CN112322581A

CN112322581A - Composition and application thereof, cell culture medium and recovery method of mesenchymal stem cells

Info

Publication number: CN112322581A
Application number: CN202010959298.4A
Authority: CN
Inventors: 陈东煌; 陈海佳; 戚康艺; 姜交华; 王小燕
Original assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Current assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2021-02-05
Anticipated expiration: 2040-09-14
Also published as: CN112322581B

Abstract

The present invention relates to a composition and its application, a cell culture medium and a method for resuscitating mesenchymal stem cells. The composition includes progesterone, estradiol, vitronectin, collagen and L-ascorbic acid; adding the composition to a serum-free culture system can improve the adherence rate of mesenchymal stem cells during recovery.

Description

Composition and application thereof, cell culture medium and recovery method of mesenchymal stem cells

Technical Field

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

Background

Mesenchymal Stem Cells (MSCs) are derived from the mesoderm in the early development stage, are a type of non-hematopoietic stem cells, and are widely present in bone marrow, subcutaneous fat, periosteum, muscle, synovium, synovial fluid, liver, peripheral tissues, umbilical cord blood, placenta and other tissues. The MSCs have high self-renewal capacity and multidirectional differentiation potential, can support the growth of hematopoietic stem cells and also have the function of immune regulation. MSCs can differentiate into bone, cartilage, muscle, nerve, cardiac muscle, endothelium and fat in vitro under different inducing conditions. The MSCs still have multidirectional differentiation potential after continuous subculture and cryopreservation, are ideal seed cells for researching tissue and organ damage repair caused by aging and lesion, are also first-choice seed cells for cell replacement therapy and tissue engineering, and have wide clinical application prospects.

The content of MSCs in human bodies is very small, and if the MSCs are used as medicines for clinical application, the MSCs need to be cultured and amplified in vitro in a large scale to obtain enough cell quantity capable of playing a role in treatment. Currently, two types of culture systems are mainly used for in vitro culture of MSCs: one is a serum culture system using a basal medium plus animal serum (e.g., fetal bovine serum, FBS), and the other is a serum-free culture system using a basal medium plus a serum substitute. The serum-free culture system has clear components and is the main flow direction of the in vitro culture of the MSCs at present.

MSCs are adherent growth type cells, and can enter normal vital metabolic processes when cultured in vitro by attaching to the surface of a culture vessel and expanding into its original form. Cell adhesion is not a process that requires energy, but rather is related to the charge carried by the cell surface. The cells firstly secrete extracellular matrix proteins to be adhered to the bottom surface of the culture vessel, and the cells are combined with the extracellular matrix proteins through the proteins on the surfaces of the cells to complete the adhesion process. When the MSCs are cultured in vitro, the MSCs frozen in liquid nitrogen often need to be recovered, and the life activities just recovered are not activated and still in a resting state, so that adhesion factors cannot be secreted through the metabolic activities of the MSCs to complete attachment.

At present, in order to improve the anchorage rate of MSCs during recovery, the following two methods are mainly adopted for recovering the MSCs: firstly, coating a layer of adhesive protein on the surface of a culture dish in advance, and then using the culture dish to recover the MSCs. Although the method has good effect when the MSCs are recovered, the operation of pre-coating the adhesion protein on the surface of the culture dish is relatively complicated, and the method is very inconvenient for the in vitro large-scale culture of the MSCs and is not beneficial to the pharmaceutical industrialization and clinical application of the MSCs. Secondly, the culture medium is supplemented with adhesion protein, for example, a traditional serum culture system is adopted, although FBS in the traditional serum culture system contains a plurality of proteins which can promote the adhesion of the MSCs, the FBS has very complex components and not only contains factors suitable for cell adhesion and growth, but also contains factors not beneficial to cell adhesion and growth, so that the effect of improving the adhesion of the recovered MSCs is not obvious when the MSCs are recovered, and the clinical application of the traditional serum culture system is limited due to the complexity of the components of the serum.

Disclosure of Invention

Therefore, the composition is required to be provided, the composition can be added into a serum-free culture system to improve the anchorage rate of the mesenchymal stem cells during recovery, has definite components and high safety, and is beneficial to large-scale culture and clinical application of the mesenchymal stem cells in vitro.

