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WO2014075634A1 - Utilisation d'une cellule de couche stroma-vasculaire et d'une cellule progénitrice mésenchymateuse pour la prévention ou le traitement de l'arthrose - Google Patents

Utilisation d'une cellule de couche stroma-vasculaire et d'une cellule progénitrice mésenchymateuse pour la prévention ou le traitement de l'arthrose Download PDF

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Publication number
WO2014075634A1
WO2014075634A1 PCT/CN2013/087284 CN2013087284W WO2014075634A1 WO 2014075634 A1 WO2014075634 A1 WO 2014075634A1 CN 2013087284 W CN2013087284 W CN 2013087284W WO 2014075634 A1 WO2014075634 A1 WO 2014075634A1
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Prior art keywords
cells
surface antigen
mesenchymal
mesenchymal progenitor
progenitor cells
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English (en)
Chinese (zh)
Inventor
曹卫
张丽
赵光宇
关战军
王新华
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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Cellular Biomedicine Group Wuxi Ltd
Cellular Biomedicine Group Shanghai Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells

Definitions

  • interstitial vascular layer cells and mesenchymal progenitor cells in preventing or treating osteoarthritis
  • the invention belongs to the field of stem cells and biomedicine.
  • the invention relates to the use of mesenchymal vascular layer cells and mesenchymal progenitor cells for the prevention or treatment of osteoarthritis. Background technique
  • Osteoarthritis is a chronic joint disease characterized by degeneration, destruction and bone hyperplasia of articular cartilage.
  • OA has a prevalence of 50% in people over 60 years of age and 80% in people over 75 years of age.
  • the disability rate of the disease can be as high as 53% (Chinese Society of Orthopaedic Branch, 2007 Revision of the Guide to Osteoarthritis).
  • OA occurs in joints that are heavy and active, such as the knee joint. Therefore, it is called knee osteoarthritis (KOA). Knee osteoarthritis is a serious health hazard to human health and is one of the major diseases that cause dysfunction in the elderly.
  • the overall treatment principle of knee osteoarthritis is to combine medical and non-pharmacological treatments, if necessary, to perform surgery to reduce or eliminate pain, correct deformity, improve or restore joint function, and improve quality of life. .
  • Drug treatment mainly includes analgesics such as non-organic anti-inflammatory drugs (KSAIDs), intra-articular injection of hyaluronic acid (HA) or glucocorticoids, improvement of disease-like drugs and cartilage protective agents.
  • KSAIDs non-organic anti-inflammatory drugs
  • HA hyaluronic acid
  • glucocorticoids glucocorticoids
  • improvement of disease-like drugs and cartilage protective agents mainly includes analgesics such as non-organic anti-inflammatory drugs (KSAIDs), intra-articular injection of hyaluronic acid (HA) or glucocorticoids, improvement of disease-like drugs and cartilage protective agents.
  • KSAIDs non-organic anti-inflammatory drugs
  • HA hyaluronic acid
  • glucocorticoids glucocorticoids
  • tissue engineering efforts such as autologous chondrocyte transplantation or matrix-induced autologous soft bone cell transplantation, offer long-term solutions to the bioremediation or regeneration of degenerative joint tissue.
  • tissue engineering efforts such as autologous chondrocyte transplantation or matrix-induced autologous soft bone cell transplantation
  • a major limitation of autologous chondrocyte transplantation or matrix-induced autologous chondrocyte transplantation is the inability to treat larger cartilage defects, its effects on donor site destruction, dedifferentiation, and limited survival time of chondrocytes, Not for KOA patients.
  • the osteoarthritis is selected from the group consisting of knee osteoarthritis, spinal osteoarthritis, hip osteoarthritis, or a combination thereof.
  • the osteoarthritis is knee osteoarthritis.
  • the mesenchymal vascular layer cells are mesenchymal vascular layer cell populations.
  • the mesenchymal progenitor cell is a mesenchymal progenitor cell population.
  • the mesenchymal vascular layer cells have any one or more of the following characteristics selected from the group consisting of:
  • more than 35% of the cells have the surface antigen CD29.
  • more than 55% of the cells have the surface antigen CD73.
  • more than 90% of the cells have the surface antigen CD49d.
  • more than 60% of the cells have the surface antigen CD90.
