CN112522191A

CN112522191A - Culture method of mesenchymal stem cells

Info

Publication number: CN112522191A
Application number: CN202011507641.8A
Authority: CN
Inventors: 李天晴; 冯春
Original assignee: Yunnan Key Lab Of Primate Biomedicine Research
Current assignee: Yunnan Key Lab Of Primate Biomedicine Research
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-03-19
Anticipated expiration: 2040-12-18
Also published as: CN112522191B

Abstract

The invention relates to the technical field of cell culture, in particular to a method for culturing mesenchymal stem cells. In the culture method, the serum-free culture medium optimized by the invention is used for culturing the mesenchymal stem cells. In addition, the invention also provides a better culture method for 3D culture. The cells obtained by the culture method have strong proliferation capacity, complete form and strong secretion capacity, and meet the international quality control standard of the mesenchymal stem cells. Meanwhile, the culture method can obtain a large amount of mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) by amplifying few mesenchymal stem cells under the conditions of occupying small space, consuming few culture media, being simpler and more convenient to operate and greatly reducing workload, and provides a solid technical scheme and theoretical basis for obtaining a large amount of high-quality mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) and secretions thereof in the clinical field.

Description

Culture method of mesenchymal stem cells

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 (MSCs) are pluripotent stem cells existing in various tissues of the human body, are immature cells, have self-replication and multidirectional differentiation capabilities, and can be differentiated into stem cells of various functional cells under certain conditions. The mesenchymal stem cells have the capacity of directionally inducing differentiation and repair under the in vivo environment, namely the possibility of realizing the repair and regeneration of damaged tissues as required.

After entering the body, the mesenchymal stem cells return to the heart through a blood circulation system, and then reach all organs and tissues of the whole body along with blood through the heart or reach the injured part under the influence of recruited factors, and the microenvironment of a specific part of the body is as follows: under the induction of conditions such as transcription factors, growth factors, mechanical pressure and the like, the gene expression of the stem cells is changed in a series, the stem cells are finally differentiated into terminal cells with specific functions, which are the same as the cells in the environment, and then, nutrient substances brought by the blood circulation of an organism are utilized to proliferate, so that the effects of repairing and regenerating pathological tissues are achieved.

In view of various biological characteristics and clinical significance of mesenchymal stem cells, cytokines and exosomes, a great number of successful application cases of the mesenchymal stem cells, the cytokines and exosomes exist at present, however, clinical application of umbilical cord mesenchymal stem cells and secretion products thereof needs a great number of high-quality umbilical cord mesenchymal stem cells, and the conventional serum and serum-free culture schemes all face the problems that the cells are easy to age, the biological characteristics cannot be maintained for a long time and the like, so that the obtaining of a great number of high-quality human umbilical cord mesenchymal stem cells is difficult. Therefore, a high-quality serum-free culture system of the umbilical cord mesenchymal stem cells also needs to meet the characteristic of scale amplification.

At present, the main mode for performing the large-scale expansion of the mesenchymal stem cells is to perform the large-scale two-dimensional cell bottle culture of the umbilical cord mesenchymal stem cells, for example, CN 109468274 a proposes a method for performing the large-scale two-dimensional expansion of the mesenchymal stem cells by using a cell factory culture bottle, however, the conventional culture mode has many defects, the use of a large number of cell culture bottles increases the cost and is easy to pollute, and the conventional two-dimensional culture mode requires strict cell density control on cells, the cells rapidly senesce at high density, and the low density severely limits the cell yield.

In order to deal with the large-scale culture of adherent cells, a microcarrier culture method is developed in recent years, wherein the culture method refers to the traditional suspension culture, and adds granular, flaky 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 method has the advantages of large surface area/volume, higher cell yield per unit volume of culture solution, high utilization rate of culture medium, easy amplification, realization of large-scale cell amplification from low density, low labor density, small occupied area of a culture system, difficult pollution and capability of obtaining more cells in unit time. Therefore, microcarrier culture of mesenchymal stem cells is considered to be the best solution for obtaining a large amount of high-quality mesenchymal stem cells in a short time to meet clinical application. For example, CN 111424011 a provides a three-dimensional culture method capable of maintaining the morphology of umbilical cord mesenchymal stem cells, however, the serum-free culture medium used in the method contains a serum substitute additive provided by a third party, and the specific components of the serum-free culture medium are unknown, and the serum-free additive purchased often contains animal-derived proteins, so that the risk of heterologous viruses cannot be completely eliminated. In addition, cells cultured in such conventional three-dimensional bioreactors are susceptible to mechanical damage and 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 basic culture medium and an additive component, wherein the basic culture medium is an IMDM culture medium;

the addition components comprise TGF-beta 3 and ascorbic acid, the concentration of the TGF-beta 3 is 1-5 mu g/L, and the dosage ratio of the ascorbic acid to the TGF-beta 3 is 25-200 mg: 1-5 mug.

The invention unexpectedly discovers that under the condition 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 is compounded with ascorbic acid according to the method, the invention can promote cells to synthesize and secrete extracellular matrixes such as fibronectin, collagen and the like, promote cell migration, enable mesenchymal stem cells to have good adhesion and adherence effects, and also have better improvement effect on proliferation. Compared with other basic culture media (such as DMEM high-sugar, low-sugar, a-MEM, DMEM/F12, RPMI1640 and the like), the IMDM culture medium has better effects on both the growth speed and the homogeneity of cells when applied in the invention.

Other components in the medium may be set by those skilled in the art according to the common general knowledge, which can all obtain the effects equivalent to those described above in the present invention.

Preferably, the culture method of the present invention is particularly effective when the mesenchymal stem cells of the present invention are preferably human umbilical cord mesenchymal stem cells.

