CN113621567B

CN113621567B - Composition and application thereof, stem cell culture medium and stem cell culture method

Info

Publication number: CN113621567B
Application number: CN202110780032.8A
Authority: CN
Inventors: 陈东煌; 陈海佳; 崔梓豪; 姜交华; 李学家; 戚康艺
Original assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Current assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Priority date: 2021-07-09
Filing date: 2021-07-09
Publication date: 2024-01-30
Anticipated expiration: 2041-07-09
Also published as: CN113621567A

Abstract

The invention relates to a composition and application thereof, a stem cell culture medium and a stem cell culture method, wherein the composition comprises serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal cell growth factor, and the composition does not contain serum. The composition has high safety when used in stem cell culture medium, and can delay stem cell aging.

Description

Composition and application thereof, stem cell culture medium and stem cell culture method

Technical Field

The invention relates to the technical field of biology, in particular to a composition and application thereof, a stem cell culture medium and a stem cell culture method.

Background

Stem cells (stem cells) are a class of pluripotent cells with self-replicating capacity. Under certain conditions, stem cells can differentiate into a variety of functional cells. Mesenchymal stem cells (Mesenchymal stem cells, MSC) are important members of the stem cell family, derived from early stages of development mesoderm and ectoderm. MSCs have the commonality of stem cells, namely the ability to self-renew, differentiate in multiple directions and home. Under a specific external differentiation environment of the organism, MSC can differentiate into various tissue cells such as nerves, hearts, bones, cartilages, fat, epithelium and the like, is considered as one of the most promising source cells of the cell therapy technology, and the MSC is taken as an important regenerative medical resource and has very important application value in the clinical disease treatment field.

Currently, in vitro expansion of MSCs is performed in medium supplemented with Fetal Bovine Serum (FBS), human autologous serum or serum analogs. However, from the perspective of potential therapeutic applications of MSC cultures, the presence of animal or human serum in MSC cultures has the following drawbacks: first, bovine serum, human serum, or other animal serum may contain blood-borne pathogens, such as viruses and mad cow disease prions, bovine Spongiform Encephalopathy (BSE), and the like. Second, animal serum may elicit the production of antibodies against xenobiotics, which may elicit an immune response in the recipient patient. Third, animal serum shows batch-to-batch variability, possibly leading to inconsistent performance.

In view of this, a large number of serum-free medium products have been developed. It is widely used in culturing mammalian and invertebrate cells to produce monoclonal antibodies, viral antigens, recombinant proteins, and the like. Currently, most serum-free media contain proteins such as transferrin that transports ions into cells, insulin and albumin that regulate glucose uptake, fibrin, fetuin, and the like, and proteins added to serum-free media perform a variety of functions in cell culture, such as providing a matrix for cell attachment, resisting bioreactor shear forces, and serving as carriers for lipids and other growth differentiation factors. However, current serum-free media are prone to rapid aging of stem cells during culture expansion.

Disclosure of Invention

Based on this, it is necessary to provide a composition which is highly safe and can slow down the aging of stem cells.

A composition comprising serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor.

The composition can slow down the aging speed of stem cells in a serum-free culture medium after being added into the serum-free culture medium through the mutual coordination of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal cell growth factor; the composition does not contain serum, and has high safety; in addition, when the serum-free culture medium added with the composition is used, the fibronectin is not needed to coat the culture container, so that the flow of culturing stem cells is simplified, and the cost is saved.

In one embodiment, the mass ratio of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor is (0.5 μg to 50 μg): (10 ng-30 ng): (0.3 ng-1 ng): (5 ng-15 ng), said composition being serum-free.

In one embodiment, the mass ratio of serum albumin to basic fibroblast growth factor is (1 μg to 10 μg): (15 ng-20 ng);

and/or the mass ratio of serum albumin to transforming growth factor beta 1 is (1 mug-10 mug): (0.5 ng-0.75 ng);

and/or the mass ratio of serum albumin to epidermal cell growth factor is (1 mu g-10 mu g): (7.5 ng-10 ng).

In one embodiment, the composition further comprises at least one of an antioxidant, an antibiotic, transferrin, dexamethasone, and a lipid.