  • the mesenchymal vascular layer cells have any one or more of the following characteristics selected from the group consisting of:
  • less than 80% of the cells have the surface antigen CD34.
  • less than 12% of the cells have the surface antigen CD45.
  • the mesenchymal vascular layer cells secrete a cytokine selected from the group consisting of stem cell growth factor (HGF), vascular endothelial growth factor (VEGF), platelet-derived factor (PDGF), human transforming growth factor P(TGF-P), macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), interleukin-10 (IL-10), or a combination thereof.
  • HGF stem cell growth factor
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived factor
  • TGF-P human transforming growth factor P(TGF-P)
  • GM-CSF macrophage colony-stimulating factor
  • IL-2 interleukin-2
  • IL-10 interleukin-10
  • the mesenchymal vascular layer cells are mesenchymal vascular layer cell populations.
  • ng/ml 0.5 ng/ml, preferably 0.8 ng/ml.
  • the concentration of vascular endothelial growth factor (VEGF) secreted by the mesenchymal vascular layer cells 35 pg/ml, preferably 40 pg/ml.
  • VEGF vascular endothelial growth factor
  • the concentration of human transforming growth factor P (TGF-P) secreted by the mesenchymal vascular layer cells is 150 pg/ml, preferably 180 pg/ml.
  • the interstitial vascular layer cells secrete interleukin-2 (IL-2) at a concentration of S3 ⁇ 4 15 pg/ml, preferably 20 pg/ml, more preferably 30 pg/ml.
  • IL-2 interleukin-2
  • the interstitial vascular layer cells secrete an interleukin-IO (IL-IO) concentration of S? 15 pg/ml, preferably 20 pg/ml, more preferably 30 pg/ml, optimally 40 pg/ml. .
  • IL-IO interleukin-IO
  • the mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:
  • more than 98% of the cells have the surface antigen CD90.
  • more than 98% of the cells have the surface antigen CD73.
  • more than 98% of the cells have the surface antigen CD29.
  • more than 98% of the cells have the surface antigen CD49d.
  • the mesenchymal progenitor cells have any one or more of the characteristics selected from the group consisting of:
  • less than 1% of the cells have the surface antigen HLA-DR.
  • less than 1% of the cells have a surface antigen Actin.
  • less than 1% of the cells have the surface antigen CD34.
  • less than 1% of the cells have the surface antigen CD45.
  • less than 1% of the cells have the surface antigen CD14.
  • the mesenchymal progenitor cells secrete a cytokine selected from the group consisting of vascular endothelial growth factor (VEGF), human transforming growth factor aCTGF-a, and human transforming growth factor P (TGF-P). ), granulocyte colony-stimulating biological factors (GM-CSF), hepatocyte growth factor (HGF), platelet-derived factor (PDGF), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10).
  • VEGF vascular endothelial growth factor
  • CTGF-a human transforming growth factor P
  • TGF-P human transforming growth factor P
  • GM-CSF granulocyte colony-stimulating biological factors
  • HGF hepatocyte growth factor
  • PDGF platelet-derived factor
  • IL-2 interleukin-2
  • IL-4 interleukin-4
  • IL-10 interleukin-10
  • the concentration of vascular endothelial growth factor (VEGF) secreted by mesenchymal progenitor cells lOpg/ml, preferably 15 pg/ml.
  • TGF-P human transforming growth factor P
  • the granulocyte colony stimulating biological factor (GM-CSF) secreted by the mesenchymal progenitor cells has a concentration of 30 ng/ml, preferably 40 ng/ml.
  • the concentration of hepatocyte growth factor (HGF) secreted by mesenchymal progenitor cells is hepatocyte growth factor (HGF) secreted by mesenchymal progenitor cells
  • the concentration of platelet-derived factor (PDGF) secreted by mesenchymal progenitor cells is PDGF.
  • 0.008 ng/ml preferably 550.01 ng/ml.
  • the concentration of interleukin-2 (IL-2) secreted by the mesenchymal progenitor cells is 25 pg/ml, preferably
  • the mesenchymal progenitor cells secrete an interleukin-IO (IL-IO) concentration of 30 pg/ml, preferably 3 ⁇ 4 40 pg/ml.
  • IL-IO interleukin-IO
  • a pharmaceutical composition for preventing and/or treating osteoarthritis comprising: an effective amount of interstitial vascular layer cells (SVF) and a charge Progenitor cells (haMPCs), and a pharmaceutically acceptable carrier.
  • SVF interstitial vascular layer cells
  • HMPCs charge Progenitor cells
  • the pharmaceutical composition is a subcutaneous injection agent.
  • the pharmaceutically acceptable carrier includes, but is not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the concentration of the mesenchymal vascular layer cells is ⁇ . ⁇ -lOOx lO 4 cells/ml, preferably
