Use of cell patch fragments in treatment of lower limb ischemia

Abstract

According to the application of the cell sheet fragments in preparation of the medicine for treating lower limb ischemia, specifically, the cell sheet fragments formed after injection administration of the cell sheet can prevent limb necrosis caused by lower limb ischemia, have a good limb protection effect, can also effectively prevent muscular atrophy of an affected limb, and can be used for preparing the medicine for treating lower limb ischemia. The limb protection effect and the effect of preventing muscle atrophy of an affected limb are equivalent to those of a cell patch, and are remarkably superior to those of a single-cell suspension.

Description

Application of cell membrane fragments in the treatment of lower limb ischemia Technical Field

[0001] The present disclosure relates to the fields of biomedicine and cell biology, and particularly to the use of cell membrane fragments in the treatment of lower limb ischemia. Background Art

[0002] Lower limb ischemia is a common peripheral arterial disease, which is a type of disease caused by stenosis or occlusion of the lower limb arteries and insufficient blood perfusion due to various reasons, resulting in symptoms such as intermittent claudication, pain, ulcers or gangrene of the lower limbs. With the aging of the population and lifestyles such as long-term smoking, type 2 diabetes, obesity and sedentary behavior, the risk of this disease has continued to increase in recent years. When lower limb ischemia develops into a severe stage, there is a high rate of amputation and risk of death. For patients who do not meet the conditions for revascularization surgery, there is currently a lack of effective treatment. In recent years, mesenchymal stem cells have been shown to restore tissue function and repair ischemic tissue through immune regulation, promotion of angiogenesis and paracrine secretion of bioactive factors, and are used to treat lower limb ischemia. Globally, the allogeneic bone marrow mesenchymal stem cell injection product of Indian Stempeutics Company It is already available in India for the treatment of lower limb ischemia. However, cell injections suffer from poor resistance to acidic and ischemic environments, short survival time, severe cell loss, inability to aggregate and act at the lesion site, and low cell utilization, resulting in suboptimal therapeutic efficacy. Recent research has also explored the use of biodegradable materials as scaffolds for stem cell transplantation, but the interaction between the material and cells, the degradation rate of the scaffold material, and the tissue compatibility of the scaffold material and its degradation products all affect the ultimate therapeutic effect. Therefore, further research and exploration is needed to effectively treat lower limb ischemia in mesenchymal stem cell preparations.

[0003] Summary of the Invention

[0004] Cell membrane technology enables cells to connect to each other through the extracellular matrix secreted by themselves during the culture process, forming a two-dimensional sheet structure without exogenous scaffolds, thereby effectively solving the problem of cell in situ retention, increasing the number of surviving cells and cell survival time, and promoting the repair of the lesion site by secreting cytokines in larger quantities and for a longer period of time, thereby enhancing the therapeutic effect. Based on this, for those skilled in the art, cell membranes are usually administered by direct transplantation to ensure the size uniformity and structural integrity of the cell membrane during administration as much as possible, and to avoid structural damage. However, the inventors of the present invention were surprised to find in experimental studies that the cell membrane fragments formed after the cell membrane of the present invention is injected can prevent limb necrosis caused by lower limb ischemia, have good limb preservation effects, and can also effectively prevent muscle atrophy of the affected limb. Moreover, the limb preservation effect and the effect of preventing muscle atrophy of the affected limb are comparable to those of cell membranes, and are significantly better than single cell suspensions. It can be seen that the cell membrane fragments formed after the cell membrane of the present invention is injected have excellent effects in treating lower limb ischemia and have broad application prospects.

[0005] To this end, in a first aspect of the present disclosure, the present disclosure provides use of cell membrane fragments in preparing a medicament for treating lower limb ischemia.

[0006] In a second aspect of the present disclosure, the present disclosure provides cell membrane fragments for use in treating lower limb ischemia.

[0007] In a third aspect of the present disclosure, the present disclosure provides a method for treating lower limb ischemia, comprising administering an effective amount of cell membrane fragments to a subject in need thereof.

[0008] In some embodiments, the cell membrane fragments are administered by injection.

[0009] In some embodiments, the injection is an intramuscular (eg, adductor or gastrocnemius) injection.

[0010] In some embodiments, during the administration by injection, the dispersant for the cell membrane fragments is physiological saline.

[0011] In some embodiments, the cell membrane fragments are obtained by disrupting cell membranes.

[0012] In some embodiments, the diameter of the cell membrane fragments is about 0.1-3 mm, for example, about 0.1-0.2 mm, 0.2-0.3 mm, 0.3-0.4 mm, 0.4-0.5 mm, 0.5-0.6 mm, 0.6-0.7 mm, 0.7-0.8 mm, 0.8-0.9 mm, 0.9-1 mm, 1-1.1 mm, 1.1-1.2 mm, 1.2-1.3 mm, 1.3-1.4 mm, 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. It should be noted that the diameter of the cell fragment refers to the longest line segment connecting two non-adjacent vertices in each cell fragment.

[0013] In some embodiments, the cell membrane fragments have a diameter of about 0.1-0.5 mm or about 0.4-3 mm.

[0014] In some embodiments, the cell membrane fragments are obtained by injecting the cell membrane fragments through a syringe needle.

