CN110934132A - Serum-free DMSO-free cell cryopreservation liquid and preparation method thereof - Google Patents

Abstract

The invention discloses a serum-free DMSO-free cell cryopreservation solution, which comprises the following components: the basal medium of DMEM/F-12 is 16.762-16.825g/L, the cell factor is 32-34mg/L, the hydroxyethyl starch is 0.2-0.8mg/L, the vitamin is 1-3mg/L, the antibiotic is 0.15-0.16g/L, the protein polypeptide is 5-6mg/L, the lipid is 1-4mg/L, and the cryoprotectant is 0.1-0.41g/L inside and outside the cell. The cell frozen stock solution which does not contain serum and is not added with DMSO components is obtained by the technical scheme of the application, and the components of the formula of the solution are determined, so that the cell frozen stock solution has very high biological safety and clinical application prospect. Meanwhile, the cell cryopreservation solution has the cell recovery survival rate of more than 85 percent, can maintain good cell activity and physiological characteristics, and is very suitable for the related research fields of primary cells and stem cells.

Description

Serum-free DMSO-free cell cryopreservation liquid and preparation method thereof

Technical Field

The invention belongs to the technical field of cell culture, and particularly relates to a serum-free DMSO-free cell cryopreservation solution and a preparation method thereof.

Background

Defining the components of the culture medium necessary for the survival and proliferation of cultured animal cells has been a very active area of research. The development of media formed from the original simple salt ion has progressed to the addition of various biological factors to the basal media. Eagle and his companion found that amino acids and vitamins are essential for the culture of mammalian cells in vitro. Later, most synthetic media were supplemented with serum to support cell survival and proliferation in vitro. However, most of the serum is derived from exogenous fetal bovine serum, and it is indicated that the synthetic medium can be used for cell culture in general mammalian cell culture as long as the growth state of the cells can be maintained.

With the development of medical technology and the progress of cell therapy, the requirement of in vitro cell culture is higher and higher. The serum-free cell culture medium and the frozen stock solution can avoid the introduction of animal-derived components and allergens on one hand, and serum components are not known to produce unpredictable influence on the growth and differentiation of cells on the other hand, and can carry out human autologous cell culture under the culture condition of definite known components on the other hand, so that the research and development of the serum-free cell culture medium and the frozen stock solution in the field of medical scientific research are extremely important, and necessary technical basis is provided for subsequent gene editing and individualized cell treatment. Early studies found that serum can be replaced by hormones, insulin, transferrin, hydrocortisone, recombinant human epidermal growth factor and fibroblast growth factor added to the culture medium during the culture of Hela cells. However, this is only a prototype of a study of serum-free cell culture media and cell lysates. Many mammalian cells, especially primary cells, have factors that are far from supporting the growth needs of the cells.

The serum-free cell freezing medium is characterized in that a certain cryoprotectant is added on the basis of a serum-free cell culture medium, so that fatal damage to cells caused by ice crystals generated in a freezing process is avoided, and the survival efficiency of the cells is improved. In addition, researches show that in the process of subsequent cell recovery, a cryoprotectant dimethyl sulfoxide (DMSO) frequently used in the traditional cell cryopreservation solution influences the differentiation potential of the stem cells, and reduces the totipotency of the stem cells to a certain extent, so that the characteristics of the stem cells are changed.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: provides a serum-free DMSO-free cell cryopreservation solution which has the recovery activity of more than 85 percent after cell cryopreservation, basically does not change the cell phenotype, and can maintain the physiological function and the biological characteristics after cell recovery.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a serum-free DMSO-free cell cryopreservation solution, which comprises the following components: the basal medium of DMEM/F-12 is 16.762-16.825g/L, the cell factor is 32-34mg/L, the hydroxyethyl starch is 0.2-0.8mg/L, the vitamin is 1-3mg/L, the antibiotic is 0.15-0.16g/L, the protein polypeptide is 5-6mg/L, the lipid is 1-4mg/L, and the cryoprotectant is 0.1-0.41g/L inside and outside the cell.

Preferably, the cytokine comprises the following components in percentage by mass: 99.955% of insulin, 0.03% of recombinant human Epidermal Growth Factor (EGF) and 0.015% of recombinant human basic fibroblast growth factor (bFGF).

Preferably, the antibiotics are penicillin and streptomycin, the content of the penicillin is 0.06g/L, and the content of the streptomycin is 0.1 g/L.

Preferably, the vitamin is vitamin C.

Preferably, the protein polypeptide is albumin and transferrin, wherein the addition amount of albumin is 0.1-0.8 μ g/L, and the addition amount of transferrin is 5.1-5.2 mg/L.

Preferably, the lipids are ethanolamine and linoleic acid, the total amount ratio of ethanolamine to linoleic acid is 1:500, and when the addition amount of ethanolamine is 1-3mg/L, the addition amount of linoleic acid is 2-6 mug/L.

Preferably, the cryoprotectant inside and outside the cells is trehalose, polyvinylpyrrolidone and methylcellulose, wherein the addition amount of the trehalose is 0.034-0.102g/L, the addition amount of the polyvinylpyrrolidone is 0.2-0.8mg/L, and the addition amount of the methylcellulose is 0.1-0.3 g/L.