  • the mesenchymal progenitor cell concentration is from 0.1 to 100 x 10 4 / ml, preferably from 1 to 10 x 10 / 4 , more preferably 2 x 10 5 / ml.
  • a method for preventing and/or treating osteoarthritis comprising the steps of: administering to a subject in need of interstitial vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs), Or a pharmaceutical composition comprising mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs).
  • SVF interstitial vascular layer cells
  • haMPCs mesenchymal progenitor cells
  • a pharmaceutical composition comprising mesenchymal vascular layer cells (SVF) and mesenchymal progenitor cells (haMPCs).
  • the subject is a human or non-human mammal, preferably a human.
  • the method includes the steps of:
  • the site of application is within the joint cavity of the subject.
  • the interval between step (1) and step (2) is 1 month or more, and/or 3 months.
  • Figure 1 shows the combination of SVF and haMPCs for the treatment of KOA.
  • Figure 2 shows the results of SVF surface antigen detection.
  • Figures 2A–21 show the results of CD34, CD29, CD73, CD49d, CD90, CD 14, CD45, Actin, and HLA-DR antigens, respectively.
  • Figure 3 shows the change in VEGF secretion of haMPCs
  • Figure 3A shows the change in VEGF secretion of haMPCs after 24 h of LPS stimulation
  • Figure 3B shows the effect of hypoxia stimulation on VEGF secretion of haMPCs.
  • Figure 4 shows the results of chondrogenic induction experiments of haMPCs.
  • the inventors have extensively and intensively studied, and for the first time, unexpectedly found that interstitial vascular layer cells and mesenchymal progenitor cells have extremely excellent effects in preventing or treating osteoarthritis.
  • the autologous adipose-derived mesenchymal vascular layer cells and mesenchymal progenitor cells of the present invention are administered to a subject in need thereof, or a pharmaceutical composition containing autologous adipose-derived mesenchymal vascular layer cells and mesenchymal progenitor cells is administered, It has a significant preventive or therapeutic effect on osteoarthritis.
  • Interstitial vascular layer cells and mesenchymal progenitor cells have the ability to differentiate into cartilage and osteogenesis.
  • the present invention also provides a method for preventing and treating osteoarthritis and a pharmaceutical composition comprising interstitial vascular layer cells and mesenchymal progenitor cells. The present invention has been completed on this basis. the term
  • osteoarthritis is used interchangeably with “osteoarthritis” or ' ⁇ ' antigen.
  • Osteoarthritis is a chronic joint disease. Its main changes are degenerative changes in the articular cartilage surface and secondary bone hyperplasia, which are manifested in joint pain and inflexibility. X-ray shows narrowing of joint space, cartilage The lower bone is dense, the trabecular bone is broken, there is hardening and cystic change; the edge of the joint has lip-like hyperplasia; the end of the bone is deformed, the joint surface is uneven; the cartilage in the joint is spalled, the bone is broken into the joint, and the joint is free. body.
  • the osteoarthritis may be osteoarthritis selected from the group consisting of knee osteoarthritis, spinal osteoarthritis, hip osteoarthritis, or a combination thereof.
  • the osteoarthritis according to the present invention is preferably knee osteoarthritis.
  • Autologous fat is an excellent source of plastic and anti-aging treatments.
  • Adipose tissue materials can be derived from the waist, hips, abdomen, thighs, upper arms and more.
  • Those skilled in the art can obtain autologous lipid tissue using a general technical method including, but not limited to, aspiration, surgical separation, and the like.
  • the adipose tissue or the fat raw material is not particularly limited, and may be an adipose tissue derived from any part of an animal or a human, preferably a human adipose tissue.
  • the adipose tissue may be tissue of the waist, buttocks, abdomen, thighs, upper arms, and the like. Interstitial vascular layer cell
  • interstitial vascular layer cells are stem cells with multi-directional differentiation potential isolated from adipose tissue.
  • SVF can stably proliferate in vitro and has a low mortality rate. It is easy to obtain, has a large amount of reserves in the body, is suitable for large-scale cultivation, has little damage to the body, has a wide range of sources, and is suitable for autologous transplantation. SVF is the most important component of stem cell-assisted fat transplantation.
