CN112522191B - Culture method of mesenchymal stem cells - Google Patents
Culture method of mesenchymal stem cells Download PDFInfo
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Abstract
The invention relates to the technical field of cell culture, in particular to a method for culturing mesenchymal stem cells. In this culture method, mesenchymal stem cells are cultured using the serum-free medium optimized according to the present invention. In addition, the invention also provides a better culture method for 3D culture. The cells obtained by the culture method have strong proliferation capability, complete morphology and strong secretion capability, and meet the international quality control standard of mesenchymal stem cells. Meanwhile, the culture method of the invention can obtain a large number of mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) through the amplification of few mesenchymal stem cells under the conditions of occupying less space, consuming little culture medium, being simpler and more convenient to operate and greatly reducing the workload, and provides a solid technical scheme and theoretical basis for obtaining a large number of high-quality mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) and secretion thereof in the clinical field.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for culturing mesenchymal stem cells.
Background
Mesenchymal stem cells (mesenchymal stem cells, MSC) are multifunctional stem cells existing in various tissues of a human body, are immature cells, have self-replication and multi-directional differentiation capabilities, and can be differentiated into various functional cells under certain conditions. The mesenchymal stem cells have the capability of directional induction differentiation and repair in an in-vivo environment, namely the possibility of realizing on-demand repair and regeneration of damaged tissues.
After entering the organism, the mesenchymal stem cells return to the heart through the blood circulation system, then reach all organs and tissues of the whole body along with blood through the heart, or reach the injury part under the influence of recruitment factors, the microenvironment at the specific part of the organism is as follows: under the induction of transcription factors, growth factors, mechanical pressure and other conditions, the gene expression of stem cells is changed in series, and finally the stem cells are differentiated into terminal cells with specific functions which are the same as the environmental cells, and then the terminal cells are proliferated by utilizing nutrient substances brought by the blood circulation of an organism, so that the pathological tissues are repaired and regenerated.
In view of various biological characteristics and clinical significance of mesenchymal stem cells, cytokines and exosomes, a large number of successful application cases of mesenchymal stem cells, cytokines and exosomes exist at present, however, in order to clinically apply umbilical cord mesenchymal stem cells and secretion products thereof, a large number of high-quality umbilical cord mesenchymal stem cells are required, and the conventional serum and serum-free culture schemes all face the problems that cells are easy to age, biological characteristics cannot be maintained for a long time and the like, so that obtaining a large number of high-quality human umbilical cord mesenchymal stem cells is difficult. Therefore, a high-quality umbilical cord mesenchymal stem cell serum-free culture system is also required to meet the characteristic of large-scale amplification.
At present, the main mode of large-scale expansion of mesenchymal stem cells is to perform large-scale two-dimensional cell bottle culture of umbilical cord mesenchymal stem cells, for example, a method for large-scale two-dimensional expansion of mesenchymal stem cells by using a cell factory culture bottle is mentioned in CN 109468274A, however, the traditional culture mode has a plurality of defects, the use of a large number of cell culture bottles increases the cost and is easy to pollute, and the traditional two-dimensional culture mode has strict cell density control, quick aging of cells under high density and serious limitation of cell yield.
In order to cope with the large-scale culture of adherent cells, a microcarrier culture method has been developed in recent years, which uses the traditional suspension culture, and adds granular, sheet-like or hollow fiber carriers made of biological materials required by cell adherence to attach cells to the carriers, and then places the carriers in a bioreactor for culture. The microcarrier is used for culturing the mesenchymal stem cells, so that the microcarrier has the advantages of large surface area/volume, higher cell yield can be obtained per unit volume of culture solution, the utilization rate of a culture medium is high, the amplification is easy, the large-scale amplification of cells can be realized from lower density, the labor density is low, the occupied area of a culture system is small, the culture system is not easy to pollute, and more cells can be obtained per unit time. Therefore, microcarrier culture of mesenchymal stem cells is considered as an optimal solution for obtaining a large amount of mesenchymal stem cells of good quality in a short time to satisfy clinical application. For example, CN 111424011A provides a three-dimensional culture method capable of maintaining the morphology of umbilical cord mesenchymal stem cells, however, the serum-free culture medium used contains serum replacement additives provided by a third party, the specific components of the serum-free culture medium are unknown, and the purchased serum-free additives often contain animal-derived proteins, so that the risk of heterologous viruses cannot be completely excluded. In addition, cells cultured in such conventional three-dimensional bioreactors are vulnerable to mechanical damage and the cells cannot sustain long-term growth thereon.
Disclosure of Invention
The invention firstly provides a method for culturing mesenchymal stem cells, which uses a serum-free culture medium to culture the mesenchymal stem cells;
the serum-free culture medium comprises a basal culture medium and additive components, wherein the basal culture medium is an IMDM culture medium;
the additive comprises TGF-beta 3 and ascorbic acid, wherein the concentration of the TGF-beta 3 is 1-5 mug/L, and the dosage ratio of the ascorbic acid to the TGF-beta 3 is 25-200mg:1-5 mug.
According to the invention, in the case of taking an IMDM culture medium as a basic culture medium, compared with other human transforming growth factors (such as TGF-beta 1), after TGF-beta 3 is used and compounded with ascorbic acid in the manner, the method can promote cell synthesis and secrete extracellular matrixes such as fibronectin, collagen and the like, promote cell migration, ensure that mesenchymal stem cells have good adhesion effect and better improvement effect on proliferation. Compared with other basic culture mediums (such as DMEM high sugar, low sugar, a-MEM, DMEM/F12, RPMI1640 and the like), the IMDM culture medium has better effect on both the cell growth speed and the cell homogeneity when the IMDM culture medium is applied.
The person skilled in the art can arrange other components in the medium according to common general knowledge, which can all achieve effects comparable to those described above in the present invention.
Preferably, the culture method of the present invention is particularly effective when applied to mesenchymal stem cells of the present invention, preferably human umbilical cord mesenchymal stem cells.