In one embodiment, the antioxidant is selected from at least one of ascorbic acid 2-phosphate, superoxide dismutase, catalase, and glutathione peroxidase.

In one embodiment, the antibiotic is selected from at least one of penicillin and streptomycin.

In one embodiment, the composition comprises an antioxidant in a molar amount to the mass of serum albumin ratio of (150 nmol to 250 nmol): (0.5-50. Mu.g);

and/or, the composition comprises an antibiotic, the ratio of the potency of the antibiotic to the mass of serum albumin (100U to 2100U): (0.5-50. Mu.g);

and/or, the composition comprises transferrin, the ratio of the volume of transferrin to the mass of serum albumin is 0.01mL: (0.5-50. Mu.g);

and/or, the composition comprises dexamethasone, the ratio of the molar quantity of dexamethasone to the mass of the serum albumin (50-150 pmol): (0.5-50. Mu.g);

and/or, the composition comprises a lipid substance, the ratio of the lipid substance to the mass of serum albumin (30 pg to 50 pg): (0.5-50. Mu.g).

The use of the above composition in a stem cell culture medium.

A stem cell culture medium comprising the composition described above and a serum-free medium;

in one embodiment, the serum-free medium is alpha-MEM.

In one embodiment, the stem cell culture medium may increase proliferation and/or delay stem cell senescence.

In one embodiment, the final concentration of serum albumin is 0.5 μg/mL-50 μg/mL, the final concentration of basic fibroblast growth factor is 10 ng/mL-30 ng/mL, the final concentration of transforming growth factor beta 1 is 0.3 ng/mL-1 ng/mL, and the final concentration of epidermal cell growth factor is 5 ng/mL-15 ng/mL.

In one embodiment, the serum-free medium is a serum-free mesenchymal stem cell medium.

In one embodiment, the serum-free medium is DMEM/F12, alpha-MEM or DMEM.

A method of culturing stem cells, comprising the steps of:

the mesenchymal stem cells are inoculated into the stem cell culture medium for culture.

In one embodiment, the stem cells are mesenchymal stem cells.

Drawings

FIG. 1 is a mesenchymal stem cell in the stem cell medium of examples 1 to 5;

FIG. 2 shows mesenchymal stem cells in the stem cell medium of examples 6 to 8;

FIG. 3 shows mesenchymal stem cells in the stem cell medium of examples 9 to 11 and comparative example 1;

FIG. 4 is a photograph showing the result of staining mesenchymal stem cells with beta-galactosidase in the stem cell culture medium of examples 1 to 8 and comparative example 1.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the invention, which may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

In one embodiment, the present invention provides a composition comprising the following components: serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor.

The three growth factors, namely the basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal cell growth factor, have bidirectional effect and time effect on cells when used independently, and particularly can promote the rapid proliferation of the cells and accelerate the aging of the cells. The composition can slow down the aging speed of the stem cells after being added into a serum-free stem cell culture medium through the mutual coordination of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal cell growth factor, and the composition does not contain serum and has high safety. The composition has been shown to slow down mesenchymal stem cell aging when added to serum-free stem cell medium.

Optionally, the mass ratio of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor is (0.5 μg-50 μg): (10 ng-30 ng): (0.3 ng-1 ng): (5 ng-15 ng), the composition being serum-free.

In this embodiment, the serum albumin is recombinant human serum albumin; the basic fibroblast growth factor is recombinant human basic fibroblast growth factor (bFGF); the recombinant human serum albumin, the recombinant human basic fibroblast growth factor, the transforming growth factor beta 1 and the epidermal growth factor can be recombinant proteins produced by adopting a fermentation technology.

In one embodiment, the mass ratio of serum albumin to basic fibroblast growth factor is (1 μg to 20 μg): (10 ng-20 ng). Further, the mass ratio of serum albumin to basic fibroblast growth factor is (1 μg-10 μg): (15 ng-20 ng). Further, the mass ratio of serum albumin to basic fibroblast growth factor is (1. Mu.g-5. Mu.g): (15 ng-20 ng).

