Efficient amplification process and anti-aging application of human-derived amniotic mesenchymal stem cells

Abstract

The invention discloses an efficient amplification process and anti-aging application of human-derived amniotic mesenchymal stem cells, and belongs to the technical field of stem cell culture. Aiming at the problems of slow cell proliferation, easy aging and pluripotency loss caused by traditional two-dimensional culture, efficient amplification is realized by constructing a modified polylactic acid-glycolic acid-chondroitin sulfate gel three-dimensional scaffold and combining with an optimized culture medium. The scaffold is formed by cross-linking a norbornene modified polylactic acid-glycolic acid copolymer and tetrazine functionalized chondroitin sulfate through an anti-Diels-Alder reaction, a natural extracellular matrix structure is simulated, the cell attachment rate is remarkably increased, and the doubling time is shortened. The culture medium contains methacrylated hyaluronic acid, growth factors and a serum substitute to synergistically support cell activity. The three-dimensional scaffold has a pH-responsive degradation characteristic, can realize biphasic slow release of basic fibroblast growth factors and platelet-derived growth factors, and maintains the dryness and differentiation potential of stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell culture, and in particular relates to a high-efficiency expansion process of human-derived amniotic mesenchymal stem cells and its anti-aging application. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell with self-renewal capacity, multilineage differentiation potential, and potent immunomodulatory functions. Human amniotic mesenchymal stem cells (hAMSCs), isolated from placental amniotic membrane tissue, are a valuable MSC subset. hAMSCs are abundant, have minimal ethical concerns, and exhibit low immunogenicity. They secrete a variety of anti-inflammatory factors (such as IL-10, IL-1RA, and TGF-β) and exhibit significant immunomodulatory effects (inducing immune tolerance by regulating the functions of T cells, B cells, NK cells, and dendritic cells). Furthermore, they secrete a variety of trophic and growth factors that promote tissue repair (such as HGF, VEGF, EGF, and FGF) and possess the potential to differentiate into osteoblasts, chondrocytes, adipocytes, and other pluripotent cells. These unique biological properties make hAMSCs extremely promising for clinical applications and research in areas such as tissue engineering and regenerative medicine, the treatment of autoimmune diseases, the prevention and treatment of graft-versus-host disease, and wound repair.

[0003] Currently, human-derived amniotic mesenchymal stem cells (hAMMSCs) primarily rely on traditional two-dimensional culture. While mature, this technique has significant drawbacks: limited cell yields, high culture costs, and, most critically, repeated subcultures can easily lead to stem cell aging (manifested by slowed proliferation, morphological changes, and increased β-galactosidase activity) and loss of pluripotency (reduced differentiation capacity). Currently, hAMMSCs exhibit limited in vitro expansion capacity, with significant issues such as slowed proliferation and reduced differentiation capacity after subculture. The effective number of passages is typically limited to 3-7, far from meeting the enormous clinical and scientific needs, resulting in a significant disparity between supply and demand. Therefore, the development of new methods for rapid, large-scale expansion and culture of hAMMSCs while effectively maintaining their stemness, viability, and function is both necessary and urgent. Summary of the Invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention constructs a modified polylactic acid-glycolic acid-chondroitin sulfate gel as a three-dimensional scaffold to improve the cell attachment rate and shorten the doubling time.

[0005] In order to achieve the above objectives, the following technical solution is adopted: The present invention provides a highly efficient expansion process of human-derived amniotic mesenchymal stem cells, comprising the following steps: S1. Tissue processing and digestion: Fresh placenta was washed three times in normal saline containing 1 mg / mL povidone-iodine, 100 U / mL penicillin, and 100 μg / mL streptomycin. The amniotic membrane was removed and minced into 1-3 mm3 tissue blocks. The tissue blocks were then digested with a mixture of 0.1% collagenase IV and 0.25% trypsin in a volume ratio of 1:1 at 37°C with shaking for 45 min. Undigested tissue was removed by filtration using a 200-mesh filter. S2. Cell separation and three-dimensional matrix seeding: The filtrate obtained in step S1 was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, the cell pellet was resuspended in physiological saline, and the centrifugation was repeated once to detect cell viability and count the number of nucleated cells. The cell suspension was mixed with modified polylactic-co-glycolic acid-chondroitin sulfate gel, seeded into a six-well plate containing basic culture medium, and cultured in a cell culture incubator; S3. Primary culture and maintenance: Culture in a 37°C, 5% CO2 incubator, with full medium replacement every 3 days. Adherent cells were retained and passaged when cell confluence reached 80%. S4. Subculture, expansion and cryopreservation: Treat the cells with a digestion solution containing 0.25% trypsin and 0.02% EDTA for 2-3 minutes. After terminating the digestion, collect the cells by centrifugation and inoculate them in flasks at a ratio of 1:3 to maintain a density of 5×10 5 The cell suspension was mixed with a freezing solution containing 10% DMSO and 90% fetal bovine serum, dispensed into cryovials, and stored in liquid nitrogen for a long time after cooling.

[0006] Furthermore, the basic culture medium components include the following components in mass percentage: modified polylactic acid-glycolic acid-chondroitin sulfate gel 3%-8%, polylysine 0.1%-2%, methacryloylated hyaluronic acid 1%-1.5%, serum substitute BIT 9500 Serum Substitute 5%-8%, folic acid 0.001%-0.002%, ascorbic acid 0.005%-0.01%, basic fibroblast growth factor 0.0005%-0.002%, platelet-derived growth factor 0.001%-0.002%, and the balance is distilled water.

