴, 3xl0⁴, 4xl0⁴, 5xl0⁴, 6xl0⁴, 7xl0⁴, 8xl0⁴, 9xl0⁴, lxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, lxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, lxlO⁸, 2xl0⁸, 3xl0⁸, 4x10^s, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, lxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, or 9xl0⁹ SVF cells. The SVF cells of the allogeneic SVF cell compositions may be mammalian SVF cells, including human SVF cells.

[0191] Further, the allogeneic SVF cell compositions described herein may comprise at least about 50,000-100,000 SVF cells/mL. The allogeneic SVF cell compositions may also comprise at least about 20,000-500,000 SVF cells/mL. Also, the allogeneic SVF cell compositions may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, and 100,000 SVF cells/mL. The allogeneic SVF cell compositions may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 SVF cells/mL. The allogeneic SVF cell compositions may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, lxlO⁴, 2xl0⁴, 3xl0⁴, 4xl0⁴, 5xl0⁴, 6xl0⁴, 7xl0⁴, 8xl0⁴, 9xl0⁴, lxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, lxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, lxlO⁸, 2xl0⁸, 3x10^s, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, lxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, 9xl0⁹, lxlO¹⁰, 2xl0¹⁰, 3xl0¹⁰, 4xl0¹⁰, 5xl0¹⁰, 6xl0¹⁰, 7xl0¹⁰, 8xl0¹⁰, or 9xl0¹⁰ SVF cells/mL. The SVF cells of the allogeneic SVF cell compositions may be mammalian SVF cells, including human SVF cells.

Stable SVF Compositions

[0192] The allogeneic SVF cell compositions described herein may comprise at least about 50,000-100,000 SVF cells/mL. The allogeneic SVF cell compositions may also comprise at least about 20,000-500,000 SVF cells/mL. Also, the allogeneic SVF cell compositions may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, and 100,000 SVF cells/mL. The allogeneic SVF cell compositions may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 SVF cells/mL. The allogeneic SVF cell compositions may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, lxlO⁴, 2xl0⁴, 3xl0⁴, 4xl0⁴, 5xl0⁴, 6xl0⁴, 7xl0⁴, 8xl0⁴, 9xl0⁴, lxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, lxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, lxlO⁸, 2xl0⁸, 3xl0⁸, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, lxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, 9xl0⁹, lxlO¹⁰, 2xl0¹⁰, 3xl0¹⁰, 4xl0¹⁰, 5xl0¹⁰, 6xl0¹⁰, 7xl0¹⁰, 8xl0¹⁰, or 9xl0¹⁰ SVF cells/mL. The SVF cells of the allogeneic SVF cell compositions may be mammalian SVF cells, including human SVF cells.

[0193] The inventor surprisingly found that the allogeneic SVF compositions may be stable at - 17°C, 4°C, or 25°C for at least 10 days. The allogeneic SVF compositions may be stable at - 17°C, 4°C, or 25°C for at least 30 days. The allogeneic SVF compositions may be stable at - 17°C, 4°C, or 25°C for at least 10-30, 1-10, 5-20, or 15-30 days. The allogeneic SVF compositions may be stable at -17°C, 4°C, or 25°C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. This unexpected stability allows for greater flexibility in matching donors and recipients and admixing compositions for SVF therapies.

[0194] The allogeneic SVF compositions may be admixed with sterile saline.

Cryopreserved Preparations of Stromal Vascular Fraction Cells

[0195] The stromal vascular fraction cells may be frozen for storage. The stromal vascular fraction cells may be stored by any appropriate method known in the art {e.g., cryogenically frozen) and may be frozen at any temperature appropriate for storage of the cells. For example, the cells may be frozen at about -20°C, -80°C, -120°C, -130°C, -135°C, -140°C, -150°C, - 160°C, -170°C, -180°C, -190°C, -196°C, at any other temperature appropriate for storage of cells. Cryogenically frozen cells may be stored in appropriate containers and prepared for storage to reduce risk of cell damage and maximize the likelihood that the cells will survive thawing. The stromal vascular fraction cells may be cryopreserved immediately following differentiation, following in vitro maturation, or after some period of time in culture. The stromal vascular fraction cells may also be maintained at room temperature, or refrigerated at, for example, about 4°C.

[0196] Similarly provided are methods of cryopreserving stromal vascular fraction cells. The stromal vascular fraction cells may be harvested, washed in buffer or media, counted, concentrated (via centrifugation), formulated in freezing media {e.g., 90% FBS/10% DMSO), or any combination of these steps. For example, the stromal vascular fraction cells may be seeded in several culture vessels and serially expanded. As the stromal vascular fraction cells are harvested and maintained in FBS at about 4°C while several flasks of stromal vascular fraction cells are combined into a single lot. The stromal vascular fraction cells may be also washed with saline solution (e.g., DPBS) at least 1, 2, 3, 4, or 5 times. The information on the label may include the type of cell (e.g., stromal vascular fraction cells), the lot number and date, the number of cells (e.g., lxlO⁶ cells/mL), the expiration date (e.g., recommended date by which the vial should be used), manufacture information (e.g., name and address), warnings, and the storage means (e.g., storage in liquid nitrogen).

[0197] Cryopreserved stromal vascular fraction (SVF) cell preparations described herein may comprise at least about 50,000-100,000 stromal vascular fraction cells. The cryopreserved stromal vascular fraction cell preparations may also comprise at least about 20,000-500,000 SVF cells. Also, the cryopreserved SVF cell preparations may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, or 100,000 SVF cells. The

cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 SVF cells. The cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, lxlO⁴, 2xl0⁴, 3xl0⁴, 4xl0⁴, 5xl0⁴, 6xl0⁴, 7xl0⁴, 8xl0⁴, 9xl0⁴, lxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, lxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, lxlO⁸, 2xl0⁸, 3xl0⁸, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, lxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, or 9xl0⁹ SVF cells. The SVF cells of the

cryopreserved SVF cell preparations may be mammalian SVF cells, including human SVF cells.

[0198] Further, the cryopreserved SVF cell preparations described herein may comprise at least about 50,000-100,000 SVF cells/mL. The cryopreserved SVF cell preparations may also comprise at least about 20,000-500,000 SVF cells/mL. Also, the cryopreserved SVF cell preparations may comprise at least about 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, and 100,000 SVF cells/mL. The cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 75,000, 80,000, 100,000, or 500,000 SVF cells/mL. The cryopreserved SVF cell preparations may comprise at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, lxlO⁴, 2xl0⁴, 3xl0⁴, 4xl0⁴, 5xl0⁴, 6xl0⁴, 7xl0⁴, 8xl0⁴, 9xl0⁴, lxlO⁵, 2xl0⁵, 3xl0⁵, 4xl0⁵, 5xl0⁵, 6xl0⁵, 7xl0⁵, 8xl0⁵, 9xl0⁵, lxlO⁶, 2xl0⁶, 3xl0⁶, 4xl0⁶, 5xl0⁶, 6xl0⁶, 7xl0⁶, 8xl0⁶, 9xl0⁶, lxlO⁷, 2xl0⁷, 3xl0⁷, 4xl0⁷, 5xl0⁷, 6xl0⁷, 7xl0⁷, 8xl0⁷, 9xl0⁷, lxlO⁸, 2xl0⁸, 3xl0⁸, 4xl0⁸, 5xl0⁸, 6xl0⁸, 7xl0⁸, 8xl0⁸, 9xl0⁸, lxlO⁹, 2xl0⁹, 3xl0⁹, 4xl0⁹, 5xl0⁹, 6xl0⁹, 7xl0⁹, 8xl0⁹, 9xl0⁹, lxlO¹⁰, 2xl0¹⁰, 3xl0¹⁰, 4xl0¹⁰, 5xl0¹⁰, 6xl0¹⁰, 7xl0¹⁰, 8xl0¹⁰, or 9xl0¹⁰ SVF cells/mL. The SVF cells of the cryopreserved SVF cell preparations may be mammalian SVF cells, including human SVF cells.

[0199] The SVF cells of the invention may be recovered from storage following

cryopreservation. The SVF cells recovered from cryopreservation also maintain their viability and differentiation status. For example, at least about 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the SVF cells may retain viability and differentiation following cryopreservation. Further, the SVF cells of the invention may be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, or 7 days. The SVF cells of the invention may also be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. The SVF cells of the invention may be cryopreserved and maintain their viability after being stored for at least about 1, 2, 3, 4, 5, 6, or 7 years. The

cryopreservation preparation comprising SVF cells may be substantially free of DMSO.

[0200] The invention also provides a method of cryopreserving stromal vascular fraction cells comprising (a) isolating stromal vascular fraction cells, (b) centrifuging said stromal vascular fraction cells, and (c) resuspending said stromal vascular fraction cells in 10% DMSO/90% FBS solution, optionally at least about 10⁴ stromal vascular fraction cells per mL.

Methods of Use— Cosmetic, Therapeutic, Research/Diagnostic

[0201] The allogeneic stromal vascular fraction compositions described herein have numerous applications including use in reconstructive and aesthetic plastic surgery, and therapies, especially indications wherein stem cells and differentiated cells derived therefrom have clinical or aesthetic efficacy. Because the subject methods avoid the use of collagenase or other substances which are undesired for infusion in humans the subject vascular fractions and cells contained therein may be directly infused into patients in need thereof. The patient may be autologous, i.e., derived from the same donor or the cells may be infused into a compatible donor. Methods of HLA tissue matching cells for infusion into patients are well known in the art. Accordingly, a recipient patient may be matched using HLA tissue matching with a donor patient (e.g., the donor tissue is harvested, subjected to ultrasonic cavitation, the SVF is isolated, optionally further processed, and then administered to an HLA-matched recipient.)

[0202] For example, major histo-compatibility complex or HLA antigens may be used for matching between a SVF donor and a SVF recipient. In this process, it is analyzed which six of the HLA antigens both individuals have and a determination of the closeness of tissue matching is made. A six-antigen match (both people have the same set of six antigens) is the best compatibility between a donor recipient pair who is not identical twins. This match occurs 25 percent of the time between siblings having the same mother and father and also occurs from time-to-time in the general population. Thus, the SVF isolated using the methods disclosed herein may be used in allogeneic matches for transplantation therapies.

[0203] Further, the stromal vascular fraction cells, optionally mesenchymal stem cells derived therefrom, unexpectedly maintain hematopoietic factors contributing to their effectiveness in therapeutic uses, methods, and compositions.

[0204] The ultrasonic cavitation methods described in U.S. Patent Application Publication No. 2012/0164113 mechanically treat tissue ex vivo in the absence of collagenase to lyse fat cells and the blood cells contained therein and the resultant sonically treated composition (from which the fat is removed) is then used to obtain a stromal vascular fraction which can be infused directly in patients in need thereof or it can be further processed to purify (and expand in culture if desired) desired cell types such as mesenchymal stem cells, endothelial cells, and other cells found in tissue.

[0205] The allogeneic SVF compositions described herein may be used in patients such as for tissue reconstruction, tissue regeneration, wound healing, breast augmentation or reconstruction, in tissue fillers for plumping areas that have lost fullness, such as via aging or because of disease such as the face, lips, the buttocks, and similar areas where such an effect is desired. In particular, contemplated uses of the allogeneic SVF cell compositions described herein include use with or in lieu of tissue fillers, e.g., for treating gum recession, loss of bone, including e.g., the jaw. Further, The allogeneic SVF compositions described herein may be used in methods of treating orthopedic problems, arthritis, migraine, multiple sclerosis, autism, diabetes, optionally Type I Diabetes, wounds, ulcers, ischemic heart failure, rheumatoid arthritis, post-infarct remodeling, chronic obstructive pulmonary disease (COPD), plantar fascitis, rotator cuff injuries, and tennis elbow. [0206] The isolated mesenchymal stem cells or other cells are derived from the allogeneic SVF cell compositions described herein may be used to promote wound healing, breast augmentation, breast reconstruction, tissue engineering, treatment of ulcers in the gastrointestinal tract, or other applications.