[0015] In some embodiments, the cell membrane fragments are obtained by the following method:

[0016] 1) Load the cell membrane sheet into the syringe needle;

[0017] 2) After the normal saline solution is drawn into the syringe barrel, the syringe needle loaded with the cell membrane sheet is installed on the syringe barrel;

[0018] 3) Pushing the syringe piston to inject the cell membrane to obtain cell membrane fragments;

[0019] Optionally, in step 1), the cell membrane sheet is pre-cut (to accommodate different administration doses).

[0020] In some embodiments, the needle gauge is 22G, 23G, 25G or 27G. The needle gauges in the present disclosure adopt international standards.

[0021] In some embodiments, the needle is 22G or 27G.

[0022] In some embodiments, the cell membrane fragments are no less than 2 pieces, for example, no less than 3 pieces, no less than 4 pieces, no less than 5 pieces, no less than 6 pieces, no less than 7 pieces, no less than 8 pieces, no less than 9 pieces, no less than 10 pieces, no less than 12 pieces, no less than 14 pieces, no less than 16 pieces, no less than 18 pieces, no less than 20 pieces, no less than 22 pieces, no less than 24 pieces, no less than 26 pieces, no less than 28 pieces, no less than 30 pieces or more pieces.

[0023] In some embodiments, the cells are mesenchymal stem cells.

[0024] In some embodiments, the cells are selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrial mesenchymal stem cells, and dental pulp mesenchymal stem cells.

[0025] In some embodiments, the cells are umbilical cord mesenchymal stem cells.

[0026] In some embodiments, the cell membrane sheet comprises multiple layers of cells, such as 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.

[0027] In some embodiments, the diameter of the cell membrane sheet is about 3-30 mm, for example, 3-4 mm, 4-5 mm, 5-6 mm, 6-7 mm, 7-8 mm, 8-9 mm, 9-10 mm, 10-12 mm, 12-14 mm, 14-16 mm, 16-18 mm, 18-20 mm, 20-22 mm, 22-24 mm, 24-26 mm, 26-28 mm or 28-30 mm.

[0028] In some embodiments, the thickness of the cell membrane sheet is about 50-1000 μm, such as 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 100-120 μm, 120-140 μm, 140-160 μm, 160-180 μm, 180-200 μm, 200-250 μm, 250-300 μm. , 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 950-1000μm.

[0029] In some embodiments, the cell membrane sheet is circular or approximately circular in shape.

[0030] In some embodiments, the cell membrane sheet is capable of secreting one or more selected from interleukin-6 (IL-6), transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor (VEGF), insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolic enzyme (indoleamine 2,3-dioxygenase, IDO), and inducible nitric oxide synthase (iNOS).

[0031] In some embodiments, the cell membrane sheet is capable of secreting at least interleukin-6 (IL-6), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).

[0032] In some embodiments, the cell sheet contains extracellular matrix secreted by cells.

[0033] In some embodiments, the extracellular matrix in the cell sheet comprises one or more of fibronectin, integrins (such as integrin-β1), and vitronectin.

[0034] In some embodiments, the extracellular matrix in the cell sheet comprises at least fibronectin and integrin-β1.

[0035] In some embodiments, the volume of the normal saline is 0.05-1 mL (e.g., 0.05-0.06 mL, 0.06-0.07 mL, 0.07-0.08 mL, 0.08-0.09 mL, 0.09-0.1 mL, 0.1-0.2 mL, 0.2-0.3 mL, 0.3-0.4 mL, 0.4-0.5 mL, 0.5-0.6 mL, 0.6-0.7 mL, 0.7-0.8 mL, 0.8-0.9 mL, or 0.9-1 mL, preferably 0.1 mL).

[0036] In some embodiments, the cell membrane sheet is obtained by the following method:

[0037] 1) Coating a layer of matrix on the surface of a temperature-sensitive culture dish;

[0038] 2) Add the cell suspension to a temperature-sensitive culture dish for culture;

[0039] 3) Lowering the temperature, the cells and the extracellular matrix secreted by them are separated into sheets, and the cell membrane sheet is obtained.

[0040] In some embodiments, the cells are mesenchymal stem cells.

[0041] In some embodiments, the cells are selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrial mesenchymal stem cells, and dental pulp mesenchymal stem cells.

[0042] In some embodiments, the cells are umbilical cord mesenchymal stem cells.

[0043] In some embodiments, the coating matrix is ​​selected from the group consisting of collagen, gelatin, fibronectin, fibrinogen, fibronectin, vitronectin, laminin, poly-ornithine, poly-lysine, and ornithine-lysine copolymer.

[0044] In some embodiments, the coating is carried out in an incubator at 35-40°C (such as 35-36°C, 36-37°C, 37-38°C, 38-39°C or 39-40°C, preferably 37°C), saturated humidity, 1-10% CO2 (such as 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9% or 9-10% CO2, preferably 5% CO2).

[0045] In some embodiments, the coating time is 0.5-48h, 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 45-48h.