[0007] Furthermore, the modified polylactic acid-glycolic acid-chondroitin sulfate gel is prepared by the following steps: a. Dissolve four-arm poly(lactic acid-co-glycolic acid) copolymer in anhydrous dichloromethane to obtain a PLGA solution; dissolve succinic anhydride in anhydrous dichloromethane to obtain a succinic anhydride solution, and add the solution dropwise to the PLGA solution. Simultaneously, add 4-dimethylaminopyridine, and stir at 40°C for 6 hours. After the reaction is completed, pour the reaction solution into icy ether, collect the precipitate, wash with deionized water, and dry to obtain succinic anhydride-terminated PLGA; Succinic anhydride-terminated PLGA was dissolved in DMF, and then citric acid was added. After stirring evenly, 10 μL of concentrated sulfuric acid was added and stirred at 60°C for 4 h. After the reaction was completed, THF was added to dilute it and the mixture was slowly added dropwise to an ice-water mixture. The precipitate was collected by centrifugation and vacuum dried for 2 h to obtain a carboxyl-activated PLGA-citric acid intermediate. Dissolve norbornene-2-methylamine in DMF, add triethylamine, and then add carboxyl-activated PLGA-citric acid intermediate. The reaction temperature is 25°C, and the mixture is stirred in the dark for 12 hours. After the reaction is completed, the reaction solution is poured into glacial ether for precipitation. After centrifugation, the precipitate is dissolved in THF, dialyzed, and freeze-dried to obtain norbornene-modified PLGA. b. Weigh (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride, dissolve it in deionized water, and stir until completely dissolved. Slowly add sodium bicarbonate powder while stirring until the pH of the solution stabilizes at 7.5-8.0. Continue stirring for 30 minutes to ensure that the hydrochloride is completely neutralized to obtain a functionalized reagent; Weigh chondroitin sulfate and dissolve it in PBS buffer at pH 7.4. After ultrasonic dissolution, centrifuge at 10,000 rpm for 10 minutes to obtain a supernatant. EDC·HCl and NHS are added to the supernatant in sequence, and stirred at room temperature for 30 minutes to obtain an activated chondroitin sulfate solution. Slowly add the functionalization reagent dropwise to the activated chondroitin sulfate solution, and fine-tune the pH to 6.5 with 1M HCl solution or NaOH solution. Stir and react at room temperature in the dark for 6-8 hours. After dialyzation, the mixture is freeze-dried to obtain tetrazine-functionalized chondroitin sulfate. c. Weigh 50 mg of norbornene-modified four-arm poly(lactic acid-glycolic acid) copolymer, dissolve it in PBS buffer at pH 7.4, and shake in a 37°C water bath for 30 minutes until completely dissolved to prepare a modified poly(lactic acid-glycolic acid) solution. Weigh tetrazine-functionalized chondroitin sulfate, dissolve it in PBS buffer at pH 7.4, and ultrasonically disperse it for 10 minutes to obtain a tetrazine-functionalized chondroitin sulfate solution. At a low temperature of 4°C, the tetrazine functionalized chondroitin sulfate solution was added dropwise to the modified polylactic acid-glycolic acid solution, immediately vortexed to mix, and incubated at a constant temperature of 37°C for 2-4 hours. The formed gel was soaked in physiological saline for 24 hours, freeze-dried for storage, and dehydrated with 20% ethanol to obtain the modified polylactic acid-glycolic acid-chondroitin sulfate gel.

[0008] Furthermore, in step a, the concentration of the PLGA solution is 0.02-0.05 g / mL, the concentration of the succinic anhydride solution is 0.02-0.05 g / mL, and the feed ratio of the PLGA solution, succinic anhydride solution and 4-dimethylaminopyridine is 3-5 mL: 5-10 mL: 5-10 mg.

[0009] Furthermore, in step a, the feed ratio of succinic anhydride-terminated PLGA, DMF, citric acid, concentrated sulfuric acid and THF is 0.1-0.5 g: 5-20 mL: 15-20 mg: 10-50 μL: 20-60 mL.

[0010] Furthermore, in the step a, the feed ratio of norbornene-2-methylamine, triethylamine and carboxyl-activated PLGA-citric acid intermediate is 30-150 mg: 50-150 μL: 0.1-1 g.

[0011] Furthermore, in the step b, the feeding ratio of (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride and deionized water is 0.5-1 g: 10-30 mL.

[0012] Furthermore, in step b, the feeding ratio of chondroitin sulfate and PBS buffer is 1-5 g: 15-40 mL.

[0013] Furthermore, in step b, the feed ratio of the supernatant, EDC·HCl and NHS is 20-40 mL: 0.15-0.25 g: 0.1-0.2 g, and the feed ratio of the functionalization reagent and the activated chondroitin sulfate solution in step b is 10-20 mL: 30-60 mL.

[0014] Furthermore, the concentration of the modified polylactic acid-glycolic acid solution in step c is 50-100 mg / mL, and the concentration of the tetrazine functionalized chondroitin sulfate solution is 20-40 mg / mL.

[0015] Furthermore, in step c, the volume ratio of the tetrazine functionalized chondroitin sulfate solution to the modified polylactic acid-glycolic acid solution is 1:1-5.

[0016] The present invention also provides applications of the human-derived amniotic mesenchymal stem cells, including applications in foods, medicines, or cosmetic skin care products for skin repair and knee joint cartilage regeneration.

[0017] The beneficial effects of the present invention are: (1) The present invention constructs a modified polylactic acid-glycolic acid-chondroitin sulfate gel as a three-dimensional scaffold to form an interpenetrating polymer network with strong mechanical stability, simulating the amino acid and glycosaminoglycan composition of the natural extracellular matrix. The sulfated polysaccharide chains of polylactic acid-glycolic acid and chondroitin sulfate form physical crosslinking points, which greatly improves the mechanical strength compared with traditional two-dimensional culture, thereby increasing the cell attachment rate and shortening the doubling time. (2) The polylactic acid-glycolic acid copolymer integrated in the gel is capped with succinic anhydride and modified with norbornene to form pH-responsive degradation characteristics. Combined with the loading system constructed by tetrazine-trans-cyclooctene click chemistry reaction, it can achieve a biphasic sustained release of basic fibroblast growth factor and platelet-derived growth factor, continuously provide antioxidant protection and mitogenic signals, effectively promote cell proliferation and differentiation, increase cell yield, and promote efficient expansion; (3) The present invention cross-links norbornene-modified PLGA and tetrazine-functionalized chondroitin sulfate through a reverse Diels-Alder reaction, avoiding the use of toxic cross-linking agents and completely retaining the natural biological activities of polylactic acid-glycolic acid copolymer and chondroitin sulfate. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a statistical chart of the cell expansion efficiency experiment in Test Example 1 of the present invention; Figure 2 This is a statistical graph of skin graft survival rate and wound contraction rate in the nude mouse full-thickness skin defect repair experiment in Test Example 2 of the present invention; Figure 3 This is a statistical chart of the evaluation of the repair results of the rat knee cartilage defect repair experiment in Test Example 3 of the present invention; Figure 4 This is a statistical graph of the mRNA expression levels of Sox9 and glycosaminoglycan (GAG) in the rat knee cartilage defect repair experiment in Test Example 3 of the present invention.