[0207] After ultrasonic cavitation the isolated mesenchymal stem cells, optionally other cells derived therefrom, may be infused or administered into a patient for a specific cosmetic or therapeutic procedure. For example, mesenchymal stem cells may be isolated from the stromal vascular faction cells isolated using the methods described herein. The inventor surprisingly found that the stromal vascular faction cells isolated using the methods described in U.S. Patent Application Publication No. 2012/0164113 produce a higher yield of mesenchymal stem cells as compared to prior art methods.

[0208] Somatic tissue stem cells can be isolated from the subject stromal vascular fraction by fractionation using fluorescence activated call sorting (FACS) with unique cell surface antigens to isolate specific subtypes of stem cells (such as adipose derived stem cells) for injection into recipients following expansion in vitro, as described herein.

[0209] The allogeneic SVF compositions described herein may be used lieu of bone marrow for treatment of conditions treated by bone marrow transplantation.

[0210] The allogeneic SVF compositions described herein may be used in allogeneic transplant methods. For example, a first patient may have tissue removed (e.g., 100 mL to 3 L of lipoaspirate by liposuction), the SVF cells may be isolated from the tissue using the methods described herein, and then transplanted to a second allogeneic patient. For example, The allogeneic SVF compositions described herein may be used in methods for treating osteoarthritis, sports medicine injuries, including but not limited to tears and sprains of the ligaments and tendons, gingival gum regeneration, dermal treatment of burns and non-healing wounds, hair loss in men and women, rheumatoid arthritis, multiple sclerosis, ALS disease, autism, tinnitus, and bone fractures.

[0211] Further the allogeneic stromal vascular fraction compositions described herein can treat numerous diseases, including, and not limited to, osteoarthritis, sports medicine injuries, including but not limited to tears and sprains of the ligaments and tendons, gingival gum regeneration, dermal treatment of burns and non-healing wounds, hair loss in men and women, rheumatoid arthritis, multiple sclerosis, ALS disease, autism, tinnitus, and bone fractures. [0212] The allogeneic stromal vascular fraction compositions described herein may be formulated into compositions for the treatment of diseases including but not limited to bone- related disorders, diseases, or injuries, including slow/non-union fractures, osteoporosis (age- related or chemotherapy-induced), inherited diseases of bone (osteogenesis imperfecta); adipose related disorders or diseases; liver related diseases, disorders, or injuries, including liver failure, hepatitis B, and hepatitis C; myocardial infarctions, including heart attack or chronic heart failures; renal diseases or kidney damage; retinal diseases or damage or necrosis; wound healing (e.g., from surgery or diabetic ulcers); skeletal muscle disorders both traumatic and inherited; cartilage and joint repair both traumatic and autoimmune; lung injuries; diabetes; intestinal disorders; nervous system disorders, diseases, or injuries, such as central nervous systems disorders, diseases, or injuries, including spinal cord injuries, Parkinson's disease, Alzheimer's disease, and stroke.

[0213] Additionally, the allogeneic stromal vascular fraction compositions described herein may be used to formulate compositions for the treatment of loss of bone, optionally the jaw, amyotrophic lateral sclerosis (ALS), arthritis, optionally rheumatoid arthritis, autism, diabetes, optionally Type I diabetes, bone fractures, chronic obstructive pulmonary disease (COPD), dermal treatment for burns and non-healing wounds, enterocutaneous fistula (HULPUTC), gingival gum regeneration, hair loss (in both men and women), gum recession, ischemic heart failure, microvascular protection treatment in a myocardial infarction, migraine, multiple sclerosis, orthopedic problems, osteoarthritis, plantar fascitis, recto-vaginal fistula, rheumatoid arthritis, rotator cuff injuries, sports medicine injuries, optionally tears and sprains of the ligaments and tendons, tennis elbow, tinnitus, ulcers, or wounds. Further, the cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, may be used to formulate compositions for the treatment of numerous diseases, including, and not limited to, osteoarthritis, sports medicine injuries, including but not limited to tears and sprains of the ligaments and tendons, gingival gum regeneration, dermal treatment of burns and non-healing wounds, hair loss in men and women, rheumatoid arthritis, multiple sclerosis, ALS disease, autism, tinnitus, and bone fractures.

[0214] Also, the allogeneic stromal vascular fraction compositions described herein may be used in methods of treatment of the loss of bone, optionally the jaw, amyotrophic lateral sclerosis (ALS), arthritis, optionally rheumatoid arthritis, autism, diabetes, optionally Type I diabetes, bone fractures, chronic obstructive pulmonary disease (COPD), dermal treatment for burns and non-healing wounds, enterocutaneous fistula (HULPUTC), gingival gum regeneration, hair loss (in both men and women), gum recession, ischemic heart failure, microvascular protection treatment in a myocardial infarction, migraine, multiple sclerosis, orthopedic problems, osteoarthritis, plantar fascitis, recto-vaginal fistula, rheumatoid arthritis, rotator cuff injuries, sports medicine injuries, optionally tears and sprains of the ligaments and tendons, tennis elbow, tinnitus, ulcers, or wounds.

[0215] By administering the allogeneic SVF compositions described herein to a patient, one can treat numerous diseases, including, and not limited to, bone-related disorders, diseases, or injuries, including slow/non-union fractures, osteoporosis (age-related or chemotherapy- induced), inherited diseases of bone (osteogenesis imperfecta); adipose related disorders or diseases; liver related diseases, disorders, or injuries, including liver failure, hepatitis B, and hepatitis C; myocardial infarctions, including heart attack or chronic heart failures; renal diseases or kidney damage; retinal diseases or damage or necrosis; wound healing (e.g., from surgery or diabetic ulcers); skeletal muscle disorders both traumatic and inherited; cartilage and joint repair both traumatic and autoimmune; lung injuries; diabetes; intestinal disorders; nervous system disorders, diseases, or injuries, such as central nervous systems disorders, diseases, or injuries, including spinal cord injuries, Parkinson's disease, Alzheimer's disease, and stroke.

[0216] The allogeneic stromal vascular fraction described herein may be used in lieu of bone marrow as a source of adult stem cells. The allogeneic stromal vascular fraction described herein has several advantages over bone marrow including a higher yield of stem cells and greater cell viability.

[0217] The subject adipose derived stromal vascular fraction or stem and endothelial precursor cells purified or derived therefrom may be induced to differentiate. In particular the following usages of cells according to the invention as described in the patent references discussed below are contemplated.

[0218] The cells obtained from the methods described herein may be used in conformable tissue implant for use in repairing or augmenting a tissue defect or injury site that may contain stem cells. The tissue implant contains a tissue carrier matrix comprising a plurality of biocompatible, bioresorbable granules and at least one tissue fragment in association with the granules. U.S. Patent No. 7,875,296. [0219] The cells obtained from the methods described herein may be used for repairing a damaged urinary tract tissue of a subject. U.S. Patent No. 7,875,276.

[0220] The cells obtained from the methods described herein may be used tissue scaffolds suitable for use in repair and/or regeneration of musculoskeletal tissue when implanted in a body. U.S. Patent No. 7,625,581.

[0221] The cells obtained from the methods described herein may be used as a tissue repair implant comprising: a tissue carrier matrix comprising a plurality of biocompatible,

bioresorbable granules and at least one tissue fragment in association with the tissue carrier matrix, the at least one tissue fragment having an effective amount of viable cells that can migrate out of the tissue fragment and populate the tissue carrier matrix, wherein the tissue carrier matrix is in the form of an injectable suspension, and wherein an average maximum outer diameter of the granules is in a range of about 150 to about 600 μπι. U.S. Patent No. 7,316,822.

[0222] The cells obtained from the methods described herein may be used in a method of implanting stem or endothelial precursor cells into a body of a patient, said method comprising the steps of: providing a support structure, harvesting a polysaccharide-based modified biofilm from bacteria, attaching viable cells for implantation to the support structure with the

polysaccharide-based modified biofilm, and connecting one portion of a blood vessel in the patient's body with a first portion of the support structure, and connecting another portion of a blood vessel in the patient's body with a second portion of the support structure. U.S. Patent No. 7,299,805.

[0223] The cells obtained from the methods described herein may be used in an implantable biodegradable device containing a fibrous matrix, the fibrous matrix being constructed from fibers A and fibers B, wherein fibers A biodegrade faster than fibers B, fibers A and fibers B are present in relative amounts and are organized such that the fibrous matrix is provided with properties useful in repair and/or regeneration of mammalian tissue, and which may contain mesenchymal or stromal stem or endothelial precursor cells. U.S. Patent No 7,192,604.

[0224] The cells obtained from the methods described herein may be induced to express at least one phenotypic characteristic of a neuronal, astroglial, hematopoietic progenitor, or hepatic cell and then used in therapy or tissue reconstruction. U.S. Patent No. 7,078,230.

[0225] The cells obtained from the methods described herein may be used in methods and compositions for directing adipose-derived stromal cells cultivated in vitro to differentiate into cells of the chondrocyte lineage. The cells obtained from the methods described herein may be used therapeutic treatment of a number of human conditions and diseases including repair of cartilage in vivo. U.S. Patent Nos. 7,033,587, 6,841,150, and 6,429,013.

Xenotransplant and Veterinary Applications

[0226] The allogeneic stromal vascular fraction described herein may be used in

xenotransplantation methods. For example, the allogeneic stromal vascular fraction described herein isolated from a human donor may be transplanted into an animal recipient. Further, the allogeneic stromal vascular fraction described herein may be isolated from an animal donor and transplanted into a human recipient. Additionally, the allogeneic stromal vascular fraction described herein have veterinary applications where the allogeneic stromal vascular fraction described herein may be isolated from one non-human animal and transplanted into another non- human animal.

[0227] Stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, may be isolated from the allogeneic stromal vascular fraction described herein. The resultant purified stem cells may be injected into desired organs to effect tissue repair, e.g. into heart muscle to effect repair of the heart muscle, after a heart attack, into brain or spinal fluid to effect neural or nerve regeneration, such as Parkinson's or Alzheimer's patients, into the bone or cartilage of individuals in need thereof such as individuals suffering from age, exertion, or disease related bone or cartilage loss. For example, isolated stem cells derived from SVF cells may be used in autograft procedures by injection into desired organs to effect tissue repair, e.g. into heart muscle to effect repair of the heart muscle, after a heart attack, into brain or spinal fluid to effect neural or nerve regeneration, such as Parkinson's or Alzheimer's patients, into the bone or cartilage of individuals in need thereof such as individuals suffering from age, exertion, or disease related bone or cartilage loss. Also, the isolated stem cells derived from the allogeneic SVF cells may be used in these procedures may be from an allogeneic donor or as a xenotransplantation. Further, the SVF cells may be formulated in compositions for allogeneic transplant. The SVF cells may be formulated in compositions for autograft transplant. The SVF cells may be formulated in compositions for xenotransplant.

[0228] The allogeneic SVF compositions described herein may be used in xenotransplant methods. For example, adipose tissue removed {e.g., by liposuction) from an animal donor, the SVF cells may be isolated from the adipose tissue using the methods described herein, and then transplanted into a human patient. Adipose tissue removed (e.g., by liposuction) from an animal donor, optionally a pig, the SVF cells may be isolated from the adipose tissue using the methods described herein, and then transplanted into a human patient to treat a disease or accelerate tissue healing. The allogeneic SVF compositions described herein may be used to grow tissue and/organs for transplant, e.g., bone, Islets of Langerhans, mitral valve. For example, methods for treating osteoarthritis, sports medicine injuries, including but not limited to tears and sprains of the ligaments and tendons, gingival gum regeneration, dermal treatment of burns and nonhealing wounds, hair loss in men and women, rheumatoid arthritis, multiple sclerosis, ALS disease, autism, tinnitus, and bone fractures.

[0229] The allogeneic SVF compositions described herein may be used in xenotransplant methods. For example, the SVF may be isolated from whole blood using the methods known in the art, admixed to form a SVF composition and then transplanted into a human patient. For example, SVF may be isolated from an animal donor, optionally a pig, using the methods known in the art, admixed to form a SVF composition, and then transplanted into a human patient to treat a disease or accelerate tissue healing.

