CN116622629A - Mesenchymal stem cell culture solution and application method thereof - Google Patents

Mesenchymal stem cell culture solution and application method thereof Download PDF

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CN116622629A
CN116622629A CN202310676923.8A CN202310676923A CN116622629A CN 116622629 A CN116622629 A CN 116622629A CN 202310676923 A CN202310676923 A CN 202310676923A CN 116622629 A CN116622629 A CN 116622629A
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mesenchymal stem
stem cell
cells
cell culture
culture solution
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李伟冰
熊伟
何驰
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Shenzhen Runke Biotechnology Co ltd
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Shenzhen Runke Biotechnology Co ltd
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Abstract

The invention discloses a mesenchymal stem cell culture solution and a use method thereof, wherein the culture solution comprises a basic culture solution and an exogenous additive, and the exogenous additive is as follows: saccharides, metformin, resveratrol, antioxidants, mTOR inhibitors, cytokines, hormones, vitamins, glutathione and nonessential amino acids. Compared with the prior art, the culture solution prepared by the invention can enhance the antioxidation capability and steady state of the mesenchymal stem cells, reduce the oxidative stress and inflammatory reaction of the mesenchymal stem cells, delay the aging and apoptosis of the mesenchymal stem cells and promote the proliferation and regeneration of the mesenchymal stem cells.

Description

Mesenchymal stem cell culture solution and application method thereof
Technical Field
The invention relates to the technical field of stem cells, in particular to a mesenchymal stem cell culture solution and a use method thereof.
Background
Mesenchymal stem cells (MesenchymalStemCells, MSCs) are a type of stem cell with self-renewal capacity and multipotent differentiation potential, originating from the mesoderm.
The mesenchymal stem cell culture fluid is a basal culture fluid comprising essential nutrients and growth factors such as insulin, transferrin, cholesterol, pyruvic acid, L-glutamic acid, vitamin C, etc. required for maintaining the growth of mesenchymal stem cells, but does not contain any derivatives or proteins. The culture solution is suitable for primary culture and amplification of MSCs.
The mesenchymal stem cell conditioned medium (MSCs-CM) is a method in which a cell component is removed from a basal medium in which mesenchymal stem cells are cultured, and the supernatant is directly used for culturing other cells or as an additive component to other cell culture media. MSCs-CM contains many cytokines secreted by mesenchymal stem cells, exosomes and other bioactive factors, and can influence the growth state and physiological functions of other cells. The types and amounts of bioactive factor components enriched in MSCs-CM derived from different stem cells vary, and thus the effects on different cells vary.
The mesenchymal stem cell culture fluid may be selected in different formulations and protocols depending on the desired application and cell type. The mesenchymal stem cell culture solution commonly used in the prior art mainly comprises DMEM/F12 culture medium, alpha-MEM culture medium and MesenCurt TM XF culture,MSC SFM culture medium and KnockOut TM DMEM/F12 medium. The DMEM/F12 medium is a basic medium which is comprehensively applicable to various cell types, and the components of the DMEM/F12 medium comprise Dulbecco Modified Eagle Medium (DMEM) and Ham F12 medium, and can be used for culturing mesenchymal stem cells. The alpha-MEM culture medium is a culture medium specially used for culturing bone marrow mesenchymal stem cells, and comprises the components of amino acid, vitamin, inorganic salt and the like, and can support proliferation and differentiation of cells. MesenCult TM The XF culture medium is a serum-free culture medium specially used for culturing mesenchymal stem cells, and the components of the serum-free culture medium comprise various growth factors and cytokines, such as FGF, TGF-beta, EGF, IGF and the like, can promote proliferation and differentiation of cells, and has higher cell survival rate and stability. />The MSC SFM culture medium is a serum-free mesenchymal stem cell culture medium, and the components of the culture medium comprise various growth factors and cytokines, such as FGF, TGF-beta, EGF, IGF and the like, can promote proliferation and differentiation of cells, and has higher cell survival rate and stability. KnockOut TM The DMEM/F12 medium is a serum-free mesenchymal stem cell medium, and its components include various growth factors and cytokines, such as FGF, TGF-beta,EGF, IGF, etc., can promote proliferation and differentiation of cells, and has high cell survival rate and stability. In addition, the culture medium can also support the culture and expansion of different types of mesenchymal stem cells by adding appropriate components.
But its composition is relatively simple, lacks specific growth factors and cytokines, and cannot meet the specific requirements of some mesenchymal stem cells. And strict aseptic conditions are required, so that the operation difficulty is high.