  • the cell cluster formed by a mixture of various cells separated from adipose tissue by collagenase digestion is called interstitial blood vessel debris. Interstitial vascular debris is rich in mesenchymal cells, which can differentiate into multiple lineage cells. It is the ideal seed cell for regenerative medicine and tissue engineering.
  • the terms "separation method”, “separation method of SVF” are used interchangeably and refer to methods and processes for obtaining isolated SVF from the original adipose tissue.
  • the first washing is performed to remove blood cells; the fat is broken and digested; the undigested tissue is removed, and the SVF-containing filtrate is obtained; and the SVF is obtained by centrifugation.
  • the resulting SVF can be used for further passage, culture or cryopreservation.
  • the separation of the SVF may include a step (but not limited to): the liposuction fat is repeatedly washed twice with PBS, and then digested with collagenase at 37 ° C for 30 min, after centrifugation at 1200 g for 10 min. That is, high-density SVF fragments are obtained, which mainly include mesenchymal cells, vascular endothelial cells, and parietal cells.
  • SVF also includes some blood vessel-derived cells, such as white blood cells and red blood cells, which have synergistic effects between various cells. Antigen detection of interstitial vascular layer cells
  • the SVF used in the present invention has a high purity and is substantially free of other types of cells or stem cells. This can be verified by detection of cell surface antigens.
  • SVF has a variety of specific antigens and receptors, mainly CD3, CD13, D29, CD34, CD45,
  • CD34 antigen is a highly glycosylated type I transmembrane protein that is selectively expressed on human hematopoietic stem (HSC), progenitor (PC) and vascular endothelial (EC) surfaces, adipose tissue progenitor cells with CD34
  • HSC human hematopoietic stem
  • PC progenitor
  • EC vascular endothelial
  • the proportion of total stem cells is preferably ⁇ 0.2%, more preferably ⁇ 0.2%.
  • CD45 is present on the surface of all hematopoietic cells, including hematopoietic stem cells and osteoclasts.
  • the proportion of adipose tissue progenitor cells bearing CD45 in total stem cells is preferably ⁇ 0.1%.
  • CD29, CD73, CD49d, CD90, etc. are mainly present on the surface of adipose mesenchymal progenitor cells.
  • the proportion of SVF with CD29 in total stem cells is preferably ⁇ 30%, more preferably ⁇ 32%, optimally ⁇ 35%.
  • the proportion of SVF with CD73 in total stem cells is preferably ⁇ 50%, more preferably ⁇ 60%, optimally ⁇ 70%.
  • the proportion of SVF with CD49d in total stem cells is preferably ⁇ 85%, more preferably ⁇ 90%, optimally ⁇ 95%.
  • the proportion of SVF with CD90 in total stem cells is preferably ⁇ 55%, more preferably ⁇ 60%, optimally ⁇ 65%.
  • One skilled in the art can use a general method to detect the purity and degree of differentiation of SVF, such as flow cytometry.
  • the antibody may be a complete monoclonal or polyclonal antibody, or may be an immunologically active antibody fragment, such as a Fab' or (Fab) 2 fragment; an antibody heavy chain; Antibody light chain; genetically engineered single chain Fv molecule (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibody, such as an antibody having murine antibody binding specificity but still retaining antibody portions from humans.
  • the antibody is added to the cell surface antigen for a certain period of time, and the cells are automatically analyzed and sorted by flow cytometry.
  • fat-derived mesenchymal progenitor cells As used herein, the terms “fat-derived mesenchymal progenitor cells”, “haMPCs” or “adipose tissue-derived mesenchymal progenitor cells” have the same meaning and are used interchangeably.
  • the adipose-derived mesenchymal progenitor cells used in the present invention are preferably human-derived adipose-derived mesenchymal progenitor cells; more preferably human autologous adipose-derived mesenchymal progenitor cells.
  • Dispensing fat The extracted fat is packed into a centrifuge tube. The extracted fat is divided into two parts. Part of the fat is digested and washed with collagenase, and then used to prepare SVF cell suspension, which is directly returned to the site; the other part is obtained by SVF. Continue to culture to obtain fat progenitor cells;
  • SVF stromal vasvular fraction Cells
  • Preparation of SVF suspension The filtered SVF cells were formulated into 5 ml of cell suspension, and the suspension was inhaled with an injection, and the suspension was poured into a 100 ml saline bag;