[0230] Cosmetic Products and Methods

[0231] The allogeneic SVF compositions described herein may be used in the manufacture of cosmetic surgery products, optionally comprising dermal fillers. For example, a method for cosmetic surgery may comprise administering the allogeneic SVF compositions described herein. A composition for cosmetic surgery may comprise an effective amount of the allogeneic SVF compositions described herein. The allogeneic SVF compositions described herein may be used in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for the reduction of a skin defect. The allogeneic SVF compositions described herein may be used in the manufacture of cosmetic surgery products including but not limited to gels, creams, lotions, and balms.

[0232] A method for formulating a topical skin cream comprising isolating SVF from a patient and compounding to form a topical skin cream may comprise autologous SVF. In another embodiment, the SVF composition may be administered topically.

[0233] The SVF compositions described herein may be a serum, lotion, or cream.

[0234] The SVF compositions described herein may be used to compound a topical skin cream comprising a patient's own growth factors derived from SVF from the same patient (autologous). The SVF compositions described herein, optionally a topical skin cream, may be patient specific and compounded by the physician at the time of the patient visit.

[0235] Compositions may be stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high SVF concentration. The carrier can be a solvent or dispersion medium containing, for example, water, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0236] Other compounds which can be included by admixture are, for example, medically inert ingredients (e.g., solid and liquid diluent), such as lactose, dextrosesaccharose, cellulose, starch, calcium phosphate, olive oil, ethyl oleate, water, or vegetable oil; lubricating agents such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; gelling agents such as colloidal clays; thickening agents such as gum tragacanth or sodium alginate; and other therapeutically acceptable accessory ingredients, such as humectants, preservatives, buffers and antioxidants, which are known additives for such formulations.

[0237] Similarly, compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, and elixirs, with suitable carriers and additives including but not limited to water, oils, glycols, and suspending agents. Typical preparations for parenteral administration comprise the active ingredient with a carrier such as sterile water or parenterally acceptable oil including but not limited to polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with other additives for aiding solubility or preservation may also be included. For dispersions and suspensions, appropriate carriers and additives include aqueous gums, celluloses, silicates, or oils.

[0238] The SVF composition may be obtained from the patient and thus, may be autologous. The SVF composition may be obtained from a donor with the same blood type and Rh factor as the recipient, and thus, may be allogeneic.

[0239] When the composition is for cosmetic use: the SVF concentration is from 0.1 to 5.0% (w/w) of the total weight of the composition. When the composition is for pharmaceutical use: the SVF concentration is from about 5.1 to 50.0% (w/w) of the total weight of the composition. The emulsion structure was studied and optimized to obtain a functional cream, which is not too oily and permits the maximum vitality to the SVF. It has characteristics of perfect skin compatibility and skin similarity with structures available in the skin tissue, as well as in the body. This cream also moisturizes, replenishes and stabilizes the lipid content. This was achieved by adding natural emolients, emulsifiers, surfactants and humectant sorbitol.

[0240] The allogeneic and autologous SVF compositions described herein may be used to formulate an antiaging serum, rosacea serum, treatment mask, post-laser treatment mask, and cleansing solution for skin brushes. These compositions may be blended by doctor and custom for the patient {e.g., comprising autologous or allogeneic SVF). The base creams, lotions and serum may be loaded into a syringe and then attached to the syringe with the SVF with a plastic transfer device and the SVF and base cream, lotion, or serum may be mixed by moving the contents of the two syringes back and forth. The final product may be delivered to the patient in the syringe or the final product can be placed into any type of container and then dispensed to the patient.

[0241] A method for reducing a skin defect may comprise administering the allogeneic SVF compositions described herein. A composition for reduction of a skin defect may comprise an effective amount of the allogeneic SVF compositions described herein. The allogeneic SVF compositions described herein may be used in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tone, improve skin texture, and tighten skin.

[0242] A method for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tone, improve skin texture, and tighten skin may comprise administering the allogeneic SVF compositions described herein.

[0243] An allogeneic composition for reducing wrinkles, improving rosacea, increase skin thickness, increase skin tone, improve skin texture, and tighten skin may comprise an effective amount of the allogeneic SVF compositions described herein matched by blood type between the donor and recipient. The use, method, or composition may be for the reduction of a skin defect, the skin defect may be a dynamic wrinkle, a fine wrinkles or a static wrinkle. The dynamic wrinkle may be a forehead crease, a brow burrow or an eye line (crow's feet). The static wrinkle may be a skin fold wrinkle resulting from sagging skin. The skin defect may be a medical condition selected from the group consisting of an acne scar, optionally a "rolling" scar, a "boxcar" scar or an "ice pick" scar, a surgical scar, trauma scar, a large pore and a soft tissue contour defect. The wrinkle or scar may be the result of loss of collagen and hyaluronic acid in the skin during the aging process. The wrinkle or scar may be the result of premature aging, optionally premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

[0244] The allogeneic SVF compositions described herein may be used in methods of treating rosacea, psoriasis, acne, eczema, and atopic dermatitis, optionally the SVF composition may be applied topically. Further, the allogeneic SVF compositions described herein may be used in cosmetic methods treat wrinkles, tone, text, large pores, dullness, or loose skin, optionally the SVF composition may be applied topically.

[0245] The compositions comprising SVF may be used in a method of augmenting soft tissue to provide of a skin defect. The compositions comprising SVF may be used in a method of augmenting soft tissue to provide reduction of a skin defect comprising topically applying to the skin defect a composition comprising SVF.

[0246] The compositions comprising SVF may be used in may be applied topically to the skin defect in a method of augmenting soft tissue to provide reduction of a skin defect. The skin defect may be a result of loss of collagen and hyaluronic acid in the skin during the aging process. The skin defect may be a result of premature aging, said premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

[0247] The compositions comprising SVF may be used in a method of augmenting soft tissue to provide reduction of a skin defect, the skin defect may be a dynamic wrinkle, a fine wrinkles or a static wrinkle. The dynamic wrinkle may be a forehead crease, a brow burrow or an eye line (crow's feet). The static wrinkle may be a skin fold wrinkle resulting from sagging skin. The skin defect is a medical condition selected from the group consisting of an acne scar, for example, a "rolling" scar, a "boxcar" scar or an "ice pick" scar, a surgical scar, trauma scar, a large pore and a soft tissue contour defect. The medical condition may be a deformity that requires re-contouring, such as a small tissue defect (e.g., after animal bite(s)) or a deformity related to trauma where the deformity is cosmetically unappealing. The augmentation may be after plastic surgery to achieve symmetry or a desired result.

[0248] The method of augmenting soft tissue to provide long-term reduction of a skin defect, a "long-term" reduction of a skin defect is of a duration of at least one year. A long-term reduction of a skin defect is of a duration of from at least one year to about five years. A long- term reduction of a skin defect is of a duration from about five years to about ten years. A long- term reduction of a skin defect is of a duration from about ten years or longer.

Differentiation of Cells from Stromal Vascular Fraction

[0249] The stromal vascular fraction produced according to the invention may be purified into desired cell types, e.g., a pure population of mesenchymal stem cells, endothelial precursor cells, hematopoietic stem cells, and these cells propagated in vitro using cell culture methods well known to those skilled in the art. As discussed herein those skilled in the art conventionally separate stem cells from other cells by FACS and other cell sorting methods based on the expression of characteristic markers.

[0250] These purified stem cells may alternatively be cultured under conditions that give rise to desired cell lineages. For example mesenchymal and stromal stem cells comprised in the subject fraction s may be differentiated into desired cell types including fibroblasts, neural cells, hematopoietic cells, myocytes, chondrocytes, and other cell types. In addition these cell types, e.g., fibroblast populations may be seeded on a scaffold, which may be used in wound healing.

[0251] The cells derived from the stromal vascular fraction, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, that have been concentrated, as described above, may be administered to a patient without further processing, or may be administered to a patient after being mixed with other tissues or cells. The concentrated cells (e.g., mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells) are mixed with one or more units of tissue that has not been similarly processed. Thus, by practicing the methods of the invention, a composition comprising tissue with an enhanced concentration of cells derived from the stromal vascular fraction, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, may be administered to the patient. The volumes of the various units of tissue may be different. For example, one volume may be at least 25% greater than the volume of another unit of tissue. Furthermore, one volume may be at least about 50%, at least about 100%, and even about 150% or more greater than the volume of another unit of tissue. In addition, the desired composition may be obtained by mixing a first unit of tissue with the concentrated cell population, which may be a cell pellet containing the desired cells from the stromal vascular fraction, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, with one or more other units of tissue. These other units will not have an increased concentration of stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, or in other words, will have an cell concentration less than that contained in the first unit of tissue. One of the units is allogeneic material that contains, for example, an increased concentration of stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells.

[0252] At least a portion of the desired cell population derived from the stromal vascular fraction may be stored for later implantation/infusion. The population may be divided into more than one aliquot or unit such that part of the population of stem cells may be retained for later application while part is applied immediately to the patient. Moderate to long-term storage of all or part of the cells in a cell bank is also within the scope of this invention. The cells may be mixed with one or more units of fresh or preserved tissue to provide a composition containing the stem cells at a higher concentration than a unit of tissue prior to processing.

[0253] At the end of processing, the concentrated cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, may be loaded into a delivery device, such as a syringe, for placement into the recipient by either subcutaneous, intravenous, intramuscular, or intraperitoneal techniques. In other words, cells may be placed into the patient by any means known to persons of ordinary skill in the art, for example, they may be injected into blood vessels for systemic or local delivery, into tissue (e.g., cardiac muscle, or skeletal muscle), into the dermis (subcutaneous), into tissue space (e.g., pericardium or peritoneum), or into tissues (e.g., periurethral emplacement), or other location, such as placement by needle or catheter, or by direct surgical implantation in association with additives such as a preformed matrix.

Automatic Systems

[0254] An automated system for separating and concentrating clinically safe regenerative cells from adipose tissue that are suitable for re-infusion into a subject may be used in conjunction with the ultrasonic cavitation methods described herein. A system for separating and

concentrating cells from tissue in accordance with the invention may include one or more of a collection chamber, a processing chamber, a waste chamber, an output chamber and a sample chamber. The various chambers are coupled together via one or more conduits such that fluids containing biological material may pass from one chamber to another in a closed, or functionally closed, sterile fluid/tissue pathway which minimizes exposure of tissue, cells, biologic and non- biologic materials with contaminants. The waste chamber, the output chamber and the sample chamber are optional. The system contains clinically irrelevant quantities of endotoxin. The system also includes a plurality of filters. The filters are effective to separate the stem cells and/or progenitor cells from, among other things, collagen, free lipids, adipocyte, that may be present in the solution after ultrasonication cavitation of the tissue sample. The filter assembly may include a hollow fiber filtration device. A filter assembly includes a percolative filtration device, which may or may not be used with a sedimentation process. The filter assembly may comprise a centrifugation device, which may or may not be used with an elutriation device and process. The system may comprise a combination of these filtering devices. The filtration functions can be two-fold, with some filters removing things from the final concentration such as collagen, free lipid, free adipocytes, and with other filters being used to concentrate the final product.

[0255] One or more components of the system are automated and include an internal processing device and associated software programs which control many of the processing functions.

Components of the system may be disposable, such that portions of the system can be disposed of after a single use. Such a system also comprises a re-usable component which includes the processing device (computer and associated software programs) and other components such as motors, pumps.

[0256] A method of treating a patient may comprise (a) collecting patient information; (b) matching the blood type to stromal vascular fraction; (c) providing a tissue removal system; (d) removing tissue from a patient using the tissue removal system, the tissue having a concentration of stem cells; (e) processing at least a part of the tissue by use of ultrasonic sonication for a time sufficient to explode all or most of the fat cells and release the stromal vascular cells into a suitable fluid medium, e.g. phosphate buffered saline solution, (f) allowing the treated solution to settle such that the fat rises to the top of the solution and the fat is removed in order to obtain a concentrated stromal vascular fraction containing regenerative cells (e.g., mesenchymal stem cells, endothelial precursor cells, hematopoietic stem cells) higher than the concentration of regenerative cells of the tissue before processing, wherein the processing occurs within a sterile, closed or functionally closed system; and (e) administering the concentrated regenerative cells to a patient, to thereby treat the patient.