The patent CN107574147B discloses a mesenchymal stem cell proliferation and differentiation culture solution, which takes DMEM/F12 culture medium solution as basic culture solution, wherein the basic culture solution contains atractylenolide II with effective concentration; the mesenchymal stem cells are bone marrow mesenchymal stem cells, the differentiation refers to osteogenic differentiation, and the concentration of the atractylenolide II is 5-60 mu M. The invention discovers that the atractylenolide II can promote proliferation and osteogenic differentiation of mesenchymal stem cells, and can be used for preparing a culture solution for inducing osteogenic differentiation of the mesenchymal stem cells so as to effectively improve the osteogenic mass of the mesenchymal stem cells. However, the mesenchymal stem cell proliferation and differentiation culture solution prepared by the invention has poor proliferation effect on mesenchymal stem cells in culture and is easy to age.
Disclosure of Invention
In view of the defects of poor proliferation effect and easiness in aging of cells of the intermediate mesenchymal stem cell culture solution in the prior art, the invention aims to provide the mesenchymal stem cell culture solution with good proliferation effect and difficulty in aging of cells and a use method thereof.
In order to achieve the above object, the present invention adopts the following technical scheme:
a mesenchymal stem cell culture solution, the culture solution comprising a basal culture solution and an exogenous additive, the exogenous additive being: saccharides, metformin, resveratrol, antioxidants, mTOR inhibitors, cytokines, hormones, vitamins, glutathione and nonessential amino acids.
The basic culture solution is a DMEM/F12 culture medium solution.
The saccharide is glucan, and the concentration of the glucan in the culture solution is 5-15 g/L.
The dimethyldiguanide is 1-2 mg/L; resveratrol is 0.3-0.5 mg/L.
The antioxidant is astaxanthin 0.4-0.6 mg/L.
The mTOR inhibitor is rapamycin 0.1-1 mg/L and lomustine 0.05-0.07 mug/L.
The components of the cytokine are as follows: the basic fibroblast growth factor (bFGF-2) is 1-10 mug/L, the Epidermal Growth Factor (EGF) is 1-5 mug/L, the transforming growth factor (TGF-beta) is 5-10 mug/L, the platelet-derived growth factor (PDGF) is 1-3 mug/L, and the stem cell growth factor (SCF) is 2-5 mug/L.
The hormone is hydrocortisone 0.05-5 μg/L, insulin 5-50 μg/L, and Growth Hormone (GH) 10-50 μg/L.
The vitamin is Vitamin C (VC) 10-100 mg/L and Vitamin E (VE) 10-50 mug/L.
The glutathione is 10-50 mug/L, and the non-essential amino acid is 0.5-2 g/L.
The invention also discloses application of the mesenchymal stem cell culture solution in preparing mesenchymal stem cell conditioned medium, namely a using method of the mesenchymal stem cell culture solution.
Specifically, the preparation method of the mesenchymal stem cell conditioned medium comprises the following steps:
step 1, placing mesenchymal stem cells in a cell incubator to culture, subculturing when the cell fusion degree reaches 70-90%, performing enzymolysis digestion on primary cultured cells by using 0.2-0.3% W/V trypsin for 1-3 min, adding prepared complete culture solution after enzymolysis is completed, adding 0.4-0.6% W/V trypsin to terminate digestion, blowing the cells, and performing subculture according to the ratio of 1:2-5 until the generation of P3-P5;
step 2, washing the P3-P5 generation cells in the step 1 with PBS for 1-3 times, adding 0.2-0.3% W/V trypsin, slightly shaking the culture dish to fully react for 10-20 s, adding 0.4-0.6% W/V trypsin to stop digestion, blowing and mixing uniformly, collecting cell suspension, centrifuging at a rotating speed of 800-1Centrifuging at 200rpm for 4-6 min, pouring out supernatant, rinsing for 1-3 times by using PBS, and using frozen stock solution with the volume ratio of 8-10: 1: DMSO composition, cell resuspension, cell counting with cell counting plate, cell concentration of at least 0.5X10 6 ～2×10 6 Adding cell suspension into a freezing tube, and placing the freezing tube into a programmed cooling box at 0.8-1.2 mL/tube, placing the freezing tube into a refrigerator at-50 to-90 ℃ for overnight, and transferring the freezing tube into a liquid nitrogen tank for storage after overnight;
step 3, preheating a constant-temperature water bath to 35-38 ℃ before resuscitating the cells, taking out the cells to be resuscitated from a liquid nitrogen tank, rapidly putting the cells into the preheated constant-temperature water bath, centrifuging after melting, centrifuging at 800-1200 rpm for 4-6 min, pouring supernatant, adding complete culture solution for resuspension cell precipitation, centrifuging again, centrifuging at 800-1200 rpm for 4-6 min, repeating the steps twice, adding complete culture solution for resuspension cell precipitation, putting the cells into a cell culture dish, shaking uniformly to ensure that the cells are uniformly distributed in the culture dish, and putting the cells into the cell culture box for culture;
step 4, when the cell fusion degree cultured in the step 3 is 70-90%; the cells are cleaned by DPBS solution, residual serum is removed, the culture solution of the mesenchymal stem cells is used for culturing for 12-24 hours, the supernatant is collected to obtain the cultured cells, the supernatant is filtered by a sterile microporous filter membrane with the size of 0.2-0.3 mu m to obtain the mesenchymal stem cell conditioned medium, and the mesenchymal stem cell conditioned medium is stored at the temperature of minus 50 ℃ to minus 90 ℃ for standby.
