CN120603598A - Application of cell membrane fragments in the treatment of lower limb ischemia - Google Patents

Abstract

The cell patch fragments are used for preparing medicines for treating lower limb ischemia, and specifically, the cell patch fragments formed after injection administration of the cell patch can prevent limb ischemia necrosis of lower limb, have good limb protecting effect, can effectively prevent muscular atrophy of affected limb, have the limb protecting effect and the muscular atrophy preventing effect equivalent to those of the cell patch, and are obviously superior to single cell suspension.

Description

Use of cell patch fragments in lower limb ischemia treatment Technical Field

The present disclosure relates to the fields of biological medicine and cell biology, in particular to the use of cell patch fragments in the treatment of lower limb ischemia.

Background

Ischemia of the lower limb is a common peripheral arterial disease, which is a disease of symptoms such as intermittent claudication, pain, ulcer or gangrene of the lower limb caused by arterial stenosis or occlusion of the lower limb and insufficient blood perfusion. The risk of developing this disease has increased in recent years with the increasing population aging and long-term smoking, type II diabetes, obesity, sedentary lifestyle, and the like. There is a high amputation rate and a high risk of death after the development of ischemia of the lower limb to the severe stage. For patients who do not meet the conditions of revascularization surgery, there is currently a lack of effective treatments. In recent years, mesenchymal stem cells have been demonstrated to restore tissue function and repair ischemic tissue through immunomodulation, promotion of angiogenesis and paracrine of bioactive factors, etc., and are used for the treatment of lower limb ischemia. Worldwide, allogenic bone marrow mesenchymal stem cell injection product of Indian Stempeutics companyHas been marketed in india for the treatment of lower limb ischemia. However, the cell injection has the problems of poor resistance of cells to acidic and ischemic environments, short survival time, serious cell loss, failure to gather and act at focus parts, low cell utilization rate and the like, so the treatment effect is poor. In recent years, there have been studies on stem cell transplantation using a degradable material as a scaffold, but the interaction between the material and cells, the degradation rate of the scaffold material, the histocompatibility of the scaffold material and its degradation products, and the like all affect the final therapeutic effect. Therefore, a mesenchymal stem cell preparation capable of effectively treating lower limb ischemia needs to be further researched and explored.

Disclosure of Invention

The cell membrane technology can enable cells to be connected with each other through the self-secreted extracellular matrix in the culture process to form a two-dimensional lamellar structure without an exogenous scaffold, so that the problem of in-situ retention of the cells is effectively solved, the number of surviving cells and the survival time of the cells are improved, the repair of lesion sites is promoted by secreting a large amount of cytokines for a long time, and the treatment effect is enhanced. Based on this, it is common for the person skilled in the art to administer the cell membrane by means of direct implantation, in order to ensure as much as possible the dimensional uniformity and structural integrity of the cell membrane during administration, avoiding structural breakage. However, the inventors of the present disclosure have surprisingly found in experimental studies that cell patch fragments formed after injection administration of the cell patch of the present disclosure can prevent ischemic limb necrosis of lower limb, have good limb-protecting effect, and can also effectively prevent muscular atrophy of affected limb, and the limb-protecting effect and the muscular atrophy preventing effect of the affected limb are equivalent to those of the cell patch, and are significantly superior to that of single cell suspension. Therefore, the cell patch fragments formed after injection administration of the cell patch disclosed by the invention have excellent effect of treating lower limb ischemia and have wide application prospect.

To this end, in a first aspect of the present disclosure, the present disclosure provides the use of cell membrane fragments in the manufacture of a medicament for the treatment of lower limb ischemia.

In a second aspect of the disclosure, the disclosure provides cell patch fragments for use in treating lower limb ischemia.

In a third aspect of the present disclosure, the present disclosure provides a method of treating ischemia of the lower extremities comprising administering to a subject in need thereof an effective amount of cell membrane fragments.

In some embodiments, the mode of administration of the cell membrane fraction is injection administration.

In some embodiments, the injection administration is intramuscular (e.g., adductor or gastrocnemius) injection administration.

In some embodiments, the dispersant of the cell membrane fraction is physiological saline upon administration by injection.

In some embodiments, the cell membrane fraction is obtained by disrupting cell membranes.

In some embodiments, the cell membrane fraction has a diameter of about 0.1-3mm, such as about 0.1-0.2mm、0.2-0.3mm、0.3-0.4mm、0.4-0.5mm、0.5-0.6mm、0.6-0.7mm、0.7-0.8mm、0.8-0.9mm、0.9-1mm、1-1.1mm、1.1-1.2mm、1.2-1.3mm、1.3-1.4mm、1.4-1.5mm、1.5-1.6mm、1.6-1.7mm、1.7-1.8mm、1.8-1.9mm、1.9-2mm、2-2.1mm、2.1-2.2mm、2.2-2.3mm、2.3-2.4mm、2.4-2.5mm、2.5-2.6mm、2.6-2.7mm、2.7-2.8mm、2.8-2.9mm or 2.9-3mm. The diameter of a cell fragment refers to the longest line segment of each cell fragment when two non-adjacent vertices are connected.

In some embodiments, the cell membrane fraction has a diameter of about 0.1-0.5mm or about 0.4-3mm.

In some embodiments, the cell patch fragments are obtained by injecting the cell patch through a syringe needle.