A composition comprising progesterone, estradiol, vitronectin, collagen, and L-ascorbic acid.

The composition enables the mesenchymal stem cells to have high adherence rate and high survival rate when the mesenchymal stem cells are revived by using the mesenchymal stem cell culture medium through the mutual matching of progesterone, estradiol, vitronectin, collagen and L-ascorbic acid. The composition has definite components and high safety, and is beneficial to in-vitro large-scale culture and clinical application of mesenchymal stem cells.

In one embodiment, the composition is a powder or solution; and/or the estradiol is beta-estradiol, and the collagen is type IV collagen.

The use of the above composition in cell culture media.

In one embodiment, the estradiol is beta-estradiol, the collagen is type iv collagen, and the working concentration of the composition is in the following range: 1 nM-10 nM of progesterone, 0.4 nM-4 nM of beta-estradiol, 20 ug/L-200 ug/L of vitronectin, 20 ug/L-200 ug/L of IV type collagen, 10 mg/L-100 mg/L of L-ascorbic acid;

preferably, the working concentration of the composition is in the following range: the working concentration of progesterone is 2 nM-5 nM; the working concentration of the beta-estradiol is 1nM to 2 nM; the working concentration of the vitronectin is 50 mug/L to 100 mug/L; the working concentration of the IV type collagen is 50 mug/L to 100 mug/L; the working concentration of the L-ascorbic acid is 25 mg/L-50 mg/L;

preferably, the working concentration of the composition when culturing cells is in the following range: the working concentration of progesterone is 5nM, the working concentration of beta-estradiol is 2nM, the working concentration of vitronectin is 100 mug/L, the working concentration of type IV collagen is 100 mug/L, and the working concentration of L-ascorbic acid is 50 mg/L.

A cell culture medium comprises the composition and a serum-free culture medium.

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

A method for resuscitating mesenchymal stem cells comprises the following steps:

the frozen mesenchymal stem cells are re-melted and then inoculated into the cell culture medium.

In one embodiment, the density of the seeding is 0.6 × 10⁴Per cm²～1×10⁴Per cm²。

In one embodiment, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells.

In one embodiment, the re-fusion is performed in a 37 ℃ water bath.

Drawings

FIG. 1 is a diagram showing the cell morphology of mesenchymal stem cells corresponding to each group of culture media after 4 hours of inoculation in example 2;

FIG. 2 is a diagram showing the cell morphology of mesenchymal stem cells corresponding to each group of culture media after 8 hours of inoculation in example 2;

FIG. 3 shows the cell morphology of mesenchymal stem cells corresponding to each group of culture media after 12h of inoculation in example 2;

FIG. 4 is a bar graph of the anchorage rate of mesenchymal stem cells corresponding to each group of media 4h after inoculation in example 2;

FIG. 5 is a bar graph of the anchorage rate of mesenchymal stem cells corresponding to each set of media 8h after inoculation in example 2;

FIG. 6 is a bar graph of the anchorage rate of mesenchymal stem cells corresponding to each group of media 12h after inoculation in example 2;

FIG. 7 is a bar graph of the viability of mesenchymal stem cells corresponding to each set of medium of example 3;

fig. 8 to 11 are flow-type detection results of surface markers of mesenchymal stem cells of control group 1, control group 3, experimental group 4 and experimental group 5, respectively, in example 4;

fig. 12 is the expression level results of Oct4, Nanog, and Sox2 in example 5.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth 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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

One embodiment of the invention provides a cell culture medium comprising a composition and a serum-free medium. The cell culture medium can improve the anchorage rate of the mesenchymal stem cells during recovery, improve the survival rate of the mesenchymal stem cells, realize the rapid proliferation of the mesenchymal stem cells, and simultaneously, does not influence the biological characteristics of the mesenchymal stem cells.

Specifically, the composition comprises progesterone, estradiol, vitronectin, collagen, and L-ascorbic acid. Further, the estradiol is beta-estradiol, and the collagen is type IV collagen. In a cell culture medium, the concentration of progesterone is 1 nM-10 nM, the concentration of beta-estradiol is 0.4 nM-4 nM, the concentration of vitronectin is 20 mug/L-200 mug/L, the concentration of IV type collagen is 20 mug/L-200 mug/L, and the concentration of L-ascorbic acid is 10 mg/L-100 mg/L. That is, the working concentration of the composition when culturing cells is in the following range: 1 nM-10 nM of progesterone, 0.4 nM-4 nM of beta-estradiol, 20-200 mug/L of vitronectin, 20-200 mug/L of IV type collagen, 10-100 mg/L of L-ascorbic acid.