[0019] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials described herein are for illustrative purposes only and are not intended to limit the scope of this application.

[0022] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and the experimental materials used in the following examples, unless otherwise specified, are all purchased from commercial channels.

[0023] Example 1: A process for efficiently expanding human amniotic mesenchymal stem cells, comprising the following steps: S1. Tissue processing and digestion: The fresh placenta was washed three times in normal saline containing 1 mg / mL povidone iodine, 100 U / mL penicillin, and 100 μg / mL streptomycin. The amniotic membrane was removed and cut into 1-3 mm pieces. 3 The tissue blocks were digested with a mixture of 0.1% collagenase IV and 0.25% trypsin in a volume ratio of 1:1 at 37°C for 45 min with shaking, and undigested tissue was removed by filtering with a 200-mesh filter. S2. Cell separation and three-dimensional matrix seeding: The filtrate obtained in step S1 was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, the cell pellet was resuspended in physiological saline, and the centrifugation was repeated once to detect cell viability and count the number of nucleated cells. The cell suspension was mixed with modified polylactic-co-glycolic acid-chondroitin sulfate gel, seeded into a six-well plate containing basic culture medium, and cultured in a cell culture incubator; S3. Primary culture and maintenance: Culture in a 37°C, 5% CO2 incubator, with full medium replacement every 3 days. Adherent cells were retained and passaged when cell confluence reached 80%. S4. Subculture, expansion and cryopreservation: Treat the cells with a digestion solution containing 0.25% trypsin and 0.02% EDTA for 2 minutes. After terminating the digestion, collect the cells by centrifugation and inoculate them in flasks at a ratio of 1:3 to maintain a density of 5×10 5 The cell suspension was mixed with a freezing solution containing 10% DMSO and 90% fetal bovine serum, dispensed into cryovials, and stored in liquid nitrogen for a long time after cooling.

[0024] The basic culture medium components include the following components by mass percentage: 3% modified polylactic acid-glycolic acid-chondroitin sulfate gel, 0.1% polylysine, 1% methacryloyl hyaluronic acid, 5% BIT9500 Serum Substitute, 0.001% folic acid, 0.005% ascorbic acid, 0.0005% basic fibroblast growth factor, 0.001% platelet-derived growth factor, and the balance is distilled water.

[0025] The modified polylactic acid-glycolic acid-chondroitin sulfate gel is prepared by the following steps: a. Dissolve four-arm poly(lactic acid-co-glycolic acid) copolymer in anhydrous dichloromethane to obtain a PLGA solution; dissolve succinic anhydride in anhydrous dichloromethane to obtain a succinic anhydride solution, and add the solution dropwise to the PLGA solution. Simultaneously, add 4-dimethylaminopyridine, and stir at 40°C for 6 hours. After the reaction is completed, pour the reaction solution into icy ether, collect the precipitate, wash with deionized water, and dry to obtain succinic anhydride-terminated PLGA; Succinic anhydride-terminated PLGA was dissolved in DMF, and then citric acid was added. After stirring evenly, 10 μL of concentrated sulfuric acid was added and stirred at 60°C for 4 h. After the reaction was completed, THF was added to dilute it and the mixture was slowly added dropwise to an ice-water mixture. The precipitate was collected by centrifugation and vacuum dried for 2 h to obtain a carboxyl-activated PLGA-citric acid intermediate. Dissolve norbornene-2-methylamine in DMF, add triethylamine, and then add carboxyl-activated PLGA-citric acid intermediate. The reaction temperature is 25°C, and the mixture is stirred in the dark for 12 hours. After the reaction is completed, the reaction solution is poured into glacial ether for precipitation. After centrifugation, the precipitate is dissolved in THF, dialyzed, and freeze-dried to obtain norbornene-modified PLGA. b. Weigh (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride, dissolve it in deionized water, and stir until completely dissolved. Slowly add sodium bicarbonate powder while stirring until the pH of the solution stabilizes at 7.5-8.0. Continue stirring for 30 minutes to ensure that the hydrochloride is completely neutralized to obtain a functionalized reagent; Weigh chondroitin sulfate and dissolve it in PBS buffer at pH 7.4. After ultrasonic dissolution, centrifuge at 10,000 rpm for 10 minutes to obtain a supernatant. EDC·HCl and NHS are added to the supernatant in sequence, and stirred at room temperature for 30 minutes to obtain an activated chondroitin sulfate solution. Slowly add the functionalization reagent dropwise to the activated chondroitin sulfate solution, and fine-tune the pH to 6.5 with 1M HCl solution or NaOH solution. Stir and react at room temperature in the dark for 6-8 hours. After dialyzation, the mixture is freeze-dried to obtain tetrazine-functionalized chondroitin sulfate. c. Weigh 50 mg of norbornene-modified four-arm poly(lactic acid-glycolic acid) copolymer, dissolve it in PBS buffer at pH 7.4, and shake in a 37°C water bath for 30 minutes until completely dissolved to prepare a modified poly(lactic acid-glycolic acid) solution. Weigh tetrazine-functionalized chondroitin sulfate, dissolve it in PBS buffer at pH 7.4, and ultrasonically disperse it for 10 minutes to obtain a tetrazine-functionalized chondroitin sulfate solution. At a low temperature of 4°C, the tetrazine functionalized chondroitin sulfate solution was added dropwise to the modified polylactic acid-glycolic acid solution, immediately vortexed to mix, and incubated at a constant temperature of 37°C for 2-4 hours. The formed gel was soaked in physiological saline for 24 hours, freeze-dried for storage, and dehydrated with 20% ethanol to obtain the modified polylactic acid-glycolic acid-chondroitin sulfate gel.