Preferably, the serum-free and DMSO-free cell frozen stock solution comprises the following components in percentage by weight: 110-116.6mg/L of anhydrous calcium chloride, 55-59.05mg/L of L-leucine, 0.03-0.042mg/L of linoleic acid, 0.0012-0.0013mg/L of blue vitriol, 90-91.25mg/L of L-lysine hydrochloride, 0.08-0.105mg/L of lipoic acid, 0.02-0.05mg/L of ferric nitrate nonahydrate, 15.00-17.24mg/L of L-methionine, 6.5-8.1mg/L of phenol red, 0.35-0.417mg/L of ferrous sulfate heptahydrate, 33-35.48mg/L of L-phenylalanine, 0.07-0.081mg/L of 1, 4-butanediamine dihydrochloride, 280-311.8mg/L of potassium chloride, 24-26.25mg/L of L-serine, 53-55mg/L of sodium pyruvate, 26.2-28.64mg/L of magnesium chloride, threonine 50.3-53.45mg/L, vitamin H0.0035-0.005 mg/L, anhydrous magnesium sulfate 47-48.84mg/L, alanine 4.45-4.50mg/L, calcium pantothenate 2.24-3.2mg/L, sodium chloride 6500-6999.5mg/L, L-asparagine 7.5-8.3mg/L, choline chloride 7.88-8.98mg/L, anhydrous sodium dihydrogen phosphate 51.2-54.35mg/L, aspartic acid 5.59-6.65mg/L, folic acid 2.65-3.25mg/L, disodium hydrogen phosphate 69.5-71.02mg/L, L-cysteine hydrochloride 16.88-17.56mg/L, I-inositol 11.3-12.6mg/L, zinc sulfate heptahydrate 0.432-0.551mg/L, glutamic acid 7.35-8.06mg/L, 2.02-2.45mg/L nicotinamide, 145-147.5 mg/L-arginine hydrochloride, 15.9-17.25 mg/L-proline, 1-2mg/L pyridoxal hydrochloride, 31.29-32.85 mg/L-cystine hydrochloride, 9.02-9.57 mg/L-tryptophan hydrochloride, 0.031-0.036mg/L pyridoxine hydrochloride, 378 mg/L-glutamine 365, 38.4-39.1 mg/L-tyrosine, 0.219-0.233mg/L riboflavin, 18.75-19.11mg/L glycine, 52.85-53.31 mg/L-valine hydrochloride, 2.17-2.22mg/L thiamine hydrochloride, 31.48-32.10 mg/L-histidine hydrochloride, 3151D-glucose and 3230mg/L, 0.365-0.445mg/L of thymidine, 54.47-55.2mg/L of L-isoleucine, 1.55-2.03mg/L of hypoxanthine, 0.68mg/L of vitamin B120.63, 8-12mg/L of insulin, 5-6mg/L of transferrin, 1-3mg/L of ethanolamine, 2-6. mu.g/L of linoleic acid, 0.1-0.8. mu.g/L of albumin, 0.1-0.5. mu.g/L of recombinant human epidermal growth factor, 0.3-0.4mg/L of hydrocortisone, 0.06g/L of penicillin, 0.1g/L of streptomycin, 3-3 mg/L of vitamin C1, 0.05-3mg/L of betaine, 0.1-0.3mM/L of trehalose, 0.1-0.3g/L of methylcellulose, 10-12.75mg/L of sodium bicarbonate, recombinant human basic fibroblast growth factor 0.1-0.2 mug/L, hydroxyethyl starch 0.2-0.8mg/L, polyvinylpyrrolidone 0.2-0.8 mg/L.