Progesterone, also known as progesterone, is a steroid hormone, and is used as a stimulating factor in the composition, and has the functions of stimulating the growth of mesenchymal stem cells and promoting the adhesion of mesenchymal stem cells. The mechanism for stimulating the growth of the mesenchymal stem cells and promoting the adherence of the mesenchymal stem cells by progesterone comprises two mechanisms: the first is that the signal pathway of ERK1/2 is activated to promote the synthesis of fibronectin by MSC; the second is to promote the cell proliferation of the mesenchymal stem cell by regulating the autocrine or paracrine function of the cell through PR reaction. In an alternative specific example, the concentration of progesterone in the cell culture medium is 1nM, 2nM, 2.5nM, 3nM, 3.5nM, 4nM, 4.5nM, 5nM, 6nM, 7nM, 8nM, 9nM, or 10 nM. Further, the concentration of progesterone in the cell culture medium is 1nM to 7 nM. Further, the concentration of progesterone in the cell culture medium is 2nM to 5 nM.

Beta-estradiol is a steroid hormone, and is used in combination with progesterone in the above composition to stimulate the adherence and growth of mesenchymal stem cells, and its action mechanism also includes two types: the first is to activate intracellular signal path (MAPK/ERK, PI3K/AKT, cAMP, PKA, PKC, etc.) by binding with ER receptor, regulate gene expression and promote mesenchymal stem cell proliferation; the second is activation of the independent signaling pathway of the ER in the cytoplasm by binding to the ER receptor to exert antioxidant effects. In an alternative embodiment, the concentration of β -estradiol in the cell culture medium is 0.4nM, 1nM, 1.5nM, 2nM, 2.5nM, 3nM, 3.5nM or 4 nM. Further, the concentration of β -estradiol in the cell culture medium is 1nM to 3 nM. Further, the concentration of β -estradiol in the cell culture medium is 1nM to 2 nM.

In the composition, the vitronectin and the type IV collagen mainly promote the adherence and the expansion of the mesenchymal stem cells, are also important mitogens and differentiation factors for maintaining normal cell functions, and play an important role in the proliferation and the differentiation of the mesenchymal stem cells.

In an alternative specific example, the concentration of vitronectin in the cell culture medium is 20 μ g/L, 40 μ g/L, 50 μ g/L, 65 μ g/L, 75 μ g/L, 85 μ g/L, 100 μ g/L, 120 μ g/L, 140 μ g/L, 150 μ g/L, 180 μ g/L, or 200 μ g/L. Further, the concentration of vitronectin in the cell culture medium is 50 to 120. mu.g/L. Further, the concentration of vitronectin in the cell culture medium is 50 to 100. mu.g/L. It should be noted that vitronectin in this document is artificially synthesized vitronectin, which is also called recombinant vitronectin.

In an alternative specific example, the concentration of type IV collagen in the cell culture medium is 20 μ g/L, 40 μ g/L, 50 μ g/L, 65 μ g/L, 75 μ g/L, 85 μ g/L, 100 μ g/L, 120 μ g/L, 140 μ g/L, 150 μ g/L, 180 μ g/L, or 200 μ g/L. Furthermore, the concentration of the type IV collagen in the cell culture medium is 40 to 150 mu g/L. Furthermore, the concentration of the type IV collagen in the cell culture medium is 50 to 100. mu.g/L.

L-ascorbic acid (vitamin C) is a hexenoic acid sugar acid, a substance that maintains healthy growth and maintenance of cells in vivo and in vitro. L-ascorbic acid has a number of functions in cell culture systems: 1) can prevent the peroxidation of esterified and non-esterified unsaturated fatty acid; 2) capable of regenerating membrane-bound alpha-tocopherol (vitamin E) which has been oxidized by lipid peroxy radicals and indirectly limiting lipid peroxidation in cell membranes; 3) is a cofactor for prolyl hydroxylase (EC1.14.11.2) that catalyzes the post-translational hydroxylation of proline residues in nascent collagen and elastin molecules, which increases the degree of intramolecular cross-linking of collagen and elastin and promotes the development of extracellular matrix, thereby increasing the adherent adhesion capacity of the cells.