[0026] The concentration of the PLGA solution in step a is 0.02 g / mL, the concentration of the succinic anhydride solution is 0.02 g / mL, and the feeding ratio of the PLGA solution, succinic anhydride solution and 4-dimethylaminopyridine is 3 mL:5 mL:5 mg.

[0027] In the step a, the feed ratio of succinic anhydride-terminated PLGA, DMF, citric acid, concentrated sulfuric acid and THF is 0.1 g: 5 mL: 15 mg: 10 μL: 20 mL.

[0028] In the step a, the feed ratio of norbornene-2-methylamine, triethylamine and carboxyl-activated PLGA-citric acid intermediate is 30 mg:50 μL:0.1 g.

[0029] In the step b, the feeding ratio of (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride and deionized water is 0.5 g:10 mL.

[0030] In step b, the feeding ratio of chondroitin sulfate and PBS buffer is 1 g:15 mL.

[0031] The feed ratio of the supernatant, EDC·HCl and NHS in step b is 20 mL: 0.15 g: 0.1 g, and the feed ratio of the functionalization reagent and the activated chondroitin sulfate solution in step b is 10 mL: 30 mL.

[0032] The concentration of the modified polylactic acid-glycolic acid solution in step c is 50 mg / mL, and the concentration of the tetrazine functionalized chondroitin sulfate solution is 20 mg / mL.

[0033] In the step c, the volume ratio of the tetrazine functionalized chondroitin sulfate solution to the modified polylactic acid-glycolic acid solution is 1:1.

[0034] Example 2: A highly efficient expansion process for human-derived amniotic mesenchymal stem cells, comprising the following steps: S1. Tissue processing and digestion: The fresh placenta was washed three times in normal saline containing 1 mg / mL povidone iodine, 100 U / mL penicillin, and 100 μg / mL streptomycin. The amniotic membrane was removed and cut into 1-3 mm pieces. 3 The tissue blocks were digested with a mixture of 0.1% collagenase IV and 0.25% trypsin in a volume ratio of 1:1 at 37°C for 45 min with shaking, and undigested tissue was removed by filtering with a 200-mesh filter. S2. Cell separation and three-dimensional matrix seeding: The filtrate obtained in step S1 was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, the cell pellet was resuspended in physiological saline, and the centrifugation was repeated once to detect cell viability and count the number of nucleated cells. The cell suspension was mixed with modified polylactic-co-glycolic acid-chondroitin sulfate gel, seeded into a six-well plate containing basic culture medium, and cultured in a cell culture incubator; S3. Primary culture and maintenance: Culture in a 37°C, 5% CO2 incubator, with full medium replacement every 3 days. Adherent cells were retained and passaged when cell confluence reached 80%. S4. Subculture, expansion and cryopreservation: Treat the cells with a digestion solution containing 0.25% trypsin and 0.02% EDTA for 3 minutes. After terminating the digestion, collect the cells by centrifugation and inoculate them in flasks at a ratio of 1:3 to maintain a density of 5×10 5 The cell suspension was mixed with a freezing solution containing 10% DMSO and 90% fetal bovine serum, dispensed into cryovials, and stored in liquid nitrogen for a long time after cooling.

[0035] The basic culture medium components include the following components by mass percentage: 8% modified polylactic acid-glycolic acid-chondroitin sulfate gel, 2% polylysine, 1.5% methacrylated hyaluronic acid, 8% BIT9500 Serum Substitute, 0.002% folic acid, 0.01% ascorbic acid, 0.002% basic fibroblast growth factor, 0.002% platelet-derived growth factor, and the balance is distilled water.

[0036] The modified polylactic acid-glycolic acid-chondroitin sulfate gel is prepared by the following steps: a. Dissolve four-arm poly(lactic acid-co-glycolic acid) copolymer in anhydrous dichloromethane to obtain a PLGA solution; dissolve succinic anhydride in anhydrous dichloromethane to obtain a succinic anhydride solution, and add the solution dropwise to the PLGA solution. Simultaneously, add 4-dimethylaminopyridine, and stir at 40°C for 6 hours. After the reaction is completed, pour the reaction solution into icy ether, collect the precipitate, wash with deionized water, and dry to obtain succinic anhydride-terminated PLGA; Succinic anhydride-terminated PLGA was dissolved in DMF, and then citric acid was added. After stirring evenly, 10 μL of concentrated sulfuric acid was added and stirred at 60°C for 4 h. After the reaction was completed, THF was added to dilute it and the mixture was slowly added dropwise to an ice-water mixture. The precipitate was collected by centrifugation and vacuum dried for 2 h to obtain a carboxyl-activated PLGA-citric acid intermediate. Dissolve norbornene-2-methylamine in DMF, add triethylamine, and then add carboxyl-activated PLGA-citric acid intermediate. The reaction temperature is 25°C, and the mixture is stirred in the dark for 12 hours. After the reaction is completed, the reaction solution is poured into glacial ether for precipitation. After centrifugation, the precipitate is dissolved in THF, dialyzed, and freeze-dried to obtain norbornene-modified PLGA. b. Weigh (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride, dissolve it in deionized water, and stir until completely dissolved. Slowly add sodium bicarbonate powder while stirring until the pH of the solution stabilizes at 7.5-8.0. Continue stirring for 30 minutes to ensure that the hydrochloride is completely neutralized to obtain a functionalized reagent; Weigh chondroitin sulfate and dissolve it in PBS buffer at pH 7.4. After ultrasonic dissolution, centrifuge at 10,000 rpm for 10 minutes to obtain a supernatant. EDC·HCl and NHS are added to the supernatant in sequence, and stirred at room temperature for 30 minutes to obtain an activated chondroitin sulfate solution. Slowly add the functionalization reagent dropwise to the activated chondroitin sulfate solution, and fine-tune the pH to 6.5 with 1M HCl solution or NaOH solution. Stir and react at room temperature in the dark for 6-8 hours. After dialyzation, the mixture is freeze-dried to obtain tetrazine-functionalized chondroitin sulfate. c. Weigh 50 mg of norbornene-modified four-arm poly(lactic acid-glycolic acid) copolymer, dissolve it in PBS buffer at pH 7.4, and shake in a 37°C water bath for 30 minutes until completely dissolved to prepare a modified poly(lactic acid-glycolic acid) solution. Weigh tetrazine-functionalized chondroitin sulfate, dissolve it in PBS buffer at pH 7.4, and ultrasonically disperse it for 10 minutes to obtain a tetrazine-functionalized chondroitin sulfate solution. At a low temperature of 4°C, the tetrazine functionalized chondroitin sulfate solution was added dropwise to the modified polylactic acid-glycolic acid solution, immediately vortexed to mix, and incubated at a constant temperature of 37°C for 2-4 hours. The formed gel was soaked in physiological saline for 24 hours, freeze-dried for storage, and dehydrated with 20% ethanol to obtain the modified polylactic acid-glycolic acid-chondroitin sulfate gel.