In one embodiment, the mass ratio of serum albumin to transforming growth factor beta 1 is (1 μg to 10 μg): (0.5 ng-1 ng). Further, the mass ratio of serum albumin to transforming growth factor beta 1 is (1. Mu.g-10. Mu.g): (0.5 ng-0.75 ng). Further, the mass ratio of serum albumin to transforming growth factor beta 1 is (1. Mu.g to 5. Mu.g): (0.5 ng-1 ng).

In one embodiment, the mass ratio of serum albumin to epidermal cell growth factor is (1 μg to 10 μg): (5 ng-10 ng). Further, the mass ratio of serum albumin to epidermal cell growth factor is (1 μg-10 μg): (7.5 ng-10 ng). Further, the mass ratio of serum albumin to epidermal growth factor is (1. Mu.g to 5. Mu.g): (5 ng-10 ng).

In one embodiment, the mass ratio of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor is (1 μg to 10 μg): (10 ng-20 ng): (0.5 ng-1 ng): (5 ng-10 ng). Further, the mass ratio of serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal cell growth factor is (1 μg-5 μg): (15 ng-20 ng): (0.5 ng-1 ng): (5 ng-10 ng).

In one embodiment, the composition is free of fibronectin (Fn).

In one embodiment, the above composition consists of the following components: serum albumin, basic fibroblast growth factor, transforming growth factor beta 1 and epidermal growth factor.

In some embodiments, the above composition further comprises at least one of an antioxidant, an antibiotic, transferrin, dexamethasone, and a lipid.

In particular, antioxidants are used to further reduce the damage of oxygen radicals to stem cells and slow down stem cell aging. Optionally, the antioxidant is selected from at least one of ascorbate 2-phosphate, superoxide dismutase, catalase, and glutathione peroxidase. Of course, in other embodiments, the antioxidant is not limited to the above-indicated substances, but may be other substances having an antioxidant effect.

Alternatively, the ratio of the molar amount of antioxidant to the mass of serum albumin is (150 nmol to 250 nmol): (0.5-50. Mu.g). Further, the ratio of the molar amount of the antioxidant to the mass of serum albumin is (150 nmol to 200 nmol): (0.5. Mu.g to 20. Mu.g) further, the ratio of the molar amount of the antioxidant to the mass of serum albumin is (150 nmol to 200 nmol): (0.5-10. Mu.g).

In particular, transferrin is used to prevent free radical production, protect cell growth, and maintain cell growth and proliferation. In this embodiment, the transferrin is recombinant transferrin.

In one example, the ratio of the volume of transferrin to the mass of serum albumin is 0.01mL: (0.5-50. Mu.g). In one specific example, the Transferrin is Insulin-Transferrin-Selenium (ITS).

Specifically, dexamethasone is an anti-inflammatory glucocorticoid that exerts a number of physiological effects on cell survival, cell signaling and gene expression, induces the production of phospholipase A2 inhibitor protein (lipo Pi Zhisu), and inhibits nitric oxide synthase-inducing activity.

In one example, the ratio of the molar amount of dexamethasone to the mass of serum albumin (50 to 150 pmol): (0.5-50. Mu.g). Further, the ratio of the molar amount of dexamethasone to the mass of serum albumin (50 to 100 pmol): (0.5-10. Mu.g). Further, the ratio of the molar amount of dexamethasone to the mass of serum albumin (50 to 90 pmol): (0.5-5. Mu.g).

In particular, antibiotics are used to inhibit the growth of bacteria in culture media. Optionally, the antibiotic is at least one selected from penicillin and streptomycin. Further, the antibiotics are a mixture of penicillin and streptomycin, and the ratio of the titer of penicillin to the mass of serum albumin (100U-200U): (0.5-50. Mu.g), the ratio of the potency of streptomycin to the mass of serum albumin (100-2000U): (0.5-50. Mu.g). Of course, in other embodiments, the antibiotics are not limited to the above, but may be other antibiotics.