Preferably, the additive component comprises 0.1-1v/v% chemical lipid.
It was found that although the conventional guiding amount of the chemical lipid is 0.1v/v% at most, the higher the content of the chemical lipid, the better the cell growth state in the range of 0.1 to 1v/v% (preferably 0.3 to 1 v/v%).
Chemical lipids in the present invention refer to a mixture of lipids comprising the following components:
arachidonic acid 1.8-2.2mg/L, cholesterol 200-240mg/L, vitamin E (tocopherol) 60-80mg/L, linoleic acid 8-12mg/L, linolenic acid 8-12mg/L, myristic acid 8-12mg/L, oleic acid 8-12mg/L, palmitic acid 8-12mg/L, palmitoleic acid 8-12mg/L, pluronic F-68 80000-100000mg/L, stearic acid 8-12mg/L.
More preferably, it consists of the following components:
arachidonic acid, cholesterol, vitamin E (tocopherol), linoleic acid, linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid, stearic acid and stearic acid in amounts of 2mg/L, 220mg/L, 70mg/L, 10mg/L, 90000mg/L, 10mg/L, and 10mg/L respectively.
In practice, chemical lipids meeting the above requirements are available in commercial ways, such as Gibco,11905 or Sigma, L0288.
More preferably, the additive component further comprises 20-35mg/L of compound ester substance, wherein the weight ratio of the compound ester substance is 1-5:5-30:2-20 of ethanolamine, cholesterol, soybean lecithin. When used together with the chemical lipid, the lipid can well provide lipids required by cell growth and metabolism and is beneficial to promoting the synthesis of cell membranes.
The present invention further found that lowering the concentration of hydrocortisone is advantageous in slowing down the aging rate of cells, and preferably the additional ingredient further comprises hydrocortisone in an amount of 0.1-2mg/L, more preferably in an amount of 0.1-1mg/L.
More preferably, the additive component further comprises insulin, progesterone and dexamethasone, wherein the dosage ratio of the insulin, the progesterone, the dexamethasone and the hydrocortisone is 8-25mg:1-10 μg:4-20 μg:0.1-2mg. When the hormone is added in the above-mentioned complex formulation, the cell can be promoted to perform substance synthesis and energy metabolism by using saccharides, amino acids, etc. in the medium.
Preferably, the additional component further comprises 1-5g/L albumin, which is recombinant human albumin obtained from a plant, preferably rice, as an expression host. In practice, recombinant human albumin meeting the above requirements can be obtained by commercial means, such as the Gramineae organism, HYC002M01.
The present invention has surprisingly found that by using the above albumin in the formulation of the present invention, the cell attachment properties and proliferation effects can be further improved, while the cell growth morphology is intact and has good uniformity, compared to recombinant human albumin obtained from other expression hosts (e.g., microorganisms, animals).
Preferably, the additive component further comprises transferrin, the dosage ratio of transferrin to recombinant human albumin is 10-30mg:1-5g, vitamins, lipids, amino acids, inorganic salt ions and the like can be combined and transported into cells, so as to provide necessary components for cell metabolism and division proliferation.
Preferably, the additive component further comprises 40-60 mug/L composite growth factors, wherein the weight ratio of the growth factors is 5-40:5-50:2-10, EGF (human epidermal growth factor), and HGF (human hepatocyte growth factor), can promote mesenchymal stem cell adherent proliferation and maintain stem cell multipotency without differentiation during proliferation.
More preferably, the additive component further includes: 2-20. Mu.g/L PDGF-BB (human platelet derived growth factor BB).
More preferably, the additive component further includes: VEGF (human vascular endothelial growth factor) 5-20 μg/L.
Preferably, the additive component further comprises IGF-1 (human insulin growth factor), and the dosage ratio of IGF-1 to insulin is 10-40 mug: 8-25mg, can better assist insulin to exert its function.
Preferably, the additive component further comprises: 1-5 mug/L CTGF (human connective tissue growth factor) can promote the accumulation of extracellular matrix and provide better environment for cell growth.
Preferably, the additive component further comprises: 1-5mg/L of reduced glutathione, 0.2-2mM of N-Acetyl-L-Cysteine and 50-100 mu M of beta-mercaptoethanol, and can be synergistic with ascorbic acid to remove active oxygen in cells well, thereby playing roles in resisting aging and protecting.
The above preferred embodiments may be combined and other components in the medium may be set according to common general knowledge by a person skilled in the art, resulting in a preferred embodiment of the present invention.
Preferably, each 1L of medium comprises the following components:
IMDM 17.662g, L-propionyl-glutamine 2mM, chemical lipid 0.1-1v/v%, cholesterol 5-30mg, insulin 8-25mg, transferrin 10-30mg, recombinant human albumin 1-5g, hydrocortisone 0.1-2mg, dexamethasone 4-20 μg, progesterone 1-10 μg, putrescine 5-15mg, ascorbic acid 25-200mg, beta-mercaptoethanol 50-100 μg, soybean lecithin 2-20mg, zinc sulfate heptahydrate 1.25-2.5mg, lipoic acid 0.1-0.5mg, N-Acetyl-L-Cysteine 0.2-2mM, reduced glutathione 1-5mg, taurine 2-10mg, Y-27632 2-10 μg, ethanolamine 1-5mg, b-FGF 5-40 μg, EGF 5-50 μg, IGF-BB 2-20 μg, IGF-110-40 μg, TGF-35 μg, IGF-5 μg, VEGF-5 μg and VEGF-5 μg.