[0037] The concentration of the PLGA solution in step a is 0.05 g / mL, the concentration of the succinic anhydride solution is 0.05 g / mL, and the feeding ratio of the PLGA solution, succinic anhydride solution and 4-dimethylaminopyridine is 5 mL:10 mL:10 mg.

[0038] In the step a, the feed ratio of succinic anhydride-terminated PLGA, DMF, citric acid, concentrated sulfuric acid and THF is 0.5 g: 20 mL: 20 mg: 50 μL: 60 mL.

[0039] In the step a, the feed ratio of norbornene-2-methylamine, triethylamine and carboxyl-activated PLGA-citric acid intermediate is 150 mg:150 μL:1 g.

[0040] In the step b, the feeding ratio of (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride and deionized water is 1 g:30 mL.

[0041] In step b, the feeding ratio of chondroitin sulfate and PBS buffer is 5 g:40 mL.

[0042] The feed ratio of the supernatant, EDC·HCl and NHS in step b is 40 mL: 0.25 g: 0.2 g, and the feed ratio of the functionalization reagent and the activated chondroitin sulfate solution in step b is 20 mL: 60 mL.

[0043] The concentration of the modified polylactic acid-glycolic acid solution in step c is 100 mg / mL, and the concentration of the tetrazine functionalized chondroitin sulfate solution is 40 mg / mL.

[0044] In the step c, the volume ratio of the tetrazine functionalized chondroitin sulfate solution to the modified polylactic acid-glycolic acid solution is 1:5.

[0045] Example 3: A process for efficiently expanding human amniotic mesenchymal stem cells, comprising the following steps: S1. Tissue processing and digestion: The fresh placenta was washed three times in normal saline containing 1 mg / mL povidone iodine, 100 U / mL penicillin, and 100 μg / mL streptomycin. The amniotic membrane was removed and cut into 1-3 mm pieces. 3 The tissue blocks were digested with a mixture of 0.1% collagenase IV and 0.25% trypsin in a volume ratio of 1:1 at 37°C for 45 min with shaking, and undigested tissue was removed by filtering with a 200-mesh filter. S2. Cell separation and three-dimensional matrix seeding: The filtrate obtained in step S1 was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, the cell pellet was resuspended in physiological saline, and the centrifugation was repeated once to detect cell viability and count the number of nucleated cells. The cell suspension was mixed with modified polylactic-co-glycolic acid-chondroitin sulfate gel, seeded into a six-well plate containing basic culture medium, and cultured in a cell culture incubator; S3. Primary culture and maintenance: Culture in a 37°C, 5% CO2 incubator, with full medium replacement every 3 days. Adherent cells were retained and passaged when cell confluence reached 80%. S4. Subculture, expansion and cryopreservation: Treat the cells with a digestion solution containing 0.25% trypsin and 0.02% EDTA for 2.5 min. After terminating the digestion, collect the cells by centrifugation and inoculate them in flasks at a ratio of 1:3 to maintain a density of 5 × 10 5 The cell suspension was mixed with a freezing solution containing 10% DMSO and 90% fetal bovine serum, dispensed into cryovials, and stored in liquid nitrogen for a long time after cooling.

[0046] The basic culture medium components include the following components by mass percentage: 5.5% modified polylactic acid-glycolic acid-chondroitin sulfate gel, 1.05% polylysine, 1.25% methacryloyl hyaluronic acid, 6.5% BIT9500 Serum Substitute, 0.0015% folic acid, 0.0075% ascorbic acid, 0.00125% basic fibroblast growth factor, 0.0015% platelet-derived growth factor, and the balance is distilled water.