[0046] In some embodiments, the density of cells in the temperature-sensitive culture dish is 1.8×10 4 ‐1.9×10 6 / cm 2 (For example, 1.8×10 4 ‐2×10 4 / cm 2 , 2×10 4-3×10 4 / cm 2 , 3×10 4 -4×10 4 / cm 2 , 4×10 4 -5×10 4 / cm 2 , 5×10 4 -6×10 4 / cm 2 , 6×10 4 -7×10 4 / cm 2 , 7×10 4 -8×10 4 / cm 2 , 8×10 4 -9×10 4 / cm 2 , 9×10 4 -1×10 5 / cm 2 , 1×10 5 ‐2×10 5 / cm 2 , 2×10 5 -3×10 5 / cm 2 , 3×10 5 -4×10 5 / cm 2 , 4×10 5 -5×10 5 / cm 2 , 5×10 5 -6×10 5 / cm 2 , 6×10 5 -7×10 5 / cm 2 , 7×10 5 -8×10 5 / cm 2 , 8×10 5 -9×10 5 / cm 2 , 9×10 5 -1×10 6 / cm 2 , 1×10 6 -1.5×10 6 / cm 2 or 1.5×10 6 -1.9×10 6 / cm 2 , preferably 1×10 6 / cm2 ).

[0047] In some embodiments, step 2) is performed by adding the cell suspension and culture medium into a temperature-sensitive culture dish for culturing.

[0048] In some embodiments, in step 2), the culture medium is a serum-free culture medium.

[0049] In some embodiments, the serum-free medium can be a basal medium plus nutrient additives, or a commercial serum-free medium.

[0050] In some embodiments, the basal culture medium can be 1640, DMEM, α-MEM, DMEM / F12, F12, etc.

[0051] In some embodiments, the nutritional additives include vitamin C, sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin (plant-expressed), progesterone, putrescine, biotin, sodium pyruvate, ethanolamine, carnitine, amino acids, vitamins, glutathione, linoleic acid, linolenic acid, etc.

[0052] In some embodiments, the commercial serum-free culture medium includes but is not limited to: CTSTM StemPro™ MSC SFM Kit, MesenCult™-ACF Medium, MesenCult™-ACF Plus Medium, MesenCult™-XF Medium, etc.

[0053] In some embodiments, in step 2), the ratio of the culture medium in the temperature-sensitive culture dish is 0.1-0.5 mL / cm 2 (e.g. 0.1-0.2 mL / cm 2 , 0.2-0.3mL / cm 2 , 0.3-0.4mL / cm 2 or 0.4-0.5 mL / cm 2 , preferably 0.3 mL / cm 2 ).

[0054] In some embodiments, in step 2), the culturing is carried out in an incubator at 35-40°C (such as 35-36°C, 36-37°C, 37-38°C, 38-39°C or 39-40°C, preferably 37°C), saturated humidity, 1-10% CO2 (such as 1-2%, 2-3%, 3-4%, 4-5%, 5-6%, 6-7%, 7-8%, 8-9% or 9-10% CO2, preferably 5% CO2).

[0055] In some embodiments, in step 2), the culturing time is 2-48 h, such as 2-3 h, 3-4 h, 4-5 h, 5-6 h, 6-7 h, 7-8 h, 8-9 h, 9-10 h, 10-15 h, 15-20 h, 20-25 h, 25-30 h, 30-35 h, 35-40 h, 40-45 h or 45-48 h.

[0056] In some embodiments, in step 3), the temperature is lowered to 4-32°C, such as 4-5°C, 5-6°C, 6-7°C, 7-8°C, 8-9°C, 9-10°C, 10-12°C, 12-14°C, 14-16°C, 16-18°C, 18-20°C, 20-22°C, 22-24°C, 24-26°C, 26-28°C, 28-30°C or 30-32°C.

[0057] In some embodiments, in step 3), no additional reagents or materials are added when the cells and the extracellular matrix secreted by them are detached in sheets.

[0058] In a fourth aspect of the present disclosure, the present disclosure provides use of cell membrane fragments in preparing a pharmaceutical composition for treating lower limb ischemia.

[0059] In a fifth aspect of the present disclosure, the present disclosure provides a pharmaceutical composition comprising cell membrane fragments for treating lower limb ischemia.

[0060] In a sixth aspect of the present disclosure, the present disclosure provides a method for treating lower limb ischemia, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising cell membrane fragments.

[0061] In some embodiments, the cell membrane fragments are as described in any of the above technical solutions.

[0062] In some embodiments, the cell is as described in any of the above technical solutions.

[0063] In some embodiments, the cell membrane sheet is as described in any of the above technical solutions.

[0064] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, such as physiological saline.

[0065] In some embodiments, the pharmaceutical composition is in the form of an injection.

[0066] In some embodiments, the pharmaceutical composition is administered by injection.

[0067] In some embodiments, the injection is an intramuscular (eg, adductor or gastrocnemius) injection.

[0068] The present disclosure also provides a method for isolating and culturing umbilical cord mesenchymal stem cells, which includes primary isolation and culture and subculture.

[0069] In some embodiments, the primary isolation culture comprises the following steps: isolating Wharton's jelly from umbilical cord tissue; mincing the Wharton's jelly to obtain tissue blocks; plating the tissue blocks in a culture container for culturing; adding an appropriate amount of complete culture medium to cover the tissue blocks and continuing to culture; when cells attached to the culture container appear around the tissue blocks and the cells grow to 70-100% confluence, removing the tissue blocks and performing a cell passaging operation.

[0070] In some embodiments, the subculture process includes the following steps: separating the cells from the culture vessel and evenly dispersing the cells in culture medium, followed by inoculation into the culture vessel; adding an appropriate amount of culture medium, replacing the culture medium with an appropriate amount of fresh medium every 1-5 days depending on the cell growth status; and repeating the subculture process when the cells reach 70-100% confluence. Each time the cells are subcultured, the number of generations increases by one. Umbilical cord mesenchymal stem cells adhere to the culture vessel and exhibit a uniform, fibroblast-like morphology.