In one embodiment, the ratio of the potency of the antibiotic to the mass of serum albumin (100U-2000U): (0.5-50. Mu.g). Further, the ratio of the potency of the antibiotic to the mass of serum albumin (100U-500U): (0.5-20. Mu.g). Further, the ratio of the potency of the antibiotic to the mass of serum albumin (100U-200U): (0.5-10. Mu.g).

Optionally, the lipid substance is at least one selected from linoleic acid, cholesterol and VE. It will be appreciated that in other embodiments, the lipid material is not limited to the above, but may be other materials.

In one embodiment, the ratio of lipid to serum albumin mass (30 pg to 50 pg): (0.5-50. Mu.g). Further, the ratio of the lipid substance to the mass of serum albumin (35 pg to 45 pg): (0.5-20. Mu.g). Further, the ratio of the lipid substance to the mass of serum albumin (35 pg to 45 pg): (0.5-10. Mu.g).

In one embodiment, the above composition consists of the following components: serum albumin, basic fibroblast growth factor, transforming growth factor beta 1. Epidermal growth factor, antioxidants, antibiotics, transferrin, dexamethasone, and lipid substances. The composition has the advantages of less component types, small batch-to-batch difference and high stability.

The composition can be added into a stem cell culture medium to slow down the aging speed of cells, and therefore, one embodiment of the invention also provides the application of the composition in the stem cell culture medium. In particular to the application of the composition in preparing a stem cell culture medium. In one example, the stem cells are mesenchymal stem cells. More specifically, the above composition is added to a stem cell culture medium to prepare the stem cell culture medium.

In addition, an embodiment of the invention also provides a preparation method of the composition, which comprises the following steps: the components of the composition are uniformly mixed to prepare the mixture.

The preparation method of the composition is simple and easy to operate, and is easy for large-scale production.

In addition, an embodiment of the invention also provides a stem cell culture medium, which comprises the composition and a serum-free culture medium.

In particular, the serum-free medium is a serum-free mesenchymal stem cell medium. Alternatively, the serum-free medium is DMEM/F12, alpha-MEM or DMEM. Of course, in other embodiments, the serum-free medium is not limited to the above, but may be other serum-free media.

In one embodiment, the final concentration of serum albumin in the stem cell culture medium is 0.5-50. Mu.g/mL, the final concentration of basic fibroblast growth factor is 10-30 ng/mL, the final concentration of transforming growth factor beta 1 is 0.3-1 ng/mL, and the final concentration of epidermal growth factor is 5-15 ng/mL. Alternatively, in the stem cell culture medium, the final concentration of serum albumin is 1 mu g/mL-10 mu g/mL, the final concentration of basic fibroblast growth factor is 15 ng/mL-20 ng/mL, the final concentration of transforming growth factor beta 1 is 0.5 ng/mL-0.75 ng/mL, and the final concentration of epidermal cell growth factor is 7.5 ng/mL-10 ng/mL. Alternatively, in the stem cell culture medium, the final concentration of serum albumin is 1 mu g/mL-5 mu g/mL, the final concentration of basic fibroblast growth factor is 10 ng/mL-20 ng/mL, the final concentration of transforming growth factor beta 1 is 0.5 ng/mL-1 ng/mL, and the final concentration of epidermal cell growth factor is 5 ng/mL-10 ng/mL.

In one embodiment, the composition includes an antioxidant, where the final concentration of antioxidant in the stem cell culture medium is 150nmol/mL to 250nmol/mL. Further, in the stem cell culture medium, the final concentration of the antioxidant is 150nmol/mL to 200nmol/mL.

In one embodiment, the composition comprises transferrin, wherein the final concentration of transferrin by volume in the stem cell culture medium is 1%.

In one embodiment, the composition comprises dexamethasone, wherein the final concentration of dexamethasone in the stem cell culture medium is in the range of 50pmol/mL to 150pmol/mL. Further, in the stem cell culture medium, the final concentration of dexamethasone is 50 to 100pmol/mL. Further, in the stem cell culture medium, the final concentration of dexamethasone is 50 to 90pmol/mL.

In one embodiment, the composition comprises an antibiotic, wherein the titer of the antibiotic in the stem cell culture medium is 100U/mL to 2100U/mL. Further, in the stem cell culture medium, the titer of the antibiotic is 100U/mL-500U/mL. Further, in the stem cell culture medium, the titer of the antibiotic is 100U/mL to 200U/mL.