[0257] The stromal vascular fraction cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, may be administered directly into the patient. In other words, the cells (e.g., the stem cells and/or endothelial precursor cells contained in the stromal vascular fraction) may be administered to the patient without being removed from the system or exposed to the external environment of the system before being administered to the patient. Providing a closed system reduces the possibility of contamination of the material being administered to the patient. Thus, processing the tissue in a closed system provides advantages because the cell population is more likely to be sterile. The only time the stem cells and/or endothelial precursor cells are exposed to the external environment, or removed from the system, is when the cells are being withdrawn into an application device and being administered to the patient. The application device can also be part of the closed system. Thus, the cells used may not processed for culturing or allogeneic.

Secondary Agents

[0258] The allogeneic SVF compositions may further comprise additional cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, and/or may be applied alone or in combination with other cells, tissue, tissue fragments, demineralized bone, growth factors (e.g., insulin or drugs, e.g., members of the thiaglitazone family), biologically active compounds, biologically inert compounds, resorbable plastic scaffolds, or other additive intended to enhance the delivery, efficacy, tolerability, or function of the population. The cell population, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells, may also be modified by insertion of DNA or by placement in cell culture in such a way as to change, enhance, or supplement the function of the cells for derivation of a cosmetic, structural, or therapeutic purpose. For example, gene transfer techniques for stem cells are known by persons of ordinary skill in the art, as disclosed in Mosca, et al. (2000) Clin Orthop (379 Suppl): S71-90, and may include viral transfection techniques, and more specifically, adeno-associated virus gene transfer techniques, as disclosed in Walther and Stein (2000) Drugs 60(2): 249-71, and Athanasopoulos, et al. (2000) Int J Mol Med 6(4): 363-75. Non-viral based techniques may also be performed as disclosed in Muramatsu, et al. (1998) Int J Mol Med 1(1): 55-62.

[0259] The cells, optionally mesenchymal stem cells, endothelial precursor cells, or

hematopoietic stem cells, may be mixed with unprocessed fragments of tissue and placed back into the recipient using a very large gauge needle or liposuction cannula. Transfer of autologous fat without supplementation with processed cells is a common procedure in plastic and reconstructive surgery. Cells derived from the stromal vascular fraction obtained by the methods described herein are, for example, substantially depleted of mature adipocytes may provide an environment that supports prolonged survival and function of the graft.

[0260] The cells, optionally mesenchymal stem cells, endothelial precursor cells, or

hematopoietic stem cells, may be placed into the recipient and surrounded by a resorbable plastic sheath such as that manufactured by MacroPore Biosurgery, Inc. U.S. Patent Nos. 6,269,716 and 5,919,234. In this setting the sheath would prevent prolapse of muscle and other soft tissue into the area of a bone fracture thereby allowing the emplaced processed tissue-derived cells to promote repair of the fracture. The beneficial effect may be enhanced by supplementation with additional components such as pro-osteogenic protein growth factors, biological scaffolds, or artificial scaffolds.

[0261] The cells, optionally mesenchymal stem cells, endothelial precursor cells, or

hematopoietic stem cells, may be combined with a gene encoding a pro-osteogenic growth factor which would allow cells to act as their own source of growth factor during bone healing or fusion. Addition of the gene could be by any technology known in the art including but not limited to adenoviral transduction, "gene guns," liposome-mediated transduction, and retrovirus or Lentivirus-mediated transduction.

[0262] Particularly when the cells and/or tissue containing the cells are administered to a patient other than the patient from which the cells and/or tissue were obtained, one or more

immunosuppressive agents may be administered to the patient receiving the cells and/or tissue to reduce, and preferably prevent, rejection of the transplant. Examples of immunosuppressive agents suitable with the methods disclosed herein include agents that inhibit T-cell/B-cell costimulation pathways, such as agents that interfere with the coupling of T-cells and B-cells via the CTLA4 and B7 pathways, as disclosed in U.S. Patent Application Publication No.

2002/0182211. Other examples include but are not limited to cyclosporin, myophenylate mofetil, rapamicin, and anti-thymocyte globulin.

[0263] The cells, optionally mesenchymal stem cells, endothelial precursor cells, or

hematopoietic stem cells, may be administered to a patient with one or more cellular

differentiation agents, such as cytokines and growth factors. Examples of various cell differentiation agents are disclosed in Gimble, et al. (1995) J Cell Biochem 58(3): 393-402; Lennon, et al. (1995) Exp Cell Res 219(1): 211-22; Majumdar, et al. (1998) J Cell Physiol 176(1): 57-66; Caplan and Goldberg (1999) Clin Orthop (367 Suppl): S12-6; Ohgushi and Caplan (1999) J Biomed Mater Res 48(6): 913-27; Pittenger, et al. (1999) Science 284(5411): 143-7; Caplan and Bruder (2001) Trends Mol Med 7(6): 259-64; Fukuda (2001) Artif Organs 25(3): 187-93; Worster, et al. (2001) J Orthop Res 19(4): 738^9; Zuk, et al. (2001) Tissue Eng 7(2): 211-28; and Mizuno, et al. (2002) Plast Reconstr Surg 109(1): 199-209.

[0264] The stromal vascular fraction cells may be formulated into a composition comprising an additional pharmaceutical or agent, or alternatively a polynucleotide that encodes for a therapeutic agent or for an inhibiting nucleic acid. Examples of nuclear acids include, a ribozyme, an antisense oligonucleotide, a double stranded RNA, a double-stranded interfering RNA (iRNA), a triplex RNA, an RNA aptamer, and at least a portion of an antibody molecule that binds to the gene product and inhibits its activity.

Biocompatible Polymers, Devices, and Implants

[0265] The cells obtained from the methods described herein may be used in methods of making bioremodelable graft prostheses prepared from cleaned tissue material derived from animal sources. The bioengineered graft prostheses of the invention are prepared using methods that preserve cell compatibility, strength, and bioremodelability of the processed tissue matrix. The bioengineered graft prostheses are used for implantation, repair, or use in a mammalian host. These prostheses may contain mesenchymal stem or endothelial precursor cells. U.S. Patent 6,986,735.

[0266] For example the allogeneic stromal vascular fraction compositions described herein may be administered alone or in combination with tissue fillers {e.g., Juvederm) or scaffolds or matrices used to promote tissue regeneration or reconstruction, e.g., breast or other cancer reconstructive surgeries, foot surgery, breast augmentation, penile implants, facial fillers, joint or cartilage surgery, neck surgery, and the like. Injectable dermal fillers provide a noninvasive option for providing a scaffold for SVF cells in therapeutic methods, uses, and compositions. The subject vascular cell fractions and stem cells derived therefrom may be used in cosmetic compositions used for topical application to the skin to effect rejuvenation and promote radiance, and reduce wrinkling, optionally in combination with a dermal filler. For example, collagen, Autologen® (autologous collagen dispersion), Isolagen® (autologous fibroblast composition), Dermalogen® (injectable human dermal implant material), hyaluronic acid, calcium

hydroxyapatite, and/or synthetic poly-lactic acid may be used as a scaffold for SVF cells in therapeutic methods, uses, and compositions. Additionally, a dermal filler composition comprising an acrylate/methacrylate copolymer may be used as scaffold for SVF cells in therapeutic methods, uses, and compositions. See U.S. Patent No. 7,910,134. Another permanent microsphere-based injectable dermal filler contains larger non-resorbable microspheres made of polymethyl methacrylate (PMMA), each having a diameter of between 30 and 42 μπι and a smooth surface, and a highly purified bovine collagen gel in a ratio of 20% PMMA to 80% bovine collagen.

[0267] In particular the invention provides a lipo-derived stem cell substantially free of adipocytes and include treatment of use with or in lieu of tissue fillers, as a gum recession, loss of bone, including the jaw, treatment of orthopedic problems, treatment of arthritis, treatment of migraine, treatment of multiple sclerosis, treatment of autism, treatment of diabetes, treatment of wounds, treatment of ulcers, treatment of COPD, treatment of plantar fascitis, treatment of rotator cuff, and treatment of tennis elbow.

[0268] The cells obtained from the methods described herein may be used for treatment of a disease, including allogeneic transplantation, in combination with a biocompatible polymer.

[0269] Biocompatible polymers, for example, include but are not limited to, homopolymers, copolymers, block polymers, cross-linkable or crosslinked polymers, photoinitiated polymers, chemically initiated polymers, biodegradable polymers, and nonbiodegradable polymers. In some embodiments, the SVF cell transplant may comprise a polymer matrix that is

nonpolymerized, to allow it to be combined with a tissue, organ, or engineered tissue in a liquid or semi-liquid state, for example, by injection. In other embodiments, the SVF cell transplant comprising liquid matrix may polymerize or substantially polymerize in situ. In still other embodiments, the SVF cell transplant may be admixed with the polymer, polymerized or substantially polymerized prior to injection. Such injectable compositions are prepared using conventional materials and methods know in the art. See, e.g., Knapp, et al. (1977) Plastic and Reconstr. Surg. 60:389-405; Fagien (2000) Plastic and Reconstr. Surg. 105: 362-73 and 2526- 28; Klein, et al. (1984) J. Dermatol. Surg. Oncol. 10: 519-22; Klein (1983) J. Amer. Acad. Dermatol. 9: 224-28; Watson, et al. (1983) Cutis 31: 543-46; Klein (2001) Dermatol. Clin. 19: 491-508; Klein (1999) Pedriat. Dent. 21: 449-50; Skorman (1987) J. Foot Surg. 26: 5115;

Burgess (1992) Facial Plast. Surg. 8: 176-82; Laude, et al. (2000) J. Biomech. Eng. 122: 231- 35; Frey, et al. (1995) J. Urol. 154: 812-15; Rosenblatt, et al. (1994) Biomaterials 15: 985-95; Griffey, et al. (2001) J. Biomed. Mater. Res. 58: 10-15; Stenburg, et al. (1999) Scfand. J. Urol. Nephroi. 33: 355-61; Sclafani, et al. (2000) Facial Plast. Surg. 16: 29-34; Spira, et al. (1993) Clin. Plast. Sure. 20: 18188; Ellis, et al. (2001) Facila Plast. Surg. Clin. North Amer. 9: 405-11 ; Alster, et al. (2000) Plastic Reconstr. Surg. 105: 2515-28; and U.S. Patent Nos. 3,949,073 and 5,709,854.

[0270] The polymerized or nonpolymerized matrix may comprise collagen, including but not limited to contracted and non-contracted collagen gels, hydrogels comprising, for example, but not limited to, fibrin, alginate, agarose, gelatin, hyaluronate, polyethylene glycol (PEG), dextrans, including dextrans that are suitable for chemical crosslinking, photocrosslinking, or both, albumin, polyacrylamide, polyglycolyic acid, polyvinyl chloride, polyvinyl alcohol, poly(n-vinyl-2-pyrollidone), poly(2-hydroxy ethyl methacrylate), hydrophilic polyurethanes, acrylic derivatives, or pluronics {e.g., polypropylene oxide and polyethylene oxide copolymer.) The fibrin or collagen is autologous or allogeneic with respect to the intended recipient. The skilled artisan will appreciate that the matrix may comprise non-degradable materials, for example, but not limited to, expanded polytetrafluoroethylene (ePTFE), polytetrafluoroethylene (PTFE), polyethyleneterephthalate (PET), polyurethane, polyethylene, polycabonate, polystyrene, silicone, or selectively degradable materials {e.g., poly (lactic-co-glycolic acid; PLGA), PLA, or PGA). See also, Middleton, et al. (2000) Biomaterials 21: 2335-2346;

Middleton, et al. Medical Plastics and Biomaterials (March/ April 1998) pages 30-37; Handbook of Biodegradable Polymers, Domb, Kost, and Domb, Eds., 1997, Harwood Academic Publishers, Australia; Rogalla (1997) Minim. Invasive Surg. Nurs. 11: 67-69; Klein (2001) Facial Plast. Surg. Clin. North Amer. 9: 205-18; Klein, et al. (1985) J. Dermatol. Surg. Oncol. 11: 337-39; Frey, et al. (1995) J. Urol. 154: 812-15; Peters, et al. (1998) J. Biomed. Mater. Res. 43: 422-27; and Kuijpers, et al. (2000) J. Biomed. Mater. Res. 51: 136-^15.