Further preferably, the serum-free and DMSO-free cell cryopreservation solution comprises the following components in percentage by weight: 116.6mg/L of anhydrous calcium chloride, 59.05mg/L of L-leucine, 0.042mg/L of linoleic acid, 00013mg/L of copper sulfate pentahydrate, 91.25mg/L of L-lysine hydrochloride, 0.105mg/L of lipoic acid, 0.05mg/L of ferric nitrate nonahydrate, 17.24mg/L of L-methionine, 8.1mg/L of phenol red, 0.417mg/L of ferrous sulfate heptahydrate, 35.48mg/L of L-phenylalanine, 0.081mg/L of 1, 4-butanediamine dihydrochloride, 311.8mg/L of potassium chloride, 26.25mg/L of L-serine, 55mg/L of sodium pyruvate, 28.64mg/L of magnesium chloride, 53.45mg/L of threonine, 0.0035mg/L of vitamin H, 48.84mg/L of anhydrous magnesium sulfate, 4.45mg/L of alanine, 2.24mg/L of calcium pantothenate, 6999.5mg/L of sodium chloride, 7.5mg/L of L-asparagine, 8.98mg/L of choline chloride, 54.35mg/L of anhydrous sodium dihydrogen phosphate, 6.65mg/L of aspartic acid, 2.65mg/L of folic acid, 71.02mg/L of disodium hydrogen phosphate, 17.56mg/L of L-cysteine hydrochloride, 12.6mg/L of I-inositol, 0.432mg/L of zinc sulfate heptahydrate, 7.35mg/L of glutamic acid, 2.02mg/L of nicotinamide, 147.5mg/L of L-arginine hydrochloride, 17.25mg/L of L-proline, 2mg/L of pyridoxal hydrochloride, 31.29mg/L of L-cystine hydrochloride, 9.02mg/L of L-tryptophan, 0.031mg/L of pyridoxine hydrochloride, 365mg/L of L-glutamine, 38.4mg/L of L-tyrosine, 0.219mg/L of riboflavin, 18.75mg/L of glycine, 52.85mg/L of L-valine, 2.17mg/L of thiamine hydrochloride, 31.48mg/L of L-histidine hydrochloride, 3151mg/L of D-glucose, 0.365mg/L of thymidine, 54.47mg/L of L-isoleucine, 2mg/L of hypoxanthine, 120.68mg/L of vitamin B, 8-12mg/L of insulin, 5-6mg/L of transferrin, 1-3mg/L of ethanolamine, 2-6. mu.g/L of linoleic acid, 0.1-0.8. mu.g/L of albumin, 0.1-0.2. mu.g/L of recombinant human epidermal growth factor, 0.3-0.4mg/L of hydrocortisone, 0.06g/L of penicillin, 0.1g/L of streptomycin, 1-3mg/L of vitamin C, 0.05-3mg/L of betaine, 0.1-0.3mM/L of trehalose, 0.1-0.3g/L of methylcellulose, 10-12.75mg/L of sodium bicarbonate, 0.1-0.2 mu g/L of recombinant human basic fibroblast growth factor, 0.2-0.8mg/L of hydroxyethyl starch and 0.4-0.8mg/L of polyvinylpyrrolidone.

The second technical problem to be solved by the invention is: provides a preparation method of serum-free and DMSO-free cell frozen stock solution.

In order to solve the second technical problem, the technical scheme is as follows: a preparation method of serum-free and DMSO-free cell frozen stock solution comprises the following steps:

1) in a cGMP clean-grade workshop, taking DMEM/F-12 as a basic culture medium, then gradually adding penicillin, streptomycin, recombinant human epidermal growth factor, basic fibroblast growth factor, ethanolamine, betaine, albumin, vitamin C and hydroxyethyl starch, finally adjusting the pH to 7.4 +/-0.1 by using 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution, then respectively adding cryoprotectant components inside and outside cells according to a proportion, and uniformly mixing;

2) filtering and sterilizing the solution obtained in the step 1) by using a filter with the pore diameter less than or equal to 0.1 mu m to obtain the serum-free and DMSO-free cell frozen stock solution.

Has the advantages that: the cell frozen stock solution which does not contain serum and is not added with DMSO components is obtained by the technical scheme of the application, and the components of the formula of the solution are determined, so that the cell frozen stock solution has very high biological safety and clinical application prospect. Meanwhile, the cell cryopreservation solution has the cell recovery survival rate of more than 85 percent, can maintain good cell activity and physiological characteristics, and is very suitable for the related research fields of primary cells and stem cells.

The serum-free DMSO-free cell frozen stock solution is safe and effective, has clear components, simple and convenient preparation method and controllable cost, has good clinical application prospect, and can be used for continuous culture or direct reinfusion of clinical cells after in vitro culture.

Drawings

FIG. 1 is a light microscope picture of human umbilical cord mesenchymal stem cells after recovery.

FIG. 2 shows the proliferation state of cells detected by a CCK-8 kit after the human umbilical cord mesenchymal stem cells are recovered.

FIG. 3 is a light microscope picture of differentiation condition of human umbilical cord mesenchymal stem cells induced into lipid.

FIG. 4 is a light microscope photograph of the differentiation of human umbilical cord mesenchymal stem cells into cartilage.