[0071] In this disclosure, "treating" generally refers to obtaining a desired pharmacological and / or physiological effect, including: (a) inhibiting the symptoms of a disease, i.e., preventing its development; or (b) alleviating the symptoms of a disease, i.e., causing regression of the disease or symptoms.

[0072] In this disclosure, a "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, a mammal refers to a human.

[0073] In this disclosure, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, the desired effect. Determination of such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other concurrently administered treatments. It will be further understood that for any particular individual, the specific dosing regimen can be adjusted over time based on individual needs and the mode of administration or the professional judgment of the person supervising administration. Beneficial effects

[0074] 1. The cell membrane disclosed herein can prevent ischemic limb necrosis of the lower limbs and has a good limb-saving effect. At the same time, cell membrane fragments can also prevent ischemic limb necrosis of the lower limbs and also have a good limb-saving effect. In fact, the limb-saving effect of cell membrane fragments is equivalent to that of cell membranes, and both are significantly better than single cell injections.

[0075] 2. Both the cell membrane sheets and cell membrane fragments disclosed herein can effectively prevent muscle atrophy in the affected limb. The cell membrane fragments and cell membrane sheets are equally effective in preventing muscle atrophy in the affected limb, and both are significantly superior to single-cell injections. In summary, the cell membrane sheets and cell membrane fragments can significantly prevent muscle atrophy in the affected limb, contributing to improved limb function in patients with lower limb ischemia, with the improvement increasing with increasing dosage.

[0076] 3. The cell membrane sheets and cell membrane fragments disclosed herein can effectively improve the symptoms of lower limb ischemia, enhance blood perfusion in the ischemic area by promoting angiogenesis, avoid muscle atrophy of the affected limb, and facilitate the preservation of the affected limb. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] In Figure 1, a shows a schematic diagram of an umbilical cord mesenchymal stem cell sheet; b shows a schematic diagram of a cell sheet being cut into 6 equal parts; c shows a schematic diagram of a cell sheet being loaded into a syringe before administration;

[0078] Figure 2 shows the typical morphology of cell membrane sheets after injection with a syringe needle (left: injection with a 27G needle; right: injection with a 22G needle);

[0079] FIG3 shows the improvement of blood perfusion in the ischemic limb of mice by umbilical cord mesenchymal stem cell membrane fragments detected by laser Doppler line scanning blood flow imaging;

[0080] FIG4 shows the gross and gastrocnemius muscle morphology of the healthy limb (left) and the affected limb (right) of the cell membrane fragment group and the normal saline group 28 days after administration. DETAILED DESCRIPTION

[0081] The present disclosure is further explained below with reference to specific embodiments, but these embodiments should not be construed as limiting the present disclosure in any way.

[0082] The culture system disclosed herein comprises a serum-free culture medium and a coating matrix matching the culture medium, wherein:

[0083] 1. The composition of serum-free medium can be basal medium + nutrient additives, or commercial serum-free medium, as follows:

[0084] (1) Basic culture medium + nutrient additives:

[0085] The basal culture medium can be 1640, DMEM, α-MEM, DMEM / F12, F12, etc.

[0086] Nutritional additives include 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.

[0087] (2) Commercial serum-free culture medium:

[0088] Including but not limited to: CTSTMStemProTMMSC SFM Kit, MesenCultTM-ACF Medium, MesenCultTM-ACF Plus Medium, MesenCultTM-XF Medium, etc.

[0089] 2. Coating matrix matched with culture medium:

[0090] Including but not limited to collagen, gelatin, fibronectin, fibrinogen, adhesion protein, vitronectin, laminin, poly-ornithine, poly-lysine, ornithine-lysine copolymer, etc.

[0091] The above culture system disclosed herein is suitable for the culture, cell sheet preparation and detection of mesenchymal stem cells from various tissue sources. Mesenchymal stem cells include but are not limited to umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrial mesenchymal stem cells, and dental pulp mesenchymal stem cells.

[0092] The present disclosure will be further explained below using umbilical cord mesenchymal stem cells as an example.

[0093] Example 1: Isolation and culture of umbilical cord mesenchymal stem cells

[0094] Umbilical cord mesenchymal stem cells are first isolated and cultured in primary culture and then subcultured to expand the cell number. The procedure is as follows:

[0095] 1. Primary Culture of Umbilical Cord Mesenchymal Stem Cells

[0096] Neonatal umbilical cords (sourced from Beijing Mingde Hospital, donated by pregnant women with signed informed consent, which was approved by the Mingde Hospital Ethics Committee) were washed with a physiological solution with an osmotic pressure equal to that of human body fluids. The arteries, veins, and adventitia were removed, and Wharton's jelly was separated. The cords were minced into 0.1-2 mm fragments and evenly plated in a matrix-coated culture vessel, with spacing of 2-30 mm between fragments. The culture vessel was placed in a cell culture incubator. After 2-7 days, an appropriate amount of complete culture medium was added to cover the fragments. Umbilical cord mesenchymal stem cells emerged from the culture vessel after 8-21 days, growing adherently and forming uniform, fibrous structures. When the cells reached 70-100% confluence, the fragments were removed and the cells were passaged.