Most preferably, each 1L of medium comprises the following components:
IMDM 17.662g, L-alanyl-glutamine 2mM, chemical lipid 0.6v/v%, cholesterol 15mg, insulin 12.5mg, transferrin 25mg, recombinant human albumin 2g, hydrocortisone 500 μg, dexamethasone 4 μg, progesterone 5.66 μg, putrescine 9mg, ascorbic acid 100mg, beta-mercaptoethanol 75 μg, soybean lecithin 10mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.2mg, N-Acetyl-L-Cysteine 1mM, reduced glutathione 2mg, taurine 5mg, Y-27632 5 μg, ethanolamine 2mg, b-FGF 20 μg, EGF 20 μg, PDGF-BB 10 μg, IGF-1 μg, TGF-beta 32 μg, HGF 10 μg, CTGF 2 μg, and VEGF 15 μg.
Preferably, the proteins used in the culture medium of the invention are recombinant proteins.
Preferably, the growth factors used in the medium of the invention are all recombinant growth factors.
The culture medium of the present invention can be obtained by conventional configuration means.
When the serum-free culture medium is prepared, IMDM powder is firstly dissolved in cell culture water, stirred uniformly, filtered by a 0.22um filter membrane, then insulin dissolved in 0.01M diluted hydrochloric acid is firstly added, and after insulin and a basic culture medium are uniformly mixed, other components are sequentially added, and the components are properly stirred in the adding process to promote uniform distribution.
Preferably, the concentration of the insulin in the 0.01M dilute hydrochloric acid is not higher than 13mg/ml, so that the dissolution effect is better, and the situation that the insulin is precipitated after other components are added can be avoided.
In some preferred embodiments, some of the agents such as cholesterol, soy lecithin, hydrocortisone, progesterone, dexamethasone are dissolved in DMSO or absolute ethanol, and care is taken to adjust the addition mode, and the addition is carried out with stirring, so as to avoid local crystallization precipitation.
The mesenchymal stem cell serum-free culture medium provided by the invention is required to be stored in a sealed state at 4 ℃ in a dark place after the preparation is finished, and is used as soon as possible within one month.
In the present invention, the cell culture may be 2D culture or 3D culture, and the above-mentioned effects can be obtained by culturing the cells using the above-mentioned medium. Furthermore, the present invention has found and provided the following preferable embodiments for 3D culture.
Preferably, the culture is 3D culture, and when the coverage rate of the mesenchymal stem cells on the microcarrier reaches 75-85%, the mesenchymal stem cells are cultured by a low-nutrition culture medium;
the low-nutrition culture medium is prepared from the serum-free culture medium and an IMDM culture medium according to a volume ratio of 1:2 to 4, more preferably 1:2.
the present inventors have unexpectedly found that the culture time of mesenchymal stem cells in a three-dimensional bioreactor can be remarkably prolonged while improving the growth state of the cells after the culture medium is changed in the above manner.
The coverage rate of mesenchymal stem cells on microcarriers can be confirmed by sampling observation or other detection means by the person skilled in the art, so that the time for changing the culture medium can be confirmed.
Preferably, the microcarriers are solid microcarriers (e.g. conventional chitosan or other types) and/or porous microcarriers.
More preferably, the microcarrier is a porous microcarrier made based on extracellular matrix, such as gelatin, collagen, etc.
Further preferred, the microcarrier is a porous microcarrier made of gelatin, such as Sigma
When the microcarrier is used, the microcarrier can be well degraded under the culture system of the invention.
When the serum-free medium of the present invention is used for culturing mesenchymal stem cells, 75-85% coverage can be achieved on porous microcarriers made based on extracellular matrix, generally on days 8-12. Therefore, preferably, the mesenchymal stem cells are cultured with a low-nutrient medium after the mesenchymal stem cells are cultured until day 8-12.
The person skilled in the art can set other steps and parameters in the cultivation method according to common general knowledge, which can obtain effects equivalent to those described above in the present invention.
Preferably, the mesenchymal stem cells are cultured by a suspension cell culture flask, which is set to stir for 15-25 minutes every 4 hours, and the rotation speed is set to 40-60rpm/min.
To obtain a large number of human umbilical cord-derived mesenchymal stem cells, it is preferable that 1g of Sigma per 1D of the cells are cultured in 3D
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The cell seeding amount of the microcarrier is 1800-2200 ten thousand, more preferably 2000 ten thousand.
Furthermore, the above-described preferred embodiments may be combined by those skilled in the art to yield preferred embodiments of the present invention.
Furthermore, the invention also provides a method for obtaining the secretion of the mesenchymal stem cells, which comprises the steps of culturing the mesenchymal stem cells by using the culture method and collecting the secretion of the mesenchymal stem cells.
Preferably, the secretion comprises one or more of cytokines, exosomes and secreted proteins.
The secretion can be concentrated and purified for scientific research, tissue injury repair, cosmetic raw material additive, etc.
Based on the technical scheme, the invention has the following beneficial effects:
(1) The cells obtained by the culture method have strong proliferation capability, complete morphology and strong secretion capability, and meet the international quality control standard of mesenchymal stem cells. And the cells cultured under the culture system are safe and ideal, and have good clinical and commercial application prospects.
(2) The culture method of the invention can obtain a large amount of mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) through few mesenchymal stem cell expansion under the conditions of occupying less space, consuming little culture medium, being simpler and more convenient to operate and greatly reducing the workload.
(3) The culture method is based on a self-grinding serum-free and animal-derived protein-free mesenchymal stem cell complete culture medium, so that virus risks brought by serum and heterologous proteins are eliminated under the culture condition, the safety is higher, and the culture method is more suitable for clinical disease treatment.
(4) By the culture method, the mesenchymal stem cells can survive for a long time, have stable cell characteristics, have no mechanical damage to cells, have larger tissue structure formed by cell growth, can truly simulate the growth environment of cells in vivo under three-dimensional conditions, can be cultured for a long time to 30-50 days, can reduce operation damage and pollution during the period, construct better cell growth microenvironment, promote cell adhesion and structure formation by self-secreted mass extracellular matrix proteins, have better secretion capacity, can better maintain cell biological characteristics, and can generate a large amount of active substances such as cytokines, exosomes and the like.