Detailed Description

Example 1

The serum-free DMSO-free cell cryopreservation solution comprises the following components: 116.6mg/L of anhydrous calcium chloride, 59.05mg/L of L-leucine, 0.042mg/L of linoleic acid, 00013mg/L of copper sulfate pentahydrate, 91.25mg/L of L-lysine hydrochloride, 0.105mg/L of lipoic acid, 0.05mg/L of ferric nitrate nonahydrate, 17.24mg/L of L-methionine, 8.1mg/L of phenol red, 0.417mg/L of ferrous sulfate heptahydrate, 35.48mg/L of L-phenylalanine, 0.081mg/L of 1, 4-butanediamine dihydrochloride, 311.8mg/L of potassium chloride, 26.25mg/L of L-serine, 55mg/L of sodium pyruvate, 28.64mg/L of magnesium chloride, 53.45mg/L of threonine, 0.0035mg/L of vitamin H, 48.84mg/L of anhydrous magnesium sulfate, 4.45mg/L of alanine, 2.24mg/L of calcium pantothenate, 6999.5mg/L of sodium chloride, 7.5mg/L of L-asparagine, 8.98mg/L of choline chloride, 54.35mg/L of anhydrous sodium dihydrogen phosphate, 6.65mg/L of aspartic acid, 2.65mg/L of folic acid, 71.02mg/L of disodium hydrogen phosphate, 17.56mg/L of L-cysteine hydrochloride, 12.6mg/L of I-inositol, 0.432mg/L of zinc sulfate heptahydrate, 7.35mg/L of glutamic acid, 2.02mg/L of nicotinamide, 147.5mg/L of L-arginine hydrochloride, 17.25mg/L of L-proline, 2mg/L of pyridoxal hydrochloride, 31.29mg/L of L-cystine hydrochloride, 9.02mg/L of L-tryptophan, 0.031mg/L of pyridoxine hydrochloride, 365mg/L of L-glutamine, 38.4mg/L of L-tyrosine, riboflavin 0.219mg/L, glycine 18.75mg/L, L-valine 52.85mg/L, thiamine hydrochloride 2.17mg/L, L-histidine hydrochloride 31.48mg/L, D-glucose 3151mg/L, thymidine 0.365mg/L, L-isoleucine 54.47mg/L, hypoxanthine 2mg/L, vitamin B120.68mg/L, insulin 10mg/L, transferrin 5mg/L, ethanolamine 1mg/L, linoleic acid 2. mu.g/L, albumin 0.8. mu.g/L, recombinant human epidermal growth factor 0.2. mu.g/L, hydrocortisone 0.3mg/L, penicillin 0.06g/L, streptomycin 0.1g/L, vitamin C1mg/L, betaine 1mg/L, trehalose 0.3mM/L, methylcellulose 0.3g/L, 10mg/L of sodium bicarbonate, 0.1 mu g/L of recombinant human basic fibroblast growth factor, 0.2mg/L of hydroxyethyl starch and 0.4mg/L of polyvinylpyrrolidone.

Example 2

The serum-free DMSO-free cell cryopreservation solution comprises the following components: 116.6mg/L of anhydrous calcium chloride, 59.05mg/L of L-leucine, 0.042mg/L of linoleic acid, 00013mg/L of copper sulfate pentahydrate, 91.25mg/L of L-lysine hydrochloride, 0.105mg/L of lipoic acid, 0.05mg/L of ferric nitrate nonahydrate, 17.24mg/L of L-methionine, 8.1mg/L of phenol red, 0.417mg/L of ferrous sulfate heptahydrate, 35.48mg/L of L-phenylalanine, 0.081mg/L of 1, 4-butanediamine dihydrochloride, 311.8mg/L of potassium chloride, 26.25mg/L of L-serine, 55mg/L of sodium pyruvate, 28.64mg/L of magnesium chloride, 53.45mg/L of threonine, 0.0035mg/L of vitamin H, 48.84mg/L of anhydrous magnesium sulfate, 4.45mg/L of alanine, 2.24mg/L of calcium pantothenate, 6999.5mg/L of sodium chloride, 7.5mg/L of L-asparagine, 8.98mg/L of choline chloride, 54.35mg/L of anhydrous sodium dihydrogen phosphate, 6.65mg/L of aspartic acid, 2.65mg/L of folic acid, 71.02mg/L of disodium hydrogen phosphate, 17.56mg/L of L-cysteine hydrochloride, 12.6mg/L of I-inositol, 0.432mg/L of zinc sulfate heptahydrate, 7.35mg/L of glutamic acid, 2.02mg/L of nicotinamide, 147.5mg/L of L-arginine hydrochloride, 17.25mg/L of L-proline, 2mg/L of pyridoxal hydrochloride, 31.29mg/L of L-cystine hydrochloride, 9.02mg/L of L-tryptophan, 0.031mg/L of pyridoxine hydrochloride, 365mg/L of L-glutamine, 38.4mg/L of L-tyrosine, riboflavin 0.219mg/L, glycine 18.75mg/L, L-valine 52.85mg/L, thiamine hydrochloride 2.17mg/L, L-histidine hydrochloride 31.48mg/L, D-glucose 3151mg/L, thymidine 0.365mg/L, L-isoleucine 54.47mg/L, hypoxanthine 2mg/L, vitamin B120.68mg/L, insulin 8mg/L, transferrin 6mg/L, ethanolamine 2mg/L, linoleic acid 4. mu.g/L, albumin 0.2. mu.g/L, recombinant human epidermal growth factor 0.2. mu.g/L, hydrocortisone 0.3mg/L, penicillin 0.6g/L, streptomycin 0.1g/L, vitamin C3 mg/L, betaine 2mg/L, trehalose 0.3mM/L, 0.3g/L of methyl cellulose, 12.75mg/L of sodium bicarbonate, 0.2 mu g/L of recombinant human basic fibroblast growth factor, 0.2mg/L of hydroxyethyl starch and 0.6mg/L of polyvinylpyrrolidone.