Preferably, the inoculation concentration of the mesenchymal stem cells in the step 1 is 1.0X10 5 ～1.0×10 6 individual/mL; the culture conditions of the step 1 and the step 3 in a cell incubator are that the temperature is 35-38 ℃ and the CO content is 4-6% 2 Culturing in a cell culture box with 85-92% humidity for 15 24h, and replacing with fresh complete culture medium for continuous culture.
Preferably, the complete culture medium consists of DMEM/F12 and serum in a volume ratio of 8-10:1.
Preferably, the mesenchymal stem cells are human umbilical cord mesenchymal stem cells obtained by digestion and extraction of a neonatal umbilical cord specimen.
The invention also discloses application of the mesenchymal stem cell conditioned medium in preparing a medicine for repairing skin wounds.
Resveratrol is a polyphenol compound with various biological activities including oxidation resistance, inflammation resistance, aging resistance and the like. Resveratrol can delay the aging of mesenchymal stem cells by regulating RELA/Sirt1 pathway. RELA is a transcription factor which can regulate the expression of various genes, including inflammation-related genes, antioxidation-related genes and the like. The activity of the RELA pathway in the prior art is related to the senescence of mesenchymal stem cells. Resveratrol can reduce inflammatory reaction and oxidative stress by inhibiting RELA pathway activity, thereby delaying aging of mesenchymal stem cells. Meanwhile, resveratrol can also enhance the antioxidation capability and the steady state of cells by activating the Sirt1 channel, thereby reducing the premature aging of mesenchymal stem cells. Sirt1 is an nad+ dependent deacetylase that can regulate a variety of biological processes including cell life cycle, DNA repair, metabolic regulation, and the like. The activity of the Sirt1 pathway in the present invention is related to proliferation and senescence of mesenchymal stem cells. Resveratrol can activate Sirt1 pathway, thereby enhancing self-renewal and regeneration capacity of cells, reducing DNA damage and apoptosis, and delaying aging of mesenchymal stem cells. Therefore, resveratrol can reduce inflammatory reaction and oxidative stress and enhance self-renewal and regeneration capacity of cells by regulating RELA/Sirt1 pathway, thereby delaying aging of mesenchymal stem cells.
Metformin is an oral hypoglycemic agent commonly used for the treatment of type 2 diabetes. Metformin can maintain the normal activity of mesenchymal stem cells through various signal paths, delay replicative senescence and reduce apoptosis. Among them, AMPK signaling pathway, mTOR signaling pathway and glycometabolism pathway are the main mechanisms of action of metformin. The metformin can activate an AMPK signal pathway, reduce the activity of an mTOR signal pathway, regulate the metabolism and proliferation of cells, enhance the oxidative stress capability and the steady state of the cells, and delay the aging and the apoptosis of mesenchymal stem cells.
Astaxanthin is a natural antioxidant with strong free radical scavenging and antioxidant effects. The addition of astaxanthin in the mesenchymal stem cell culture solution can enhance the antioxidant capacity and the steady state of cells and reduce the oxidative stress and damage of cells, thereby promoting the proliferation and the regeneration of cells.
Rapamycin is a cholesterol-lowering agent that can regulate the metabolism and proliferation of cells by activating PPAR-gamma signaling pathways, thereby affecting mesenchymal stem cells. The addition of rapamycin to the mesenchymal stem cell culture fluid can promote proliferation and differentiation of cells and enhance the metabolic capacity and homeostasis of cells.
Lomustine is a statin which can affect mesenchymal stem cells by inhibiting HMG-CoA reductase, lowering cholesterol and lipid levels.