(4) As the low-nutrition culture medium is used in the later stage, the cost is greatly reduced, and a solid technical scheme and theoretical basis are provided for obtaining a large number of high-quality mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) and secretion thereof in the clinical field.
In addition, the method of the present invention has the advantages shown in Table 1 compared with the conventional two-dimensional culture method, as summarized by a plurality of experiments.
TABLE 1
Drawings
FIG. 1 shows various generations of human umbilical cord mesenchymal stem cells cultured in serum-free medium according to example 1 of the present invention.
FIG. 2 shows cells obtained by culturing using the culture medium of example 3 and comparative example 1.
FIG. 3 is a schematic technical flow chart of the cultivation method in example 4.
The human umbilical cord-derived mesenchymal stem cells of example 4 were inoculated with microcarriers and stained for Calcein AM.PI dead cells after days 2, 5, 7, 25, 30, 35 from left to right and from top to bottom in FIG. 4;
FIG. 5 shows the results of digestion of human umbilical cord mesenchymal stem cells cultured with the three-dimensional microcarrier in example 4.
FIG. 6 is a graph showing the secretion capacity of human umbilical cord mesenchymal stem cells compared with that of 2D culture by the culture method of example 4.
FIG. 7 shows the proliferation of human fibroblasts after concentration of secreted proteins from human umbilical cord-derived mesenchymal stem cells in three-dimensional culture in example 4 (albumin was added to the blank and low protein groups to a final protein concentration of 0.025 mg/ml).
FIG. 8 is a graph showing the effect of different concentrations of secreted proteins and albumin of human umbilical cord-derived mesenchymal stem cells on cell survival after 20 minutes of UV irradiation in three dimensions; in the figure, A. No protein; 3mg/ml albumin; c.6mg/ml albumin; 3mg/ml secreted protein; d.6mg/ml secreted protein.
FIG. 9 shows the culture results of the three-dimensional culture method in example 5.
FIG. 10 shows the culture results of the three-dimensional culture method in comparative example 2.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention.
In order to facilitate the comparison effect, the proteins used in the following examples are recombinant proteins, and the growth factors used are recombinant growth factors, wherein the recombinant human albumin is obtained by taking rice as an expression host, and the specific product is a cereal organism, HYC002M01; the chemical lipids were all Gibco,11905.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or equipment used were conventional products available for purchase by regular vendors without the manufacturer's attention.
Example 1
The present example provides a 2D culture method of mesenchymal stem cells, and the formula of the serum-free medium used is as follows:
each 1L of medium consists of the following components:
17.662g of IMDM, 2mM of L-alanyl-glutamine, 0.6v/v of chemical lipid, 15mg of cholesterol, 12.5mg of insulin, 25mg of transferrin, 2g of recombinant human albumin, 500 μg of hydrocortisone, 4 μg of dexamethasone, 5.66 μg of progesterone, 9mg of putrescine, 100mg of ascorbic acid, 75 μg of beta-mercaptoethanol, 10mg of soybean lecithin, 2.5mg of zinc sulfate heptahydrate, 0.2mg of lipoic acid, 1mM of N-Acetyl-L-Cysteine, 2mg of reduced glutathione, 5mg of taurine, Y-27632 5 μg, 2mg of ethanolamine, 20 μg of b-FGF, 20 μg of EGF, 10 μg of PDGF-BB, 1 μg of IGF-15 μg, 10 μg of TGF-beta 32 μg of HGF, 2 μg of CTGF and 15 μg of VEGF; the balance being water for cell culture.
The preparation method of the serum-free culture medium comprises the following steps:
dissolving IMDM powder in water for cell culture, stirring uniformly, filtering with 0.22um filter membrane, adding recombinant human insulin dissolved in 0.01M diluted hydrochloric acid, mixing insulin with basic culture medium uniformly, sequentially adding other components, and stirring properly to promote uniform distribution during the addition process of each component. Among other components, cholesterol, soybean lecithin, hydrocortisone, progesterone, and dexamethasone were dissolved in DMSO and added dropwise with stirring.
The specific process is as follows:
1. primary isolated culture of umbilical cord-derived mesenchymal stem cells
Umbilical cord tissue was soaked with sterile PBS containing 2.2% cyan/streptomycin for 2min, residual blood stain was washed off, residual blood was removed as much as possible, the tissue was soaked with 75% medical alcohol for 1min, the tissue was immediately cut off at both ends with high temperature sterilized and cooled ophthalmic scissors, and washed twice with sterile PBS. Cutting longer umbilical cord tissue into small pieces of about 2cm using an ophthalmic scissors, carefully removing two arteries and a vein, and then cutting the target tissue to 1mm 3 The tissue blocks with the size are transferred into 10cm culture dishes, 0.5-1 g of tissue is added into each dish, 12ml of fresh serum-free culture medium is added into each dish, the culture dish is gently shaken to uniformly distribute cells and the tissue blocks, and the culture is carried out in a 5% CO2 incubator at 37 ℃ and is marked as P0.
After 72 hours, each dish is supplemented with 3ml of fresh serum-free culture medium, then the old culture medium is removed every 72 hours, the tissue blocks are reserved, 10ml of fresh culture medium is added, the tissue blocks are uniformly distributed in the dish by shaking, the culture medium and the tissue blocks are shaking once every day, the crawled cells are uniformly distributed in the dish, the culture medium is replaced every 3-4 days, about 12-14 days, the old culture medium and the tissue blocks are discarded, 10ml of fresh culture medium is added in the old dish for continuous culture, and then most of the areas in the dish are full of cells, and the continuous culture can be carried out by passage or liquid change as appropriate.