Example 3

The serum-free DMSO-free cell cryopreservation solution comprises the following components: 116.6mg/L of anhydrous calcium chloride, 59.05mg/L of L-leucine, 0.042mg/L of linoleic acid, 00013mg/L of copper sulfate pentahydrate, 91.25mg/L of L-lysine hydrochloride, 0.105mg/L of lipoic acid, 0.05mg/L of ferric nitrate nonahydrate, 17.24mg/L of L-methionine, 8.1mg/L of phenol red, 0.417mg/L of ferrous sulfate heptahydrate, 35.48mg/L of L-phenylalanine, 0.081mg/L of 1, 4-butanediamine dihydrochloride, 311.8mg/L of potassium chloride, 26.25mg/L of L-serine, 55mg/L of sodium pyruvate, 28.64mg/L of magnesium chloride, 53.45mg/L of threonine, 0.0035mg/L of vitamin H, 48.84mg/L of anhydrous magnesium sulfate, 4.45mg/L of alanine, 2.24mg/L of calcium pantothenate, 6999.5mg/L of sodium chloride, 7.5mg/L of L-asparagine, 8.98mg/L of choline chloride, 54.35mg/L of anhydrous sodium dihydrogen phosphate, 6.65mg/L of aspartic acid, 2.65mg/L of folic acid, 71.02mg/L of disodium hydrogen phosphate, 17.56mg/L of L-cysteine hydrochloride, 12.6mg/L of I-inositol, 0.432mg/L of zinc sulfate heptahydrate, 7.35mg/L of glutamic acid, 2.02mg/L of nicotinamide, 147.5mg/L of L-arginine hydrochloride, 17.25mg/L of L-proline, 2mg/L of pyridoxal hydrochloride, 31.29mg/L of L-cystine hydrochloride, 9.02mg/L of L-tryptophan, 0.031mg/L of pyridoxine hydrochloride, 365mg/L of L-glutamine, 38.4mg/L of L-tyrosine, riboflavin 0.219mg/L, glycine 18.75mg/L, L-valine 52.85mg/L, thiamine hydrochloride 2.17mg/L, L-histidine hydrochloride 31.48mg/L, D-glucose 3151mg/L, thymidine 0.365mg/L, L-isoleucine 54.47mg/L, hypoxanthine 2mg/L, vitamin B120.68mg/L, insulin 8mg/L, transferrin 5mg/L, ethanolamine 2mg/L, linoleic acid 4-5. mu.g/L, albumin 0.5. mu.g/L, recombinant human epidermal growth factor 0.2. mu.g/L, hydrocortisone 0.35mg/L, penicillin 0.06g/L, streptomycin 0.1g/L, vitamin C2 mg/L, betaine 2mg/L, trehalose 0.3mM/L, 0.2g/L of methyl cellulose, 10mg/L of sodium bicarbonate, 0.2 mu g/L of recombinant human basic fibroblast growth factor, 0.8mg/L of hydroxyethyl starch and 0.8mg/L of polyvinylpyrrolidone.

The preparation method of the serum-free and DMSO-free cell frozen stock solution comprises the following steps of feeding according to the raw material formula of the embodiment 1-3:

1) in a cGMP clean-grade workshop, taking DMEM/F-12 as a basic culture medium, then gradually adding penicillin, streptomycin, recombinant human epidermal growth factor, basic fibroblast growth factor, ethanolamine, betaine, albumin, vitamin C and hydroxyethyl starch, finally adjusting the pH to 7.4 by using 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution, then respectively adding cryoprotectant components inside and outside cells according to a proportion, and uniformly mixing;

2) filtering and sterilizing the solution obtained in the step 1) by using a filter with the aperture of 0.1 mu m to obtain the serum-free and DMSO-free cell frozen stock solution.

The serum-free and DMSO-free cell frozen stock solution obtained by the formula in the example 2 is randomly selected for application comparison with a common cell frozen stock solution:

the first step,

1) Preparing a cell frozen stock solution, namely a DMEM basic culture medium, 10% serum and 10% DMSO solution, and mixing uniformly for later use, wherein the DMEM basic culture medium is sequentially added with the serum accounting for 10% of the volume of the basic culture medium and the DMSO solution accounting for 10% of the volume of the basic culture medium;

the serum-free and DMSO-free cell frozen stock solution is the serum-free and DMSO-free cell frozen stock solution prepared according to the proportion listed in example 1.

2) Human umbilical cord mesenchymal stem cells whose growth phase was in the logarithmic phase were digested with pancreatin and then centrifuged at 4000rpm for 2 minutes to collect the cells. Each cell line (1-5X 10)⁶Cells) is added with 1ml of common cell frozen stock solution or the serum-free and DMSO-free cell frozen stock solution, the mixture is repeatedly and uniformly blown by a pipette tip to prepare cell suspension, and the cell density is 1-5 multiplied by 10⁶Cells/ml are preferred.

3) Subpackaging into a freezing tube, and marking information such as the name of the cell, the freezing date, the type of the cell freezing solution and the like.

4) Slowly freezing the cells according to a programmed cooling mode, namely standing at 4 ℃ for 30min, standing at-20 ℃ for 30min, then freezing and storing at-80 ℃ overnight, and finally storing the cells in a liquid nitrogen tank.

Step two,

1) The water bath was preheated to 37 ℃ 10 minutes in advance. And the clean bench is cleaned with 75% alcohol for standby.