[0097] 2. Passaging of Umbilical Cord Mesenchymal Stem Cells

[0098] When the cells grow to 70-100% confluence, the cells are passaged: the cells are separated from the culture container by methods including but not limited to digestion with trypsin or similar substances, cell scraping, etc. The cells are dispersed in the culture medium by stirring, vortexing, etc. (including but not limited to) and the cells are plated at a density of 500-100,000 / cm 2 Cells were seeded at a suitable density in a culture vessel. An appropriate amount of culture medium was added, and fresh medium was replaced every 1-5 days depending on the cell growth status. The subculture procedure was repeated when the cells reached 70-100% confluence. The cultured umbilical cord mesenchymal stem cells adhered to the vessel and exhibited a uniform, fibrous morphology.

[0099] Example 2: Detection and identification of umbilical cord mesenchymal stem cells

[0100] The obtained umbilical cord mesenchymal stem cells were detected and identified as follows:

[0101] 1. Umbilical cord mesenchymal stem cell growth curve detection

[0102] The method for determining the growth curve includes but is not limited to the MTT method, WST method, DNA content detection method, ATP detection method, etc., and the WST method is taken as an example here for further explanation. The umbilical cord mesenchymal stem cells are dispersed and inoculated into the culture well plate at a certain density with reference to the reagent instructions, and the liquid is changed according to normal culture conditions. At a fixed time every day for a period of time, the cell activity is detected according to the instructions to obtain data on cell activity or quantity. The WST reagent is used to detect the activity of the cells every day within 7 days. The method is to add the WST reagent to the cell well plate being cultured according to the recommended ratio in the instructions, and after incubation for a fixed time in the cell culture incubator, use a microplate reader or an ultraviolet spectrophotometer to detect the absorbance value of the well plate chamber liquid at a wavelength of 450nm, which is positively correlated with the number of cells. The results show that as the culture time increases, the cell activity in the chamber increases, and it can be inferred that the cell number increases with the culture time.

[0103] 2. Identification of Umbilical Cord Mesenchymal Stem Cells

[0104] Methods for identifying umbilical cord mesenchymal stem cells include, but are not limited to, flow cytometry detection of cell surface marker proteins, three-way differentiation of umbilical cord mesenchymal stem cells, and PCR detection of cell expressed genes. Flow cytometry and three-way differentiation are used as examples for illustration.

[0105] 2.1 Identification of surface markers of umbilical cord mesenchymal stem cells

[0106] Umbilical cord mesenchymal stem cells were dispersed in culture medium and centrifuged. Cell surface markers, including but not limited to CD73, CD90, CD105, CD34, CD11B, CD19, CD45, and HLA-DR, were stained using serum or a physiological solution with a serum protein content of 1-20% and an osmotic pressure equivalent to that of human body fluids according to the purchased reagent instructions. The phenotype for CD73, CD90, and CD105 was considered positive, with a ratio of no less than 95%. The phenotype for CD34, CD11B, CD19, CD45, and HLA-DR was considered negative, with a ratio of no more than 2%. The results showed that the ratios for positive markers were all greater than 99%, and the ratios for negative markers were no more than 0.1%.

[0107] 2.2 Three-way differentiation of umbilical cord mesenchymal stem cells

[0108] Umbilical cord mesenchymal stem cells have the ability to differentiate into bone, cartilage, and adipogenesis. To perform osteogenic and adipogenic differentiation assays, cells are plated in appropriate culture vessels according to the ratios specified in the three-way differentiation reagent instructions. When cells reach 50-90% confluence for osteogenic induction and 90% or greater confluence for adipogenic induction, osteogenic and adipogenic induction media, respectively, are added. For chondrogenic induction, a defined number of cells are centrifuged to the bottom of the centrifuge tube, followed by the addition of chondrogenic induction media. Once the cells have pelleted, the pellets are lifted off the tube bottom to ensure complete contact with the induction media. Cells are then assayed after at least seven days of induction culture. Osteogenic induction staining can be performed with, but is not limited to, Alizarin Red and anti-hOsteocalcin; adipogenic induction staining can be performed with, but is not limited to, Oil Red O and anti-mFABP4; and chondrogenic induction staining can be performed with, but is not limited to, Alcian Blue, Safranin O, and anti-hAggrecan. The results showed that umbilical cord mesenchymal stem cells could be stained after adipogenic differentiation, osteogenic differentiation and chondrogenic differentiation.

[0109] Example 3: Preparation of umbilical cord mesenchymal stem cell membrane

[0110] The procedure for preparing the umbilical cord mesenchymal stem cell membrane sheet is as follows:

[0111] 1. Thermosensitive smart culture dish coating

[0112] Before preparing membranes using a serum-free culture system and mesenchymal stem cells, the surface of the thermosensitive smart culture dish should be coated with a matrix that facilitates mesenchymal stem cell attachment. This matrix includes, but is not limited to, collagen, gelatin, fibronectin, fibrinogen, fibronectin, vitronectin, laminin, poly-ornithine, poly-lysine, ornithine-lysine copolymers. During coating, dilute the coating matrix in a physiological buffer such as saline or PBS, then add it to the thermosensitive smart culture dish and cover it with a lid. The thermosensitive smart culture dish should then be placed in an incubator at 37°C, saturated humidity, and 5% CO2 for 0.5-48 hours.