Preferably, the additional ingredient comprises 0.1-1 v/v% chemical lipid.

The present inventors have found that, although the conventionally directed amount of the chemical lipid is 0.1 v/v% at most, the higher the content of the chemical lipid is, the better the growth state of the cells is, in the range of 0.1 to 1 v/v% (preferably 0.3 to 1 v/v%).

The chemical lipid in the invention refers to a mixture of various lipids, comprising the following components:

arachidonic acid with the content of 1.8-2.2mg/L, cholesterol with the content of 200-240mg/L, vitamin E (tocopherol) with the content of 60-80mg/L, linoleic acid with the content of 8-12mg/L, linolenic acid with the content of 8-12mg/L, myristic acid with the content of 8-12mg/L, oleic acid with the content of 8-12mg/L, palmitic acid with the content of 8-12mg/L, palmitoleic acid with the content of 8-12mg/L, Pluronic F-68 with the content of 80000-100000mg/L and stearic acid with the content of 8-12 mg/L.

More preferably, it consists of:

arachidonic acid with a content of 2mg/L, cholesterol with a content of 220mg/L, vitamin E (tocopherol) with a content of 70mg/L, linoleic acid with a content of 10mg/L, linolenic acid with a content of 10mg/L, myristic acid with a content of 10mg/L, oleic acid with a content of 10mg/L, palmitic acid with a content of 10mg/L, palmitoleic acid with a content of 10mg/L, Pluronic F-68 with a content of 90000mg/L, stearic acid with a content of 10 mg/L.

For practical use, chemical lipids meeting the above requirements are commercially available, such as Gibco, 11905 or Sigma, L0288.

More preferably, the additive component further comprises 20-35mg/L of composite ester substances, and the weight ratio of the composite ester substances is 1-5: 5-30: 2-20 of ethanolamine, cholesterol, and soybean lecithin. When the lipid is used together with the chemical lipid, the lipid required by cell growth and metabolism can be well provided, and the synthesis of cell membranes can be promoted.

The invention further discovers that the reduction of the concentration of hydrocortisone is beneficial to slowing down the aging speed of cells, and preferably, the additive component also comprises 0.1-2mg/L of hydrocortisone, and more preferably, the content of the hydrocortisone is 0.1-1 mg/L.

More preferably, the additive component further comprises insulin, progesterone and dexamethasone, and the dosage ratio of the insulin, the progesterone, the dexamethasone and the hydrocortisone is 8-25 mg: 1-10. mu.g: 4-20 μ g: 0.1-2 mg. When the hormone is added in the above-mentioned combination mode, it can promote cell to utilize saccharide and amino acid in culture medium to make substance synthesis and energy metabolism.

Preferably, the additive component further comprises 1-5g/L albumin, wherein the albumin is recombinant human albumin obtained by using a plant as an expression host, and the plant is preferably rice. In practical applications, recombinant human albumin meeting the above requirements, such as proc 002M01, is commercially available.

The invention discovers that compared with recombinant human albumin obtained by other expression hosts (such as microorganisms and animals), the albumin is used in the formula of the invention, so that the adherence performance and the proliferation effect of cells can be further improved, and the growth form of the cells is complete and has good uniformity.

Preferably, the additive component further comprises transferrin, and the dosage ratio of the transferrin to the recombinant human albumin is 10-30 mg: 1-5g, it can combine and transport vitamins, lipids, amino acids and inorganic salt ions into cells, and provide essential components for cell metabolism and division and proliferation.

Preferably, the additive component further comprises 40-60 mug/L of composite growth factor, and the weight ratio of the growth factor is 5-40: 5-50: 2-10 of b-FGF (human basic fibroblast growth factor), EGF (human epidermal growth factor) and HGF (human hepatocyte growth factor) can promote adherent proliferation of mesenchymal stem cells and keep the pluripotency of the stem cells without differentiation in the proliferation process.

More preferably, the additive components further comprise: 2-20. mu.g/L of PDGF-BB (human platelet-derived growth factor BB).

More preferably, the additive components further comprise: 5-20. mu.g/L VEGF (human vascular endothelial growth factor).

Preferably, the additive component also comprises IGF-1 (human insulin growth factor), and the dosage ratio of the IGF-1 to the insulin is 10-40 μ g: when the dosage is 8-25mg, the insulin can be better assisted to play the function.

Preferably, the additive components further comprise: CTGF (human connective tissue growth factor) at 1-5 mug/L can promote the accumulation of extracellular matrix and provide a better environment for cell growth.

Preferably, the additive components further comprise: 1-5mg/L reduced glutathione, 0.2-2mM N-Acetyl-L-Cysteine and 50-100 mu M beta-mercaptoethanol, and has synergistic effect with ascorbic acid, can well scavenge active oxygen in cells, and has antiaging and protecting effects.

The skilled person can combine the above preferred embodiments and set the other components in the medium according to common general knowledge to obtain preferred embodiments of the invention.

Preferably, each 1L of the culture medium comprises the following components:

IMDM 17.662g, L-propylamine-glutamine 2mM, chemical lipid 0.1-1 v/v%, cholesterol 5-30mg, insulin 8-25mg, transferrin 10-30mg, recombinant human albumin 1-5g, hydrocortisone 0.1-2mg, dexamethasone 4-20 μ g, progesterone 1-10 μ g, putrescine 5-15mg, ascorbic acid 25-200mg, beta-mercaptoethanol 50-100 μ M, 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-276322-10 μ M, ethanolamine 1-5mg, FGF b-FGF 5-40 μ g, and, EGF 5-50 μ g, PDGF-BB 2-20 μ g, IGF-110-40 μ g, TGF-beta 31-5 μ g, HGF 2-10 μ g, CTGF 1-5 μ g and VEGF 5-20 μ g.