2) The human umbilical cord mesenchymal stem cells which are respectively preserved by the ordinary cell frozen stock solution and the serum-free DMSO-free cell frozen stock solution in the embodiment 1 and are frozen for 6 months are taken out of the liquid nitrogen, immediately placed in a water bath kettle at 37 ℃, and gently shaken for being melted rapidly.

3) And centrifuging the thawed cells at the speed of 1000rpm for 2 minutes, removing the upper layer of cell frozen stock solution, adding a proper amount of special culture medium for the human umbilical cord mesenchymal stem cells, re-suspending the cell precipitate, inoculating the cell precipitate into a cell culture dish, and culturing in a 5% carbon dioxide incubator at 37 ℃.

4) After 12 hours of culture, the recovery state and the growth condition of the cells are observed, and the culture medium is replaced with fresh one in time for continuous culture.

As shown in FIG. 1, FIG. 1A is the growth state of human umbilical cord mesenchymal stem cells recovered after cryopreservation of common cell cryopreservation liquid; FIG. 1B is the growth state of human umbilical cord mesenchymal stem cells recovered after cryopreservation of the serum-free and DMSO-free cell cryopreservation solution of the invention.

Compared with the common cell cryopreservation solution of DMEM + 10% serum + 10% DMSO, the recovery effect (figure 1B) of the cell cryopreservation solution after cryopreservation of human umbilical cord mesenchymal stem cells is superior to that of the common cell cryopreservation solution (figure 1A) in terms of recovery effect and cell state of the human umbilical cord mesenchymal stem cells after 6 months of cryopreservation of the serum-free DMSO-free cell cryopreservation solution, namely the survival rate of the cells is up to more than 80% and the morphology of the cells is closer to the standard morphology, which shows that the cell cryopreservation solution can replace the traditional cell cryopreservation solution in the aspect of cryopreservation of human umbilical cord mesenchymal stem cells.

Step three,

1) The human umbilical cord mesenchymal stem cells recovered in example 2 were seeded in a 96-well plate, and 100. mu.l of cell suspension was prepared per well.

2) After the plates were incubated at 37 ℃ for 24 hours, 48 hours and 72 hours in a 5% carbon dioxide incubator (FIG. 2), respectively, 10. mu.l of CCK8 solution (cell proliferation assay-8 solution) was added to the plates. The plates were then incubated in an incubator for 2 hours.

3) The absorbance is measured at 450nm of the microplate reader, and the reference wavelength is 600 nm.

4) The method for calculating the cell proliferation activity comprises the following steps:

cell viability (%) ([ a (culture time 2) -a (blank) ]/[ a (culture time 1) -a (blank) ] × 100

Wherein, a (culture time): absorbance of culture wells with cells and CCK8 solution at different culture times;

a (blank): absorbance of culture wells with medium and CCK8 solution without cells;

as shown in fig. 2, fig. 2 shows the proliferation of human umbilical cord mesenchymal stem cells treated by the serum-free and DMSO-free cell cryopreservation solution of the present invention (dotted line-round black dots), and the proliferation of human umbilical cord mesenchymal stem cells treated by the common cell cryopreservation solution (solid line-gray square dots).

Compared with the common cell frozen stock solution of DMEM + 10% serum + 10% DMSO, the cell proliferation condition of the cells treated by the serum-free and DMSO-free cell frozen stock solution is better than the effect of the cells treated by the common cell frozen stock solution.

Step four,

1) The resuscitated human umbilical cord mesenchymal stem cell suspension in example 2 is inoculated in a 10cm cell culture dish.

2) And (3) carrying out induced differentiation on the human umbilical cord mesenchymal stem cells by using the mesenchymal stem cell adipogenic induced differentiation kit. The relevant procedures were performed strictly in accordance with commercial induction instructions.

3) After 7-10 days of induction, staining the induced fat droplets by using an oil red O staining kit (as shown in fig. 3A and 3B, fig. 3A is the induced adipogenic differentiation condition of the human umbilical mesenchymal stem cells recovered after the cryopreservation of the common cell cryopreservation liquid; FIG. 3B is the induced adipogenic differentiation of human umbilical cord mesenchymal stem cells that were recovered after cryopreservation with the serum-free and DMSO-free cell cryopreservation solution of the present invention. It can be seen from fig. 3 that the number of oil drops in fig. 3B is significantly greater than that in fig. 3A, indicating that the cells treated with the cell cryopreservation solution of the present invention have better totipotency and more prominent adipogenic induction effect. )

4) The recovered cell suspension of example 2 was seeded in a 10cm cell culture dish.

5) And (3) carrying out induced differentiation on the human umbilical cord mesenchymal stem cells by using a commercial chondrogenic induction kit. The relevant procedures were performed strictly in accordance with commercial induction instructions.