Clinical Methods

[0271] The present invention provides for a method of providing an allogeneic stromal vascular fraction composition to a clinical site comprising (a) collecting patient information; (b) matching the blood type to stromal vascular fraction; (c) thawing vials of allogeneic stromal vascular fraction cells that match the patient's blood type, (d) resuspending the stromal vascular fraction cells in media, (e) centrifuging the stromal vascular fraction cells, (f) resuspending the stromal vascular fraction cells in media, (g) aliqouting the stromal vascular fraction cells into vials, and (h) transferring to the clinical site. In one embodiment, the resuspension and centrifugation steps may be repeated at least 1, 2, 3, 4, or 5 times. In another embodiment, the stromal vascular fraction product is transported to the clinical site within at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours of completion of step (e). In a further embodiment, the vials may be labeled.

[0272] The present invention also provides a method for providing allogeneic stromal vascular fraction cell preparation for sale comprising (a) producing stromal vascular fraction cells; (b) collecting patient information; (c) matching the blood type to stromal vascular fraction; and (d) preparing said stromal vascular fraction cell preparations for transfer to a customer. In one embodiment, the method may comprise cryopreserving the stromal vascular fraction cells. In another embodiment, the method comprises offering said stromal vascular fraction cell preparations for sale. In a further embodiment, the method comprises advertising the stromal vascular fraction cell preparations.

[0273] A method for preparing an allogeneic stromal vascular fraction cell composition may comprise (a) isolating stromal vascular fraction cells by ultrasonic cavitation; (b) removing white blood cells from the stromal vascular fraction cells; (c) cryopreserving the stromal vascular fraction cells; (b) collecting patient information; (c) matching the blood type to stromal vascular fraction; and (d) preparing a stromal vascular fraction cell composition for the patient. In another embodiment, the method comprises offering said stromal vascular fraction cell preparations for sale. In a further embodiment, the method comprises advertising the stromal vascular fraction cell preparations. In one embodiment, the stromal vascular fraction cells may be preparing in accordance with cGMP and/or cGTP standards for in-human and research use.

[0274] The allogeneic SVF compositions described herein may be stored in liquid nitrogen (i.e., -70°C) or refrigerated (i.e., 4°C). The SVF may be tested to match common blood types and Rh factors to recipients and then allogeneic and frozen to be stored for use by patients requiring cellular therapies in physician's offices and hospitals at future dates. The present invention provides for a method of providing a SVF compositions described herein to a clinical site comprising (a) thawing vials of allogeneic SVF, (b) admixing SVF to form a SVF composition, (c) aliqouting The allogeneic SVF compositions described herein into vials, and (d) transferring to the clinical site. The allogeneic SVF compositions described herein product is transported to the clinical site within at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours of completion of step (d). The vials may be labeled. [0275] A method for a providing SVF compositions for sale may comprise (a) producing SVF; (b) admixing the SVF to form a SVF composition; (c) preparing said SVF composition for transfer to a customer. The method may comprise cryopreserving the SVF. The method comprises offering said SVF compositions for sale. The method may comprise advertising the allogeneic SVF compositions.

[0276] Further, a method for matching a donor with a recipient for a SVF therapy may comprise (a) selecting SVF donor; (b) determining the SVF donor's HLA type; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's HLA type; (e) matching the donor with a compatible recipient; (f) isolating the SVF from the SVF donor; (g) admixing the SVF to form a SVF composition; and (h) administering the SVF composition to the SVF recipient. The physician may select a SVF donor, determine the SVF donor's HLA type, and isolate the SVF for later use. The physician may then select a SVF recipient, determine the SVF recipient's HLA type, select the stored SVF based on the recipient's HLA type, and administer the SVF to the recipient. The SVF may be admixed to form a composition and stored for 1-10 days or 1-6 days, optionally 10 days or 3 months.

[0277] A method for matching a donor with a recipient for a SVF therapy may comprise (a) selecting SVF donor; (b) determining the SVF donor's blood type and Rh factor; (c) selecting a SVF recipient in need of SVF therapy; (d) determining the SVF recipient's blood type and Rh factor; (e) matching the donor with a compatible recipient; (f) isolating the SVF from the SVF donor; (g) admixing the SVF to form a SVF composition; and (h) administering the SVF composition to the SVF recipient. The physician may select a SVF donor, determine the SVF donor's blood type and Rh status, and isolate the SVF for later use. The physician may then select a SVF recipient, determine the SVF recipient's blood type and Rh status, select the stored SVF based on the recipient's blood type and Rh status, and administer the SVF to the recipient. The SVF may be admixed to form a composition and stored for 1-10 days or 1-6 days, optionally 10 days or 3 months.

[0278] The stromal vascular fraction obtained by the ultrasonic cavitation methods described herein may be stored in liquid nitrogen (i.e., -70°C) or refrigerated (i.e., 4°C). For example, one may harvest tissue from cadaveric donors less than 24 hours post-mortem. SVF cells will be isolated from the cadaveric tissue and processed using the standard ultrasonic cavitation method. The SVF cells will then be sorted/graded with immuno-affinity columns to isolate/remove allogeneic antigens that would cause a transplant reaction. The cells would be tested to match common blood types and Rh factors to recipients and then cryo-preserved and frozen to be stored for use by patients requiring cellular therapies in physician's offices and hospitals at future dates.

[0279] All publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All such publications (e.g., Non-Patent Literature), patents, patent application publications, and patent applications are herein incorporated by reference to the same extent as if each individual publication, patent, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference.

[0280] Although methods and materials similar or equivalent to those described herein may be used in the invention or testing of the present invention, suitable methods and materials are described herein. The materials, methods and examples are illustrative only, and are not intended to be limiting.

[0281] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLES

Example 1

Ultrasonic Cavitation Protocol for Isolation of Stromal Vascular Fraction

[0282] The following laboratory protocol was used to process adipose tissue and derive a stromal vascular fraction containing stem cells from adipose tissue (e.g., collected from patients as taught in the examples). It is to be understood that the protocol is exemplary and that the specifics may be modified by a skilled artisan in order to further optimize. Using this protocol, the inventor has processed hundreds of samples with consistently good results. As disclosed herein, and substantiated by Millipore studies (See Figures 1A-E, 2A-E, 3A-E, 4A-E and 5A-E) the subject ultrasonication protocol results in about 10-fold more viable cells than comparable adipose samples treated with collagenase. Also, the inventive methods result in the same cell population and cell types as collagenase isolation procedures, suggesting that the inventive methods preserve the integrity of all the desired stromal vascular fraction cells, and especially the cell types identified herein.

[0283] Turn on Laminar Flow hood 3 minutes prior to procedure. Set up Laminar Flow hood with sterile disposable drapes and tubes. Turn on Millipore Guava and check software. Check Gauge on Laminar Flow hood.

[0284] Attach the probe#14 (with a rode size of about 13-14 mm) to the Ultrasonic machine with a 200W generator and tighten it with a wrench to be secured in place. Log in fat into Guava flow cytometer computer. Add about 50 mL of lipoaspirate (adipose tissue) to tube. A timer is used on 10 min. preset. Place probe into fat (make sure the probe does not touch the plastic). Slowly increase Cycle and Amplitude once the probe is submerged into syringe with fat, until reach Cycle 0.9 and Amplitude 90%. The ultrasonic cavitation was performed at 24 kHz. After 5 min. stop the ultrasonic process and raise the probe to a level of 40 cc on the syringe; check the sample and make sure that is not overflowing.

[0285] Remove specimen from Ultrasonic and pour the contents into a red top sterile conical specimen tube for filtering. Divide in equal amounts in two sterile red top conical specimen tubes, then add equal amounts of 0.9% Sodium Chloride. Centrifuge both specimens for 3 min. at 500 RCF (relative centrifugal force). When spinning is complete you will have a specimen that is layered, liquid on the bottom (with a pellet) and fat on top. Using a 20 cc syringe and metal infusion cannula attachment (spinal needles) submerge to bottom of specimen tube and remove liquid stem cells solution including the pellet (from this sample take approximately 2cc of liquid to be used for testing with Flow Cytometer).

[0286] Pipette sample SVF into specimen tube. Pipette Guava reagent into sample and mix. Place sample into dark for 5-20 minutes. Place sample into flow cytometer. Run Guava Soft program. Record all results. Exemplary results are shown in Tables 3 and 4:

Table 3: Yields

Table 4: Cell Markers Experiment CD14 CD31 CD4S CD34 CD29 CD73 CD90 CD166 CD105 LIN1 CD44

A Lipoaspirate 40% 15% v.; 1 % 60% 4% 60% 30% 70% 50% 40%

B Lipoaspirate 10% 4% Wo 1 % 20% 1% 50% 4% 30% 8% 3%

C Lipoaspirate 9% 8% 1% 1% 35% 1% 55% 7% 50% 10% 5%

[0287] The results therein show that the subject ultrasonication protocol results in about 10-fold more viable cells than comparable adipose samples (same amount of adipose tissue) which were treated with an enzyme that breaks down collagen (collagenase). The results therein further show that the inventive methods result in the same cell population and cell types as collagenase isolation procedures, suggesting that the inventive methods preserve the integrity of all the desired stromal vascular fraction cells, and especially the cell types identified herein.

[0288] In contrast, U.S. Patent Application Publication No. 2006/0051865 describes the use of ultrasonic methods to release adult stem cells from adipose tissue. See Example 2. When the inventor reproduced their methods and disclosed operating conditions they were ineffective, i.e., they yielded few stromal vascular fraction cells. As measured by flow cytometry, the

unprocessed lipoaspirate yields about 500,000 cells/ml, the 2006/0051865 protocol is a slight improvement at 700,000 cells/ml. In contrast, using the methods described herein, the inventor has been able to isolate 2,000,000 up to 22,000,000 cells/ml. Thus, the claimed method provides a yield of four times up to thirty times as much as the 2006/0051865 protocol. Thus, by contrast, the present invention consistently produces results in very high numbers of viable stromal vascular fraction cells, which are well suited for use in cell therapy or cosmetic procedures.

[0289] Thus, using the ultrasonic cavitation methods described herein one may isolate high levels of viable stromal vascular fraction cells from adipose tissue.

Example 2

Preparation of Adipose Tissue from Human Donor

Method 1: Preparation of an Aspirate Containing Adipose Tissue by Liposuction

[0290] An excess amount of Tumescent solution (saline containing 0.0001% adrenalin), which exceeds the amount of liposuction to be aspirated prior to the liposuction operation, is infused into hypodermic fat layer (tumescent method), and thereafter cannulae having 2-3 mm of inner diameter (made of metal with aspirator) are used for the liposuction operation. Liposuction operations are well known in the art, and for example, can be referred to in Safe Liposuction and Fat Transfer Rhoda Narins (Ed) Marcel Dekker, Inc (2005) and Textbook of Liposuction Hanke, et al. Informa Pic. (2007).

[0291] Aspirated fat is washed with saline. About five to ten liters of washed aspirate was generated, and the resultant adipose tissue derived cellular materials are used for derivation of stromal vascular fractions.

Method 2: Preparation of Adipose Fat Tissue by Surgery

[0292] Fat tissue was obtained by surgery from human subjects who had given their informed consent. Separation was conducted with techniques well known in the art. Briefly, human fat tissue was aseptically separated from fat tissue suctioned from human subjects who had given their informed consent. The resultant adipose tissue derived cellular materials are used for derivation of stromal vascular fractions.