[0047] The modified polylactic acid-glycolic acid-chondroitin sulfate gel is prepared by the following steps: a. Dissolve four-arm poly(lactic acid-co-glycolic acid) copolymer in anhydrous dichloromethane to obtain a PLGA solution; dissolve succinic anhydride in anhydrous dichloromethane to obtain a succinic anhydride solution, and add the solution dropwise to the PLGA solution. Simultaneously, add 4-dimethylaminopyridine, and stir at 40°C for 6 hours. After the reaction is completed, pour the reaction solution into icy ether, collect the precipitate, wash with deionized water, and dry to obtain succinic anhydride-terminated PLGA; Succinic anhydride-terminated PLGA was dissolved in DMF, and then citric acid was added. After stirring evenly, 10 μL of concentrated sulfuric acid was added and stirred at 60°C for 4 h. After the reaction was completed, THF was added to dilute it and the mixture was slowly added dropwise to an ice-water mixture. The precipitate was collected by centrifugation and vacuum dried for 2 h to obtain a carboxyl-activated PLGA-citric acid intermediate. Dissolve norbornene-2-methylamine in DMF, add triethylamine, and then add carboxyl-activated PLGA-citric acid intermediate. The reaction temperature is 25°C, and the mixture is stirred in the dark for 12 hours. After the reaction is completed, the reaction solution is poured into glacial ether for precipitation. After centrifugation, the precipitate is dissolved in THF, dialyzed, and freeze-dried to obtain norbornene-modified PLGA. b. Weigh (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride, dissolve it in deionized water, and stir until completely dissolved. Slowly add sodium bicarbonate powder while stirring until the pH of the solution stabilizes at 7.5-8.0. Continue stirring for 30 minutes to ensure that the hydrochloride is completely neutralized to obtain a functionalized reagent; Weigh chondroitin sulfate and dissolve it in PBS buffer at pH 7.4. After ultrasonic dissolution, centrifuge at 10,000 rpm for 10 minutes to obtain a supernatant. EDC·HCl and NHS are added to the supernatant in sequence, and stirred at room temperature for 30 minutes to obtain an activated chondroitin sulfate solution. Slowly add the functionalization reagent dropwise to the activated chondroitin sulfate solution, and fine-tune the pH to 6.5 with 1M HCl solution or NaOH solution. Stir and react at room temperature in the dark for 6-8 hours. After dialyzation, the mixture is freeze-dried to obtain tetrazine-functionalized chondroitin sulfate. c. Weigh 50 mg of norbornene-modified four-arm poly(lactic acid-glycolic acid) copolymer, dissolve it in PBS buffer at pH 7.4, and shake in a 37°C water bath for 30 minutes until completely dissolved to prepare a modified poly(lactic acid-glycolic acid) solution. Weigh tetrazine-functionalized chondroitin sulfate, dissolve it in PBS buffer at pH 7.4, and ultrasonically disperse it for 10 minutes to obtain a tetrazine-functionalized chondroitin sulfate solution. At a low temperature of 4°C, the tetrazine functionalized chondroitin sulfate solution was added dropwise to the modified polylactic acid-glycolic acid solution, immediately vortexed to mix, and incubated at a constant temperature of 37°C for 2-4 hours. The formed gel was soaked in physiological saline for 24 hours, freeze-dried for storage, and dehydrated with 20% ethanol to obtain the modified polylactic acid-glycolic acid-chondroitin sulfate gel.

[0048] The concentration of the PLGA solution in step a is 0.035 g / mL, the concentration of the succinic anhydride solution is 0.035 g / mL, and the feeding ratio of the PLGA solution, succinic anhydride solution and 4-dimethylaminopyridine is 4 mL:7.5 mL:7.5 mg.

[0049] In the step a, the feed ratio of succinic anhydride-terminated PLGA, DMF, citric acid, concentrated sulfuric acid and THF is 0.3 g: 12.5 mL: 17.5 mg: 30 μL: 40 mL.

[0050] In the step a, the feed ratio of norbornene-2-methylamine, triethylamine and carboxyl-activated PLGA-citric acid intermediate is 90 mg:100 μL:0.55 g.

[0051] In the step b, the feeding ratio of (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride and deionized water is 0.75 g:20 mL.

[0052] In step b, the feeding ratio of chondroitin sulfate and PBS buffer is 3 g:27.5 mL.

[0053] The feed ratio of the supernatant, EDC·HCl and NHS in step b is 30 mL: 0.2 g: 0.15 g, and the feed ratio of the functionalization reagent and the activated chondroitin sulfate solution in step b is 15 mL: 45 mL.

[0054] The concentration of the modified polylactic acid-glycolic acid solution in step c is 75 mg / mL, and the concentration of the tetrazine functionalized chondroitin sulfate solution is 30 mg / mL.

[0055] In the step c, the volume ratio of the tetrazine functionalized chondroitin sulfate solution to the modified polylactic acid-glycolic acid solution is 1:3.

[0056] Test Example 1: Cell proliferation efficiency experiment Experimental group: The inoculation experiment was carried out according to the method in Examples 1-3. Control group: traditional two-dimensional culture (DMEM / F12+10% FBS); On days 1, 3, 5, and 7 after seeding, CCK-8 reagent (10 μL / well) was added to each well and incubated at 37°C for 2 hours. The absorbance at a wavelength of 450 nm (OD value) was measured using a microplate reader to plot cell growth curves.

[0057] The above data are expressed as mean ± standard deviation, and the difference between the groups was considered significant when *p* < 0.001.

[0058] See the results Figure 1 , Figure 1It shows that the OD value of the three-dimensional culture system of the present invention is significantly higher than that of the traditional two-dimensional culture, indicating that the cell proliferation efficiency is significantly improved.

[0059] Test Example 2: Nude Mouse Skin Full-Thickness Defect Repair Experiment (1) The rat tail tendon was sterilized and minced, and then collagen was extracted by acid extraction. 6-Chondroitin sulfate C solution was added to the collagen solution, and the solution was pre-frozen at -30 °C for 4 hours. The freeze-dried film was prepared using a vacuum freeze dryer. The freeze-dried film was initially cross-linked at high temperature in a vacuum, and then cross-linked with 0.05% glutaraldehyde at 4 °C for 24 hours. The collagen film was then dried in a vacuum to prepare the collagen film.

[0060] (2) The collagen membrane was repeatedly rinsed with PBS, disinfected with 75% ethanol, and then soaked in IMDM culture medium for 24 hours. The hAMSCs cultured in Example 3 of the present invention were cultured at a rate of 1×10 6 pieces / cm 2 The cells were seeded at a density of 10 × 10 on one side of the collagen membrane and cultured in IMDM medium containing 10% fetal bovine serum at 37°C and 5% carbon dioxide incubator for 4 days. The collagen membrane was flipped over and 2 × 10 cells were seeded on the other side. 5 pieces / cm 2 Epidermal stem cells were seeded at a high density and cultured in stem cell culture medium. The cells were first cultured under liquid surface for 3 weeks. After the epidermal stem cells became fused, they were switched to air-liquid interface culture for 12 days.