2. Subculture of umbilical cord-derived mesenchymal stem cells
The human umbilical cord-derived mesenchymal stem cells in log phase with good growth state are removed from the old culture medium. Adaptations for useThe amount (10 cm dish 4 ml) of PBS at room temperature was used to wash off the residual medium in the dish, to facilitate cell digestion, and the PBS was discarded. An appropriate amount (about 2ml in a 10cm dish) of 1:2 diluted TrypLE was added TM Express Enzyme digestive Enzyme, gently shaking the culture dish to enable the digestive Enzyme to uniformly cover the bottom of the dish, digesting for about 1min in the culture box, tapping the culture dish to enable cells to fall off after cell contact feet shrink and round, transferring the culture dish into an operation table, stopping digestion by using a culture medium, gently blowing the cells to completely fall off by using a pipette, blowing away cell clusters, transferring the suspension into a centrifuge tube, centrifuging at room temperature after balancing, 1200rpm/min, about 300g centrifuging for 5min, discarding the supernatant after centrifugation, dispersing the cell clusters at the bottom of the flick, adding a proper amount of culture medium to resuspend cells, uniformly mixing with a 5/10ml pipette, performing cell counting, inoculating according to the cell counting, and recommending the cell inoculation density to be 4000-12000 cells/cm 2 Wherein the recommended inoculation density of P0-P4 is 4000-8000cells/cm 2 The recommended inoculation density of P5-P10 is 10000cells/cm 2 . The cell type, algebra and passage date are marked, the culture dish is gently slid in the cross direction, and the process is repeated for 4-6 times to ensure that the cells are uniformly distributed, the culture dish is carefully transferred into a culture dish with 37 ℃ and 5% CO2 for culture, and the cell proliferation is about 3.5-9 times every 48 hours according to the passage times of the cells.
The cell states of different generations are recorded by photographing, and the figure shows that the culture medium can obtain primary human umbilical cord mesenchymal stem cells by a tissue mass suspension culture method, and can maintain the uniformity and proliferation capacity of cells for more than ten generations of long-term passage.
Example 2
The present example provides a 2D culture method of mesenchymal stem cells, which is different from example 1 in that the serum-free medium is used in the following formulation:
each 1L of medium consists of the following components:
17.662g of IMDM, 2mM of L-propionyl-glutamine, 0.1v/v of chemical lipid, 5mg of cholesterol, 8mg of insulin, 10mg of transferrin, 1g of recombinant human albumin, 0.1mg of hydrocortisone, 4 μg of dexamethasone, 1 μg of progesterone, 5mg of putrescine, 25mg of ascorbic acid, 50 μM of beta-mercaptoethanol, 2mg of soybean lecithin, 1.25mg of zinc sulfate heptahydrate, 0.1mg of lipoic acid, 0.2mM of N-Acetyl-L-Cysteine, 1mg of reduced glutathione, 2mg of taurine, Y-27632 2 μM, 1mg of ethanolamine, 5 μg of b-FGF, 5 μg of EGF, 2 μg of PDGF-BB, 1 μg of IGF-10 μg, 2 μg of TGF-beta 31 μg, 1 μg of HGF, and 5 μg of VEGF; the balance being water for cell culture.
The configuration method is the same as that of example 1.
Example 3
The present example provides a 2D culture method of mesenchymal stem cells, which is different from example 1 in that the serum-free medium is used in the following formulation:
IMDM 17.662g, L-propionyl-glutamine 2mM, chemical lipid 1v/v%, cholesterol 30mg, insulin 25mg, transferrin 30mg, recombinant human albumin 5g, hydrocortisone 2mg, dexamethasone 20 μg, progesterone 10 μg, putrescine 15mg, ascorbic acid 200mg, beta-mercaptoethanol 100 μM, soybean lecithin 20mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.5mg, N-Acetyl-L-Cysteine 2mM, reduced glutathione 5mg, taurine 10mg, Y-27632 10 μM, ethanolamine 5mg, b-FGF 40 μg, EGF 50 μg, PDGF-BB 20 μg, IGF-1 40 μg, TGF-beta 35 μg, HGF 10 μg, GF5 μg and VEGF 20 μg; the balance being water for cell culture.
The configuration method is the same as that of example 1.
The same P1-generation human umbilical cord mesenchymal stem cells were taken and tested, 3 groups of repeated experiments were performed in each of examples 1-3, and the inoculation density was 10000cells/cm 2 Cells were harvested after 48 hours and counted and fold expansion was recorded. Then inoculating again with the same density until the CELLs are harvested at the time of P5, calculating the total amount of the CELLs harvested under the culture conditions of different embodiments according to the expansion times of the CELLs of each generation, simultaneously detecting the diameter distribution of the CELLs of the P5 generation by using a Beckmann Coulter Vi-CELL XR counter, obtaining proliferation and diameter data of the mesenchymal stem CELLs of the human umbilical cord in different embodiments according to the statistical results, wherein the statistical results are shown in Table 2, and the results show that the proliferation speed of the mesenchymal stem CELLs of the human umbilical cord under the culture conditions of the embodiment 1 is faster and the uniformity of the CELL diameters is better, while the proliferation speed of the mesenchymal stem CELLs cultured in the embodiments 2 and 3 is slightly slowerIn particular, the large number of cells with larger diameters were likely to appear during the culture, which suggests that the mesenchymal stem cells cultured in example 2 and example 3 were less uniform, and that aging and differentiation were more likely to occur during the long-term culture.
TABLE 2
Comparative example 1
This comparative example provides a 2D culture method of mesenchymal stem cells, which is different from example 3 in that TGF- β3 of serum-free medium used therein is replaced with TGF- β1 in equal amount.
The configuration method is the same as that of example 1.
The results of cells cultured using the culture medium of example 3 and comparative example 1 and a control group without human transforming growth factor are shown in FIG. 2, and from the results, it is known that TGF-. Beta.1, in combination with ascorbic acid, can promote the secretion of extracellular matrix by mesenchymal stem cells, and many documents have reported that this combination can stimulate the production of collagen and fibronectin by mesenchymal stem cells, and it was also seen in this experiment that this combination can indeed promote the adherence of mesenchymal stem cells under serum-free conditions. The combined action of TGF-beta 3 and ascorbic acid can promote the mesenchymal stem cells to secrete extracellular matrix, reduce the degradation of the extracellular matrix, form extracellular matrix accumulation, and is more beneficial to the adherence and proliferation of cells. Furthermore, long-term passaging with TGF- β1 is far less effective than TGF- β3.