Most preferably, each 1L of medium comprises the following components:

IMDM 17.662g, L-propylamino-glutamine 2mM, chemical lipid 0.6 v/v%, cholesterol 15mg, insulin 12.5mg, transferrin 25mg, recombinant human albumin 2g, hydrocortisone 500 μ g, dexamethasone 4 μ g, progesterone 5.66 μ g, putrescine 9mg, ascorbic acid 100mg, beta-mercaptoethanol 75 μ M, soybean lecithin 10mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.2mg, N-Acetyl-L-Cysteine 1mM, reduced glutathione 2mg, taurine 5mg, Y-276325 μ M, ethanolamine 2mg, b-FGF 20 μ g, EGF 20 μ g, PDGF-BB 10 μ g, IGF-115 μ g, TGF- β 32 μ g, HGF 10 μ g, CTGF 2 μ g and VEGF 15 μ g.

Preferably, the proteins used in the medium of the invention are all recombinant proteins.

Preferably, the growth factors used in the medium of the invention are all recombinant growth factors.

The medium of the present invention can be obtained by a conventional preparation means.

Preferably, when the serum-free medium is prepared, the IMDM powder is dissolved in water for cell culture, the mixture is stirred uniformly, after the mixture is filtered by using a 0.22-micron filter membrane, insulin dissolved in 0.01M dilute hydrochloric acid is added, other components are added in sequence after the insulin and the basal medium are mixed uniformly, and proper stirring is needed to promote uniform distribution in the adding process of each component.

Preferably, when the concentration of insulin in 0.01M dilute hydrochloric acid is not higher than 13mg/ml, the dissolution effect is better, and precipitation after other components are added can be avoided.

In some preferred embodiments, some reagents such as cholesterol, soy lecithin, hydrocortisone, progesterone, and dexamethasone are dissolved in DMSO or absolute ethanol, and care is taken to adjust the addition mode so as to avoid local crystallization and precipitation while stirring.

The mesenchymal stem cell serum-free culture medium disclosed by the invention needs to be stored in a sealed and light-proof manner at 4 ℃ after the preparation is finished, and can be used as soon as possible within one month.

In the present invention, the culture method of the cells may be 2D culture or 3D culture, and the above-mentioned effects can be obtained by using the above-mentioned medium. In addition, the present invention also finds and provides the following preferable embodiments for 3D culture.

Preferably, the culture is 3D culture, and after the coverage rate of the mesenchymal stem cells on the microcarriers reaches 75-85%, the mesenchymal stem cells are cultured by using a low-nutrient medium;

the low-nutrient medium is prepared by mixing the serum-free medium and the IMDM medium according to the volume ratio of 1: 2-4, more preferably 1: 2.

the invention unexpectedly discovers that the culture time of the mesenchymal stem cells in the three-dimensional bioreactor can be remarkably prolonged and the growth state of the cells can be improved after the culture medium is replaced according to the mode.

The coverage rate of the mesenchymal stem cells on the microcarrier and the time for changing the culture medium can be confirmed by sampling observation or other detection means.

Preferably, the microcarrier is a solid microcarrier (e.g.conventional chitosan or other type) and/or a porous microcarrier.

More preferably, the microcarrier is a porous microcarrier made of an extracellular matrix such as gelatin and collagen.

Further preferably, the microcarrier is a porous microcarrier made of gelatin, such as Sigma

When the microcarrier is used, the microcarrier can be degraded well under the culture system of the invention.

When mesenchymal stem cells are cultured using the serum-free medium of the present invention, a coverage of 75-85% on porous microcarriers made based on extracellular matrix is typically achieved on days 8-12. Therefore, preferably, the mesenchymal stem cells are cultured with a low nutrient medium after the mesenchymal stem cells are cultured to 8 to 12 days.

Other steps and parameters in the cultivation process can be set by those skilled in the art according to the common general knowledge, which can all obtain the effects equivalent to those described above in the present invention.

Preferably, the mesenchymal stem cells are cultured by suspending the cells in a spinner flask set to stir for 15 to 25 minutes every 4 hours at a rotation speed of 40 to 60 rpm/min.

To obtain a large amount of human umbilical cord-derived mesenchymal stem cells, as a preferable embodiment, Sigma is added every 1g in the 3D culture

Or

The cell inoculation amount of the microcarrier is 1800-2200 ten thousand, and more preferably 2000 ten thousand.

Furthermore, one skilled in the art can combine the above-described preferred embodiments to create 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 includes one or more of cytokine, exosome and secretory protein.

The secretion can be used for scientific research, tissue injury repair, cosmetic raw material additives and the like after being concentrated and purified.

Based on the technical scheme, the invention has the following beneficial effects:

(1) the cells obtained by the culture method have strong proliferation capacity, complete form and strong secretion capacity, and meet the international quality control standard of the 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 can obtain a large amount of mesenchymal stem cells (especially human umbilical cord mesenchymal stem cells) by amplifying 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.

(3) Because the culture method is based on the self-researched serum-free animal-derived protein-free mesenchymal stem cell complete culture medium, the virus risks caused by serum and heterologous protein are eliminated under the culture condition, the safety is higher, and the method is more suitable for clinical disease treatment.

(4) By the culture method, the mesenchymal stem cells can survive for a long time, the cell characteristics are stable, mechanical damage of the cells does not exist, the tissue structure formed by cell growth is larger, the growth environment of the cells under in-vivo three-dimensional conditions can be simulated really, the mesenchymal stem cells can be cultured for a long time to 30-50 days, operation damage and pollution can be reduced in the period, a better cell growth microenvironment is constructed, a large amount of autocrine extracellular matrix proteins promote cell adhesion and structure formation, the cells have better secretion capacity, and the cell biological characteristics can be better maintained, so that a large amount of active substances such as cell factors, exosomes and the like can be generated.