In some embodiments, the cell membrane fraction is obtained by:

1) Loading a cell patch into a syringe needle;

2) After physiological saline is sucked into the syringe cylinder, a syringe needle loaded with a cell membrane is mounted on the syringe cylinder;

3) Pushing the injector piston to inject the cell membrane to obtain cell membrane fragments;

Optionally, in step 1), the cell membrane sheet is cut (so as to accommodate different doses of administration) in advance.

In some embodiments, the needle is 22G, 23G, 25G, or 27G in size. The needle model in this disclosure is of international standard.

In some embodiments, the needle is 22G or 27G in size.

In some embodiments, the cell membrane fraction is not less than 2 pieces, e.g., not less than 3 pieces, not less than 4 pieces, not less than 5 pieces, not less than 6 pieces, not less than 7 pieces, not less than 8 pieces, not less than 9 pieces, not less than 10 pieces, not less than 12 pieces, not less than 14 pieces, not less than 16 pieces, not less than 18 pieces, not less than 20 pieces, not less than 22 pieces, not less than 24 pieces, not less than 26 pieces, not less than 28 pieces, not less than 30 pieces, or more.

In some embodiments, the cell is a mesenchymal stem cell.

In some embodiments, the cells are selected from the group consisting of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrium mesenchymal stem cells, dental pulp mesenchymal stem cells.

In some embodiments, the cell is an umbilical cord mesenchymal stem cell.

In some embodiments, the cell membrane comprises multiple layers of cells, e.g., at least 2 layers, at least 3 layers, at least 4 layers, at least 5 layers, at least 6 layers, at least 7 layers, at least 8 layers, at least 9 layers, at least 10 layers, at least 12 layers, at least 14 layers, at least 16 layers, at least 18 layers, at least 20 layers, or more.

In some embodiments, the cell membrane has a diameter of about 3-30mm, such as 3-4mm、4-5mm、5-6mm、6-7mm、7-8mm、8-9mm、9-10mm、10-12mm、12-14mm、14-16mm、16-18mm、18-20mm、20-22mm、22-24mm、24-26mm、26-28mm or 28-30mm.

In some embodiments, the cell patch has a thickness of about 50-1000 μm, such as 50-60μm、60-70μm、70-80μm、80-90μm、90-100μm、100-120μm、120-140μm、140-160μm、160-180μm、180-200μm、200-250μm、250-300μm、300-350μm、350-400μm、400-450μm、450-500μm、500-550μm、550-600μm、600-650μm、650-700μm、700-750μm、750-800μm、800-850μm、850-900μm、900-950μm or 950-1000 μm.

In some embodiments, the cell patch is circular or nearly circular in shape.

In some embodiments, the cell membrane is capable of secreting one or more selected from interleukin-6 (interleukin-6, il-6), transforming growth factor-beta (transforming growth factor-beta, TGF-beta), prostaglandin E2 (prostaglandin E2, PGE 2), hepatocyte growth factor (hepatocyte growth factor, HGF), epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor (vascularendothelial growth factor, VEGF), insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolizing enzyme (indoleamine 2,3-dioxygenase, IDO), nitric oxide synthase (inducible nitric oxidesynthase, iNOS).

In some embodiments, the cell membrane is capable of secreting at least interleukin-6 (interleukin-6, IL-6), hepatocyte growth factor (hepatocyte growth factor, HGF), vascular endothelial growth factor (vascular endothelial growthfactor, VEGF).

In some embodiments, the cell membrane contains extracellular matrix secreted by the cell.

In some embodiments, the extracellular matrix in the cell membrane comprises one or more of Fibronectin, integrin family (e.g., integrin- β1), vitronectin.

In some embodiments, the extracellular matrix in the cell patch comprises at least fibronectin and integrin- β1.

In some embodiments, the volume of physiological saline is 0.05 to 1mL (e.g., 0.05-0.06mL、0.06-0.07mL、0.07-0.08mL、0.08-0.09mL、0.09-0.1mL、0.1-0.2mL、0.2-0.3mL、0.3-0.4mL、0.4-0.5mL、0.5-0.6mL、0.6-0.7mL、0.7-0.8mL、0.8-0.9mL or 0.9 to 1mL, preferably 0.1 mL).

In some embodiments, the cell membrane is obtained by:

1) Coating a layer of matrix on the surface of the temperature-sensitive culture dish;

2) Adding the cell suspension into a temperature-sensitive culture dish for culturing;

3) And (3) reducing the temperature, and separating the cells and the extracellular matrix secreted by the cells into lamellar layers to obtain the cell membrane.

In some embodiments, the cell is a mesenchymal stem cell.

In some embodiments, the cells are selected from the group consisting of umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrium mesenchymal stem cells, dental pulp mesenchymal stem cells.

In some embodiments, the cell is an umbilical cord mesenchymal stem cell.

In some embodiments, the coating matrix is selected from collagen, gelatin, fibronectin, fibrinogen, fibronectin, vitronectin, laminin, polyornithine, polylysine, ornithine lysine copolymer.

In some embodiments, the coating is performed in an incubator at 35-40 ℃ (e.g., 35-36 ℃, 36-37 ℃, 37-38 ℃, 38-39 ℃, or 39-40 ℃, preferably 37 ℃), saturated humidity, 1-10% co ₂ (e.g., 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10% co ₂, preferably 5% co ₂).

In some embodiments, the coating is for a period of time ranging from 0.5 to 48 hours, such as 0.5-1h、1-2h、2-3h、3-4h、4-5h、5-6h、6-7h、7-8h、8-9h、9-10h、10-15h、15-20h、20-25h、25-30h、30-35h、35-40h、40-45h or 45 to 48 hours.