[0061] (3) Sixty 7-8 week old nude mice were randomly divided into three groups: Experimental group: transplanted composite skin substitute containing hAMSCs and epidermal stem cells; Control group 1: transplantation of composite skin substitute containing epidermal cells and hAMSCs (containing only hAMSCs, without epidermal stem cells); Control group 2: transplanted collagen sponge membrane without cell inoculation.

[0062] After anesthesia, nude mice were anesthetized and full-thickness skin defects of 1.5 cm × 1.5 cm were prepared on the back. Corresponding substitutes were implanted and fixed with sterile gauze under pressure.

[0063] Penicillin was injected intramuscularly daily for 3 days after surgery to prevent infection.

[0064] (4) Observe the wound healing, calculate the skin graft survival rate, measure the change in wound area, and calculate the wound shrinkage rate.

[0065] The above data are expressed as mean ± standard deviation, and the difference between the groups was considered significant when *p* < 0.001.

[0066] The above results can be seen in Figure 2 , Figure 2The above results show that the skin graft survival rate in the experimental group was significantly higher and the wound contraction rate was lower, indicating that hAMSCs synergistically with epidermal stem cells can significantly promote skin repair.

[0067] Test Example 3: Rat Knee Cartilage Defect Repair Experiment (1) Sodium alginate was dissolved in PBS to prepare a 0.5% solution, and allowed to stand to remove bubbles. The hAMSCs cultured in Example 3 of the present invention were diluted with PBS to a concentration of 1×10 10 The cell suspension of 100 cells / L was mixed evenly with sodium alginate solution at a volume ratio of 1:1, and 2% calcium chloride solution was added. The mixture was allowed to stand for 15 minutes, and the hAMSCs-sodium alginate gel was filtered out for later use.

[0068] (2) Forty-eight 6-week-old SPF male Sprague-Dawley rats (weighing 145-160 g) were selected and anesthetized with intraperitoneal injection of 10% chloral hydrate; The knee joint was exposed through a left parapatellar medial incision, and a full-thickness cartilage defect model with a diameter of 2 mm and a depth of 1.5 mm was prepared. The rats were randomly divided into three groups (16 rats in each group): Experimental group: 25 μL of hAMSCs-sodium alginate gel (containing hAMSCs, cell concentration 1×10 10 pieces / L).

[0069] Control group: 25 μL of sodium alginate hydrogel without hAMSCs was implanted; Injury group: no treatment; Penicillin was injected intramuscularly for 3 days after surgery, and there was no restriction on activity.

[0070] (3) Twelve weeks after surgery, eight rats in each group were randomly killed, and the knee joints were removed and evaluated according to the International Cartilage Repair Society (ICRS) scoring criteria: Defect repair degree: observe the defect filling situation (0-4 points); Boundary integration: assesses the degree of fusion between the repair tissue and the surrounding cartilage (0–4 points); Gross appearance: Check surface smoothness and texture (0-4 points).

[0071] The total score is 12 points (normal), 8-11 points are close to normal, 4-7 points are abnormal, and 1-3 points are severely abnormal.

[0072] The above results can be seen in Figure 3 .

[0073] (4) Knee joint cartilage tissues were extracted from the control group and the observation group, and the mRNA expression levels of cartilage-specific genes Sox9 and glycosaminoglycan (GAG) were detected by qRT-PCR; total RNA was extracted according to the kit instructions, reverse transcribed into cDNA, and the relative expression levels were calculated by the 2-ΔΔCt method, with GAPDH as the internal reference gene. The above results are shown in Figure 4 .

[0074] The above data are expressed as mean ± standard deviation, and the difference between the groups was considered significant when *p* < 0.001.

[0075] from Figure 3 and Figure 4 It can be seen that the ICRS score of the experimental group was significantly higher, and the expression level of cartilage-specific genes was significantly upregulated, indicating that hAMSCs can effectively promote cartilage regeneration.

[0076] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

[0077] The present invention and its embodiments are described above. Such description is not restrictive. The drawings show only one embodiment of the present invention, and actual applications are not limited thereto. In short, if a person skilled in the art is inspired by the above, and does not deviate from the purpose of the present invention, any method and embodiment similar to the technical solution without creative design shall fall within the scope of protection of the present invention.