(4) Because the low-nutrition culture medium is used in the later period, the cost is greatly reduced, and a solid technical scheme and a theoretical basis are provided for obtaining a large amount of high-quality mesenchymal stem cells (particularly human umbilical cord mesenchymal stem cells) and secretions thereof in the clinical field.

In addition, the method of the present invention has advantages shown in table 1 compared to the conventional two-dimensional culture method, as summarized by a plurality of experiments.

TABLE 1

Drawings

FIG. 1 shows different 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 cultured in the medium of example 3 and comparative example 1.

FIG. 3 is a schematic view of the technical flow of the cultivation method in example 4.

In FIG. 4, staining of Calcein AM & PI dead and alive cells after 2, 5, 7, 25, 30 and 35 days of the inoculation of the microcarrier with the human umbilical cord-derived mesenchymal stem cells in example 4 is performed sequentially from left to right and from top to bottom;

FIG. 5 shows the digestion results of human umbilical cord mesenchymal stem cells cultured in three-dimensional microcarriers in example 4.

FIG. 6 is the comparison of the secretion capacity of human umbilical cord mesenchymal stem cells in the culture method of example 4 with that in 2D culture.

FIG. 7 shows the effect of the human umbilical cord-derived mesenchymal stem cell-secreted protein after concentration on the human fibroblast proliferation (blank and low protein group with albumin added to a final added protein concentration of 0.025mg/ml) in the three-dimensional culture of example 4.

FIG. 8 shows the effect of different concentrations of secreted proteins and albumin of human umbilical cord-derived mesenchymal stem cells cultured in three dimensions on cell survival after UV irradiation for 20 minutes; in the figure, a. no protein; b.3mg/ml albumin; c.6mg/ml albumin; c.3mg/ml secreted protein; d.6mg/ml secreted protein.

FIG. 9 shows the results of the three-dimensional cultivation in example 5.

FIG. 10 shows the results of the three-dimensional culturing method in comparative example 2.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

In order to facilitate comparison effects, all proteins used in the following examples are recombinant proteins, and all 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 gramineous organism, HYC002M 01; the chemical lipids are all Gibco, 11905.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1

The present embodiment 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 consisted of the following components:

IMDM 17.662g, L-propylamino-glutamine 2mM, chemical lipid 0.6 v/v%, cholesterol 15mg, insulin 12.5mg, transferrin 25mg, recombinant human albumin 2g, hydrocortisone 500 μ g, dexamethasone 4 μ g, progesterone 5.66 μ g, putrescine 9mg, ascorbic acid 100mg, beta-mercaptoethanol 75 μ M, soybean lecithin 10mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.2mg, N-Acetyl-L-Cysteine 1mM, reduced glutathione 2mg, taurine 5mg, Y-276325 μ M, ethanolamine 2mg, b-FGF 20 μ g, EGF 20 μ g, PDGF-BB 10 μ g, IGF-115 μ g, TGF- β 32 μ g, HGF 10 μ g, CTGF 2 μ g and VEGF 15 μ g; the balance being water for cell culture.

The preparation method of the serum-free culture medium comprises the following steps:

firstly, IMDM powder is dissolved in water for cell culture, the mixture is stirred uniformly, after the filtration by using a 0.22um filter membrane, recombinant human insulin dissolved in 0.01M dilute hydrochloric acid is added, when the insulin is mixed uniformly with a basic culture medium, other components are added in sequence, and in the adding process of each component, the mixture is stirred properly to promote the uniform distribution. Among the other components, cholesterol, soybean lecithin, hydrocortisone, progesterone, dexamethasone were dissolved in DMSO and added while stirring while dropping.

The specific process is as follows:

primary isolated culture of umbilical cord-derived mesenchymal stem cells

Soaking umbilical cord tissue in sterile PBS containing 2.2% penicillin/streptomycin for 2min, washing off residual blood stain, removing residual blood as much as possible, soaking tissue in 75% medical alcohol for 1min, immediately cutting off two ends of tissue with ophthalmic scissors sterilized at high temperature and cooled, and washing twice with sterile PBS. Using an ophthalmic scissors, long umbilical cord tissue was cut into small pieces of about 2cm in length, two arteries and one vein were carefully removed, and then the target tissue was cut into pieces of 1mm³Tissue pieces of size, the tissue pieces were transferred to 10cm petri dishes, 0.5 per dish1g of tissue, 12ml of fresh serum-free medium was added to each dish, the dishes were gently shaken to distribute the cells and tissue mass evenly, and cultured in a 5% CO2 incubator at 37 ℃ and designated as P0.

After 72 hours, 3ml of fresh serum-free culture medium is supplemented to each dish, then, the culture medium is changed every 72 hours, on day 6, the old culture medium is removed, the tissue block is reserved, 10ml of fresh culture medium is added, the tissue block is shaken to be uniformly distributed in the dish, then, the culture medium and the tissue block are shaken every day to uniformly distribute the climbed cells in the dish, the culture medium is changed every 3-4 days, about day 12-14, the old culture medium and the tissue block are discarded, 10ml of fresh culture medium is added to the old dish to continue the culture, and then, most of the area in the dish is fully grown with the cells after about 3-4 days, and the continuous culture can be carried out by passage or liquid change as required.