6) After 21 days of induction, induced chondrocytes are stained by using an alcian blue staining kit (as shown in fig. 4A and 4B, fig. 4A shows the induced chondrogenic differentiation condition of human umbilical cord mesenchymal stem cells recovered after cryopreservation of common cell cryopreservation liquid; FIG. 4B shows the induced chondrogenic differentiation of human umbilical cord mesenchymal stem cells (indicated by the arrow is the induced cartilage nodule) that has been restored after cryopreservation with the serum-free and DMSO-free cell cryopreservation solution of the present invention; as can be seen from the figure, the number and degree of cartilage formed in fig. 4B are significantly better than the cartilage effect in fig. 4A, indicating that the serum-free and DMSO-free cell culture medium of the present invention has more excellent effect of maintaining the characteristics of stem cells).

Claims (10)

1. A serum-free DMSO-free cell cryopreservation solution, which comprises the following components: the basal medium of DMEM/F-12 is 16.762-16.825g/L, the cell factor is 32-34mg/L, the hydroxyethyl starch is 0.2-0.8mg/L, the vitamin is 1-3mg/L, the antibiotic is 0.15-0.16g/L, the protein polypeptide is 5-6mg/L, the lipid is 1-4mg/L, and the cryoprotectant is 0.1-0.41g/L inside and outside the cell.

2. The serum-free DMSO-free cell cryopreservation solution of claim 1, wherein: the cytokine comprises the following components in percentage by mass: 99.955% of insulin, 0.03% of recombinant human epidermal growth factor and 0.015% of recombinant human basic fibroblast growth factor.

3. The serum-free DMSO-free cell cryopreservation solution of claim 1, wherein: the antibiotic is penicillin and streptomycin, the content of the penicillin is 0.06g/L, and the content of the streptomycin is 0.1 g/L.

4. The serum-free DMSO-free cell cryopreservation solution of claim 1, wherein: the vitamin is vitamin C.

5. The serum-free DMSO-free cell cryopreservation solution of claim 1, wherein: the protein polypeptide is albumin and transferrin, the adding amount of the albumin is 0.1-0.8 mug/L, and the adding amount of the transferrin is 5.1-5.2 mg/L.

6. The serum-free DMSO-free cell cryopreservation solution of claim 1, wherein: the lipid is ethanolamine and linoleic acid, the addition amount of the ethanolamine is 1-3mg/L, and the addition amount of the linoleic acid is 2-6 mug/L.

7. The serum-free DMSO-free cell cryopreservation solution of claim 1, wherein: the cryoprotectant inside and outside the cells is trehalose, polyvinylpyrrolidone and methylcellulose; the addition amount of the trehalose is 0.034-0.102g/L, the addition amount of the polyvinylpyrrolidone is 0.2-0.8mg/L, and the addition amount of the methylcellulose is 0.1-0.3 g/L.

8. A serum-free DMSO-free cell cryopreservation solution according to any one of claims 1 to 7 wherein: the serum-free DMSO-free cell cryopreservation solution comprises the following components: 110-116.6mg/L of anhydrous calcium chloride, 55-59.05mg/L of L-leucine, 0.03-0.042mg/L of linoleic acid, 0.0012-0.0013mg/L of blue vitriol, 90-91.25mg/L of L-lysine hydrochloride, 0.08-0.105mg/L of lipoic acid, 0.02-0.05mg/L of ferric nitrate nonahydrate, 15.00-17.24mg/L of L-methionine, 6.5-8.1mg/L of phenol red, 0.35-0.417mg/L of ferrous sulfate heptahydrate, 33-35.48mg/L of L-phenylalanine, 0.07-0.081mg/L of 1, 4-butanediamine dihydrochloride, 280-311.8mg/L of potassium chloride, 24-26.25mg/L of L-serine, 53-55mg/L of sodium pyruvate, 26.2-28.64mg/L of magnesium chloride, threonine 50.3-53.45mg/L, vitamin H0.0035-0.005 mg/L, anhydrous magnesium sulfate 47-48.84mg/L, alanine 4.45-4.50mg/L, calcium pantothenate 2.24-3.2mg/L, sodium chloride 6500-6999.5mg/L, L-asparagine 7.5-8.3mg/L, choline chloride 7.88-8.98mg/L, anhydrous sodium dihydrogen phosphate 51.2-54.35mg/L, aspartic acid 5.59-6.65mg/L, folic acid 2.65-3.25mg/L, disodium hydrogen phosphate 69.5-71.02mg/L, L-cysteine hydrochloride 16.88-17.56mg/L, I-inositol 11.3-12.6mg/L, zinc sulfate heptahydrate 0.432-0.551mg/L, glutamic acid 7.35-8.06mg/L, nicotinamide 2.02-2.45mg/L, 145-147.5mg/L of L-arginine hydrochloride, 15.9-17.25mg/L of L-proline, 1-2mg/L of pyridoxal hydrochloride, 31.29-32.85mg/L of L-cystine hydrochloride, 9.02-9.57mg/L of L-tryptophan hydrochloride, 0.031-0.036mg/L of pyridoxine hydrochloride, 378mg/L of L-glutamine 365, 38.4-39.1mg/L of L-tyrosine, 0.219-0.233mg/L of riboflavin, 18.75-19.11mg/L of glycine, 52.85-53.31mg/L of L-valine, 2.17-2.22mg/L of thiamine hydrochloride, 31.48-32.10mg/L of L-histidine hydrochloride, 3151mg/L of D-glucose, 3230mg/L of thymidine, 0.365-0.445mg/L of thymidine, l-isoleucine 54.47-55.2mg/L, hypoxanthine 1.55-2.03mg/L, vitamin B120.63-0.68mg/L, insulin 8-12mg/L, transferrin 5-6mg/L, ethanolamine 1-3mg/L, linoleic acid 2-6. mu.g/L, albumin 0.1-0.8. mu.g/L, recombinant human epidermal growth factor 0.1-0.5. mu.g/L, hydrocortisone 0.3-0.4mg/L, penicillin 0.06g/L, streptomycin 0.1g/L, vitamin C1-3mg/L, betaine 0.05-3mg/L, trehalose 0.1-0.3mM/L, methyl cellulose 0.1-0.3g/L, sodium bicarbonate 10-12.75mg/L, recombinant human basic fibroblast growth factor 0.1-0.2. mu.g/L, 0.2-0.8mg/L of hydroxyethyl starch and 0.2-0.8mg/L of polyvinylpyrrolidone.