  • e. Cell culture adjust the inoculation density according to the amount of counted cells, inoculate into the culture flask and set up C0 2 incubator culture;
  • Passage a small amount of adherent mesenchymal progenitor cells appear in the inoculation for about 3 days, and culture the adherent cells for 5-7 days.
  • primary adherence 1 1-2 ratio subculture, subculture for 2 to 3 weeks, collecting cells (autologous fat derived progenitor cells);
  • Preparation of adipose-derived progenitor cell suspension After centrifugation of the collected adipose-derived progenitor cells, physiological saline is injected to prepare a cell suspension. Antigen detection of adipose-derived mesenchymal progenitor cells
  • the adipose-derived mesenchymal progenitor cells used in the present invention have extremely high purity and activity.
  • One of ordinary skill in the art can detect mesenchymal progenitor cell surface antigens using conventional methods, such as flow cytometry.
  • Adipose-derived mesenchymal progenitor cells have a variety of specific antigens and receptors, mainly including: CD29, CD73, CD90, CD49d and the like.
  • the proportion of mesenchymal progenitor cells bearing CD73 antigen in total mesenchymal progenitor cells is ⁇ 95%, more preferably ⁇ 98%, more preferably ⁇ 99%, most preferably 100%.
  • the proportion of mesenchymal progenitor cells bearing CD90 antigen in total mesenchymal progenitor cells is ⁇ 95%, more preferably ⁇ 98%, more preferably ⁇ 99%, most preferably 100%.
  • the proportion of mesenchymal progenitor cells with CD29 antigen in total mesenchymal progenitor cells is ⁇ 95%, more preferably ⁇ 98
  • the proportion of mesenchymal progenitor cells with CD49d antigen in total mesenchymal progenitor cells is ⁇ 95%, more preferably ⁇ 98 %, more preferably ⁇ 99%, optimally 100%.
  • Negative indicators of adipose-derived mesenchymal progenitor cells include: HLA-DR, Actin, CD34, CD45, CD14, and the like.
  • the proportion of mesenchymal progenitor cells with HLA-DR antigen in total mesenchymal progenitor cells is ⁇ 2%, more preferably ⁇ 1%, more preferably ⁇ 0.5%, and optimally no HLA-DR antigen.
  • the proportion of mesenchymal progenitor cells with Actin antigen in total mesenchymal progenitor cells is ⁇ 2%, more preferably ⁇ 1%, more preferably ⁇ 0.5%, optimally free of Actin antigen.
  • the proportion of mesenchymal progenitor cells bearing CD34 in total mesenchymal progenitor cells is < 2%, more preferably < 1%, more preferably < 0.5%, optimally free of CD34.
  • the proportion of mesenchymal progenitor cells bearing CD45 in total mesenchymal progenitor cells is < 2%, more preferably < 1%, more preferably < 0.5%, optimally without CD45.
  • the proportion of mesenchymal progenitor cells bearing CD14 in total mesenchymal progenitor cells is < 2%, more preferably < 1%, more preferably < 0.5%, optimally free of CD14.
  • the haMPCs used in the present invention are preferably human-derived haMPCs capable of secreting a large amount of VEGF, TGF-a, TGF- ⁇ , GM-CSF, HGF, PDGF, IL-2, IL-4, IL-10 and other gene factors. Strong colony forming ability and extremely low immunogenicity.
  • haMPCs can be used, process, apply, etc. using conventional methods. For example, each batch of haMPCs must be tested for sterility, endotoxin and mycoplasma, and DNA identification before delivery or use. The cells should be in compliance with cell viability ⁇ 95% and cell purity (positive index >95%, negative index ⁇ 2%). The haMPCs were all acute and allergic to the test results, and there was a corresponding test report.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of mesenchymal vascular layer cells and mesenchymal progenitor cells, and a pharmaceutically acceptable carrier.
  • interstitial vascular layer cells and mesenchymal progenitor cells can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, such as physiological saline, wherein the pH is usually about 5-8, preferably. Ground, pH is about 7-8.
  • a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium such as physiological saline, wherein the pH is usually about 5-8, preferably. Ground, pH is about 7-8.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals.
  • a “pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, a substance having a reasonable benefit/risk ratio .
  • Term "medicine "Study acceptable carrier” means a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of the active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.