The mesenchymal stem cell culture solution can produce synergistic effect on cells by adding substances such as metformin, resveratrol, astaxanthin, rapamycin, lomustine and the like, and the metformin is used together with the substances such as resveratrol, astaxanthin, rapamycin, lomustine and the like, so that the antioxidation capability and the steady state of cells can be enhanced, the oxidative stress and the inflammatory reaction of the cells are reduced, the aging and the apoptosis of the cells are delayed, and the proliferation and the regeneration of the cells are promoted. The mesenchymal stem cell culture fluid contains a plurality of exogenous additives, and the additives have different effects and related synergistic effects, and are helpful for promoting cell proliferation, increasing self-renewal and regeneration capacity of stem cells, promoting implantation and survival of stem cells, and the like.
Saccharides (glucans) provide energy and nutrients for cells, promoting cellular metabolism and proliferation. Glutathione can protect cells from oxidative stress, promote cell proliferation and stem cell survival. Cytokines, hormones, and growth factors can promote stem cell proliferation, differentiation, and engraftment. Vitamin C and vitamin E have antioxidant and antiinflammatory effects, and can promote proliferation, self-renewal and regeneration of stem cells. The nonessential amino acids provide essential amino acids to the cells, promoting cellular metabolism and proliferation.
Compared with the prior art, the invention has the beneficial effects that:
1) According to the invention, the metformin is used together with resveratrol, astaxanthin, rapamycin, lomustine and other substances, so that the antioxidation capability and the steady state of the mesenchymal stem cells can be enhanced, the oxidative stress and the inflammatory reaction of the mesenchymal stem cells are reduced, the aging and the apoptosis of the mesenchymal stem cells are delayed, and the proliferation and the regeneration of the mesenchymal stem cells are promoted.
2) The invention can promote the proliferation, differentiation and implantation of stem cells by adopting cytokines, hormones and growth factors. Vitamin C and vitamin E have antioxidant and antiinflammatory effects, and can promote proliferation, self-renewal and regeneration of stem cells. The nonessential amino acids provide essential amino acids to the cells, promoting cellular metabolism and proliferation.
3) The mesenchymal stem cell culture solution prepared by the invention contains a plurality of exogenous additives, and the additives have different effects and related synergistic effects, and are helpful for promoting cell proliferation, increasing self-renewal and regeneration capacity of stem cells and promoting implantation and survival of stem cells.
Detailed Description
The main material sources are as follows:
resveratrol: the stock name of the company, inc.: CQBLLC-02.
Astaxanthin: shanxi Hao Biotech Co., ltd., product number: HY6035241.
Non-essential amino acids: merck company, product number: TMS-001.
Trypsin: vitality: 4178BAEE units/mg.
Example 1
A mesenchymal stem cell culture solution consists of the following raw materials: the DMEM/F12 culture medium solution, based on the final concentration, is added with 10g/L of glucan, 1.6mg/L of metformin, 0.4mg/L of resveratrol, 0.55mg/L of astaxanthin, 0.5mg/L of rapamycin, 0.06 μg/L of lomustine, 5 μg/L of basic fibroblast growth factor (bFGF-2), 3 μg/L of Epidermal Growth Factor (EGF), 8 μg/L of transforming growth factor (TGF-beta), 2 μg/L of Platelet Derived Growth Factor (PDGF), 3 μg/L of stem cell growth factor (SCF), 0.2 μg/L of hydrocortisone, 20 μg/L of insulin, 30 μg/L of Growth Hormone (GH), 30mg/L of vitamin C, 30 μg/L of vitamin E, 30 μg/L of glutathione and 1g/L of nonessential amino acid.