Claims

1. A highly efficient expansion process for human amniotic mesenchymal stem cells, characterized by: The following steps are involved: S1. Tissue processing and digestion: The fresh placenta was washed three times in normal saline containing 1 mg / mL povidone iodine, 100 U / mL penicillin, and 100 μg / mL streptomycin. The amniotic membrane was removed and cut into 1-3 mm pieces. 3 The tissue blocks were digested with a mixture of 0.1% collagenase IV and 0.25% trypsin in a volume ratio of 1:1 at 37°C for 45 min with shaking, and undigested tissue was removed by filtering with a 200-mesh filter. S2. Cell separation and three-dimensional matrix seeding: The filtrate obtained in step S1 was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, the cell pellet was resuspended in physiological saline, and the centrifugation was repeated once to detect cell viability and count the number of nucleated cells. The cell suspension was mixed with modified polylactic-co-glycolic acid-chondroitin sulfate gel, seeded into a six-well plate containing basic culture medium, and cultured in a cell culture incubator; S3. Primary culture and maintenance: Culture in a 37°C, 5% CO2 incubator, with full medium replacement every 3 days. Adherent cells were retained and passaged when cell confluence reached 80%. S4. Subculture, expansion and cryopreservation: Treat the cells with a digestion solution containing 0.25% trypsin and 0.02% EDTA for 2-3 minutes. After terminating the digestion, collect the cells by centrifugation and inoculate them in flasks at a ratio of 1:3 to maintain a density of 5×10 5 cells / mL, mix the cell suspension with a freezing solution containing 10% DMSO and 90% fetal bovine serum, dispense into cryovials, and store in liquid nitrogen for a long time after cooling; The basic culture medium comprises the following components by mass percentage: 3%-8% modified polylactic acid-glycolic acid-chondroitin sulfate gel, 0.1%-2% polylysine, 1%-1.5% methacryloyl hyaluronic acid, 5%-8% BIT 9500 Serum Substitute, 0.001%-0.002% folic acid, 0.005%-0.01% ascorbic acid, 0.0005%-0.002% basic fibroblast growth factor, 0.001%-0.002% platelet-derived growth factor, and the balance is distilled water; The modified polylactic acid-glycolic acid-chondroitin sulfate gel is prepared by the following steps: a. Dissolve four-arm poly(lactic acid-co-glycolic acid) copolymer in anhydrous dichloromethane to obtain a PLGA solution; dissolve succinic anhydride in anhydrous dichloromethane to obtain a succinic anhydride solution, and add the solution dropwise to the PLGA solution. Simultaneously, add 4-dimethylaminopyridine, and stir at 40°C for 6 hours. After the reaction is completed, pour the reaction solution into icy ether, collect the precipitate, wash with deionized water, and dry to obtain succinic anhydride-terminated PLGA; Succinic anhydride-terminated PLGA was dissolved in DMF, and then citric acid was added. After stirring evenly, 10 μL of concentrated sulfuric acid was added and stirred at 60°C for 4 h. After the reaction was completed, THF was added to dilute it and the mixture was slowly added dropwise to an ice-water mixture. The precipitate was collected by centrifugation and vacuum dried for 2 h to obtain a carboxyl-activated PLGA-citric acid intermediate. Dissolve norbornene-2-methylamine in DMF, add triethylamine, and then add carboxyl-activated PLGA-citric acid intermediate. The reaction temperature is 25°C, and the mixture is stirred in the dark for 12 hours. After the reaction is completed, the reaction solution is poured into glacial ether for precipitation. After centrifugation, the precipitate is dissolved in THF, dialyzed, and freeze-dried to obtain norbornene-modified PLGA. b. Weigh (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride, dissolve it in deionized water, and stir until completely dissolved. Slowly add sodium bicarbonate powder while stirring until the pH of the solution stabilizes at 7.5-8.

0. Continue stirring for 30 minutes to ensure that the hydrochloride is completely neutralized to obtain a functionalized reagent; Weigh chondroitin sulfate and dissolve it in PBS buffer at pH 7.

4. After ultrasonic dissolution, centrifuge at 10,000 rpm for 10 minutes to obtain a supernatant. EDC·HCl and NHS are added to the supernatant in sequence, and stirred at room temperature for 30 minutes to obtain an activated chondroitin sulfate solution. Slowly add the functionalization reagent dropwise to the activated chondroitin sulfate solution, and fine-tune the pH to 6.5 with 1M HCl solution or NaOH solution. Stir and react at room temperature in the dark for 6-8 hours. After dialyzation, the mixture is freeze-dried to obtain tetrazine-functionalized chondroitin sulfate. c. Weigh 50 mg of norbornene-modified four-arm poly(lactic acid-glycolic acid) copolymer, dissolve it in PBS buffer at pH 7.4, and shake in a 37°C water bath for 30 minutes until completely dissolved to prepare a modified poly(lactic acid-glycolic acid) solution. Weigh tetrazine-functionalized chondroitin sulfate, dissolve it in PBS buffer at pH 7.4, and ultrasonically disperse it for 10 minutes to obtain a tetrazine-functionalized chondroitin sulfate solution. At a low temperature of 4°C, the tetrazine functionalized chondroitin sulfate solution was added dropwise to the modified polylactic acid-glycolic acid solution, immediately vortexed to mix, and incubated at a constant temperature of 37°C for 2-4 hours. The formed gel was soaked in physiological saline for 24 hours, freeze-dried for storage, and dehydrated with 20% ethanol to obtain the modified polylactic acid-glycolic acid-chondroitin sulfate gel.

2. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: The concentration of the PLGA solution in step a is 0.02-0.05 g / mL, the concentration of the succinic anhydride solution is 0.02-0.05 g / mL, and the feeding ratio of the PLGA solution, succinic anhydride solution and 4-dimethylaminopyridine is 3-5 mL: 5-10 mL: 5-10 mg.

3. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: In the step a, the feed ratio of succinic anhydride-terminated PLGA, DMF, citric acid, concentrated sulfuric acid and THF is 0.1-0.5 g: 5-20 mL: 15-20 mg: 10-50 μL: 20-60 mL.

4. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: In the step a, the feed ratio of norbornene-2-methylamine, triethylamine and carboxyl-activated PLGA-citric acid intermediate is 30-150 mg: 50-150 μL: 0.1-1 g.

5. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: In the step b, the feeding ratio of (1,2,4,5-tetrazine-3-yl)methylamine hydrochloride and deionized water is 0.5-1 g: 10-30 mL.

6. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: In the step b, the feeding ratio of chondroitin sulfate and PBS buffer is 1-5 g: 15-40 mL.

7. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: The feeding ratio of the supernatant, EDC·HCl and NHS in step b is 20-40 mL: 0.15-0.25 g: 0.1-0.2 g, and the feeding ratio of the functionalization reagent and the activated chondroitin sulfate solution in step b is 10-20 mL: 30-60 mL.

8. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: The concentration of the modified polylactic acid-glycolic acid solution in step c is 50-100 mg / mL, and the concentration of the tetrazine functionalized chondroitin sulfate solution is 20-40 mg / mL.

9. The process for efficiently expanding human amniotic mesenchymal stem cells according to claim 1, wherein: In the step c, the volume ratio of the tetrazine functionalized chondroitin sulfate solution to the modified polylactic acid-glycolic acid solution is 1:1-5.

10. A use of human-derived amniotic mesenchymal stem cells, characterized by: The human-derived amniotic mesenchymal stem cells are obtained by the efficient expansion process of human-derived amniotic mesenchymal stem cells according to any one of claims 1 to 9, and are used in foods, medicines or beauty products for skin repair and knee cartilage regeneration.

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