  • the effective amount of the mesenchymal vascular layer cells and mesenchymal progenitor cells of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like.
  • the selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials).
  • the factors include, but are not limited to, the pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.
  • the pharmaceutical composition of the present invention is preferably a subcutaneous injection agent.
  • the concentration of the interstitial vascular layer cells of the subcutaneous injection agent is 0.1-100 ⁇ 10 4 /ml, preferably 1-lOx10 4 /ml, more preferably 2 ⁇ 10 5 /
  • the concentration of the mouth and/or mesenchymal progenitor cells is 0.1-100 ⁇ 10 4 /ml, preferably 1-10 ⁇ 10 4 /ml, more preferably 2 ⁇ 10 5 /ml.
  • the invention also provides a method of using the pharmaceutical composition of the invention, in a specific embodiment, comprising the steps of:
  • the mesenchymal progenitor cells are administered to a subject in need thereof, and the preferred site of administration is within the joint cavity of the subject; the preferred period of use is 1 month, and/or 2 months after step (1).
  • haMPCs After the stimulation of LPS, hypoxia, etc., the expression levels of various cytokines are significantly increased in haMPCs used in the present invention, haMPCs have high cytokine secretion ability, and can repair body damage under suitable conditions in vivo. ;
  • the haMPCs of the present invention have typical mesenchymal stem cell characteristics and have potential differentiation ability under suitable conditions in vivo, thereby satisfying the relative needs of the body;
  • haMPCs can be converted into chondrocytes under specific induction conditions, and are used for preventing or treating osteoarthritis.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.
  • Example 1 Example 1
  • the cells were collected into a centrifuge tube by enzymatic digestion.
  • the cell suspension was adjusted to a density of lx l0 5 /mL, 800 r/min (120 g), centrifuged for 5 min, the supernatant was discarded, and washed with cold D-Hanks at 4 °C.
  • the cells were resuspended, and the cell suspension was again centrifuged at 800 r/mm for 5 min, after which the supernatant was discarded.
  • the cells were then resuspended to 1 mL with D-Hanks, 5 to 10 L of antibody was added, protected from light, and placed on ice for 30 min.
  • Table 1 The expression of cell surface antigen marker expression by SVF was analyzed by flow cytometry. The results showed that the ratio of SVF in freshly isolated progenitor cells was 60%, and the content of hematopoietic progenitor cells was 70%, and there were more mixed cells.
  • Fig. 3A The results showed (Fig. 3A) that the concentration of VEGF decreased with the increase of LPS concentration in fresh haMPCs cultured in complete medium; the concentration of VEGF in the fresh haMPCs group cultured in 5% FBS medium was basically the same as that in the control group at 200 ng/ml. 100ng/ml and 300ng/ml were decreased respectively.
  • the concentration of VEGF in the cryopreserved haMPCs group in complete medium culture did not change much with the increase of LPS concentration. Overall, serum culture is higher than VEGF in complete medium.
  • the chondrogenic differentiation medium was prepared using the GIBCO STEMPRO into a chondrogenic differentiation kit, and the p3 generation cells were obtained by digesting the haMPCs by centrifugation, and a cell suspension of 1.6 X 10 ⁇ 7 viable cells/mL was prepared using standard medium.
  • a 5 L cell suspension (cell volume 8 x l0 4 ) was pipetted with a sample gun and inoculated into the center of the well of a multi-well plate to be placed. Pre-culture for 2 hours in a 0 2 incubator.
  • the medium was aspirated, washed once with DPBS, and the cells were fixed with a 4% formalin solution for 30 minutes. After fixation, it was washed with DPBS, and then stained with a 1% Alcian Blue solution (dissolved in 0.1 N of HC1) for 30 minutes. The cells were washed three times with 0.1 N of HC1, added to distilled water and acidic, and then observed under light microscope for each experimental group and control group, and the pictures were saved. The experimental group that successfully differentiated into chondrocytes stained with Alcian Blue and became blue due to the proteoglycan synthesized by chondrocytes.
  • this example shows that the haMPCs of the present invention exhibit chondrocyte characteristics in cell morphology and classical chemical staining under the culture conditions of the differentiation medium, indicating that haMPCs have the differentiation potential to the chondrocytes.