The steps of culturing the mesenchymal stem cells by adopting the mesenchymal stem cell culture solution are as follows:
step 1, placing mesenchymal stem cells in a cell incubator for culture by using a complete culture medium, wherein the mesenchymal stem cells are human umbilical cord mesenchymal stem cells extracted by digestion of a neonatal umbilical cord specimen; the complete culture medium consists of DMEM/F12 and serum in the volume ratio of 9 to 1, and the inoculation concentration of the mesenchymal stem cells is 1.0X10 5 individual/mL; the culture conditions were 37℃and 5% CO 2 Culturing in a cell culture box with 90% humidity, and continuously culturing after 24 hours by replacing the cell culture box with a fresh complete culture medium; when the cell fusion degree reaches 85%, carrying out subculture, carrying out enzymolysis digestion on primary cultured cells by using 0.25% W/V trypsin for 2min, adding prepared complete culture solution after enzymolysis is finished, adding 0.5% W/V trypsin to terminate digestion, blowing the cells, and carrying out subculture according to the ratio of 1:3 until the generation of P5;
step 2, washing the P5 generation cells in the step 1 with PBS for 2 times, adding 0.25% W/V trypsin, gently shaking the culture dish to fully react for 15 seconds, adding 0.5% W/V trypsin to stop digestion, blowing and mixing uniformly, collecting cell suspension, centrifuging at 1000rpm for 5 minutes, pouring out the supernatant, rinsing with PBS for 2 times, and using a frozen solution with the volume ratio of 9: 1: DMSO composition, cell resuspension, cell counting with cell counting plate, cell concentration of at least 1×10 6 Adding cell suspension into a freezing tube at a concentration of 1 mL/tube, placing the freezing tube into a programmed cooling box, placing the freezing tube in a refrigerator at a temperature of-80 ℃ overnight, and transferring the freezing tube into a liquid nitrogen tank for storage after overnight;
step 3, preheating a constant-temperature water bath to 37 ℃ before resuscitating the cells, taking out the cells needing resuscitating from a liquid nitrogen tank, rapidly putting the cells into the preheated constant-temperature water bath, centrifuging after melting, centrifuging at 1000rpm for 5min, pouring supernatant, adding complete culture solution for resuspension cell precipitation, centrifuging again, centrifuging at 1000rpm for 5min, repeating the steps for two times, adding complete culture solution for resuspension cell precipitation, putting the cells into a cell culture dish, and shaking to culture the cells uniformlyUniformly distributed in culture dish, placing at 37deg.C, 5% CO 2 Culturing in a cell culture box with 90% humidity;
step 4, when the cell fusion degree cultured in the step 3 is 80%; the cells are washed once by DPBS solution, residual serum is removed, the culture solution of the mesenchymal stem cells is replaced, the cells are cultured for 24 hours, the supernatant is collected to obtain the cultured cells, the supernatant is filtered by a sterile microporous filter membrane with the size of 0.22 mu m to obtain the mesenchymal stem cell conditioned medium, and the mesenchymal stem cell conditioned medium is stored at the temperature of minus 80 ℃ for standby.
Example 2
A mesenchymal stem cell culture solution consists of the following raw materials: the DMEM/F12 culture medium solution, based on the final concentration, is added with 10g/L of glucan, 0.4mg/L of resveratrol, 0.55mg/L of astaxanthin, 0.5mg/L of rapamycin, 0.06 μg/L of lomustine, 5 μg/L of basic fibroblast growth factor (bFGF-2), 3 μg/L of Epidermal Growth Factor (EGF), 8 μg/L of transforming growth factor (TGF-beta), 2 μg/L of platelet-derived growth factor (PDGF), 3 μg/L of stem cell growth factor (SCF), 0.2 μg/L of hydrocortisone, 20 μg/L of insulin, 30 μg/L of Growth Hormone (GH), 30mg/L of vitamin C, 30 μg/L of vitamin E, 30 μg/mL of glutathione and 1g/L of nonessential amino acid.
The procedure for culturing mesenchymal stem cells using the mesenchymal stem cell culture medium was the same as in example 1.
Example 3
A mesenchymal stem cell culture solution consists of the following raw materials: the DMEM/F12 culture medium solution, based on the final concentration, is added with 10g/L of glucan, 1.6mg/L of metformin, 0.55mg/L of astaxanthin, 0.5mg/L of rapamycin, 0.06 μg/L of lomustine, 5 μg/L of basic fibroblast growth factor (bFGF-2), 3 μg/L of Epidermal Growth Factor (EGF), 8 μg/L of transforming growth factor (TGF-beta), 2 μg/L of Platelet Derived Growth Factor (PDGF), 3 μg/L of stem cell growth factor (SCF), 0.2 μg/L of hydrocortisone, 20 μg/L of insulin, 30 μg/L of Growth Hormone (GH), 30mg/L of vitamin C, 30 μg/L of vitamin E, 30 μg/mL of glutathione and 1g/L of nonessential amino acid.
The procedure for culturing mesenchymal stem cells using the mesenchymal stem cell culture medium was the same as in example 1.
Example 4
A mesenchymal stem cell culture solution consists of the following raw materials: the DMEM/F12 culture medium solution, based on the final concentration, is added with 10g/L of glucan, 1.6mg/L of metformin, 0.4mg/L of resveratrol, 0.5mg/L of rapamycin, 0.06 g/L of lomustine, 5g/L of basic fibroblast growth factor (bFGF-2), 3 g/L of Epidermal Growth Factor (EGF), 8 g/L of transforming growth factor (TGF-beta), 2g/L of platelet-derived growth factor (PDGF), 3 g/L of stem cell growth factor (SCF), 0.2 g/L of hydrocortisone, 20 g/L of insulin, 30 g/L of Growth Hormone (GH), 30mg/L of vitamin C, 30 g/L of vitamin E, 30 g/mL of glutathione and 1g/L of nonessential amino acid.