9. The serum-free DMSO-free cell cryopreservation solution of claim 8, wherein: the serum-free DMSO-free cell cryopreservation solution comprises the following components: 116.6mg/L of anhydrous calcium chloride, 59.05mg/L of L-leucine, 0.042mg/L of linoleic acid, 00013mg/L of copper sulfate pentahydrate, 91.25mg/L of L-lysine hydrochloride, 0.105mg/L of lipoic acid, 0.05mg/L of ferric nitrate nonahydrate, 17.24mg/L of L-methionine, 8.1mg/L of phenol red, 0.417mg/L of ferrous sulfate heptahydrate, 35.48mg/L of L-phenylalanine, 0.081mg/L of 1, 4-butanediamine dihydrochloride, 311.8mg/L of potassium chloride, 26.25mg/L of L-serine, 55mg/L of sodium pyruvate, 28.64mg/L of magnesium chloride, 53.45mg/L of threonine, 0.0035mg/L of vitamin H, 48.84mg/L of anhydrous magnesium sulfate, 4.45mg/L of alanine, 2.24mg/L of calcium pantothenate, 6999.5mg/L of sodium chloride, 7.5mg/L of L-asparagine, 8.98mg/L of choline chloride, 54.35mg/L of anhydrous sodium dihydrogen phosphate, 6.65mg/L of aspartic acid, 2.65mg/L of folic acid, 71.02mg/L of disodium hydrogen phosphate, 17.56mg/L of L-cysteine hydrochloride, 12.6mg/L of I-inositol, 0.432mg/L of zinc sulfate heptahydrate, 7.35mg/L of glutamic acid, 2.02mg/L of nicotinamide, 147.5mg/L of L-arginine hydrochloride, 17.25mg/L of L-proline, 2mg/L of pyridoxal hydrochloride, 31.29mg/L of L-cystine hydrochloride, 9.02mg/L of L-tryptophan, 0.031mg/L of pyridoxine hydrochloride, 365mg/L of L-glutamine, 38.4mg/L of L-tyrosine, 0.219mg/L of riboflavin, 18.75mg/L of glycine, 52.85mg/L of L-valine, 2.17mg/L of thiamine hydrochloride, 31.48mg/L of L-histidine hydrochloride, 3151mg/L of D-glucose, 0.365mg/L of thymidine, 54.47mg/L of L-isoleucine, 2mg/L of hypoxanthine, 0.120.68mg/L of vitamin B, 8-12mg/L of insulin, 5-6mg/L of transferrin, 1-3mg/L of ethanolamine, 2-6. mu.g/L of linoleic acid, 0.1-0.8. mu.g/L of albumin, 0.1-0.2. mu.g/L of recombinant human epidermal growth factor, 0.3-0.4mg/L of hydrocortisone, 0.06g/L of penicillin, 0.1g/L of streptomycin, 1-3mg/L of vitamin C, 0.05-3mg/L of betaine, 0.1-0.3mM/L of trehalose, 0.1-0.3g/L of methylcellulose, 10-12.75mg/L of sodium bicarbonate, 0.1-0.2 mu g/L of recombinant human basic fibroblast growth factor, 0.2-0.8mg/L of hydroxyethyl starch and 0.4-0.8mg/L of polyvinylpyrrolidone.

10. A method of preparing a serum-free DMSO-free cell culture medium according to claims 1-9, comprising the steps of:

1) in a cGMP clean-grade workshop, taking DMEM/F-12 as a basic culture medium, then gradually adding penicillin, streptomycin, recombinant human epidermal growth factor, basic fibroblast growth factor, ethanolamine, betaine, albumin, vitamin C and hydroxyethyl starch, finally adjusting the pH to 7.4 +/-0.1 by using a 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, then respectively adding cryoprotectant components inside and outside cells according to a proportion, and uniformly mixing;

2) filtering and sterilizing the solution obtained in the step 1) by using a filter with the pore diameter less than or equal to 0.1 mu m to obtain the serum-free and DMSO-free cell frozen stock solution.

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