In an alternative embodiment, the concentration of L-ascorbic acid in the cell culture medium is from 10mg/L to 100 mg/L. Further, the concentration of L-ascorbic acid in the cell culture medium is 25mg/L to 50 mg/L.

In one embodiment, the concentration of progesterone is 2nM to 5nM, the concentration of beta-estradiol is 1nM to 2nM, the concentration of vitronectin is 50 μ g/L to 100 μ g/L, the concentration of type IV collagen is 50 μ g/L to 100 μ g/L, and the concentration of L-ascorbic acid is 25mg/L to 50mg/L in the cell culture medium.

In one embodiment, the composition comprises progesterone, beta-estradiol, vitronectin, type iv collagen, and L-ascorbic acid. The ratio of the molar amount of progesterone to the molar amount of beta-estradiol, the mass of vitronectin, the mass of type IV collagen and the mass of L-ascorbic acid is (1nmol to 10 nmol): (0.4nmol to 4 nmol): (20. mu.g-200. mu.g): (20. mu.g-200. mu.g): (10 mg-100 mg). Further, the ratio of the molar amount of progesterone to the molar amount of β -estradiol, the mass of vitronectin, the mass of type iv collagen and the mass of L-ascorbic acid is (2 to 5 nmol): (1nmol to 2 nmol): (50. mu.g-100. mu.g): (50. mu.g-100. mu.g): (25 mg-50 mg).

In one embodiment, the composition comprises progesterone, beta-estradiol, vitronectin, type IV collagen, L-ascorbic acid and a buffer, wherein the working concentration of the progesterone is 1nM to 10nM, the working concentration of the beta-estradiol is 0.4nM to 4nM, the working concentration of the vitronectin is 20 mug/L to 200 mug/L, the working concentration of the type IV collagen is 20 mug/L to 200 mug/L, and the working concentration of the L-ascorbic acid is 10mg/L to 100 mg/L. Specifically, the buffer is DBPS (duller phosphate buffer). Furthermore, the composition consists of progesterone, beta-estradiol, vitronectin, type IV collagen, L-ascorbic acid and buffer solution, wherein the working concentration of the progesterone is 2 nM-5 nM, the working concentration of the beta-estradiol is 1 nM-2 nM, the working concentration of the vitronectin is 50 mug/L-100 mug/L, the working concentration of the type IV collagen is 50 mug/L-100 mug/L, and the working concentration of the L-ascorbic acid is 25 mg/L-50 mg/L.

In one embodiment, the composition comprises 1 to 10 μ M progesterone, 0.4 to 4 μ M β -estradiol, 20 to 200mg/L vitronectin, 20 to 200mg/L type IV collagen, and 10 to 100g/L ascorbic acid. Furthermore, the composition consists of 1 to 10 mu M of progesterone, 0.4 to 4 mu M of beta-estradiol, 20 to 200mg/L of vitronectin, 20 to 200mg/L of IV type collagen, 10 to 100g/L of L-ascorbic acid and buffer solution. The composition is convenient for storage and transportation, and can be diluted for use.

In one embodiment, the composition is a powder or solution. In an alternative specific example, the composition is a lyophilized powder.

In one embodiment, the composition is a solution comprising 5 μ M progesterone, 2 μ M β -estradiol, 100mg/L vitronectin, 100mg/L type IV collagen, and 50g/L ascorbic acid.

It should be noted that the "working concentration of progesterone" herein refers to the concentration of progesterone in the cell culture medium; the "working concentration of β -estradiol" herein refers to the concentration in the cell culture medium of β -estradiol; the "working concentration of vitronectin", "type IV collagen" and "working concentration of L-ascorbic acid" in the present text and so on refer to the concentration of the corresponding substances in the cell culture medium.

Specifically, serum-free medium refers to a medium for culturing cells that is free of animal serum, including serum replacement and nutrients necessary for cell growth. In this embodiment, the serum-free medium is a serum-free mesenchymal stem cell medium for culturing mesenchymal stem cells. In one embodiment, the serum-free medium is a serum-free medium of national cell technology, Inc

hMSC-SFM. Of course, in other embodiments, the serum-free medium is not limited to the above, and may be other serum-free media that can be used for culturing mesenchymal stem cells.