Method 3: Harvesting Adipose Fat Tissue from Cadaver

[0293] Fat tissue may be obtained from a human cadaver using methods known in the art. The adipose tissue may be refrigerated (i.e., 4°C), frozen (i.e., -20°C), stored in liquid nitrogen (i.e., - 70°C).

Example 3

Preparation of a Stem Cell Suspension from an Aspirate of Liposuction

[0294] Adipose tissue derived from liposuction aspirates or surgically as described in the previous example are placed in a suitable tube and a biologic solution if desired (e.g., phosphate buffered saline solution or normal saline solution) and the adipose tissue in the composition is placed contact with the ultrasonic probe of an ultrasonic cavitation device as described in the Materials and Methods section above.

[0295] In particular, the Amplitude is set at about 50-100%, typically about 100%, Cycle 0.1- 1.0 and about 50 cc fat lipoaspirate is placed into a tube, 60 cc tube size, 28 mm diameter and 110 mm length and is treated by ultrasonic cavitation for about 10 minutes where at 5 minutes, the ultracaviation is stopped and the probe is adjust upward towards the middle of the sample and continued for the remaining 5 minutes using a 14 mm ultrasonic rod.

[0296] The device may be set at about 50-100% intensity and frequency of about 10-100% for about 5-60 minutes for about 50 cc of adipose tissue. This treatment explodes the fat cells and thereby releases the stromal vascular fraction into the biologic solution, e.g., phosphate buffered or normal saline. As noted this treatment does not include the addition of collagenase or equivalent enzyme intended to break down collagen as cell dissociation is instead accomplished by ultrasonic sonication.

[0297] Preferably after ultrasonication the resultant solution is allowed to settle over time or is treated by centrifugation. The fat will float to the top. This solution will contain the stromal vascular fraction at the bottom which includes adipose-derived stem cells, endothelial cell precursors and other cells and this fraction is uncontaminated by exogenous enzymes such as collagenases.

[0298] The fat containing supernatant may be discarded. In addition as the desired cells may also float, an aspirator may be used to carefully perform suction without damaging the cells.

Example 4

Characterization of Recovered Stem Cells

[0299] The stromal vascular fraction containing stem cells recovered in Example 2 and using the Protocol above is characterized by known methods, e.g., flow cytometry or FACS, e.g., using antibodies that detect markers expressed on mesenchymal and stromal adipose derived stem cells. These methods will detect the presence of viable stem cells.

[0300] It is to be understood that the protocols disclosed herein are exemplary and that the specifics may be modified by a skilled artisan in order to further optimize. Using the specific protocol reported in the Example 1, the applicant has processed over 200 samples with consistently good results. The stem cells resulting therefrom have been used to treat patients. In addition, the applicant has compared the stem cell containing cell samples derived according to the invention to those derived by conventional procedures (collagenase derived samples). More specifically, adipose-derived stem cell samples produced according to the invention were compared to those obtained in a study by Millipore. The comparison revealed that the inventive ultrasonic cavitation procedures result in the same cell population. Unexpectedly, the inventive procedure is much more efficient, i.e., it consistently results in about 10 times the number of cells for the same amount of fat.

[0301] For example, the inventor compared three methods of isolating stromal vascular or mesenchymal vascular cells including lipoaspirate, the protocol of U.S. Patent Application Publication No. 2006/0051865, and the method described herein. As measured by flow cytometry, lipoaspirate yields about 500,000 cells/ml, the U.S. Patent Application Publication No. 2006/0051865 protocol is a slight improvement at 700,000 cells/ml. In contrast, using the method described herein, the inventor isolated 2,000,000 up to 22,000,000 cells/ml. This was an unexpected result because sonication is considered in the art for lysing cells and the length of ultracaviation (i.e., 10 minutes) was unusual as compared to what was tried in the art.

EXAMPLE 5

Post-mortem Adipose Tissue from Animals

[0302] Chicken fat was purchased from the butcher. The chicken was killed the day before and kept on ice chips. The fat was exposed to ultrasonic cavitation protocol described in Example 1 and the Stromal Vascular Fraction counts were as follows: Viability: 86.5%, Cell Count: 1.28 x 10⁷ cells per mL, and Debris: 5.57%.

[0303] Beef fat was purchased from the butcher. It is estimated that it was aged for

approximately 28 days at refrigerated temperatures (4°C). The fat was exposed to ultrasonic cavitation protocol described in Example 1 and the Stromal Vascular Fraction counts were as follows: Viability: 96.6%, Cell Count: 3.49 x 10⁶ cells per mL, Debris: 1.28%.

[0304] Blend approximately 2 ounces of chicken fat to a uniform smooth consistency for approximately 60 seconds. Place 30 cc's of the fat into a 60 cc syringe. Place the syringe into the holding arm in a vertical position on the sonicator unit. The ultrasound cavitation rod is placed into the syringe at the 15 cc mark (or 1/2 the depth of the sample). Make sure the sonicator settings are set to the lowest settings and then turn on the unit. Slowly turn up the Amplitude setting to 90% then slowly turn up the Cycle setting to .9 Set the timer and let the unit run for 5 minutes. After 5 minutes is up, turn off the sonicator and turn the Amplitude and Cycle knobs back to the lowest settings.

[0305] Remove the 60 cc syringe from the holding arm and pour equal amounts of the sonicated lipo-aspirate tissue into two 50 cc conical tubes. Calculate the amount of specimen there is (usually 30 cc's) and add an equal amount of sterile 0.9% NaCl injectable solution to each of the two specimens in the 50 cc conical tubes. Filter each specimen by screwing on a Millipore steriflip 100 micro-meter filter and use a syringe to create suction to draw the liquid solution across the membrane. Once all of the liquid has been transferred from one conical tube to the other, unscrew the filter and put the cap back on the conical tube.

[0306] Transfer the SVF into four (4) 15 cc conical tubes. Put the conical tubes inside of the centrifuge and spin them for 6 minutes at 2800 RPM. The SVF cells to be used are contained in a pellet that drops to the bottom of the conical tube via gravity. The top of the conical contains the centrifuged fat and a white membrane like sub-structure that contains primarily white blood cells.

[0307] Use a 16 gauge blunt tip spinal needle attached to a 25 cc syringe to draw up the SVF cells that made a pellet on the bottom of the conical tubes. This will be approximately 20 cc. Aliquot approximately 1 cc of cells from each syringe into an Eppendorf® tube for flow cytometer testing.

TABLE 5

[0308] Thus the ultrasonic cavitation method described herein may be used on post-mortem sources of adipose tissue to isolate a stromal vascular fraction with high cell yields and high cell viability.

EXAMPLE 6

Homogenization with Beads

[0309] Stromal vascular fraction cells may be isolated from adipose tissue by means of homogenization with beads. A stored lipoaspirate sample from a human donor was defrosted and aliquoted into tube samples containing 7 mL of adipose tissue and 3 mL of tumescent fluid (a mixture of saline, Lidocaine®, and a vasoconstrictor, i.e., epinephrine).

[0310] One level scoop, approximately 5 mL of 2 mm zirconium oxide beads, was added to the sample. The tube was then placed into the Bullet Blender® Blue 50 instrument manufactured by Next Advance. The sample was homogenized on speed 1 for 3 minutes.

[0311] Approximately 10 mL of saline was then added to the sample. The sample was then filtered with a 100 μπι steriflip filter (100 to 50 micron nylon material filters). The sample was then prepared for testing on the Millipore easyCyte flow cytometer, the results of which are described in Table 6.

TABLE 6: Cell Yield from Homogenization with Beads

Sample Volume (mL) Cell Count Viability Debris 7 2.3xl0^b 88% 3.68%

[0312] Thus the homogenization with beads method described herein may be used on adipose tissue to isolate a stromal vascular fraction with high cell yields and high cell viability.

EXAMPLE 7

IN SITU EXTRACTION AND ISOLATION OF STROMAL VASCULAR FRACTION

[0313] A patient is selected to undergo identification and extraction of tissue, optionally connective, muscle, adipose, nervous, or epithelial tissue. The tissue is identified using an ultrasound method. The tissue is thereby extracted by and subject to ultrasonic cavitation using an ultrasound flow cell. The stromal vascular fraction is then isolated.

EXAMPLE 8

Indirect Sonication Protocol

[0314] Indirect Sonicator from Q Sonica was used to obtain SVF from lipoaspirate. 60 cc of lipoaspirate was placed in a metal bowl which was then submerged in water in order to facilitate the transfer of sound waves from the probe to the lipoaspirate. The sample was sonicated for about 30 minutes at an amplitude of about 100. The power was set to about 200W. After the sonication, normal saline was added to the sample in a 50/50 ratio and the sample was then spun to harvest the pellet. The pellet was tested on flow cytometer. Some of the results include a cell count of 1.67 x 10⁷ and 2.24 x 10⁷. Viabilities were 98.9% and 97.1%

Table 7: Human Samples (adipose tissue)

Table 8: Other tissue (nonliving) Abdomen adipose 1.26 x 10' 90.8

Omental adipose 9.1 x 10^b 84.9

Gluteal adipose 3.09 x 10" 89.2

Dog adipose 5.51 x 10⁵ 88.0

Chicken adipose 8.9 x 10" 87.5

[0315] Thus indirect cavitation may be used to isolate stromal vascular fraction from tissues, optionally adipose, different species, and different species with high cell yields and high cell viability.

[0316] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

A method for matching a donor with a recipient for a SVF transplantation comprising

(a) selecting SVF donor;

(b) determining the SVF donor's blood type;

(c) selecting a SVF recipient in need of SVF therapy;

(d) determining the SVF recipient's blood type; and

(e) matching the donor with a compatible recipient based on blood type.

A method for stromal vascular fraction transplantation therapy comprising

(a) selecting SVF donor;

(b) determining the SVF donor's blood type;

(c) selecting a SVF recipient in need of SVF therapy;

(d) determining the SVF recipient's blood type;

(e) matching the donor with a compatible recipient based on blood type;

(f) isolating the SVF from the SVF donor; and

(h) administering the SVF composition to the SVF recipient.

(a) selecting a SVF recipient in need of SVF therapy;

(b) determining the SVF recipient's blood type; and

(c) matching the SVF recipient with a SVF composition from a donor with a compatible blood type.

A method for stromal vascular fraction transplantation therapy comprising

(a) selecting SVF donor;

(b) determining the SVF donor's blood type;

(c) selecting a SVF recipient in need of SVF therapy;

(d) determining the SVF recipient's blood type;

(e) matching the donor with a compatible recipient;

(f) isolating the SVF from the SVF donor; and

(h) administering the SVF composition to the SVF recipient.

(a) selecting SVF donor;

(b) determining the SVF donor's HLA; (c) selecting a SVF recipient in need of SVF therapy;

(d) determining the SVF recipient's HLA and;

(e) matching the donor with a compatible recipient.

6. A method for stromal vascular fraction transplantation comprising

(a) selecting SVF donor;

(b) determining the SVF donor's HLA type;

(c) selecting a SVF recipient in need of SVF therapy;

(d) determining the SVF recipient's HLA type;

(e) matching the donor with a compatible recipient;

(f) isolating the SVF from the SVF donor;

(g) admixing the SVF to form a SVF composition; and

(h) administering the SVF composition to the SVF recipient.

7. An allogeneic SVF composition for use in the manufacture of cosmetic surgery products, wherein the donor of the SVF is matched to the recipient by blood type.

8. An allogeneic SVF composition for use in the manufacture of cosmetic surgery products, wherein the donor of the SVF is matched to the recipient by HLA.

9. A method for cosmetic surgery comprising

(a) selecting SVF donor;

(b) determining the SVF donor's blood type;

(c) selecting a SVF recipient in need of SVF therapy;

(d) determining the SVF recipient's blood type;

(e) matching the donor with a compatible recipient;

(f) isolating the SVF from the SVF donor;

(g) admixing the SVF to form a SVF composition; and

(h) administering the SVF composition to the SVF recipient.