[0113] 2. Preparation of Umbilical Cord Mesenchymal Stem Cell Sheets

[0114] After the coating is completed, the remaining coating matrix is ​​removed from the smart culture dish, and the mesenchymal stem cell suspension is added to the temperature-sensitive smart culture dish. In this embodiment, a temperature-sensitive smart culture dish with a diameter of 35 mm is used, and the amount of cells added is 1.8×10 5 ‐1.8×10 7 (The most preferred is 1×10 7), add 1-4 ml of culture medium (preferably 3 ml). Then place the thermosensitive smart culture dish in a 37°C, saturated humidity, 5% CO2 incubator for 2-48 hours. After the culture is completed, move the thermosensitive smart culture dish to a 4-32°C environment. The cells will detach from the bottom of the thermosensitive smart culture dish in sheets without adding any additional reagents or materials, forming cell membrane sheets. Collect the liquid in the culture dish to obtain the cell membrane sheet supernatant. The cell membrane sheet is off-white, with a dense structure and a smooth surface. The size is a circle with a diameter of 3-30 mm and a thickness of 50-1000 μm. Tissue section observation shows that the cell membrane sheet contains a multi-layer cell structure and contains extracellular matrix components secreted by the cells.

[0115] Example 4: Functional testing of umbilical cord mesenchymal stem cell membranes

[0116] The functional assays of the formed cell membrane sheets are as follows:

[0117] 1. Detection of cell membrane secretion factors by enzyme-linked immunosorbent assay

[0118] The supernatant of the cell membrane preparation process was tested according to the instructions of the enzyme-linked immunosorbent assay kit to determine the content of various factors secreted by the cell membrane sheets. The factors referred to here include but are not limited to interleukin-6 (IL-6), transforming growth factor-β (TGF-β), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor (VEGF), insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolic enzyme indoleamine 2,3-dioxygenase (IDO), and inducible nitric oxide synthase (iNOS). Here, we examined the secretion of factors from a umbilical cord mesenchymal stem cell sheet, using interleukin-6 (IL-6), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF) as examples. This experiment was repeated three times (samples 1, 2, and 3). The secretion of factors from a human mesenchymal stem cell suspension and a human mesenchymal stem cell sheet was compared using these three samples. The results showed that the human mesenchymal stem cell sheet exhibited superior factor secretion compared to the human mesenchymal stem cell suspension.

[0119] 2. Observe the protein content in cell membrane by tissue section

[0120] The cell membrane sheets obtained above are fixed with paraformaldehyde or formalin fixative and then prepared into 4-10 μm thick tissue sections using paraffin or frozen sections. The sections are then stained to observe the presence of proteins in the extracellular matrix within the cell membrane sheets. These proteins include, but are not limited to, fibronectin, integrins, and vitronectin. Cell nuclei are typically stained with fluorescent dyes such as DAPI or Hoechst 33258 to aid localization. In addition to immunofluorescence, other staining methods, including but not limited to immunohistochemistry, can also be used for observation. Here, fibronectin and integrin-β1 are used as examples. Both fibronectin and integrin-β1 are stained with fluorescein-labeled antibodies, and cell nuclei are stained with DAPI. The results show that the prepared cell membrane sheets contain significant amounts of fibronectin and integrin-β1.

[0121] 3. Scanning electron microscopy to observe the surface structure of cell membrane

[0122] The cell membrane sheets obtained above were fixed with 2.5% glutaraldehyde fixative, then dehydrated through a gradient of alcohol and air-dried to obtain dried cell membrane sheets for scanning electron microscopy analysis. The dried cell membrane sheets were affixed to the surface of a sample stage using conductive double-sided tape and then gold-sprayed using vacuum magnetron sputtering to make the surface conductive. The sample stage was placed in a scanning electron microscope (Hitachi S-4800) for observation. The results showed that the surface morphology of the cell membrane sheets was relatively flat and smooth, with visible mesenchymal stem cells, intercellular protein connections, and cell stacking.

[0123] Example 5: Activity test of umbilical cord mesenchymal stem cell membrane and its fragments

[0124] 1. Animal modeling:

[0125] (1) Mice of appropriate age (7 weeks in this experiment) (can be ICR / NODSCID / C57 male mice, in this experiment, ICR male mice) are anesthetized with isoflurane gas anesthesia or sodium pentobarbital intravenously or hydroxyproline hydrate intraperitoneally;

[0126] (2) Fix the mouse's hind limbs, shave the hair, prepare the skin, and disinfect. Then, make a 1 cm incision from the knee to the inner thigh of the left leg of the mouse, separate the subcutaneous tissues and muscle tissues, and expose the blood vessels of the lower limbs.

[0127] (3) Modeling surgical method 1: ligating both ends of the femoral artery with surgical sutures (nylon, 7-0) from the inguinal ligament to the bifurcation of the femoral artery, and performing collateral and central severing with a scalpel; Modeling surgical method 2: separating the femoral artery with pointed forceps, then moving the forceps along the femoral artery to destroy the collaterals and microvessels, and ligating the proximal end of the femoral artery with surgical sutures (nylon, 7-0);

[0128] (4) Suture the incision with nylon suture (4-0 suture in this experiment);

[0129] (5) Use a laser Doppler line scanning blood flow imager (the instrument model in this experiment is: Moor LDLS) to detect blood flow signals and calculate the postoperative ipsilateral / contralateral blood flow ratio;

[0130] (6) The animal was sent to the feeding area and waited for it to recover from anesthesia. Meloxicam was injected daily for three days after surgery.