The cell culture medium comprises the composition, the composition comprises progesterone, beta-estradiol, vitronectin, type IV collagen and L-ascorbic acid, and the progesterone, the beta-estradiol, the vitronectin, the type IV collagen and the L-ascorbic acid are matched with each other, so that the mesenchymal stem cells are high in adherence rate and survival rate when being recovered by using the cell culture medium.

An embodiment of the present invention also provides a method for preparing a cell culture medium, comprising the steps of: mixing the above composition with serum-free medium to prepare cell culture medium. In particular, the ratio of the composition to serum-free medium when mixed can be determined by the fold between the concentration of each component in the composition and its corresponding working concentration.

In one embodiment, the method further comprises the step of preparing the composition. Specifically, the components of the composition are mixed uniformly to obtain the composition. In some embodiments, the step of preparing the composition comprises: the composition is prepared at a high multiple concentration and then diluted at the time of use. It will be appreciated that the preparation of the composition may also include the step of sterilization. Further, filtration was performed with a 0.22 μm filter to remove microorganisms from the composition. Of course, the composition is stored at-20 ℃ to-80 ℃.

The preparation method of the cell culture medium is simple and convenient, and is easy for large-scale production.

The embodiment of the invention also provides a method for recovering the mesenchymal stem cells, which comprises the following steps: and (3) re-melting the frozen mesenchymal stem cells, and inoculating the mesenchymal stem cells into the cell culture medium.

Specifically, frozen mesenchymal stem cells taken out from liquid nitrogen are thawed in water bath at 37 ℃, counted and inoculated into the mesenchymal stem cell culture medium, wherein the inoculation density is 0.6 multiplied by 10⁴Per cm²～1×10⁴Per cm²。

In one embodiment, the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells. It is understood that in other embodiments, the mesenchymal stem cells are not limited to umbilical cord-derived mesenchymal stem cells, but may be mesenchymal stem cells of other sources.

According to the recovery method of the mesenchymal stem cells, the mesenchymal stem cell culture medium is used as the recovery culture medium, so that the adherence rate during recovery is high, the mesenchymal stem cell culture medium is definite in components and high in safety, and the method is beneficial to large-scale culture and clinical application of the mesenchymal stem cells in vitro.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. The components and reagents referred to in the following examples are all conventional commercial products. Of these, progesterone (cat # P0130), β -estradiol (cat # E2758), vitronectin (cat # SRP3186), collagen IV (cat # C5533) and L-ascorbic acid (cat # A4544) were purchased from Sigma. DBPS (cat # SH30256.01) was purchased from HyClone. Mesenchymal stem cells (UC-MSCs) derived from umbilical cord were purchased from glala stem cell technologies, guangzhou;

hMSC-SFM was purchased from national cell Co., Ltd, DMEM/F12 complete medium containing 10% FBS; MSC

XF Medium is available from BI corporation.

Example 1

Preparation of mesenchymal Stem cell Medium

(1) 1000X compositions of each group were prepared according to the concentration of progesterone, beta-estradiol, vitronectin, type IV collagen and L-ascorbic acid in the mesenchymal stem cell culture medium of each group in Table 1, and were sterilized by filtration using a 0.22 μm filter membrane and stored at-20 ℃ for future use. Wherein, the composition of the experimental group 1 consists of progesterone, beta-estradiol and DBPS; the composition of experiment group 2 consists of vitronectin, type IV collagen and DBPS, and the compositions of experiment groups 3-6 consist of progesterone, beta-estradiol, vitronectin, type IV collagen, L-ascorbic acid and DBPS.

TABLE 1

(2) The 1000X compositions of each group were separately combined with

hMSC-SFM (serum-free mesenchymal stem cell culture medium) was performed according to 1: mixing the mixed solution according to the volume ratio of 1000 to obtain the mesenchymal stem cell culture medium of each group.