Example 4
The embodiment provides a three-dimensional culture method of mesenchymal stem cells derived from human umbilical cord (technical flow diagram is shown in fig. 3).
Wherein the serum-free medium used was the same as that in example 1.
The technical scheme comprises the following specific steps:
1. three-dimensional culture inoculation of human umbilical cord-derived mesenchymal stem cells:
(1) Purchased Sigma
Sterile water bottles (also other brands or types of cell culture microcarriers) are autoclaved;
(2) Taking 3-7 generation human umbilical cord source mesenchymal stem cells in logarithmic growth phase, wherein the density is at most not more than 95%, and digesting the cells;
(3) Incubation in a water bath at 37 ℃ for about 3min with a 1:3 diluted tryple digestive enzyme in an incubator at 37 ℃;
(4) Stopping digestion, centrifuging at 1200rpm/min for 5min at about 300g, and re-suspending cells in a completely serum-free medium for cell counting;
(5) Taking 1g of microcarrier, adding deionized water, autoclaving, transferring into a 50ml centrifuge tube, centrifuging 300g for 5min, and removing water;
(6) Diluting 2000 Wankel of serum-free culture medium to 12ml, adding into a centrifuge tube containing 1g of microcarrier, mixing, inoculating into 250 rotary bottle (3-5 dishes with low adsorption of 10cm and 15ml of culture medium per dish), supplementing serum-free culture medium to 200ml, stirring for 20 min every 4 hr at 50rpm/min, and 5% CO at 7deg.C 2 Placing and culturing the incubator.
1.1 procedure for obtaining a large number of human umbilical cord-derived mesenchymal stem cells:
(1) The corresponding inoculated cell quantity of each 1g of microcarrier in the inoculated three-dimensional culture system is 2000 ten thousand;
(2) After 72 hours the cells had attached to the microcarriers and proliferated rapidly, at which time if it was necessary to increase the cell harvest, carefully transferring the microcarriers and culture medium that had been aggregated into small clumps into a 50ml centrifuge tube, centrifuging at 100g for 3min to remove the supernatant;
(3) Adding 200mg or a proper amount of new microcarrier and 20ml of fresh serum-free culture medium, gently mixing the new microcarrier, the old microcarrier and the culture medium by a pipette, transferring into a 250ml rotary bottle, supplementing the culture medium to 200ml, continuing to culture, and observing the growth state and the density of cells under a microscope after the cells are stained by a Calcein AM-PI dead living cell staining kit after sampling;
(4) When harvesting, taking out the microcarrier containing cells, washing the cell mass for 2 times by using calcium-magnesium-free PBS, and removing the PBS;
(5) 1mg/ml collagenase IV dissolved in a proper amount of basal medium was used to digest the tissue pieces in an incubator at 37℃for 25min, with light shaking every 5min to make contact more complete;
(6) After 25min, 1/2 volume of TrypLE was added TM Express Enzyme digestive Enzyme (Gibco, 12605010), mixing, digesting at 37deg.C for 2min, gently beating with pipette for several times to obtain single cell suspension, centrifuging at 300g for 5min, and removing supernatant to obtain cells.
1.2 is the operation of harvesting human umbilical cord-derived mesenchymal stem cell secreted proteins and exosomes:
(1) Three-dimensional culture of human umbilical cord-derived mesenchymal stem cells can be replaced with fresh culture medium every 3-5 days, and after 10 days of inoculation, old culture medium is discarded, and microcarrier containing cells is reserved;
(2) 250ml of newly prepared 3 times IMDM basal medium and a complete serum-free medium are added to mix and dilute the obtained low-nutrition medium (namely 2 parts of IMDM basal medium and one part of serum-free medium), 37 ℃ and 5 percent CO 2 Placing and culturing the incubator.
(3) After 72 hours, the medium turns yellow, the old medium is sucked out by a pipette for preservation, 250ml of low-nutrition medium is added for continuous culture, and the secreted protein in the collected old medium can be used for other experiments after concentration and purification.
2. Staining and observation of three-dimensional cultured human umbilical cord-derived mesenchymal stem cells:
(1) The mesenchymal stem cells growing in the microcarrier cannot be directly observed under a microscope, and cell staining is required;
(2) Taking out a small amount of microcarriers needing to be dyed, placing the microcarriers in a micro-pore plate, sucking the microcarriers twice by PBS, and removing the PBS;
(3) Using a Calcein AM-PI dead living cell staining kit, and staining the dead living cells according to a reagent protocol;
(4) After the staining solution acts for about 15-30 minutes, observing the cell state under a fluorescence microscope;
(5) After staining, living cells are green under a fluorescence microscope, dead cells are red, and whether the cell secretion protein can be continued is determined according to the growth condition of the cells.
After about 30-45 days, partial cells start to have the phenomena of contact foot fracture and enlarged cytoplasms, and dead cells dyed red by PI appear, and the conditions of contact foot fracture of the cells and the proportion of the dead cells dyed red gradually rise along with the continuous extension of the culture time, so that the collection of secreted proteins or exosomes can be stopped. The staining conditions of cells on days 2, 5, 7, 25, 30 and 35 of three-dimensional culture are shown in the figure 4 from left to right and from top to bottom, wherein the proportion of dead cells on and before day 25 is extremely low and does not exceed 2%, and the cell contact foot breakage starts to occur after 35 days, so that the thunderbolt of dead cells stained red gradually rises to between 5 and 10%. It can be seen that after the proliferation in the microcarrier is completed early, the cells start to proliferate on the surface of the microcarrier, and gather around the microcarrier to form larger spheres by the adhesion property of extracellular matrix, then the cells continue to proliferate on the surface of the larger spheres to form larger spheres, at this time, the cell viability is very high, after more than 35 days, the cells start to have contact breakage, and a small proportion of dead cells stained red is about 5% -10%, and if the culture is continued, the contact breakage of the cells and the proportion of dead cells stained red gradually rise.