In one embodiment, the composition includes lipid material at a final concentration of 30pg/mL to 50pg/mL in the stem cell culture medium. Further, in the stem cell culture medium, the final concentration of the lipid substance is 35pg/mL to 45pg/mL.

In one embodiment, the stem cell culture medium is free of fibronectin (Fn).

In one embodiment, the stem cell culture medium consists of the composition and a serum-free medium.

The stem cell culture medium does not contain serum, comprises the composition, and can slow down cell aging and has high safety when used for culturing cells. In addition, when the stem cell culture medium is used, the culture container is not required to be pretreated with fibronectin so that the fibronectin is coated on the culture container, and the stem cell culture medium can be directly cultured by using an empty culture container, so that the time and the cost are saved. In addition, the stem cell culture medium has definite components, few component types, small batch-to-batch difference and high stability.

In addition, an embodiment of the invention also provides a preparation method of the stem cell culture medium, which comprises the following steps: the above composition was added to a serum-free medium to prepare the above stem cell medium. The amount of the above composition added may be adjusted according to the final concentration of each component in the stem cell culture medium to be prepared.

The preparation method of the stem cell culture medium is simple and easy to implement, and is easy for industrial production, and the prepared stem cell culture medium can be used for stem cell culture and can slow down the aging of stem cells.

In addition, an embodiment of the present invention provides a method for culturing stem cells, comprising the steps of: the stem cells are inoculated into the above-mentioned stem cell medium for culturing, wherein the culture vessel for culturing the stem cells does not have to be pre-coated with fibronectin.

Alternatively, the stem cells are mesenchymal stem cells. Further, the mesenchymal stem cells are umbilical cord mesenchymal stem cells. Of course, the mesenchymal stem cells are not limited to umbilical cord mesenchymal stem cells, but may be other mesenchymal stem cells.

Alternatively, the mesenchymal stem cells may be seeded at 0.6X10 ⁴ Individual/cm ² ～1×10 ⁴ Individual/cm ² 。

In one embodiment, the cryopreserved stem cells are thawed and inoculated into a culture vessel containing the stem cell culture medium described above for culturing, wherein the culture vessel is not subjected to pretreatment with coated fibronectin (i.e., the culture vessel is not coated with fibronectin).

The stem cell culture method is simple and quick, and the stem cells cultured by the stem cell culture method are not easy to age.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer. TL-108 of Wo Kawei recombinant human serum albumin in the following examples and comparative examples), 100-18B of Peprotech as recombinant human basic fibroblast factor, 100-36E of Peprotech as transforming growth factor beta 1, GF316 of Sigma-Aldrich as epidermal cell growth factor, gibco as lipid substance, unless otherwise specified ^TM Chemically Defined Lipid Concentrate Insulin-Transferrin-Selenium (ITS) product under the trade designation 11905031 is the PB 180131 of the Procell.

Examples 1 to 11

The stem cell culture medium of each example consists of the composition and the α - Μ culture medium, and the components of the composition and the final concentrations in the stem cell culture medium of each example are shown in table 1. Wherein the compositions in the stem cell culture medium of examples 1 to 10 all consist of recombinant human serum albumin, recombinant human basic fibroblast factor, transforming growth factor beta 1, epidermal cell growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and ITS; the stem cell culture medium of example 11 consists of recombinant human serum albumin, recombinant human basic fibroblast factor, transforming growth factor beta 1, epidermal growth factor and alpha- Μ E.

The preparation method of the stem cell culture medium of each embodiment includes, but is not limited to, the following steps:

the components of the compositions were weighed according to the final concentrations shown in table 1 and mixed with α - Μ, e.g., m medium, respectively, to prepare stem cell culture media of each example.

Comparative example 1

The stem cell culture medium of comparative example 1 consists of a composition and an alpha- Μ culture medium, and the specific composition and content are shown in table 1.