The procedure for culturing mesenchymal stem cells using the mesenchymal stem cell culture medium was the same as in example 1.
Example 5
A mesenchymal stem cell culture solution consists of the following raw materials: the DMEM/F12 culture medium solution, based on the final concentration, is added with 10g/L of glucan, 1.6mg/L of metformin, 0.4mg/L of resveratrol, 0.55mg/L of astaxanthin, 5 mug/L of basic fibroblast growth factor (bFGF-2), 3 mug/L of Epidermal Growth Factor (EGF), 8 mug/L of transforming growth factor (TGF-beta), 2 mug/L of platelet-derived growth factor (PDGF), 3 mug/L of stem cell growth factor (SCF), 0.2 mug/L of hydrocortisone, 20 mug/L of insulin, 30 mug/L of Growth Hormone (GH), 30mg/L of vitamin C, 30 mug/L of vitamin E, 30 mug/L of glutathione and 1g/L of nonessential amino acid.
The procedure for culturing mesenchymal stem cells using the mesenchymal stem cell culture medium was the same as in example 1.
Comparative example 1
A mesenchymal stem cell culture solution consists of the following raw materials: the culture medium solution of DMEM/F12 comprises 10g/L dextran, 5 μg/L basic fibroblast growth factor (bFGF-2), 3 μg/L Epidermal Growth Factor (EGF), 8 μg/L transforming growth factor (TGF-beta), 2 μg/L Platelet Derived Growth Factor (PDGF), 3 μg/L stem cell growth factor (SCF), 0.2 μg/L hydrocortisone, 20 μg/L insulin, 30 μg/L Growth Hormone (GH), 30mg/L vitamin C, 30 μg/L vitamin E, 30 μg/mL glutathione and 1g/L nonessential amino acid added to the culture medium solution of DMEM/F12 based on final concentration.
The procedure for culturing mesenchymal stem cells using the mesenchymal stem cell culture medium was the same as in example 1.
Comparative example 2
The steps of culturing mesenchymal stem cells are as follows:
step 1, placing mesenchymal stem cells in a cell incubator for culture by using a complete culture medium, wherein the mesenchymal stem cells are human umbilical cord mesenchymal stem cells extracted by digestion of a neonatal umbilical cord specimen; the complete culture medium consists of DMEM/F12 and serum in the volume ratio of 9:1, and the inoculation concentration of the mesenchymal stem cells is 1.0X105/mL; culturing in a cell culture box with 37 ℃,5% CO2 and 90% humidity for 24 hours, and continuously culturing after replacing with a fresh complete culture medium; when the cell fusion degree reaches 85%, carrying out subculture, carrying out enzymolysis digestion on primary cultured cells by using 0.25% W/V trypsin for 2min, adding prepared complete culture solution after enzymolysis is finished, adding 0.5% W/V trypsin to terminate digestion, blowing the cells, and carrying out subculture according to the ratio of 1:3 until the generation of P5;
step 2, washing the P5 generation cells in the step 1 with PBS for 2 times, adding 0.25% W/V trypsin, gently shaking the culture dish to fully react for 15 seconds, adding 0.5% W/V trypsin to stop digestion, blowing and mixing uniformly, collecting cell suspension, centrifuging at 1000rpm for 5 minutes, pouring out the supernatant, rinsing with PBS for 2 times, and using a frozen solution with the volume ratio of 9: 1: DMSO composition, cell resuspension, cell counting with cell counting plate, cell concentration of at least 1×10 6 Adding cell suspension into a freezing tube at a concentration of 1 mL/tube, placing the freezing tube into a programmed cooling box, placing the freezing tube in a refrigerator at a temperature of-80 ℃ overnight, and transferring the freezing tube into a liquid nitrogen tank for storage after overnight;
step 3, preheating the constant-temperature water bath box to be before cell resuscitatingTaking out the cells to be revived from the liquid nitrogen tank at 37 ℃, rapidly putting the cells into a preheated constant-temperature water bath, centrifuging after melting, centrifuging at 1000rpm for 5min, pouring supernatant, adding the complete culture solution for cell precipitation, centrifuging again at 1000rpm for 5min, repeating the steps for two times, adding the complete culture solution for cell precipitation, placing the cells into a cell culture dish, shaking uniformly to ensure that the cells are uniformly distributed in the culture dish, placing the cells into 37 ℃ and 5% CO 2 Culturing in a cell culture box with 90% humidity;
step 4, when the cell fusion degree cultured in the step 3 is 80%; the cells are washed once by DPBS solution, residual serum is removed, the whole culture medium is continuously replaced, the culture is continued for 24 hours, the supernatant is collected to obtain the cultured cells, the supernatant is filtered by a sterile microporous filter membrane with the diameter of 0.22 mu m to obtain mesenchymal stem cell conditioned medium, and the mesenchymal stem cell conditioned medium is stored at the temperature of minus 80 ℃ for standby.