In some embodiments, the density of cells in the temperature sensitive culture dish is 1.8x10 ⁴-1.9×10⁶/cm² (e.g., 1.8×10⁴-2×10⁴/cm²、2×10⁴-3×10⁴/cm²、3×10⁴-4×10⁴/cm²、4×10⁴-5×10⁴/cm²、5×10⁴-6×10⁴/cm²、6×10⁴-7×10⁴/cm²、7×10⁴-8×10⁴/cm²、8×10⁴-9×10⁴/cm²、9×10⁴-1×10⁵/cm²、1×10⁵-2×10⁵/cm²、2×10⁵-3×10⁵/cm²、3×10⁵-4×10⁵/cm²、4×10⁵-5×10⁵/cm²、5×10⁵-6×10⁵/cm²、6×10⁵-7×10⁵/cm²、7×10⁵-8×10⁵/cm²、8×10⁵-9×10⁵/cm²、9×10⁵-1×10⁶/cm²、1×10⁶-1.5×10⁶/cm² or 1.5x10 ⁶-1.9×10⁶/cm², preferably 1 x 10 ⁶/cm²).

In some embodiments, step 2) is performed by adding the cell suspension and culture broth to a temperature sensitive culture dish for culturing.

In some embodiments, in step 2), the culture broth is a serum-free medium.

In some embodiments, the serum-free medium may be basal medium + nutritional supplement, or a commercial serum-free medium.

In some embodiments, the basal medium may be 1640, DMEM, alpha-MEM, DMEM/F12, etc.

In some embodiments, the nutritional supplement includes a plurality of vitamin C, sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin (plant expression), progesterone, putrescine, biotin, sodium pyruvate, ethanolamine, carnitine, amino acids, vitamins, glutathione, linoleic acid, linolenic acid, and the like.

In some embodiments, the commercial serum-free Medium includes, but is not limited to CTSTMStemProTMMSC SFM Kit, mesenCultTM-ACF Medium, mesenCultTM-ACF Plus Medium, mesenCultTM-XF Medium, and the like.

In some embodiments, in step 2), the ratio of culture fluid in the temperature sensitive culture dish is 0.1-0.5mL/cm ² (e.g., 0.1-0.2mL/cm ²、0.2-0.3mL/cm²、0.3-0.4mL/cm² or 0.4-0.5mL/cm ², preferably 0.3mL/cm ²).

In some embodiments, step 2), the culturing is performed in an incubator at 35-40 ℃ (e.g., 35-36 ℃, 36-37 ℃, 37-38 ℃, 38-39 ℃, or 39-40 ℃, preferably 37 ℃), saturated humidity, 1-10% co ₂ (e.g., 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9%, or 9-10% co ₂, preferably 5% co ₂).

In some embodiments, in step 2), the incubation time is 2-48 hours, such as 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-15 hours, 15-20 hours, 20-25 hours, 25-30 hours, 30-35 hours, 35-40 hours, 40-45 hours, or 45-48 hours.

In some embodiments, in step 3), the temperature is reduced to 4-32 ℃, such as 4-5℃、5-6℃、6-7℃、7-8℃、8-9℃、9-10℃、10-12℃、12-14℃、14-16℃、16-18℃、18-20℃、20-22℃、22-24℃、24-26℃、26-28℃、28-30℃ or 30-32 ℃.

In some embodiments, in step 3), no additional reagents or materials are added when the cells and their secreted extracellular matrix are exfoliated.

In a fourth aspect of the disclosure, the disclosure provides the use of cell membrane fragments in the manufacture of a pharmaceutical composition for treating ischemia of the lower limb.

In a fifth aspect of the disclosure, the disclosure provides a pharmaceutical composition comprising cell membrane fragments for use in treating lower limb ischemia.

In a sixth aspect of the present disclosure, the present disclosure provides a method of treating ischemia of the lower extremities comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising cell membrane fragments.

In some embodiments, the cell membrane fraction is as described in any of the above claims.

In some embodiments, the cell is as described in any one of the above claims.

In some embodiments, the cell membrane is as described in any of the above claims.

In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, such as physiological saline.

In some embodiments, the pharmaceutical composition is in the form of an injection.

In some embodiments, the pharmaceutical composition is administered by injection.

In some embodiments, the injection administration is intramuscular (e.g., adductor or gastrocnemius) injection administration.

In the present disclosure, there is also provided a method of isolated culture of umbilical cord mesenchymal stem cells, comprising primary isolated culture and subculture.

In some embodiments, the primary isolation culture comprises the steps of isolating Wharton's jelly from umbilical cord tissue, chopping the Wharton's jelly to obtain a tissue mass, spreading the tissue mass in a culture vessel for culture, adding an appropriate amount of complete medium to cover the tissue mass and continuing the culture, removing the tissue mass when cells adhering to the culture vessel appear around the tissue mass and the cells grow to 70-100% confluence, and performing passaging operation on the cells.

In some embodiments, the subculture comprises the steps of separating cells from a culture container, uniformly dispersing the cells in a culture medium, inoculating the cells into the culture container, adding a proper amount of culture medium, replacing a proper amount of fresh culture medium every 1-5 days according to the growth state of the cells, and repeating the subculture operation when the cells grow to 70-100% of confluence. The algebra increases by 1 for each passage operation of the cells. Umbilical cord mesenchymal stem cells grow on the wall, are fibrous and have uniform morphology.