The preparation method of the stem cell culture medium of comparative example 1 includes, but is not limited to, the following steps:

the components were weighed according to the final concentrations shown in table 1 and mixed with an α - Μ culture medium to prepare the stem cell culture medium of comparative example 1.

Comparative example 2

The composition of the stem cell culture medium of this comparative example was substantially the same as that of example 11, except that the epidermal growth factor of example 11 was replaced with the same amount of DGEA peptide. In this comparative example, the composition of the stem cell medium is: recombinant serum albumin, recombinant basic fibroblast growth factor, transforming growth factor beta 1, DGEA peptide and alpha-MEm culture medium, wherein the final concentration of the recombinant serum albumin is 2 mug/mL, the final concentration of the recombinant basic fibroblast growth factor is 20ng/mL, the final concentration of the transforming growth factor beta 1 is 0.5ng/mL, and the final concentration of the DGEA peptide is 5ng/mL.

The preparation method of the stem cell culture medium of comparative example 2 includes, but is not limited to, the following steps:

the components were weighed according to the final concentration and mixed with the α - Μ -culture media to prepare the stem cell culture media of this comparative example.

Comparative example 3

The composition of the stem cell culture medium of this comparative example was substantially the same as in example 1, except that recombinant serum albumin was omitted. In this comparative example, the stem cell culture medium consisted of recombinant human basic fibroblast factor, transforming growth factor beta 1, epidermal growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and ITS, and the contents of the respective components are shown in table 1 in detail.

Comparative example 4

The composition of the stem cell culture medium of this comparative example was substantially the same as in example 1, except that the recombinant human basic fibroblast growth factor was omitted. In this comparative example, the stem cell culture medium consisted of recombinant human serum albumin, transforming growth factor beta 1, epidermal growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and ITS, and the contents of the respective components are shown in table 1 in detail.

Comparative example 5

The composition of the stem cell culture medium of this comparative example was substantially the same as in example 1, except that the transforming growth factor β1 was omitted. In this comparative example, the stem cell culture medium consisted of recombinant human serum albumin, recombinant human basic fibroblast growth factor, epidermal cell growth factor, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and ITS, and the contents of the components are shown in table 1 in detail.

Comparative example 6

The composition of the stem cell culture medium of this comparative example was substantially the same as in example 1, except that the epidermal growth factor was omitted. In this comparative example, the stem cell culture medium consisted of recombinant human serum albumin, recombinant human basic fibroblast growth factor, transforming growth factor beta 1, ascorbic acid 2-phosphate, penicillin, streptomycin, dexamethasone, lipid substances and ITS, and the contents of the components are shown in table 1 in detail.

TABLE 1

Testing

(1) The frozen 3 rd generation umbilical cord mesenchymal stem cells are taken out and thawed in a water bath at 37 ℃ to form a cell suspension.

(2) Slowly adding the cell suspension obtained in the step (1) into a prepared centrifuge tube (10 mL of DPBS is added in advance) in an ultra clean bench; sampling and counting after uniform re-suspension.

(3) Centrifuging the cell suspension re-suspended in the step (2) at 1200rpm for 5min, and discarding the supernatant; then adding a proper amount of DPBS to resuspend the cells, centrifuging at 1200rpm for 5min, and discarding the supernatant to remove residual FBS;

(4) Based on the counting results, the respective examples and comparative examples were usedCorresponding cell suspensions were prepared from the medium of each of the examples and comparative examples at a cell concentration of 1.0X10 ⁶ Individual cells/mL.

(5) According to 1.0X10 ⁴ Individual cells/cm ² The cells were inoculated uniformly into 6-well plates (uncoated fibronectin) and 3 parallel wells were simultaneously set.

(6) Placing at 37 deg.C, 5% CO ₂ Culturing in a cell culture incubator, and changing liquid every day on the 2 nd day. And the following evaluation tests were performed:

cell morphology observations

The operation method comprises the following steps: the cells cultured to day 1, day 2 and day 3 of each group were inverted and observed under a microscope for each group of cell morphology and image acquisition, and the results are shown in fig. 1 to 3.