Subculturing umbilical cord-derived mesenchymal stem cells

Removing old culture medium from the human umbilical cord-derived mesenchymal stem cells in the logarithmic phase in a good growth state. Residual medium in the dish was washed away with an appropriate amount (10cm dish 4ml) of room temperature PBS to facilitate cell digestion, and the PBS was discarded. Adding proper amount (about 2ml in a 10cm dish) of 1:2 diluted TrypLE^TMExpress Enzyme digestive Enzyme, slightly shaking the culture dish to enable the digestive Enzyme to uniformly cover the bottom of the dish, digesting in an incubator for about 1min, slightly beating the culture dish to enable cells to fall off after cell pins shrink and become round, transferring the culture dish into an operation table, stopping digestion by using a culture medium, slightly blowing and beating the cells by using a pipette to enable the cells to completely fall off, blowing cell aggregates to disperse, transferring the suspension into a centrifugal tube, balancing, centrifuging at room temperature, 1200rpm/min, centrifuging for 5min at about 300g, discarding centrifugation supernatant, slightly bouncing the bottom to disperse the cell aggregates, adding an appropriate amount of culture medium to resuspend the cells, uniformly mixing by using an 5/10ml straw, counting the cells, inoculating according to the cell count, recommending the cell inoculation density to be 4000-12000 cells/cm²Wherein the P0-P4 recommends that the inoculation density is 4000-²P5-P10 recommend a seeding density of 10000cells/cm². Labeling cell type, generation number, and passage date, gently sliding the dish in a cross direction, repeating for 4-6 times to distribute the cells evenly, transferring the dish carefully to a dish containing 5% CO2 at 37 deg.CAnd culturing, wherein the cell proliferation is about 3.5-9 times per 48 hours according to the number of cell passages.

The cell states of different generations are recorded by photographing, and as shown in figure 1, the primary human umbilical cord mesenchymal stem cells can be obtained by the culture medium through a tissue mass suspension culture method, and the cell uniformity and the proliferation capacity of people can be maintained after long-term passage of more than ten generations.

Example 2

This example provides a 2D culture method of mesenchymal stem cells, which is different from example 1 in that the formula of the serum-free medium used is as follows:

each 1L of medium consisted of the following components:

IMDM 17.662g, L-propylamino-glutamine 2mM, chemical lipid 0.1 v/v%, cholesterol 5mg, insulin 8mg, transferrin 10mg, recombinant human albumin 1g, hydrocortisone 0.1mg, dexamethasone 4 μ g, progesterone 1 μ g, putrescine 5mg, ascorbic acid 25mg, beta-mercaptoethanol 50 μ M, soybean lecithin 2mg, zinc sulfate heptahydrate 1.25mg, lipoic acid 0.1mg, N-Acetyl-L-Cysteine 0.2mM, reduced glutathione 1mg, taurine 2mg, Y-276322 μ M, ethanolamine 1mg, b-FGF 5 μ g, EGF 5 μ g, PDGF-BB 2 μ g, IGF-110 μ g, TGF- β 31 μ g, HGF 2 μ g, CTGF 1 μ g and VEGF 5 μ g; the balance being water for cell culture.

The method of arrangement is the same as in example 1.

Example 3

IMDM 17.662g, L-propylamino-glutamine 2mM, chemical lipid 1 v/v%, cholesterol 30mg, insulin 25mg, transferrin 30mg, recombinant human albumin 5g, hydrocortisone 2mg, dexamethasone 20 μ g, progesterone 10 μ g, putrescine 15mg, ascorbic acid 200mg, beta-mercaptoethanol 100 μ M, soybean lecithin 20mg, zinc sulfate heptahydrate 2.5mg, lipoic acid 0.5mg, N-Acetyl-L-Cysteine 2mM, reduced glutathione 5mg, taurine 10mg, Y-2763210 μ M, ethanolamine 5mg, b-FGF 40 μ g, EGF 50 μ g, PDGF-BB 20 μ g, IGF-140 μ g, TGF- β 35 μ g, HGF 10 μ g, CTGF5 μ g and VEGF 20 μ g; the balance being water for cell culture.

The method of arrangement is the same as in example 1.

The same strain P1 generation human umbilical cord mesenchymal stem cells are taken for testing, 3 groups of repeated experiments are carried out on each of examples 1-3, and the inoculation density is 10000cells/cm²Cells were harvested and counted after 48 hours and fold amplifications recorded. Then inoculating is carried out again at the same density, the cells are harvested till P5, the total cell harvest amount under the culture conditions of different examples is calculated according to the expansion multiple of the cells of each generation, meanwhile, a Beckmann Coulter Vi-CELL XR counter is used for detecting the distribution condition of the diameters of the P5 generation CELLs, obtaining the proliferation and diameter data of the human umbilical cord mesenchymal stem cells in different embodiments according to the statistical results, the statistical results are shown in table 2, and it is understood from the results that the human umbilical cord mesenchymal stem cells proliferated faster and cell diameter uniformity was better under the culture conditions of example 1, the mesenchymal stem cells cultured in the examples 2 and 3 have a slightly slower proliferation speed, and are easy to appear more cells with larger diameters in the culture process, this indicates that the mesenchymal stem cells cultured in examples 2 and 3 have poor homogeneity and are more likely to undergo aging and differentiation during 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- β 3 of the serum-free medium used therein is replaced with TGF- β 1 in equal amounts.

The method of arrangement is the same as in example 1.