In this disclosure, "treating" refers generally to obtaining a desired pharmacological and/or physiological effect, including (a) inhibiting the symptoms of a disease, i.e., arresting its development, or (b) alleviating the symptoms of a disease, i.e., causing the disease or symptoms to degenerate.

In this disclosure, "subject" refers to a vertebrate. In certain embodiments, a vertebrate refers to a mammal. Mammals include, but are not limited to, livestock (such as cattle), pets (such as cats, dogs, and horses), primates, mice, and rats. In certain embodiments, the mammal refers to a human.

In this disclosure, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, the desired effect. Determination of such effective amounts is well within the ability of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, and the like. It is further understood that for any particular individual, the particular dosage regimen may be adjusted over time according to the individual needs and the manner of administration or the professional judgment of the person supervising the administration.

Advantageous effects

1. The cell patch disclosed by the invention can prevent the necrosis of the ischemic limb of the lower limb, has a good limb-protecting effect, and meanwhile, the cell patch fragments can also prevent the necrosis of the ischemic limb of the lower limb, also have a good limb-protecting effect, even the limb-protecting effect of the cell patch fragments is equivalent to the limb-protecting effect of the cell patch, and is obviously superior to that of a single-cell injection.

2. The cell patch and the cell patch fragments disclosed by the invention can be used for effectively preventing the muscular atrophy of the affected limb, and the cell patch fragments and the cell patch have equivalent effects on preventing the muscular atrophy of the affected limb, and are obviously superior to single-cell injection. In a word, cell membrane and cell membrane piece can prevent suffering limb muscular atrophy notably, be favorable to the improvement of low limbs ischemia limbs function, and improve effect and strengthen along with the dose increases.

3. The cell membrane and the cell membrane fragments can effectively improve the symptom of ischemia of lower limbs, enhance the blood flow perfusion of the ischemia part by promoting angiogenesis, avoid muscular atrophy of the affected limb and are beneficial to the preservation of the affected limb.

Drawings

In FIG. 1, a shows a schematic of umbilical cord mesenchymal stem cell membrane, b shows a schematic of cell membrane cut into 6 equal parts, c shows a schematic of cell membrane loaded into a syringe prior to administration;

FIG. 2 shows a typical morphology of a cell patch after injection through a syringe needle (left: injection through a 27G needle; right: injection through a 22G needle);

FIG. 3 shows that the patch of umbilical cord mesenchymal stem cells detected by a laser Doppler line scanning blood flow imager improves blood perfusion of an ischemic limb of a mouse;

Figure 4 shows the general and gastrocnemius morphology of healthy (left) and diseased (right) limbs of the cell patch group and saline group 28 days after dosing.

Detailed Description

The present disclosure is further illustrated below in connection with specific examples, but should not be construed as limiting the disclosure in any way.

The culture system of the present disclosure comprises a serum-free medium and a coating matrix mated to the medium, wherein:

1. the serum-free medium composition may be basal medium + nutritional supplement, or commercial serum-free medium, as follows:

(1) Basal medium + nutritional additives:

the basal medium can be 1640, DMEM, alpha-MEM, DMEM/F12, F12 and the like;

The nutritional supplement includes a plurality of vitamin C, sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin (plant expression), progesterone, putrescine, biotin, sodium pyruvate, ethanolamine, carnitine, amino acids, vitamins, glutathione, linoleic acid, linolenic acid, etc.

(2) Commercial serum-free medium:

including but not limited to CTSTMStemProTMMSC SFM Kit, mesenCultTM-ACF Medium, mesenCultTM-ACF Plus Medium, mesenCultTM-XF Medium, and the like.

2. Coating matrix matched with culture medium:

including but not limited to collagen, gelatin, fibronectin, fibrinogen, fibronectin, vitronectin, laminin, polyornithine, polylysine, ornithine lysine copolymer, and the like.

The culture system disclosed above is suitable for culture, cell patch preparation and detection of mesenchymal stem cells derived from various tissues, including but not limited to umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrium mesenchymal stem cells, dental pulp mesenchymal stem cells.

The present disclosure is further explained below using umbilical cord mesenchymal stem cells as an example only.

Example 1 isolation and culture of umbilical cord mesenchymal Stem cells

Umbilical cord mesenchymal stem cells are subjected to primary separation culture and subculture to carry out cell number expansion, and the procedure is as follows:

1. Primary culture of umbilical cord mesenchymal stem cells

The obtained neonatal umbilical cord (from Beijing Minde hospital, pregnant and parturient women sign informed consent to donate, the informed consent has been approved by Minde hospital ethical committee) is washed with physiological solution with the same osmotic pressure as human body fluid, artery, vein and adventitia are removed, whatman's jelly is separated, sheared into small tissue blocks of 0.1-2mm, and the tissue blocks are uniformly spread in a culture container coated with matrix, wherein the interval between the tissue blocks is 2-30mm. Placing the culture container in a cell incubator, adding a proper amount of complete culture medium to cover the tissue blocks after 2-7 days, and allowing umbilical cord mesenchymal stem cells to climb out in 8-21 days, and allowing the umbilical cord mesenchymal stem cells to grow by adhering to the walls, so that the umbilical cord mesenchymal stem cells are fibrous and uniform in morphology. When the cells climb out to 70-100% confluence, removing tissue blocks, and carrying out passage operation on the cells.