Test example 1
Cell proliferation potency assay
The cells cultured in step 4 of examples 1 to 5 and comparative example 1 were selected for cell proliferation capacity test, and comparative example 2 was replaced with the complete medium, and the mesenchymal stem cells were cultured for 15 hours as a control group. Washing cells with PBS in a clean bench, gently beating to wash floating cells, repeating for 2 times, adding a proper amount of digestive solution containing 0.25% pancreatin and 0.02% EDTA, putting into an incubator for digestion for 4min, after the mesenchymal stem cells are observed to fall off from the bottom of a cell culture dish under a microscope, adding 0.5% pancreatin inhibitor with the same volume to terminate digestion, beating uniformly, collecting cell suspension, centrifuging at 1000rpm for 5min, pouring out supernatant, washing with PBS for 2 times, adding DKSFM to resuspension cell pellet, counting cells, inoculating the cells into 96-well cell culture plate with cell density of 1×10 4 Holes, each group is provided with 6 compound holes, and the compound holes are placed at 37 ℃ and 5 percent CO 2 Culturing in a cell culture incubator with 90% humidity. MTT assay was performed on each group of samples taken at day 2, 5, 7 post inoculation: adding 20 mu L of 0.5% MTT solution into each hole, and placing the mixture into an incubator to keep out of the sun for culturing for 4 hours; sucking out the culture solution in the hole, adding 150uLDMSO, and placing the cell culture plate on a microplate shaker for low-speed shaking for 10min, accelerating the complete dissolution of the crystals in the wells, the above procedure was followed in the absence of light. And measuring the absorbance of each hole at 490nm by using an enzyme-labeled instrument, taking the absorbance of each group of uninoculated cell holes as a blank control for zeroing, measuring the relation between the OD value and time, taking the average value of six holes, and repeating the experiment three times. And taking an average value. The test results are shown in Table 1.
Table 1: results of cell proliferation potency test
Test example 2
Cell senescence detection
The cells cultured in step 4 of examples 1 to 5 and comparative example 1 were selected for cell senescence detection, and comparative example 2 was changed to a complete medium, and mesenchymal stem cells cultured for 15 hours were used as a control group for cell senescence detection. Washing cells with PBS in a clean bench, gently beating to wash floating cells, repeating for 2 times, adding a proper amount of digestion solution containing 0.25% pancreatin and 0.02% EDTA, putting into an incubator for digestion for 4min, taking out for observing digestion conditions, stopping digestion after hKCs are observed under a mirror to fall off from the bottom of a cell culture dish, adding 0.5% pancreatin inhibitor with the same volume, beating, mixing uniformly, collecting cell suspension, centrifuging at a speed of 1000rpm for 5min, pouring supernatant, washing with PBS for 2 times, adding DKSFM for cell precipitation, counting cells, inoculating cells into a 6-hole cell culture plate, culturing in a cell culture incubator with a cell density of 2×10 holes, each group being provided with 3 compound holes, and placing in a cell culture incubator with a humidity of 37 ℃ and 5% CO 2. After cells grew to 75% confluence, the cell culture supernatant was blotted off, washed 1 time with pre-warmed PBS, 1mL of β -galactosidase staining fixative was added to each well, fixed at room temperature for 15min, the cell fixative was discarded, and washed 3 times with PBS for 3 min/time. 1mL of staining working solution is added to each well, the wells are incubated overnight in a 37 ℃ incubator under dark conditions, 2mL of PBS is added, and the staining results are observed under a common optical microscope and counted.
Each group was tested three times and averaged. The test results are shown in Table 2.
Table 2: cell senescence test results
From the test results of test examples 1 and 2, it can be seen that the best overall performance of example 1 may be due to the fact that metformin of the present invention can activate AMPK signaling pathway, reduce activity of mTOR signaling pathway, regulate metabolism and proliferation of cells, enhance oxidative stress and homeostasis of cells, and delay aging and apoptosis of mesenchymal stem cells. The addition of astaxanthin in the mesenchymal stem cell culture solution can enhance the antioxidant capacity and the steady state of cells and reduce the oxidative stress and damage of cells, thereby promoting the proliferation and the regeneration of cells. Rapamycin can regulate cellular metabolism and proliferation by activating PPAR-gamma signaling pathways, thereby affecting mesenchymal stem cells. Lomustine has an effect on mesenchymal stem cells by inhibiting HMG-CoA reductase, reducing cholesterol and lipid levels.