As shown in FIG. 2, it is understood from the results of the cells cultured using the medium of example 3 and comparative example 1 and the control group containing no human transforming growth factor that the secretion of extracellular matrix by mesenchymal stem cells is promoted by the combined action of TGF-. beta.1 and ascorbic acid, and many reports have been made on the fact that the combination stimulates the production of collagen and fibronectin by mesenchymal stem cells, and it has been found in this experiment that the combination can indeed promote the adherence of mesenchymal stem cells under serum-free conditions. And the combined action of the TGF-beta 3 and the ascorbic acid can promote the mesenchymal stem cells to secrete extracellular matrix, reduce the degradation of the extracellular matrix, form the accumulation of the extracellular matrix and be more favorable for the adherence and proliferation of the cells. Furthermore, long-term passaging using TGF-. beta.1 is far less effective than TGF-. beta.3.

Example 4

The embodiment provides a three-dimensional culture method of human umbilical cord-derived mesenchymal stem cells (a technical flow diagram is shown in fig. 3).

The serum-free medium used therein 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

(also other brands or types of cell culture microcarriers) sterile water bottles are autoclaved;

(2) taking 3-7 generation human umbilical cord source mesenchymal stem cells in logarithmic growth phase, wherein the density of the mesenchymal stem cells is not more than 95% at most, and digesting the cells;

(3) incubating in water bath at 37 ℃ for about 3min with 1:3 diluted tryple digestive enzyme in an incubator at 37 ℃;

(4) terminating digestion, centrifuging at 1200rpm/min for 5min at about 300g, resuspending cells in complete serum-free medium, and counting cells;

(5) taking 1g of microcarrier, adding deionized water for autoclaving, transferring into a 50ml centrifuge tube, centrifuging by 300g for 5min, and removing water;

(6) diluting the serum-free culture medium to 12ml in 2000 ten thousand cells, adding into a centrifuge tube containing 1g of microcarrier, mixing with a pipette, inoculating into a 250-roller bottle (or 3-5 low adsorption 10cm dishes, 15ml culture medium per dish), supplementing the serum-free culture medium to 200ml, stirring for 20 min every 4 hours in a roller bottle at a rotation speed of 20 min50rpm/min，7℃，5％CO₂The incubator is used for placing and culturing.

1.1 operation for obtaining a large amount of human umbilical cord-derived mesenchymal stem cells:

(1) the quantity of inoculated cells corresponding to each 1g of microcarriers in the inoculated three-dimensional culture system is 2000 ten thousand;

(2) after 72 hours, the cells are attached to the microcarriers and rapidly proliferate, at which time the cell harvest is increased, the microcarriers and the culture medium which have aggregated into small clumps are carefully transferred to a 50ml centrifuge tube and centrifuged for 3min at 100g 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 the mixture into a 250ml spinner flask, supplementing the culture medium to 200ml, and continuing to culture, wherein the growth state and the density of cells can be observed under a microscope after being dyed by a Calcein AM & PI dead and live 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) digesting the tissue mass with a suitable amount of 1mg/ml collagenase IV dissolved in a basal medium in an incubator at 37 ℃ for 25min, while shaking gently every 5 minutes to make the contact more complete;

(6) after 25min, 1/2 volumes of TrypLE were added^TMExpress Enzyme digestive Enzyme (Gibco, 12605010), mixing well, digesting for 2min at 37 ℃, gently blowing and beating a pipette for several times to obtain a single cell suspension, centrifuging for 5min at 300g, and removing supernatant to obtain cells.

1.2 is the operation of harvesting human umbilical cord source mesenchymal stem cell secretory protein and exosome:

(1) the three-dimensional culture of the human umbilical cord-derived mesenchymal stem cells can change a fresh culture medium every 3 to 5 days, and after 10 days of inoculation, the old culture medium is discarded, and a microcarrier containing cells is reserved;

(2) adding 250ml of newly prepared 3 times IMDM basal medium and completely serum-free medium, mixing and diluting to obtain low-nutrient medium (i.e. 2 parts IMDM basal medium and one part serum-free medium), 37 deg.C, and 5% CO₂The incubator is used for placing and culturing.

(3) After 72 hours, the culture medium turns yellow, the pipette sucks the old culture medium out for storage, 250ml of low-nutrient medium is added for continuous culture, and the secreted protein in the collected old culture medium can be used for other experiments after being concentrated and purified.

2. Staining observation of three-dimensionally cultured human umbilical cord-derived mesenchymal stem cells:

(1) the human umbilical cord-derived mesenchymal stem cells growing in the microcarriers cannot be directly observed under a microscope and need to be subjected to cell staining;

(2) taking out a small amount of microcarrier to be dyed and placing the microcarrier in a micropore plate, sucking PBS twice, and removing the PBS;

(3) staining dead and live cells according to a reagent protocol by using a Calcein AM & PI dead and live cell staining kit;

(4) after the staining solution acts for about 15-30 minutes, observing the cell state under a fluorescence microscope;

(5) after staining, live cells are green under a fluorescence microscope, dead cells are red, and whether the secretion of proteins by the cells can be continued is determined according to the growth condition of the cells.

After about 30-45 days, part of cells begin to have broken tentacles, the cell quality becomes large, and dead cells stained red by PI appear, and as the culture time continues to be prolonged, the broken tentacles of the cells and the proportion of the dead cells stained red gradually increase, and at the moment, the collection of secretory 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 from left to right in sequence in FIG. 4, wherein the proportion of dead cells on and before day 25 is extremely low and is not more than 2%, and after day 35, breaking of cell tentacles begins to occur, and thunderbolt, which is the dead cells stained red, also gradually rises to 5-10%. As can be seen, after the early proliferation inside the microcarrier is completed, the cells begin to proliferate on the surface of the microcarrier and gather the surrounding microcarrier to form a larger sphere by virtue of the adhesion property of the extracellular matrix, then the cells continue to proliferate on the surface of the larger sphere to form a larger cell sphere, the cell survival rate is very high, after more than 35 days, the cells begin to have broken contact pins and have a small proportion of dead cells stained red, the proportion is about 5-10%, and if the culture is continued, the broken contact pins of the cells and the proportion of dead cells stained red gradually increase.