2. Umbilical cord mesenchymal stem cell passage

When the cells grow to 70-100% confluence, the cells are subjected to a passaging operation, wherein the cells are separated from the culture vessel by a method including but not limited to digestion with pancreatin and the like, scraping of the cells and the like. The cells are dispersed in the culture medium by stirring, vortexing, etc., including but not limited to, and the cells are inoculated into the culture vessel at a density of 500-100000/cm ². Adding a proper amount of culture medium, changing a proper amount of fresh culture medium every 1-5 days according to the growth state of the cells, and repeating the passage operation when the cells grow to 70-100% confluence. Umbilical cord mesenchymal stem cells cultured by the culture method grow on the wall, are fibrous and have uniform morphology.

Example 2 detection and identification of umbilical cord mesenchymal Stem cells

The obtained umbilical cord mesenchymal stem cells are detected and identified by the following method:

1. umbilical cord mesenchymal stem cell growth curve detection

The measurement method of the growth curve includes, but is not limited to, MTT method, WST method, DNA content detection method, ATP detection method, etc., and will be further described herein by taking the WST method as an example. Umbilical cord mesenchymal stem cells are dispersed, inoculated into a culture pore plate at a certain density according to a reagent instruction book, and subjected to liquid exchange operation according to normal culture conditions. Cell activity was measured according to the instructions at fixed times of day over a period of time to obtain data on cell activity or number. The activity of cells was measured daily within 7 days using WST reagent by adding WST reagent to the cell well plate being cultured according to the recommended ratio of the specification, incubating for a fixed time in the cell incubator, and measuring the absorbance value of the cell liquid of the porous plate at 450nm wavelength by using an enzyme-labeled instrument or an ultraviolet spectrophotometer, which is positively correlated with the cell number. The results showed that the cell activity in the cells increased with the increase in culture time, and it was concluded that the cell number increased with the increase in culture time.

2. Umbilical cord mesenchymal stem cell identification

The identification method of the umbilical cord mesenchymal stem cells comprises, but is not limited to, detection of cell surface marker proteins by flow cytometry, three-way differentiation of umbilical cord mesenchymal stem cells, detection of cell expression genes by a PCR method and the like, and is exemplified by flow cytometry and three-way differentiation.

2.1 Identification of umbilical cord mesenchymal Stem cell surface markers

Umbilical cord mesenchymal stem cells are dispersed in a culture medium and centrifuged, and cell surface marker proteins including, but not limited to, CD73, CD90, CD105, CD34, CD11B, CD, CD45, HLA-DR are stained according to the purchased reagent instructions with serum or a physiological solution having a serum protein content of 1-20% equivalent to the osmotic pressure of human body fluid. Wherein the phenotype of CD73, CD90, CD105 is positive, the ratio thereof should be not less than 95%, the phenotype of CD34, CD11B, CD, CD45, HLA-DR is negative, the ratio thereof should be not more than 2%. The results showed that the ratio of positive markers was greater than 99% and the ratio of negative markers was no greater than 0.1%.

2.2 Three-way induced differentiation of umbilical mesenchymal Stem cells

Umbilical cord mesenchymal stem cells have the ability to induce differentiation in bone, cartilage, and fat directions. The specific operation of the osteogenesis and adipogenesis differentiation detection is that cells are inoculated into a proper culture vessel according to the proportion of a three-way induced differentiation reagent instruction book, and when the cells detected by osteogenesis induction grow to be 50-90% confluence, the cells detected by adipogenesis induction grow to be more than 90% confluence, the cells are respectively added into osteogenesis and adipogenesis induction culture media. And centrifuging a certain number of cells to the bottom of a centrifuge tube during chondrogenesis, adding a chondrogenesis culture medium, and after the cells are clustered into pellets, enabling the pellets to leave the bottom of the tube, ensuring complete contact with the induction culture medium, and detecting the cells after the cells are subjected to induction culture for more than 7 days. Osteogenic induction may be stained with alizarin red, anti-hOsteocalcin, fat induction may be stained with oil red O, anti-mFABP4, and chondrogenic induction may be stained with oil red O, anti-HAGGRECAN, including but not limited to alcian blue. The results show that umbilical cord mesenchymal stem cells can be stained after undergoing adipogenic differentiation, osteogenic differentiation and chondrogenic differentiation.

Example 3 preparation of umbilical cord mesenchymal Stem cell Membrane

The umbilical cord mesenchymal stem cell membrane preparation procedure is as follows:

1. Temperature-sensitive intelligent culture dish coating

Before membrane preparation, a layer of matrix which is favorable for attaching mesenchymal stem cells, including but not limited to collagen, gelatin, fibronectin, fibrinogen, mucin, vitronectin, laminin, polyornithine, polylysine, ornithine lysine copolymer and the like, is coated on the surface of a temperature-sensitive intelligent culture dish in advance by utilizing a serum-free culture system and the mesenchymal stem cells. When in coating, physiological buffer solution such as physiological saline, PBS and the like is used for diluting the coating matrix, then the diluted coating matrix is added into a temperature-sensitive intelligent culture dish, and a culture dish cover is covered. And then placing the temperature-sensitive intelligent culture dish in a culture box with saturated humidity and 5% CO ₂ for coating for 0.5-48h.