10. A method for cosmetic surgery comprising

(a) selecting SVF donor;

(b) determining the SVF donor's HLA type;

(c) selecting a SVF recipient in need of SVF therapy;

(d) determining the SVF recipient's HLA type;

(e) matching the donor with a compatible recipient; (f) isolating the SVF from the SVF donor;

(g) admixing the SVF to form a SVF composition; and

(h) administering the SVF composition to the SVF recipient.

11. A method for allogeneic transplantation of SVF comprising matching the SVF donor and SVF recipient by blood type, isolating SVF from said donor, and administering said isolated allogeneic SVF to said recipient.

12. A method for allogeneic transplantation of SVF comprising matching the SVF donor and SVF recipient by HLA type, isolating SVF from said donor, and administering said isolated allogeneic SVF to said recipient.

13. An allogeneic composition for cosmetic surgery comprising an effective amount of an allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by blood type.

14. An allogeneic composition for cosmetic surgery comprising an effective amount of an allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by HLA type.

15. An allogeneic composition for cosmetic surgery comprising an effective amount of an allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by blood type.

16. An allogeneic composition for cosmetic uses comprising an effective amount of an

allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by blood type.

17. An allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, optionally for the reduction of a skin defect, wherein the donor of the SVF is matched to the recipient by blood type.

18. A method for reducing a skin defect comprising administering an allogeneic SVF

composition, wherein the donor of the SVF is matched to the recipient by blood type.

19. An allogeneic composition for reduction of a skin defect comprising an effective amount of an allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by blood type.

20. An allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin, wherein the donor of the SVF is matched to the recipient by blood type.

21. A method for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin comprising administering an allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by blood type.

22. An allogeneic composition for reducing wrinkles, improving rosacea, increasing skin

thickness, increasing skin tone, improve skin texture, and tighten skin comprising an effective amount of the SVF composition, wherein the donor of the SVF is matched to the recipient by blood type.

23. An allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, optionally for the reduction of a skin defect, wherein the donor of the SVF is matched to the recipient by HLA type.

24. A method for reducing a skin defect comprising administering an allogeneic SVF

composition, wherein the donor of the SVF is matched to the recipient by HLA type.

25. An allogeneic composition for reduction of a skin defect comprising an effective amount of an allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by HLA type.

26. An allogeneic SVF composition for use in the manufacture of cosmetic surgery products, optionally comprising dermal fillers, for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin, wherein the donor of the SVF is matched to the recipient by HLA type.

27. A method for reducing wrinkles, improving rosacea, increasing skin thickness, increasing skin tone, improving skin texture, or tightening skin comprising administering an allogeneic SVF composition, wherein the donor of the SVF is matched to the recipient by HLA type.

28. An allogeneic composition for reducing wrinkles, improving rosacea, increasing skin

thickness, increasing skin tone, improve skin texture, and tighten skin comprising an effective amount of the SVF composition, wherein the donor of the SVF is matched to the recipient by HLA type.

29. Use of the stromal vascular fraction in the manufacture of allogeneic cosmetic surgery products.

30. A method for cosmetic surgery comprising administering an allogeneic stromal vascular fraction composition.

31. A composition for cosmetic surgery comprising an effective amount of an allogeneic stromal vascular fraction composition.

32. Use of an allogeneic stromal vascular fraction composition in the manufacture of a

medicament for the treatment of a disease.

33. A cosmetic composition comprising an effective amount of an allogeneic stromal vascular fraction.

34. The composition of claim 33, wherein said composition is in a form selected from the group consisting of a balm, solution, suspension, emulsion, ointment, foam, paste, gel, cream, lotion, powder, salve, soap, surfactant-containing cleansing, oil, serum, drops, liposomes, nanoparticles, nanoboots, and spray.

35. The composition of claim 33, wherein said composition is formulated for topical

administration.

36. A method of treating a disease comprising administering an allogeneic stromal vascular fraction composition.

37. A composition for treating a disease comprising an effective amount of an allogeneic stromal vascular fraction composition.

38. Use of an allogeneic stromal vascular fraction composition in the manufacture of

medicament for allogeneic transplantation to treat a disease.

39. A method for treating a disease comprising administering an allogeneic transplant

comprising an allogeneic stromal vascular fraction composition.

40. A composition for allogeneic transplantation comprising an effective amount of an

allogeneic stromal vascular fraction composition.

41. A method for treating a disease comprising obtaining stromal vascular fraction from a patient and administering the stromal vascular fraction to the same patient to treat said disease.

42. A method for allogeneic transplantation comprising transplanting an allogeneic stromal vascular fraction composition.

43. A pharmaceutical composition for the treatment of a disease comprising an allogeneic stromal vascular fraction composition.

44. A pharmaceutical composition for the treatment of a disease comprising an allogeneic stromal vascular fraction composition.

45. A method for treating rosacea, psoriasis, acne, eczema, and atopic dermatitis comprising administering an allogeneic stromal vascular fraction composition, optionally the SVF composition is applied topically.

46. A cosmetic method for treating wrinkles, tone, text, large pores, dullness, or loose skin, comprising administering an effective amount of an allogeneic stromal vascular fraction composition, optionally the allogeneic SVF composition may be applied topically.

47. A method for augmenting soft tissue to provide relief of a skin defect comprising

administering an effective amount of an allogeneic stromal vascular fraction composition.

48. A method for augmenting soft tissue to provide reduction of a skin defect comprising

topically applying to the skin defect an allogeneic stromal vascular fraction composition.

49. A method for augmenting soft tissue to provide reduction of a skin defect, optionally

wherein the skin defect is a dynamic wrinkle, a fine wrinkles or a static wrinkle, comprising administering an effective amount of an allogeneic stromal vascular fraction composition.

50. A method of augmenting soft tissue to provide long-term reduction of a skin defect

comprising administering an effective amount of an allogeneic stromal vascular fraction composition.

51. The method, use, or composition of any one of claims 1-50, wherein the skin defect is the result of loss of collagen and hyaluronic acid in the skin during the aging process.

52. The method, use, or composition of any one of claims 1-50, wherein the skin defect is the result of premature aging, said premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

53. The method, use, or composition of any one of claims 1-50, wherein the stromal vascular fraction are used in a cosmetic surgery application, to promote wound healing, are used in a tissue filler or in association with breast augmentation, breast reconstruction, tissue engineering, or burn treatment.

54. The method, use, or composition of any one of claims 1-50, wherein the SVF is allogeneic and matched with the donor by blood type.

55. The method, use, or composition of any one of claims 1-50, wherein the SVF is allogeneic and matched with the donor by HLA.

56. The method, use, or composition of any one of claims 1-50, wherein said SVF composition does not include the addition of an endopeptidase, optionally collagenase.

57. The method, use, or composition of any one of claims 1-50, wherein said animal is a

mammal, optionally a human.

58. The method, use, or composition of any one of claims 1-50, wherein the SVF is obtained from the stromal or mesenchymal compartment of a human cadaver, tissue bank, organ donation, solid fat obtained from a human cadaver, or a liposuction derived aspirate.

59. The method of claim 58, wherein said animal is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post-mortem.

60. The method of claim 58, wherein said animal is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-mortem.

61. The method, use, or composition of any one of claims 1-60, wherein the stromal vascular fraction comprises mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts, megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, CD29+ cells, CD166+ cells, Thy-1+ stem cells, CD90+ stem cells, CD44+ cells, monocytes, leukocytes, lymphocytes, B cells, T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, or neutrophil granulocytes.

62. The method, use, or composition of any one of claims 1-60, wherein allogeneic

hematopoietic stem cells are isolated from the allogeneic stromal vascular fraction.

63. The method, use, or composition of claim 62, wherein said allogeneic hematopoietic stem cells are used in lieu of bone marrow for therapeutic uses.

64. The method, use, or composition of any one of claims 1-60, wherein the SVF is isolated by mechanical, enzymatic, and/or chemical treatment.

65. The method, use, or composition of any one of claims 1-60, wherein said SVF composition comprises at least about lxlO⁶ to lxlO⁷ stromal vascular cells per mL.

66. The method, use, or composition of any one of claims 1-60, wherein at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of said stromal vascular fraction cells are viable.

67. The method, use, or composition of any one of claims 1-60, wherein said method further comprises administering stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells.

68. The method, use, or composition of any one of claims 1-60, wherein said isolated stromal vascular fraction does not comprise any exogenous collagenase.

69. The method, use, or composition of any one of claims 1-60, wherein said stromal vascular fraction comprises hematopoietic elements.

70. The method, use, or composition of any one of claims 1-60, wherein said stromal vascular fraction comprises extracellular matrix (ECM) materials.

71. The method, use, or composition of any one of claims 1-70, wherein white blood cells have been removed from the stromal vascular fraction.

72. The method, use, or composition of claim 71, wherein said stromal vascular fraction is essentially free of white blood cells.

73. The method, use, or composition of any one of claims 1-72, wherein said disease is gum recession, loss of bone, including the jaw, amyotrophic lateral sclerosis (ALS), arthritis, optionally rheumatoid arthritis, autism, diabetes, optionally Type I diabetes, bone fractures, chronic obstructive pulmonary disease (COPD), dermal treatment for burns and nonhealing wounds, enterocutaneous fistula (HULPUTC), gingival gum regeneration, hair loss (in both men and women), gum recession, ischemic heart failure, microvascular protection treatment in a myocardial infarction, migraine, multiple sclerosis, orthopedic problems, osteoarthritis, plantar fascitis, recto-vaginal fistula, rheumatoid arthritis, rotator cuff injuries, sports medicine injuries, optionally tears and sprains of the ligaments and tendons, tennis elbow, tinnitus, ulcers, or non-healing wounds.

74. The method, use, or composition of any one of claims 1-72, wherein said composition further comprises tissue fillers.

75. The method, use, or composition of any one of claims 1-72 wherein said use, method, or composition comprises treatment alone or in combination with tissue fillers.

76. The method, use, or composition of any one of claims 1-72, wherein said method, use, or composition is for the reduction of a skin defect.

77. The method, use, or composition of claim 76, wherein the skin defect is a dynamic wrinkle, a fine wrinkles or a static wrinkle.

78. The method, use, or composition of claim 76, wherein the dynamic wrinkle is a forehead crease, a brow burrow or an eye line (crow's feet).

79. The method, use, or composition of claim 76, wherein the static wrinkle may be a skin fold wrinkle resulting from sagging skin.

80. The method, use, or composition of claim 76, wherein the skin defect is a medical

condition selected from the group consisting of an acne scar, optionally a "rolling" scar, a "boxcar" scar or an "ice pick" scar, a surgical scar, trauma scar, a large pore and a soft tissue contour defect.

81. The method, use, or composition of claim 76, wherein the wrinkle or scar is the result of loss of collagen and hyaluronic acid in the skin during the aging process.

82. The method, use, or composition of claim 76, wherein the wrinkle or scar is the result of premature aging, optionally premature aging caused by overexposure to sunlight, overexposure to environmental pollutants, smoking tobacco products, exposure to cigarette smoke, poor nutrition, and skin disorders.

83. The method, use, or composition of any one of claims 1-72, wherein the medical condition is a deformity that requires re-contouring, such as a small tissue defect, optionally after animal bite(s)) or a deformity related to trauma wherein the deformity is cosmetically unappealing.

84. The method, use, or composition of any one of claims 1-72, wherein the augmentation is done after plastic surgery to achieve symmetry or a desired result.

85. The method, use, or composition of any one of claims 1-72, wherein said long-term

reduction of a skin defect is of a duration of at least one year, one year to about five years, five years to about ten years, or ten years or longer.

86. The method, use, or composition of any one of one of claims 1-85, wherein the stromal vascular faction is isolated from a tissue or organ comprising subjecting said tissue to mechanical, chemical, and/or enzymatic treatment to release the stromal vascular fraction.

87. The method, use, or composition of claim 86, wherein said method comprises subjecting said tissue to ultrasonic cavitation, wherein the cells in the tissue and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction.

88. The method, use, or composition of claim 86, wherein said method comprises subjecting a tissue to ultrasonic cavitation comprising bringing said tissue into contact with a powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate.