4. Harvesting of three-dimensionally cultured human umbilical cord mesenchymal stem cells
Whether static or dynamic (roller bottle, reactor) culture, if the purpose of the culture is to harvest a large amount of mesenchymal stem cells, the culture time is preferably not longer than 12 days (depending on the cell seeding density and growth rate in particular). Mesenchymal stem cells cultured in three dimensions for a long period of time may produce fibrous materials that cannot be degraded by collagenase or tryple enzyme digestion, resulting in reduced quality and efficiency of cell harvest. Thus, the culture was stopped as much as possible before microcarriers had aggregated into larger spheres.
The cells harvested in this example are shown in FIG. 5, and the digested cells can still be used for two-dimensional culture or three-dimensional culture.
5. Human umbilical cord mesenchymal stem cell secreted protein detection cultured in two-dimensional and three-dimensional states
Collecting P3 generation human umbilical cord mesenchymal stem cells, collecting cell culture supernatant, filtering with a 0.45um filter membrane to remove impurities on 15 days of three-dimensional culture, filtering with a 30Kda ultrafiltration tube to obtain filtrate, removing albumin, filtering the filtrate with a 3Kda ultrafiltration tube again to obtain protein concentrate between 3 and 30Kda mainly containing protein of secreted protein, collecting the secreted protein by the same method as collecting the culture supernatant of P3 generation of the two-dimensional culture mesenchymal stem cells, and performing protein mass spectrometry detection on the obtained secreted protein, wherein compared with the two-dimensional culture mesenchymal stem cells, collagen, insulin-like growth factor, high migration histone, calcium-dependent transporter and other various protein secretion are obviously improved, apoptosis-related protein apoptosis induction factors, autophagy regulatory proteins, annexin and the like are obviously reduced, and the activity of the mesenchymal stem cells under the three-dimensional culture condition is excellent.
6. Three-dimensional culture of mesenchymal stem cell secretory protein for promoting proliferation of human skin fibroblast
In order to verify the function of mesenchymal stem cells secreting protein in a three-dimensional culture state, the invention provides an experiment for promoting proliferation of the protein on fibroblasts, the fibroblast proliferation experiment needs to be inoculated with low-density fibroblasts, so P3 human skin fibroblasts in a logarithmic phase are taken, cell counting is carried out after digestion, the cells are inoculated into 6 pore plates at the density of 2000cells per square centimeter, each group is repeated three times, the addition concentration of each group of secreted proteins is respectively 0, 0.0156, 0.03125, 0.0625, 0.0125 and 0.025mg/ml, and a low-concentration protein group is supplemented to 0.025mg/ml by albumin. The growth of cells was observed under a microscope every 24 hours after inoculation and recorded by photographing, and after 72 hours, each group of cells was harvested and the amount of harvested cells was counted. As can be seen from fig. 7, the mesenchymal stem cell secretin under three-dimensional culture conditions has a good effect on proliferation of fibroblasts, and this effect is enhanced with increasing concentration of addition.
7. Human skin fibroblast repair with three-dimensional culture of mesenchymal stem cell secreted protein for promoting ultraviolet loss
Taking P3 human skin fibroblast in logarithmic phase, digesting, performing cell count, and re-suspending cells according to cell count result with 10% FBS-containing high sugar DMEM medium to obtain 3×10 4 cell/ml cell suspension cells were seeded into six well plates, each well of about 6 ten thousand cells, 2ml medium. After the cells grew to 60-70%, each group was replaced with 10% fbs-containing high-sugar DMEM medium containing 0g/L HSA (recombinant human serum albumin), 3g/L HSA, 6g/L HSA, 3g/L secreted protein, 6g/L secreted protein, respectively, after mixing well, added to the corresponding well plate, 2ml per well, 3 controls were established per group, when the cells grew to 70-80%, the medium was removed, replaced with 37 ℃ incubated PBS medium containing 2% fbs, about 700ul per well, with the bottom of the plate just covered, the plate cover was opened, the dish was placed in a biosafety cabinet, uv radiation was opened for 20 minutes (pre-experiments had been done), PBS was removed, replaced with the corresponding fresh medium per group, after which cell death, morphology differences and cell recovery and proliferation conditions were observed, the corresponding fresh medium was replaced as appropriate, and the total number of cells was counted after 96 hours after photographing and recording. In fig. 8, a to E correspond to the results of the following test groups, respectively: A. no protein; 3mg/ml albumin; c.6mg/ml albumin; 3mg/ml secreted protein; d.6mg/ml secreted protein. As can be seen from fig. 8, although albumin with a high concentration has a certain repairing effect on human skin fibroblasts damaged by ultraviolet, the three-dimensional culture of mesenchymal stem cell secretes protein has a stronger repairing effect, and the repairing effect is more obvious at a high concentration.
Example 5
The present example provides a three-dimensional culture method of human umbilical cord-derived mesenchymal stem cells, which is different from example 4 in that the human umbilical cord-derived mesenchymal stem cells are cultured using a serum-free medium with normal nutrition in the whole course without using a low-nutrition medium.
The culture results are shown in FIG. 9, and when human umbilical cord mesenchymal stem cells in a three-dimensional state are continuously cultured using a medium of normal nutrition, the mesenchymal stem cells are expanded inside and on the surface of the microcarrier in the early period, and the contact feet are stretched. As the cell density increases, the growth area decreases, the cells start to undergo competitive inhibition, the proportion of dead cells gradually increases, the free dead cells gradually increase in the culture solution while proliferating and dying, the proportion of cells dyed with PI to red gradually increases (green and red cannot be distinguished under black and white, so that part of red cells are indicated by white arrows), and the proportion of red cells is about 2% and 10% respectively in the culture results on days 12 and 18. The proportion of dead cells was significantly increased compared to example 4, which replaced the low nutrient medium on the same day and on day 12.