2. Umbilical cord mesenchymal stem cell membrane preparation

After coating, the residual coating matrix is sucked out of the intelligent culture dish, and the mesenchymal stem cell suspension is added into the temperature-sensitive intelligent culture dish. For this example, a 35mm diameter temperature sensitive smart dish was used with a 1.8X10 ⁵-1.8×10⁷ (most preferably 1X 10 ⁷) cell addition and a 1-4ml (most preferably 3 ml) culture volume. And then placing the temperature-sensitive intelligent culture dish in a culture box with saturated humidity and 5% CO ₂ for culture for 2-48h at the temperature of 37 ℃. After the culture is completed, the temperature-sensitive intelligent culture dish is moved to the environment of 4-32 ℃, and cells are separated from the bottom of the temperature-sensitive intelligent culture dish in a lamellar manner, so that the cells become cell membranes without adding any additional reagent or material. The liquid in the dish was collected to obtain a cell membrane supernatant. The cell membrane is grey white, compact in structure and smooth and flat in surface. The size is round with diameter of 3-30mm, and the thickness is 50-1000 μm. The cell patch is seen to comprise a multi-layered cell structure and contains extracellular matrix components secreted by the cells as seen by tissue section observation.

Example 4 functional detection of umbilical cord mesenchymal Stem cell Membrane

The formed cell membrane was subjected to functional assays as follows:

1. ELISA method for detecting cell membrane secretion factor

The supernatant from the preparation of the cell membrane is assayed for the secretion of factors including, but not limited to, interleukin-6 (interleukin-6, IL-6), transforming growth factor-beta (transforming growth factor-beta, TGF-beta), prostaglandin E2 (prostaglandin E, PGE 2), hepatocyte growth factor (hepatocyte growth factor, HGF), epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor (vascularendothelial growth factor, VEGF), insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolizing enzyme (indoleamine, 3-dioxygenase, IDO), nitric oxide synthase (inducible nitric oxidesynthase, iNOS), and the like, according to the ELISA kit instructions. The detection of certain umbilical cord mesenchymal stem cell membrane secreted factors is performed by taking interleukin-6 (interleukin-6, IL-6), hepatocyte growth factor (hepatocyte growth factor, HGF) and vascular endothelial growth factor (vascular endothelial growthfactor, VEGF) as examples. The experiment was repeated for three experiments (sample 1, sample 2, sample 3). And comparing secretion factors of the human mesenchymal stem cell suspension and the human mesenchymal stem cell membrane with three samples. The results show that the human mesenchymal stem cell membrane has better factor secretion function compared with the human mesenchymal stem cell suspension.

2. Tissue section for observing protein condition in cell membrane

The cell membrane obtained above is fixed with paraformaldehyde or formalin fixed solution, and then the cell membrane is made into tissue section with thickness of 4-10 μm by paraffin section method or frozen section method for staining, and the condition of protein contained in extracellular matrix in the cell membrane is observed, wherein the protein includes but is not limited to Fibronectin (Fibronectin), integrin family, vitronectin, etc., and the cell nucleus is usually stained with fluorescent dye such as DAPI or Hoechst33258 for auxiliary positioning at the same time of staining. In addition to immunofluorescence methods, staining observations can be performed using methods including, but not limited to, immunohistochemical methods, and the like. Here, fibronectin and integrin- β1 are exemplified, and both fibronectin and integrin- β1 are stained with a fluorescein-labeled antibody dye, and nuclei are stained with DAPI, and the results show that the prepared cell membrane contains a large amount of fibronectin and integrin- β1.

3. Scanning electron fiber mirror for observing surface structure of cell membrane

Fixing the obtained cell membrane with 2.5% glutaraldehyde fixing solution, dehydrating with gradient alcohol, and naturally air drying to obtain dried cell membrane for analysis by scanning electron microscope. And (3) sticking the dried cell membrane on the surface of a sample stage by using a conductive double-sided adhesive tape, and then performing surface metal spraying treatment by using a vacuum magnetron sputtering method to conduct electricity on the surface of the observed sample. The sample stage was placed in a scanning electron microscope (Hitachi S-4800 for use) for observation. The results show that the cell patch surface morphology is relatively flat and smooth, mesenchymal stem cells are visible, intercellular protein connection is also visible, and cell stacking is also visible.

Example 5 Activity test of umbilical mesenchymal Stem cell Membrane and fragments thereof

1. Animal modeling:

(1) Mice of age-appropriate age (7 weeks in this experiment) (which may be ICR/nodscd/C57 male mice, in this experiment ICR male mice), anesthetized with isoflurane, or anesthetized with sodium pentobarbital, or with hydrogalactal, i.p. injection;

(2) Fixing hind limbs of the mice, shaving Mao Bei skins, sterilizing, cutting an incision of about 1cm from knees of lower limbs of left side legs to inner sides of thighs of the mice, separating subcutaneous tissues and muscle tissues, and exposing vessels of lower limbs;

(3) The modeling operation method 1 comprises ligating two ends of femoral artery with surgical suture (nylon thread, 7-0) from above inguinal ligament to femoral artery bifurcation, and performing lateral branch and medial branch with surgical blade; the modeling operation method 2 comprises separating femoral artery with pointed forceps, moving forceps along femoral artery to destroy collateral branch and microvasculature, and ligating with surgical suture (nylon thread, 7-0) at proximal end of femoral artery;

(4) The incision was closed with nylon suture (4-0 suture in this experiment);

(5) Detecting blood flow signals by using a laser Doppler line scanning blood flow imager (model of the experimental instrument is Moor LDLS), and calculating the ratio of the affected side to the opposite side;

(6) Animals were sent to the feeding area and awakened after anesthesia. The analgesic drug meloxicam is injected every day three days after operation.