In conclusion, the mesenchymal stem cell culture solution can produce synergistic effect on cells by adding substances such as metformin, resveratrol, astaxanthin, rapamycin, lomustine and the like, and the metformin is used together with the substances such as resveratrol, astaxanthin, rapamycin, lomustine and the like, so that the antioxidation capability and the steady state of the cells can be enhanced, the oxidative stress and the inflammatory reaction of the cells can be reduced, the aging and the apoptosis of the cells can be delayed, and the proliferation and the regeneration of the cells can be promoted. The mesenchymal stem cell culture fluid contains a plurality of exogenous additives, and the additives have different effects and related synergistic effects, and are helpful for promoting cell proliferation, increasing self-renewal and regeneration capacity of stem cells, promoting implantation and survival of stem cells, and the like.

Claims (10)

1. The mesenchymal stem cell culture solution is characterized by comprising a basic culture solution and an exogenous additive, wherein the exogenous additive is as follows: saccharides, metformin, resveratrol, antioxidants, mTOR inhibitors, cytokines, hormones, vitamins, glutathione, nonessential amino acids;
the basic culture solution is a DMEM/F12 culture medium solution.
2. A mesenchymal stem cell culture medium according to claim 1, wherein the saccharide is dextran and the concentration in the culture medium is 5-15 g/L.
3. A mesenchymal stem cell culture medium according to claim 1, wherein the metformin is present in an amount of 1-2 mg/L; resveratrol is 0.3-0.5 mg/L.
4. A mesenchymal stem cell culture fluid according to claim 1, wherein the antioxidant is astaxanthin at 0.4-0.6 mg/L.
5. A mesenchymal stem cell culture medium according to claim 1, wherein the mTOR inhibitor is rapamycin 0.1-1 mg/L and lomustine 0.05-0.07 μg/L.
6. The mesenchymal stem cell culture medium of claim 1, wherein the cytokine-added components are: the basic fibroblast growth factor is 1-10 mug/L, the epidermal growth factor is 1-5 mug/L, the transforming growth factor is 5-10 mug/L, the platelet-derived growth factor is 1-3 mug/L, and the stem cell growth factor is 2-5 mug/L.
7. A mesenchymal stem cell culture medium according to claim 1, wherein the hormone is hydrocortisone in an amount of 0.05 to 5 μg/L, insulin in an amount of 5 to 50 μg/L and growth hormone in an amount of 10 to 50 μg/L.
8. The mesenchymal stem cell culture fluid of claim 1, wherein the vitamins are 10-100 mg/L vitamin C and 10-50 μg/L vitamin E.
9. The mesenchymal stem cell culture medium of claim 1, wherein the glutathione is 10-50 μg/L and the nonessential amino acid is 0.5-2 g/L.
10. Use of the mesenchymal stem cell culture solution according to any one of claims 1 to 9 for preparing a mesenchymal stem cell conditioned culture solution.
CN202310676923.8A 2023-06-08 2023-06-08 Mesenchymal stem cell culture solution and application method thereof Pending CN116622629A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114591902A (en) * 2022-03-30 2022-06-07 深圳市茵冠生物科技有限公司 Culture method and application of mesenchymal stem cells
CN117138028A (en) * 2023-10-30 2023-12-01 北京岷德生物科技有限公司 Composition containing epidermal stem cell factor and application thereof in diabetic foot repair
CN117946529A (en) * 2024-01-26 2024-04-30 广东壹加再生医学研究院有限公司 Hydrogel and application thereof in amplification culture of mesenchymal stem cells

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114591902A (en) * 2022-03-30 2022-06-07 深圳市茵冠生物科技有限公司 Culture method and application of mesenchymal stem cells
CN114591902B (en) * 2022-03-30 2024-03-22 深圳市茵冠生物科技有限公司 Culture method and application of mesenchymal stem cells
CN117138028A (en) * 2023-10-30 2023-12-01 北京岷德生物科技有限公司 Composition containing epidermal stem cell factor and application thereof in diabetic foot repair
CN117138028B (en) * 2023-10-30 2024-02-13 北京岷德生物科技有限公司 Composition containing epidermal stem cell factor and application thereof in diabetic foot repair
CN117946529A (en) * 2024-01-26 2024-04-30 广东壹加再生医学研究院有限公司 Hydrogel and application thereof in amplification culture of mesenchymal stem cells

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