As can be seen from fig. 1 to 3, the stem cell culture media of examples 1 to 11 can be used for culturing the mesenchymal stem cells after resuscitation, wherein the increase in the number of the mesenchymal stem cells is remarkable in the stem cell culture media of examples 3, 5, 7 and 8.

(II) cell expansion fold determination

The operation method comprises the following steps: cells were collected after 3 days of culture for each group, the collection amount was counted using a hemocytometer, and the expansion ratio of the collection amount to the inoculation amount, which is the expansion ratio of cells, was calculated, and the results are shown in table 2.

TABLE 2

As can be seen from table 2, the stem cell culture media of examples 1 to 11 can be used for culturing the recovered mesenchymal stem cells, wherein the expansion ratio of the mesenchymal stem cells in the stem cell culture medium of example 5 is the highest, followed by example 3.

(III) cell senescence assay

The operation method comprises the following steps: after 3 days of culture, each group was stained with beta-galactosidase assay to determine the degree of cell senescence, and the cell surface antigen CD146 was detected using a flow cytometer. The results are shown in FIG. 4 and Table 3.

TABLE 3 Table 3

In FIG. 4, the black part (blue in the color chart) shows senescent cells (stained with β -galactosidase), and as can be seen from FIG. 4, the stem cell culture medium of example 5 shows minimal senescent cells, and examples 3 and 7 times.

CD146 is a surface antigen marker associated with cellular senescence, with lower numbers indicating faster cellular senescence. As can be seen from Table 3, the CD146 ratio was higher in examples 1 to 11, and the CD146 ratio was the highest in example 5.

In summary, it was found that the mesenchymal stem cells cultured using the stem cell medium of examples 1 to 11 had high expansion factor and slow aging, and it was not necessary to coat fibronectin in advance in the culture vessel.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A stem cell culture medium, comprising a composition and a serum-free medium;

the composition consists of recombinant human serum albumin, recombinant human basic fibroblast growth factor, transforming growth factor beta 1, epidermal cell growth factor, antioxidant, antibiotic, transferrin, dexamethasone and lipid substances;

the serum-free medium is alpha-MEM; the antioxidant is selected from ascorbic acid 2-phosphate; the antibiotic is selected from penicillin and streptomycin; the transferrin is selected from insulin-transferrin-selenium; the lipid material is a lipid concentrate;

the final concentration of the recombinant human serum albumin is 0.5-50 mu g/mL, the final concentration of the recombinant human basic fibroblast growth factor is 10-30 ng/mL, the final concentration of the transforming growth factor beta 1 is 0.3-1 ng/mL, and the final concentration of the epidermal cell growth factor is 5-15 ng/mL.

2. The stem cell culture medium according to claim 1, wherein the mass ratio of recombinant human serum albumin, recombinant human basic fibroblast growth factor, transforming growth factor β1 and epidermal cell growth factor is 2 μg:20ng:0.5ng:5ng.

3. The stem cell culture medium according to claim 1 or 2, wherein the ratio of the molar amount of the antioxidant to the mass of the recombinant human serum albumin is (150 nmol to 250 nmol): (0.5-50. Mu.g);

and/or the ratio of the titer of the antibiotic to the mass of the recombinant human serum albumin (100U-2100U): (0.5-50. Mu.g);

and/or, the ratio of the volume of the transferrin to the mass of the recombinant human serum albumin is 0.01mL: (0.5-50. Mu.g);

and/or, the ratio of the molar amount of dexamethasone to the mass of the recombinant human serum albumin (50-150 pmol): (0.5-50. Mu.g);

and/or, the ratio (30 pg to 50 pg) of the lipid substance to the mass of the recombinant human serum albumin: (0.5-50. Mu.g).

4. The stem cell culture medium of claim 1 or 2, wherein the stem cells are umbilical mesenchymal stem cells.

5. The stem cell culture medium according to claim 1 or 2, wherein the stem cell culture medium can increase proliferation and/or delay senescence of umbilical mesenchymal stem cells.

6. A method of culturing stem cells, comprising the steps of:

inoculating stem cells into the stem cell culture medium according to any one of claims 1-5 for culturing; the stem cells are umbilical cord mesenchymal stem cells.