[0131] 2. Patching:

[0132] A single dose of medication is administered within 0-72 hours after animal modeling. Specific steps:

[0133] (1) After modeling, the mice were anesthetized with isoflurane gas, intravenous injection of sodium pentobarbital, or intraperitoneal injection of hydroxyproline, and the mice were locally disinfected with iodine before administration;

[0134] (2) Cut the prepared cell membrane sheet into the required size according to the dosage. For example, for mouse administration, a cell membrane sheet with a diameter of 1.8 cm is cut into 6 equal parts for administration;

[0135] (3) The administration method can be direct transplantation or injection administration: the direct transplantation method is: after the animal modeling step (3), before the incision is sutured, the cell membrane sheet is spread flat near the femoral artery, and the incision is sutured after waiting for 2 minutes to complete the administration; the injection administration method is: remove the syringe needle (it can be a needle of various types, taking the mouse administration as an example, a 27G needle can be used), load the cut cell membrane sheet into the syringe needle (see Figure 1), and after 0.1mL of normal saline (0.05-1mL) is sucked into the syringe barrel, the loaded cell membrane sheet is injected into the syringe needle. The syringe needle of the cell membrane sheet is gently installed on the syringe barrel, and the needle is inserted into the thigh muscle of the affected limb (it can be the adductor muscle or gastrocnemius muscle. The injection site in this experiment is the adductor muscle). The syringe is pushed to inject the cell membrane sheet into the mouse muscle through the needle. The needle is withdrawn to complete the drug administration. (The size of the membrane sheet after injection into the adductor muscle varies depending on the size of the syringe needle. Taking a 27-gauge needle as an example, the size of the membrane sheet after injection is about 0.1-0.5mm; taking a 22-gauge needle as an example, the size of the membrane sheet after injection is about 0.4-3mm, see Figure 2).

[0136] 3. Effectiveness test:

[0137] Multiple experimental groups with multiple doses were set up, including a low-dose group (i.e., a dose group with 1*10^6 equivalent cells) and a high-dose group (i.e., a dose group with 2*10^6 equivalent cells): (Group 1: low-dose cell membrane, Group 2: low-dose cell membrane fragments, Group 3: low-dose single cell injection, Group 4: low-dose cell membrane supernatant group; Group 5: high-dose cell membrane, Group 6: high-dose cell sheet fragments, Group 7: high-dose single cell injection, Group 8: high-dose cell sheet supernatant), and the improvement effect of each experimental group on lower limb ischemic disease was tested; animals that were only modeled without medication (Group 9) or injected with an equal amount of normal saline after modeling (Group 10) were used as experimental control groups to verify the effectiveness of umbilical cord mesenchymal stem cell sheets and their fragments in treating lower limb ischemia.

[0138] (1) Improvement of lower limb blood perfusion: Laser Doppler line scanning blood flow imaging was used to detect blood flow signals on days 3, 7, 14, 21, and 28 after administration, and the ipsilateral / contralateral blood flow ratio was calculated to evaluate the improvement of lower limb blood perfusion. After comparison, the blood flow of animals in the cell membrane fragment group (Group 2) showed significant improvement starting from day 14, with an increase of approximately 16.4% on day 14, approximately 17.5% on day 21, and approximately 19.3% on day 28 compared with the control group (Group 10) (see Figure 3).

[0139] (2) Evaluation of limb salvage effect: The limb damage of mice in the control group and the experimental group was observed on the 7th, 14th, 21st and 28th day after administration, and the proportion of mice in each group with normal modeling side or only toe discoloration, toe necrosis and foot ring death was calculated. The proportions of the three conditions in each group were statistically analyzed, and it was found that the proportions of the three conditions in groups 1 to 10 were: Group 1: 80%, 12%, 8%; Group 2: 83%, 8%, 9%; Group 3: 58%, 28%, 14%; Group 4: 60%, 24%, 16%; Group 5: 87%, 8%, 5%; Group 6: 89%, 5%, 6%; Group 7: 68%, 18%, 14%; Group 8: 68%, 22%, 10%; Group 9: 50%, 25%, 25%; Group 10: 45%, 23%, 32% respectively. It can be seen that cell membranes can prevent ischemic limb necrosis of the lower limbs and have a good limb-saving effect. At the same time, cell membrane fragments can also prevent ischemic limb necrosis of the lower limbs and have a good limb-saving effect. In fact, the limb-saving effect of cell membrane fragments is equivalent to that of cell membranes, and both are significantly better than the single cell injection group.

[0140] (3) Preventing muscle atrophy of the affected limb: On the 28th day after administration, the mice were dissected to remove the gastrocnemius muscles of the affected and healthy limbs (see Figure 4), the weight of the gastrocnemius muscles on both sides was weighed, and the weight ratio of the gastrocnemius muscles of the affected limb / healthy limb was calculated. The weight ratios of the gastrocnemius muscles of the affected limb / healthy limb in each group were 82.2%, 80.4%, 53.1%, 57.8%, 85.2%, 84.8%, 68.3%, 67.8%, 51.4%, and 50.2%, respectively. This shows that both cell membranes and cell membrane fragments can effectively prevent muscle atrophy of the affected limb, and the cell membrane fragments and cell membranes are equally effective in preventing muscle atrophy of the affected limb, and are significantly better than the single cell injection group. In short, cell membranes and cell membrane fragments can significantly prevent muscle atrophy of the affected limb, which is beneficial to the improvement of the function of the lower limb ischemic limb, and the improvement effect is enhanced with increasing dose.