4. Harvesting of three-dimensionally cultured human umbilical cord mesenchymal stem cells

Whether static or dynamic (spinner flask, reactor) culture, if the aim of the culture is to harvest a large amount of mesenchymal stem cells, the culture time is preferably not more than 12 days (depending on the cell seeding density and growth rate). Mesenchymal stem cells cultured for a long time in a three-dimensional state can generate a fibrous substance which cannot be digested and degraded by collagenase or tryple enzyme, so that the quality and efficiency of cell harvesting are reduced. Therefore, it is attempted to stop the culture before the microcarriers aggregate into larger spheres.

The cells harvested in this example are shown in FIG. 5, and the digested cells can still be used in two-dimensional culture or in three-dimensional culture.

5. Detection of human umbilical cord mesenchymal stem cell secretory protein cultured in two-dimensional and three-dimensional states

Taking human umbilical cord mesenchymal stem cells of P3 generation, collecting cell culture supernatant when the cells are cultured in three dimensions on day 15, filtering with a 0.45um filter membrane to remove impurities, filtering with a 30Kda ultrafiltration tube, collecting filtrate, removing albumin, filtering the filtrate again with a 3Kda ultrafiltration tube to obtain a protein concentrate of 3-30Kda, mainly containing secreted protein, collecting culture supernatant of P3 generation which is cultured for 72 hours for the mesenchymal stem cells cultured in two dimensions, collecting secreted protein by the same method, and performing protein mass spectrometry on the obtained secreted protein, as shown in FIG. 6, compared with two-dimensional culture, the secretion of various proteins such as collagen, insulin-like growth factor, high mobility histone and calcium dependent transporter of the mesenchymal stem cells cultured in three dimensions is obviously increased, and apoptosis-related proteins such as apoptosis-inducing factor, autophagy regulatory protein, annexin and the like are obviously reduced, the mesenchymal stem cells have more excellent activity under the three-dimensional culture condition.

6. Three-dimensional culture mesenchymal stem cell secretory protein for promoting human skin fibroblast proliferation

In order to verify the function of protein secreted by mesenchymal stem cells in a three-dimensional culture state, the invention provides a proliferation promotion experiment of the protein on fibroblasts, wherein the fibroblast proliferation experiment needs to be inoculated with low-density fibroblasts, P3 human skin fibroblasts in a logarithmic growth phase are taken, cell counting is carried out after digestion, the cells are inoculated into a 6-well plate at the density of 2000cells per square centimeter, each group is repeated three times, the addition concentrations of each group of secreted protein are respectively 0, 0.0156, 0.03125, 0.0625, 0.0125 and 0.025mg/ml, and the addition concentration of the low-concentration protein group is 0.025mg/ml by using albumin. After inoculation, the growth of the cells is observed under a microscope every 24 hours and recorded by photographing, and groups of cells are harvested after 72 hours, and the harvested cell amount is counted. As can be seen from FIG. 7, the mesenchymal stem cell secreted protein under the three-dimensional culture condition has a good environmental effect on the proliferation of fibroblasts, and the environmental effect is enhanced along with the increase of the addition concentration.

7. Human skin fibroblast repair method for promoting ultraviolet loss by three-dimensionally culturing mesenchymal stem cell secretory protein

Collecting P3 human skin fibroblast cells in logarithmic growth phase, digesting, counting cells, and resuspending the cells in 10% FBS-containing high-glucose DMEM medium to obtain 3 x 10⁴cells/ml cell suspension, cells were seeded into six-well plates, approximately 6 million cells per well, 2ml medium. When the cells grow to 60-70%, each group is changed into high-sugar DMEM culture medium containing 10% FBS and respectively containing 0g/L HSA (recombinant human serum albumin), 3g/L HSA, 6g/L HSA, 3g/L secretory protein and 6g/L secretory protein, the high-sugar DMEM culture medium is uniformly mixed and added into a corresponding pore plate, 2ml of the high-sugar DMEM culture medium is added into each pore plate, 3 controls are set in each group, when the cells grow to 70-80%, the culture medium is removed, the high-sugar DMEM culture medium containing 2% FBS and incubated at 37 ℃ is changed into PBS culture medium containing 2% FBS, each pore is about 700ul, the cover of the culture plate is opened just covering the bottom surface of the culture plate, the culture dish is placed in a biological safety cabinet, ultraviolet radiation is opened for 20 minutes (pre-experiment is performed), PBS is removed, the fresh culture medium corresponding to each group is changed, and then cell death, the corresponding fresh culture medium was replaced as appropriate, and recorded by photographing, and the total number of cells in each group was counted after 96 hours. In FIG. 8, A to E correspond to the respective components A to EResults for the following test groups: A. protein-free; b.3mg/ml albumin; c.6mg/ml albumin; c.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 dermal fibroblasts damaged by ultraviolet light, the protein secreted from the mesenchymal stem cells cultured in three dimensions has a stronger repairing effect, and the repairing effect is more remarkable at a high concentration.

Example 5

This example provides a three-dimensional culture method of human umbilical cord-derived mesenchymal stem cells, which is different from example 4 in that a low nutrient medium is not used, and a serum-free medium with normal nutrition is used throughout the culture of human umbilical cord-derived mesenchymal stem cells.