Comparative example 2
This comparative example provides a three-dimensional culture method of human umbilical cord-derived mesenchymal stem cells, which is different from example 4 in that the medium used is IMDM medium supplemented with 10% FBS.
As a result, as shown in FIG. 10, when three-dimensional microcarrier culture of mesenchymal stem cells was performed using a medium containing FBS, comparative example 4, the culture method containing FBS had different degrees of dead cells (green and red cannot be distinguished in black and white, and thus some red cells were indicated by white arrows) present in the culture results at days 2 and 8, and the proportion of red cells was about 3% and 15%, respectively, throughout the culture. In the case of the FBS-containing examples, when there is a large growth space for the microcarriers, a large number of dead cells are present, and the content of dead cells increases with the increase of the culture time, and even if the fresh medium is replaced, the situation is not improved. Thus, the serum-free medium mentioned in example 4 is clearly more suitable for porous microcarrier culture of human umbilical cord mesenchymal stem cells than FBS-containing medium, and example 4 can reduce the generation of dead cells during three-dimensional culture while harvesting more human umbilical cord mesenchymal stem cells.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (8)
1. A method for culturing mesenchymal stem cells, characterized in that the mesenchymal stem cells are cultured by using a serum-free medium;
each 1L of medium contained the following components:
IMDM 17.662g, L-propionyl-glutamine 2mM, chemical lipid 0.1-1v/v%, cholesterol 5-30mg, insulin 8-25mg, transferrin 10-30mg, recombinant human albumin 1-5g, hydrocortisone 0.1-2mg, dexamethasone 4-20 μg, progesterone 1-10 μg, putrescine 5-15mg, ascorbic acid 25-200mg, beta-mercaptoethanol 50-100 μg, soybean lecithin 2-20mg, zinc sulfate heptahydrate 1.25-2.5mg, lipoic acid 0.1-0.5mg, N-Acetyl-L-Cysteine 0.2-2mM, reduced glutathione 1-5mg, taurine 2-10mg, Y-27632 2-10 μg, ethanolamine 1-5mg, b-FGF 5-40 μg, EGF 5-50 μg, IGF-BB 2-20 μg, IGF-110-40 μg, TGF-35 μg, IGF-5 μg, VEGF-5 μg and VEGF-5 μg;
the chemical lipid comprises the following components:
arachidonic acid 1.8-2.2mg/L, cholesterol 200-240mg/L, vitamin E (tocopherol) 60-80mg/L, linoleic acid 8-12mg/L, linolenic acid 8-12mg/L, myristic acid 8-12mg/L, oleic acid 8-12mg/L, palmitic acid 8-12mg/L, palmitoleic acid 8-12mg/L, pluronic F-68 80000-100000mg/L, stearic acid 8-12mg/L;
the albumin is recombinant human albumin obtained by taking plants as expression hosts;
the culture mode is 3D culture, and when the coverage rate of the mesenchymal stem cells on the microcarrier reaches 75-85%, the mesenchymal stem cells are cultured by using a low-nutrition culture medium;
the low-nutrition culture medium is prepared from the serum-free culture medium and an IMDM culture medium according to a volume ratio of 1: and 2-4 dilution.
2. The method of claim 1, wherein the plant is rice.
3. The culture method according to claim 1, wherein each 1L of the medium comprises the following components:
IMDM 17.662g, L-alanyl-glutamine 2mM, chemical lipid 0.6v/v%, cholesterol 15mg, insulin 12.5mg, transferrin 25mg, recombinant human albumin 2g, hydrocortisone 500 μg, dexamethasone 4 μg, progesterone 5.66 μg, putrescine 9mg, ascorbic acid 100mg, beta-mercaptoethanol 75 μg, soybean lecithin 10mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.2mg, N-Acetyl-L-Cysteine 1mM, reduced glutathione 2mg, taurine 5mg, Y-27632 5 μg, ethanolamine 2mg, b-FGF 20 μg, EGF 20 μg, PDGF-BB 10 μg, IGF-1 μg, TGF-beta 32 μg, HGF 10 μg, CTGF 2 μg, and VEGF 15 μg.
4. A culture method according to any one of claims 1 to 3, wherein the microcarriers are solid microcarriers and/or porous microcarriers.
5. The method according to claim 4, wherein the microcarrier is a porous microcarrier made based on an extracellular matrix.
6. The culture method according to claim 5, wherein when the microcarrier is a porous microcarrier made based on an extracellular matrix, the mesenchymal stem cells are cultured with a low-nutrient medium after the mesenchymal stem cells are cultured until day 8-12.
7. A method for obtaining a secretion of mesenchymal stem cells, comprising culturing mesenchymal stem cells by the culture method according to any one of claims 1 to 6, and collecting the secretion of mesenchymal stem cells.
8. The method for obtaining a secretion of mesenchymal stem cells according to claim 7, wherein the secretion comprises one or more of cytokines, exosomes and secreted proteins.
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| CN113502261B (en) * | 2021-07-16 | 2023-08-29 | 上海东方星际干细胞科技有限公司 | Large-scale 3D low-oxygen mesenchymal stem cell culture system capable of efficiently differentiating into lipid |
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| CN114591901B (en) * | 2022-03-24 | 2023-11-24 | 和携科技有限公司 | Extraction method of animal umbilical cord mesenchymal stem cell exosome |
| CN115029306A (en) * | 2022-06-21 | 2022-09-09 | 长春科技学院 | Method for efficiently preparing antler stem cell exosomes by applying three-dimensional culture system |
| CN115232787B (en) * | 2022-08-10 | 2024-04-02 | 北京积水潭医院 | Preparation method of extracellular body based on acellular matrix three-dimensional scaffold culture |
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