89. The method, use, or composition of claim 86, wherein said method is performed in situ in a patient.

90. The method, use, or composition of claim 86, wherein said method further comprises removing the lysed tissue comprising the lysed blood vessels and the stromal vascular fraction, optionally adipose tissue.

91. The method, use, or composition of claim 86, wherein said method further comprises identifying and targeting the tissue using ultrasound.

92. The method, use, or composition of claim 86, wherein said ultrasonic cavitation is applied directly externally to the tissue, optionally applied to the patient's skin and the blood vessels are lysed to release the stromal vascular fraction.

93. The method, use, or composition of claim 86, wherein the ultrasonic cavitation rod is inserted through a puncture or incision in the skin into the patient's tissue and lyse the blood vessels to release stromal vascular fraction.

94. The method, use, or composition of claim 86, wherein the tissue is located using ultrasound device and then removed using indirect cavitation device and subject to ultrasonic cavitation to release the stromal vascular fraction.

95. The method, use, or composition of claim 86, wherein the tissue is, optionally located using ultrasound device, removed subject to ultrasonic cavitation by an ultrasonic flow cell device, optionally, an Ultrasonic Mini Row Cell, to release the stromal vascular fraction, optionally, wherein the stromal vascular fraction is further isolated.

96. The use, composition, or method of claim 86, wherein said stromal vascular fraction is isolated by a method comprising

providing about 40-60 mL of adipose tissue obtained from a nonliving animal; treating said adipose tissue with ultrasonic cavitation using an about 13-14 mm probe for about 10 minutes at about 24 kHz, wherein the adipose cells and blood vessels in the adipose tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated adipose tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction.

97. The method of claim 96, wherein the ultrasonic cavitation is effected for about 5 minutes, paused, and then continued for another 5 minutes for a total of 10 minutes.

98. The method of claim 97, wherein the probe is placed towards the bottom of the tissue

sample for a first 5 minute period, paused, and then the probe is moved upwards to about half-way in the tissue sample and continued for the second 5 minute period.

99. The method of any one of claims 96-98, wherein said ultrasonic cavitation is at a frequency of about 20-30 kHz, optionally about 20, 21, 22, 23, 24, 24, or 25 kHz, or optionally about 20-23 kHz or 23-25 kHz.

100. The method of any one of claims 96-98, wherein said ultrasonic cavitation is performed using an ultrasonic probe of about 10-15 mm, about 13 or 14 mm probe, optionally about 14 mm.

101. The method of claim 100, wherein said probe is 14 mm.

102. The method of any one of claims 96-101, wherein said ultrasonic cavitation device may have a 200W-500W generator, optionally a 200W generator.

103. The method, use, or composition of claim 86, wherein said stromal vascular fraction is isolated by a method comprising providing a tissue obtained from an animal; mixing said a tissue with tumescent fluid; and homogenizing said tissue with beads for about 30 seconds to 6 minutes, wherein the tissue cells and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction.

104. The method of claim 103, wherein said method further comprises isolating the stromal vascular fraction (SVF), optionally removing the beads.

105. The method of claim 103, wherein the adipose tissue is comminuted prior to

homogenization with beads.

106. The method of claim 103, wherein said beads are stainless steel, zirconium oxide, tungsten carbide, ceramic, zirconium silicate, or glass beads, optionally zirconium oxide beads.

107. The method of claim 103, wherein said beads are about 0.01-2 mm beads, optionally 2 mm beads.

108. The method of any one of claims 103-107, wherein the beads are added at a bead-to-sample ratio of about 1:1-1:4, optionally 1:4 (25% beads by volume).

109. The method of any one of claims 103-107, wherein the sample comprises about 7 mL of tissue, optionally adipose tissue.

110. The method of any one of claims 103-107, wherein the sample is mixed with about 3 mL of tumescent fluid.

111. The method of claim 86, wherein said method comprising providing about 7 mL of tissue obtained from an animal; mixing said tissue with about 3 mL of tumescent fluid;

homogenizing said tissue with about 5 mL of 2 mm zirconium oxide beads for about 3 minutes at a bead-to-sample ratio of 1:3, wherein the tissue cells and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction; and isolating the stromal vascular fraction (SVF) cells, optionally removing the beads.

112. The method of any one of claims 103-111, wherein the sample is homogenized with beads for about 3 minutes.

113. The method of claim 86, wherein said tissue is mechanically treated using a homogenizer, optionally a rotor stator homogenizer; a dounce; mortar and pestle; tissue mill, mixer-mill, or bead-beater assembly; blender; spin column homogenizer; or a sonicator.

114. The method of claim 113, wherein said tissue is mechanically comminuted, optionally by grinding, dicing, slicing, chopping up, comminuting, grinding with mortar and pestal, granulating, pressing, cubing, mincing, milling, grating, grading, crushing, rolling, shearing, dividing, or hewing.

115. The method of claim 114, wherein after mechanical comminuting, the tissue is further treated with enzymes, chemicals, or ultrasonic cavitation.

116. The method of any one of claims 86-115, wherein said amount of tissue is about 50 mL.

117. The method of any one of claims 86-115, wherein the sample comprises about 40, 45, 50, 55, or 60 cc of tissue.

118. The method of any one of claims 86-115, wherein said method further comprises isolating the stromal vascular fraction (SVF).

119. The method of any one of claims 86-115, wherein said method does not include the

addition of an endopeptidase, optionally collagenase.

120. The method of any one of claims 86-115, wherein said animal is a mammal, optionally a human.

121. The method of any one of claims 86-115, wherein the tissue is obtained from the stromal or mesenchymal compartment of a human cadaver, tissue bank, organ donation, solid fat obtained from a human cadaver, or a liposuction derived aspirate.

122. The method of claim 121, wherein said animal is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours post-mortem.

123. The method of claim 121, wherein said animal is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days post-mortem.

124. The method of claim 121, wherein the tissue is comprised in phosphate buffered saline, normal saline, or another biologically acceptable liquid.

125. The method of any one of claims 86-124, wherein said tissue comprises blood vessels.

126. The method of claim 125, wherein said tissue is selected from the group consisting of connective tissue, muscle tissue, adipose tissue, nervous tissue, and epithelial tissue.

127. The method of claim 126, wherein said connective tissue is selected from the group

consisting of blood, bone, and extracellular matrix.

128. The method of claim 126, wherein said nervous tissue is selected from the group consisting of neural tissue selected from the group consisting of central nervous system comprising the brain and spinal cord and peripheral nervous system comprising cranial nerves and spinal nerves comprising motor neurons.

129. The method of claim 126, wherein said muscle tissue is selected from the group consisting of skeletal (striated) muscle, cardiac muscle, and smooth muscle.

130. The method of claim 126, wherein said epithelial tissue is selected from the group consisting of squamous epithelium, cuboidal epithelium, columnar epithelium, glandular epithelium, and ciliated epithelium.

131. The method of any one of claims 86-124, wherein said organ comprises blood vessels.

132. The method of claim 131, wherein said organ is heart, lung, liver, bladder, kidney,

pancreas, or stomach.

133. The method of any one of claims 86-132, wherein the method further comprises allowing the treated tissue, optionally adipose tissue, to settle or is centrifuged, optionally for about 3 minutes at 500 RCF (relative centrifugal force), resulting in the fat rising to the top of the sample.

134. The method of any one of claims 86-132, wherein the stromal vascular fraction comprises mesenchymal stem cells, hematopoietic cells, hematopoietic stem cells, platelets, Kupffer cells, osteoclasts, megakaryocytes, granulocytes, NK cells, endothelial precursor or progenitor cells, CD34+ cells or mesenchymal stem cells, CD29+ cells, CD166+ cells, Thy-1+ stem cells, CD90+ stem cells, CD44+ cells, monocytes, leukocytes, lymphocytes, B cells, T cells, NK cells, macrophages, neutrophil leukocytes, neutrophils, and neutrophil granulocytes.

135. The method of any one of claims 86-132, wherein, after mechanical, enzymatic, and/or chemical treatment, the sample is assayed, optionally by flow cytometry, for the presence of adipose-derived stem cells including CD34 and/or Thy-1 or CD90 expressing stem cells.

136. The method of any one of claims 86-132, wherein, after mechanical, enzymatic, and/or chemical treatment, the sample is fractionated using fluorescence activated call sorting (FACS) based on cell surface antigens which are specific to stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or hematopoietic stem cells.

137. The method of any one of claims 86-132, wherein said method further comprises isolating the stromal vascular fraction and cryopreserving said stromal vascular fraction.

138. The method of any one of claims 86-132, wherein said method results in a yield of at least about 1x10 to 1x10 stromal vascular cells per mL of adipose tissue.

140. The method of any one of claims 86-132, wherein at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of said stromal vascular fraction cells isolated are viable.

141. The method of any one of claims 86-132, wherein said method further comprises isolating stem cells, optionally mesenchymal stem cells, endothelial precursor cells, or

hematopoietic stem cells, from said stromal vascular fraction.

142. The method of claim 86, wherein said stromal vascular fraction are isolated from a tissue comprising

(a) subjecting said tissue to ultrasonic cavitation comprising bringing said tissue into contact with a powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate, optionally wherein said method is performed in situ in a patient, wherein the cells in the tissue and blood vessels in the tissue are lysed, thereby dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction, and

(b) removing the lysed tissue comprising the lysed blood vessels and the stromal vascular fraction.

143. The method of claim 142, wherein said tissue is adipose tissue.

144. The method of claim 86, wherein said stromal vascular fraction are isolated from a tissue comprising

(a) identifying the tissue using ultrasound;

(b) subjecting said tissue to ultrasonic cavitation comprising bringing said tissue into contact with a powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate, wherein said ultrasonic cavitation is applied directly externally to the tissue, optionally applied to the patient's skin, and wherein the blood vessels are lysed, dissociating or releasing substantial numbers of intact stromal vascular fraction cells from the lysed blood vessels contained in the ultrasonicated tissue while substantially maintaining the viability of the cells constituting the stromal vascular fraction, and

(c) removing the lysed tissue comprising the lysed blood vessels and the stromal vascular fraction.

145. The method of claim 144, wherein said method is performed in situ in a patient.

146. The method of claim 144, wherein said tissue is adipose tissue.

147. The method of claim 86, wherein said stromal vascular fraction are isolated from a tissue comprising (a) identifying the tissue using ultrasound;

(b) subjecting said tissue to ultrasonic cavitation comprising inserting a cavitation rod through a puncture or incision in the skin into the patient's tissue and lyse the blood vessels to release stromal vascular fraction while substantially maintaining the viability of the cells constituting the stromal vascular fraction, and

148. The method of claim 86, wherein said stromal vascular fraction are isolated from a tissue comprising

(a) subjecting said tissue to ultrasonic cavitation comprising inserting a cannulae through a puncture or incision in the skin into the patient's tissue, and

(c) extracting the tissue comprising blood vessels and the stromal vascular fraction.

149. The method of claim 148, wherein the tissue is subject to indirect ultrasonic cavitation to release the stromal vascular fraction.

150. The method of claim 149, wherein the ultrasonic cavitation is by an ultrasonic flow cell device, optionally an Ultrasonic Mini Flow Cell, powerful ultrasonic cup horn, ultrasonic probe (rod), ultrasonic flow cell, or ultrasonic microtiter plate.

151. A composition comprising the stromal vascular fraction obtained by the method of any one of claims 86-150.

152. The composition of claim 151, wherein said composition is in a form selected from the group consisting of a balm, solution, suspension, emulsion, ointment, foam, paste, gel, cream, lotion, powder, salve, soap, surfactant-containing cleansing, oil, serum, drops, liposomes, nanoparticles, nanoboots, and spray.

153. The composition of claim 152, wherein said composition is a cream, lotion, or solution.

154. A method for formulating an autologous skin cream comprising isolating SVF from a

patient and compounding to form a skin cream comprising autologous SVF for said patient.

155. The use, method, or composition of any one of claims 43-85, wherein said SVF

composition is administered topically.

156. A method for formulating an allogeneic SVF topical skin cream comprising matching a SVF composition with a patient by blood type and compounding to form a topical skin cream comprising allogeneic SVF.