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Abstract

L'invention concerne des utilisations de cellules de couche stroma-vasculaire et de cellules progénitrices mésenchymateuses pour la prévention et le traitement de l'arthrose. L'invention concerne également une composition pharmaceutique de cellules de couche stroma-vasculaire et de cellules progénitrices mésenchymateuses et un procédé pour la prévention et le traitement de l'arthrose.
PCT/CN2013/087284 2012-11-16 2013-11-16 Utilisation d'une cellule de couche stroma-vasculaire et d'une cellule progénitrice mésenchymateuse pour la prévention ou le traitement de l'arthrose Ceased WO2014075634A1 (fr)

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CN201210466717.6A CN103816183A (zh) 2012-11-16 2012-11-16 间质血管层细胞和间充质祖细胞在预防或治疗骨性关节炎中的应用
CN201210466717.6 2012-11-16

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WO2014075634A1 true WO2014075634A1 (fr) 2014-05-22

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CN106031792A (zh) * 2015-03-10 2016-10-19 西比曼生物科技（上海）有限公司 治疗关节软骨缺损的组合物
CN109355251A (zh) * 2018-09-20 2019-02-19 瑞梦德医药科技(北京)有限公司 脂肪组织来源复合细胞群及其制备方法与应用

Citations (2)

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CN101103101A (zh) * 2004-09-24 2008-01-09 成血管细胞系统公司 增强间充质前体细胞（mpc）的增生和/或存活的方法
CN102144028A (zh) * 2008-09-03 2011-08-03 成血管细胞系统公司 造血前体的扩增

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CN102144028A (zh) * 2008-09-03 2011-08-03 成血管细胞系统公司 造血前体的扩增

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LINDA L.BLACK ET AL.: "Effect of Adipose-Derived Mesenchymal Stem and Regenerative Cells on Lameness in Dogs with Chronic Osteoarthritis of the Coxofemoral Joints: A Randomized, Double-Blinded, Multicenter, Controlled Trial.", VETERINARY THERAPEUTICS., vol. 8, no. 4, December 2007 (2007-12-01), pages 272 - 284, XP009190607 *
RICHARD TULI ET AL., HUMAN MESENCHYMAL PROGENITOR CELL -BASED TISSUE ENGINEERING OF A SINGLE-UNIT OSTEOCHONDRAL CONSTRUCT., vol. 10, no. 7/8, 2004, pages 1169 - 1179 *

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