2. Membrane drug administration:

Single administration is carried out within 0-72h after animal molding operation, and the specific steps are as follows:

(1) Carrying out gas anesthesia on the molded mice by isoflurane, intravenous injection by using sodium pentobarbital or intraperitoneal injection by using hydrated sharp aldehyde, and carrying out local disinfection by using iodophor for administration;

(2) Cutting the prepared cell membrane into required size according to dosage, taking administration of mice as an example, and cutting cell membrane with diameter of 1.8cm into 6 equal parts for administration;

(3) The administration method comprises the steps of directly transplanting or injecting, wherein after the animal molding step (3), spreading the cell membrane near femoral artery before incision suturing, waiting for 2min, performing incision suturing to finish administration, taking down the syringe needle (which can be a needle of various types and can be used for administration of mice, for example, a 27G needle) to load the cut cell membrane into the syringe needle (see figure 1), sucking 0.1mL of physiological saline (0.05-1 mL can be used) into the syringe barrel, lightly installing the syringe needle loaded with the cell membrane onto the syringe needle cylinder, penetrating the needle into thigh muscle of affected limb (can be adductor muscle and gastrocnemius muscle), pushing the syringe to inject the cell membrane into muscle of mice through the needle, withdrawing the needle, and finishing administration, (the size of the broken cell membrane after injection into the adductor muscle is different according to the size of the syringe needle, for example, the size of the 27 needle is about 0.05-1 mm after injection, and the size of the membrane after injection is about 0.4mm, and the size of the membrane after injection is about 0.4 mm).

3. Validity test:

Setting a plurality of test groups with a plurality of doses, namely a low-dose group with 1 x 10-6 equivalent cells and a high-dose group with 2x 10-6 equivalent cells, wherein (group 1: low-dose cell membrane sheet, group 2: low-dose cell membrane sheet fragments, group 3: low-dose single-cell injection, group 4: low-dose cell membrane supernatant, group 5: high-dose cell membrane sheet, group 6: high-dose cell membrane sheet fragments, group 7: high-dose single-cell injection, group 8: high-dose cell membrane supernatant), testing the improvement effect of each test group on lower limb ischemic diseases, and respectively adopting animals which are not administered with the same amount of normal saline (group 9) or injected with the same amount of normal saline (group 10) after the same amount of the same is molded as test control groups to verify the effectiveness of umbilical cord mesenchymal stem cell membrane and fragments thereof for treating lower limb ischemia.

(1) Improvement of blood flow in lower limbs by measuring blood flow signals with a laser Doppler line scanning blood flow imager on days 3, 7, 14, 21 and 28 after administration, calculating a post-patient side/side blood flow ratio, evaluating improvement of blood flow in lower limbs, and comparing, starting from day 14, the blood flow of animals in cell patch fragment group (group 2) shows remarkable improvement, and on day 14, the improvement is about 16.4%, the improvement is about 17.5% on day 21 and the improvement is about 19.3% on day 28 (see fig. 3) compared with the control group (group 10).

(2) Evaluation of the limb-protecting effect, namely, observing limb injury conditions of mice in a control group and a test group on days 7, 14, 21 and 28 after administration, and calculating the proportion of normal or only toe discoloration, toe necrosis and foot ring death of the molding sides of the mice in each group. The proportion of the three cases in each group was counted, and the occurrence proportion of the three cases in groups 1 to 10 was found to be respectively 1:80%, 12%, 8%, 2:83%, 8%, 9%, 3:58%, 28%, 14%, 4:60%, 24%, 16%, 5:87%, 8%, 5%, 6:89%, 5%, 6%, 7:68%, 18%, 14%, 8:68%, 22%, 10%, 9:50%, 25%, and 10:45%, 23%, 32%. Therefore, the cell membrane can prevent the necrosis of the ischemic limb of the lower limb, has good limb-protecting effect, and meanwhile, the cell membrane fragments can also prevent the necrosis of the ischemic limb of the lower limb, have good limb-protecting effect, even the limb-protecting effect of the cell membrane fragments is equivalent to the limb-protecting effect of the cell membrane, and are obviously superior to that of a single-cell injection group.

(3) Prevention of muscle atrophy in affected limbs mice were dissected from the affected limbs and the gastrocnemius of the healthy limb on day 28 post-dose (see fig. 4), the weights of the two side gastrocnemius were weighed and the weight ratio of the affected limb/healthy limb gastrocnemius was calculated for each group at 82.2%, 80.4%, 53.1%, 57.8%, 85.2%, 84.8%, 68.3%, 67.8%, 51.4%, 50.2%, respectively. Therefore, the cell patch and the cell patch fragments can effectively prevent the muscular atrophy of the affected limb, and the cell patch fragments and the cell patch have the same effect on preventing the muscular atrophy of the affected limb, and are obviously superior to the single-cell injection group. In a word, cell membrane and cell membrane piece can prevent suffering limb muscular atrophy notably, be favorable to the improvement of low limbs ischemia limbs function, and improve effect and strengthen along with the dose increases.

While specific embodiments of the present disclosure have been described above, it will be appreciated by those skilled in the art that these are by way of example only, and the scope of the disclosure is defined by the appended claims. Various alterations and modifications may be made to these embodiments without departing from the principles and spirit of the disclosure, which are intended to be within the scope of the disclosure.