[0141] While specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that the scope of protection of the present disclosure is defined by the appended claims. Those skilled in the art may make various changes or modifications to these embodiments without departing from the principles and essence of the present disclosure, and such changes and modifications are intended to fall within the scope of protection of the present disclosure.

Claims

1. Use of cell membrane sheet fragments in the preparation of a medicament for treating lower limb ischemia.

2. Use according to claim 1, wherein, The cell membrane sheet fragments are obtained by crushing cell membrane sheets.

3. Use according to any one of claims 1-2, wherein, The diameter of the cell membrane sheet fragments is about 0.1 - 3 mm, preferably about 0.1 - 0.5 mm or about 0.4 - 3 mm.

4. Use according to any one of claims 1 to 3, wherein, The cell membrane sheet fragments are obtained when the cell membrane sheets are injected via a syringe needle. Use according to any one of claims 1-4, wherein, The cell membrane sheet fragments are obtained by the following method: 1) Loading the cell membrane sheet at the syringe needle; 2) After sucking physiological saline into the syringe barrel, installing the syringe needle loaded with the cell membrane sheet onto the syringe barrel; 3) Pushing the syringe piston to inject the cell membrane sheet to obtain cell membrane sheet fragments; Optionally, in step 1), the cell membrane sheet is pre-cut.

6. Use according to any one of claims 4 - 5, wherein, The model of the needle is 22G, 23G, 25G or 27G, preferably 22G or 27G.

7. Use according to any one of claims 1-6, wherein, There are no less than 2 cell membrane sheet fragments.

8. Use according to any one of claims 1-7, wherein, The cells are mesenchymal stem cells, preferably selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrial mesenchymal stem cells, dental pulp mesenchymal stem cells, and more preferably umbilical cord mesenchymal stem cells.

9. Use according to any one of claims 1-8, wherein, The cell membrane sheet further has one or more technical features selected from the following (i) - (vi): (i) The cell membrane sheet contains multiple layers of cells; (ii) The diameter of the cell membrane sheet is about 3 - 30 mm; (iii) The thickness of the cell membrane sheet is about 50 - 1000 μm; (iv) The shape of the cell membrane sheet is circular or approximately circular; (v) The cell membrane sheet can secrete one or more selected from interleukin - 6 (IL - 6), transforming growth factor - β (TGF - β), prostaglandin E2 (PGE2), hepatocyte growth factor (HGF), epidermal growth factor, fibroblast growth factor, platelet - derived growth factor, vascular endothelial growth factor (VEGF), insulin - like growth factor, stromal cell - derived factor - 1, indoleamine 2,3 - dioxygenase (IDO), inducible nitric oxide synthase (iNOS); Preferably, the cell membrane sheet can secrete at least interleukin - 6 (IL - 6), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF); (vi) The cell membrane sheet contains the extracellular matrix secreted by cells; Preferably, the extracellular matrix in the cell membrane sheet comprises one or more of fibronectin, integrin family (such as integrin-β1), and vitronectin; More preferably, the extracellular matrix in the cell membrane sheet comprises at least fibronectin and integrin-β1.

10. Use according to any one of claims 1-9, wherein, The cell membrane sheet is obtained by the following method: 1) Coating a layer of matrix on the surface of a thermosensitive culture dish; 2) Adding a cell suspension into the thermosensitive culture dish for culturing; 3) Lowering the temperature, and the cells and the extracellular matrix secreted by them are detached in a lamellar shape to obtain the cell membrane sheet.

11. The use according to claim 10, wherein, The method for preparing the cell membrane sheet 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, placental mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, endometrial mesenchymal stem cells, dental pulp mesenchymal stem cells, and 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 °C (preferably 37 °C), saturated humidity, and 1-10% CO2 (preferably 5% CO2); (iv) The coating time is 0.5-48 h; (v) The density of the cells in the temperature-sensitive culture dish is 1.8×10 4 -1.9×10 6 / cm 2 (preferably 1×10 6 / cm 2 ); (vi) Step 2) is carried out by the following method: adding a cell suspension and a culture medium into the thermosensitive culture dish for culturing; Preferably, the culture medium is a serum-free medium; Preferably, the proportion of the culture solution in the temperature-sensitive culture dish is 0.1-0.5 mL / cm 2 (preferably 0.3 mL / cm 2 ); (vii) The culturing is carried out in an incubator at 35-40 °C (preferably 37 °C), saturated humidity, and 1-10% CO2 (preferably 5% CO2); (viii) The culturing time is 2-48 h; (ix) In step 3), the temperature is lowered to 4-32 °C; (x) When the cells and the extracellular matrix secreted by them are detached in a lamellar shape, no additional reagent or material is added.

12. Use of cell membrane sheet fragments in the preparation of a pharmaceutical composition for treating lower limb ischemia.

13. Use according to claim 12, wherein, The cell membrane sheet fragments are as defined in any one of claims 1-7.

14. Use according to any one of claims 12 - 13, wherein, The cells are as defined in claim 8.

15. Use according to any one of claims 12 - 14, wherein, The cell membrane sheet is as defined in any one of claims 9-11.

16. Use according to any one of claims 12-15, wherein, The pharmaceutical composition further comprises a pharmaceutically acceptable excipient, such as physiological saline.

17. Use according to any one of claims 12 - 16, wherein, The dosage form of the pharmaceutical composition is an injection.

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