The culture results are shown in fig. 9, when the human umbilical cord mesenchymal stem cells in the three-dimensional state are continuously cultured by using the culture medium with normal nutrition degree, the early mesenchymal stem cells are amplified in the interior and the surface of the microcarrier, and the contact feet are extended. As the cell density increased, the area available for growth decreased, cells began to be competitively inhibited, the proportion of dead cells gradually increased, cells died while proliferating, and free dead cells gradually increased in the culture medium, and the proportion of cells stained red by PI gradually increased after staining (green and red cannot be distinguished under black and white, and thus a part of red cells are indicated by white arrows), and the proportion of red cells was about 2% and 10% in the culture results on day 12 and day 18, respectively. The proportion of dead cells was significantly increased compared to example 4, which had been on the same day and had been replaced with low nutrient medium 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 culture medium used is IMDM culture medium supplemented with 10% FBS.

As a result, as shown in FIG. 10, in the case of culturing mesenchymal stem cells using a three-dimensional microcarrier using a medium containing FBS, in comparative example 4, the culturing method containing FBS had different degrees of dead cells (green and red cannot be distinguished in black and white, and thus a part of red cells is indicated by white arrows) during the whole culturing process, and the proportion of red cells was about 3% and 15% in the culturing results on

days

2 and 8, respectively. Moreover, the FBS-containing example shows a large number of dead cells when the microcarriers still have a large growth space, and the content of the dead cells increases with the increase of the culture time, which cannot be improved even if the culture medium is replaced with fresh medium. Therefore, compared with a FBS-containing culture medium, the serum-free culture medium mentioned in example 4 is obviously more suitable for the culture of the porous microcarrier of the human umbilical cord mesenchymal stem cells, and example 4 can harvest more human umbilical cord mesenchymal stem cells and simultaneously reduce the generation of dead cells in the three-dimensional culture process.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A method for culturing mesenchymal stem cells is characterized in that the mesenchymal stem cells are cultured by using a serum-free culture medium;

2. The culture method according to claim 1, wherein the additive component comprises 0.1-1 v/v% of a chemical lipid;

the chemical lipid comprises the following components:

arachidonic acid with the content of 1.8-2.2mg/L, cholesterol with the content of 200-240mg/L, vitamin E (tocopherol) with the content of 60-80mg/L, linoleic acid with the content of 8-12mg/L, linolenic acid with the content of 8-12mg/L, myristic acid with the content of 8-12mg/L, oleic acid with the content of 8-12mg/L, palmitic acid with the content of 8-12mg/L, palmitoleic acid with the content of 8-12mg/L, Pluronic F-68 with the content of 80000-100000mg/L and stearic acid with the content of 8-12 mg/L;

preferably, the additive component further comprises 20-35mg/L of composite ester substances, and the weight ratio of the composite ester substances is 1-5: 5-30: 2-20 of ethanolamine, cholesterol, and soybean lecithin.

3. The culture method according to claim 1 or 2, wherein the additive further comprises hydrocortisone at 0.1-2 mg/L;

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 8-25 mg: 1-10. mu.g: 4-20 μ g: 0.1-2 mg;

more preferably, the additive component further comprises IGF-1, and the dosage ratio of the IGF-1 to insulin is 10-40 μ g: 8-25 mg.

4. The culture method according to any one of claims 1 to 3, wherein the additional components further comprise 1 to 5g/L of albumin, which is a recombinant human albumin obtained from a plant as an expression host, preferably rice;

preferably, the additive component further comprises transferrin, and the dosage ratio of the transferrin to the recombinant human albumin is 10-30 mg: 1-5 g.

5. The culture method according to any one of claims 1 to 4, wherein the additional components further comprise 40 to 60 μ g/L of a complex growth factor comprising a mixture of 5 to 40: 5-50: 2-10 of b-FGF, EGF and HGF;

and/or, the additive components further comprise: 1-5 μ g/L of CTGF;

and/or, the additive components further comprise: 1-5mg/L of reduced glutathione, 0.2-2mM of N-Acetyl-L-Cysteine and 50-100 mu M of beta-mercaptoethanol.

6. The culture method according to any one of claims 1 to 5, wherein each 1L of the culture medium comprises the following components:

IMDM 17.662g, L-propylamine-glutamine 2mM, chemical lipid 0.1-1 v/v%, cholesterol 5-30mg, insulin 8-25mg, transferrin 10-30mg, recombinant human albumin 1-5g, hydrocortisone 0.1-2mg, dexamethasone 4-20 μ g, progesterone 1-10 μ g, putrescine 5-15mg, ascorbic acid 25-200mg, beta-mercaptoethanol 50-100 μ M, 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-276322-10 μ M, ethanolamine 1-5mg, FGF b-FGF 5-40 μ g, and, EGF 5-50 μ g, PDGF-BB 2-20 μ g, IGF-110-40 μ g, TGF-beta 31-5 μ g, HGF 2-10 μ g, CTGF 1-5 μ g and VEGF 5-20 μ g;

preferably, each 1L of medium comprises the following components:

7. The culture method according to any one of claims 1 to 6, wherein the culture mode is 3D culture, and after the coverage of the mesenchymal stem cells on the microcarriers reaches 75-85%, the mesenchymal stem cells are cultured by using a low nutrient medium;

the low-nutrient medium is prepared by mixing the serum-free medium and the IMDM medium according to the volume ratio of 1: diluting the mixture for 2-4 to obtain the product.

8. The culture method according to claim 7, wherein the microcarrier is a solid microcarrier and/or a porous microcarrier;

preferably, the microcarrier is a porous microcarrier made on the basis of an extracellular matrix.

9. The culture method according to claim 8, 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 for 8 to 12 days.

10. A method for obtaining a secretion of a mesenchymal stem cell, comprising culturing a mesenchymal stem cell by the culture method according to any one of claims 1 to 9, and collecting the secretion of the mesenchymal stem cell;