Claims (17)

1. Use of cell membrane fragments in the manufacture of a medicament for the treatment of lower limb ischemia.
2. The use of claim 1, wherein the cell membrane fraction is obtained by disrupting cell membranes.
3. The use according to any one of claims 1-2, wherein the cell membrane fraction has a diameter of about 0.1-3mm, preferably about 0.1-0.5mm or about 0.4-3mm.
4. The use according to any one of claims 1-3, wherein the cell membrane fraction is obtained by injecting cell membranes through a syringe needle.
5. The use according to any one of claims 1 to 4, wherein the cell membrane fraction is obtained by:

   1) Loading a cell patch into a syringe needle;

   2) After physiological saline is sucked into the syringe cylinder, a syringe needle loaded with a cell membrane is mounted on the syringe cylinder;

   3) Pushing the injector piston to inject the cell membrane to obtain cell membrane fragments;

   Optionally, in step 1), the cell membrane sheet is cut in advance.
6. Use according to any one of claims 4 to 5, wherein the needle is 22G, 23G, 25G or 27G, preferably 22G or 27G.
7. The use of any one of claims 1-6, wherein the cell membrane fraction is not less than 2.
8. The use according to any one of claims 1-7, wherein the cells are mesenchymal stem cells, preferably selected from umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrium mesenchymal stem cells, dental pulp mesenchymal stem cells, more preferably umbilical cord mesenchymal stem cells.
9. The use of any one of claims 1-8, wherein the cell membrane further has one or more technical features selected from the following (i) - (vi):

   (i) The cell patch comprises a plurality of layers of cells;

   (ii) The cell membrane has a diameter of about 3-30mm;

   (iii) The cell patch has a thickness of about 50-1000 μm;

   (iv) The shape of the cell membrane is circular or approximate circular;

   (v) The cell membrane is capable of secreting one or more selected from interleukin-6 (interleukin-6, il-6), transforming growth factor-beta (transforming growth factor-beta, TGF-beta), prostaglandin E2 (prostaglandin E, PGE 2), hepatocyte growth factor (hepatocyte growth factor, HGF), epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor (vascularendothelial growth factor, VEGF), insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolizing enzyme (indoleamine, 3-dioxygenase, IDO), nitric oxide synthase (inducible nitric oxidesynthase, iNOS);

   Preferably, the cell membrane is capable of secreting at least interleukin-6 (interleukin-6, il-6), hepatocyte growth factor (hepatocyte growth factor, HGF), vascular endothelial growth factor (vascular endothelial growthfactor, VEGF);

   (vi) The cell membrane contains extracellular matrix secreted by the cells;

   preferably, the extracellular matrix in the cell membrane comprises one or more of Fibronectin (Fibronectin), integrin family (such as integrin- β1), vitronectin;

   More preferably, the extracellular matrix in the cell patch comprises at least fibronectin and integrin- β1.
10. The use according to any one of claims 1 to 9, wherein the cell membrane is obtained by:

    1) Coating a layer of matrix on the surface of the temperature-sensitive culture dish;

    2) Adding the cell suspension into a temperature-sensitive culture dish for culturing;

    3) And (3) reducing the temperature, and separating the cells and the extracellular matrix secreted by the cells into lamellar layers to obtain the cell membrane.
11. The use of claim 10, wherein the method of preparing the cell membrane further has one or more technical features selected from the following (i) - (x):

    (i) The cells are mesenchymal stem cells, preferably selected from umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrium mesenchymal stem cells, dental pulp mesenchymal stem cells, more preferably umbilical cord mesenchymal stem cells;

    (ii) The coating matrix is selected from collagen, gelatin, fibronectin, fibrinogen, fibronectin, vitronectin, laminin, polyornithine, polylysine, ornithine lysine copolymer;

    (iii) The coating is carried out in an incubator at 35-40 ℃, preferably 37 ℃, saturated humidity, 1-10% co ₂, preferably 5% co ₂;

    (iv) The coating time is 0.5-48h;

    (v) The density of cells in the temperature sensitive culture dish is 1.8X10 ⁴-1.9×10⁶/cm² (preferably 1X 10 ⁶/cm²);

    (vi) Step 2) is carried out by adding the cell suspension and the culture solution into a temperature-sensitive culture dish for culturing;

    Preferably, the culture solution is a serum-free medium;

    preferably, the proportion of the culture solution in the temperature-sensitive culture dish is 0.1-0.5mL/cm ² (preferably 0.3mL/cm ²);

    (vii) The cultivation is carried out in an incubator at 35-40 ℃, preferably 37 ℃, saturated humidity, 1-10% co ₂, preferably 5% co ₂;

    (viii) The culture time is 2-48h;

    (ix) In step 3), the temperature is reduced to 4-32 ℃;

    (x) No additional reagents or materials are added when the cells and their secreted extracellular matrix are detached in lamellar form.
12. Use of cell membrane fragments in the manufacture of a pharmaceutical composition for the treatment of lower limb ischemia.
13. The use according to claim 12, wherein the cell membrane fraction is as defined in any one of claims 1 to 7.
14. The use according to any one of claims 12 to 13, wherein the cells are as defined in claim 8.
15. The use according to any one of claims 12 to 14, wherein the cell membrane is as defined in any one of claims 9 to 11.
16. The use according to any one of claims 12-15, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, such as physiological saline.
17. The use of any one of claims 12-16, wherein the pharmaceutical composition is in the form of an injection.