Example 2

DMEM/F12 complete medium containing 10% FBS (control 1), MSC from BI company

XF Medium (control group 2), of national cell Co

The mesenchymal stem cell culture media of the hMSC-SFM (control group 3) and the experimental groups 1-6 prepared in the example 1 are respectively used as recovery culture media to recover mesenchymal stem cells (UC-MSCs). The operation of each group is as follows:

and (3) taking out the frozen UC-MSCs from the liquid nitrogen, rapidly thawing in a water bath at 37 ℃, transferring to an ultra-clean workbench after complete thawing, diluting the cell suspension by 10 times of volume of DPBS, and taking a proper amount of suspension for cell counting and calculating the survival rate. Then, the cells were resuspended at 0.6X 10 with DPBS as a result of viable cell count by centrifugation at 1200rpm for 5min and discarding the supernatant⁶Suspension in 0.6X 10/mL⁵The quantity of individual/well was inoculated in 6-well plates previously supplemented with 2mL of the corresponding group medium, with 3 replicates per group set up. Then put in 5% CO₂Culturing at 37 ℃ in an incubator, counting the adherent cells of each group 4h, 8h and 12h after inoculation, and calculating the wall adhesion rate of the mesenchymal stem cells among groups.

The adherent conditions of the mesenchymal stem cells corresponding to the groups of culture media after 4h, 8h and 12h of inoculation are shown in fig. 1-6, and the statistical results of the adherent conditions of the mesenchymal stem cells corresponding to the groups of culture media after 4h, 8h and 12h of inoculation are shown in table 2. FIG. 1 shows the cell morphology (microscope 100X) of mesenchymal stem cells corresponding to each group of culture media 4h after inoculation; FIG. 2 shows the cell morphology (microscope 100X) of mesenchymal stem cells corresponding to each group of culture media after 8h of inoculation; FIG. 3 shows the cell morphology (microscope 100X) of mesenchymal stem cells corresponding to each group of culture media after 12h of inoculation; FIG. 4 is a bar graph of the anchorage rate of mesenchymal stem cells corresponding to each set of culture medium 4 hours after inoculation; FIG. 5 is a bar graph of the anchorage rate of mesenchymal stem cells corresponding to each set of culture medium after 8h of inoculation; fig. 6 is a bar graph of the anchorage rate of mesenchymal stem cells corresponding to each group of culture media 12h after inoculation.

TABLE 2

In table 2, p <0.01 and p < 0.05.

As can be seen from table 2 and fig. 4, after 4 hours of inoculation, the anchorage rate of the mesenchymal stem cells of the experimental groups 3 to 6 is higher than that of the control groups 1 to 3, the experimental group 1 and the experimental group 2; compared with control groups 1-3, experiment group 1 and experiment group 2, the anchorage rates of the mesenchymal stem cells in the experiment groups 3-6 are very different (p is less than 0.01).

As can be seen from table 2 and fig. 5, after 8h of inoculation, the anchorage rate of the mesenchymal stem cells of the experimental groups 3-6 is higher than that of each of the control group, the experimental group 1 and the experimental group 2; the anchorage rates of the mesenchymal stem cells of the experimental group 3 and the experimental group 6 are respectively significantly different (p is less than 0.05) compared with those of the control group, the experimental group 1 and the experimental group 2, and the anchorage rates of the mesenchymal stem cells of the experimental group 4 and the experimental group 5 are respectively significantly different (p is less than 0.01) compared with those of the control group, the experimental group 1 and the experimental group 2.

As can be seen from table 2 and fig. 6, after 12h of inoculation, the anchorage rate of the mesenchymal stem cells of the experimental groups 3-6 is higher than that of each of the control group, the experimental group 1 and the experimental group 2; the adherence rates of the mesenchymal stem cells of the experimental groups 4 and 5 are respectively compared with those of the control group, the experimental group 1 and the experimental group 2, and have significant difference (p is less than 0.05), and the adherence rates of the mesenchymal stem cells of the experimental groups 3 and 6 are respectively compared with those of the control group 1 and the control group 2, and have significant difference (p is less than 0.05), but the adherence rates of the mesenchymal stem cells of the experimental group 3 and the experimental group 6 are respectively compared with those of the control group 3, the experimental group 1 and the experimental group 2, and have no significant difference (p is greater than 0.05).

The results show that the mesenchymal stem cell culture medium of the experimental group 3-6 can obviously improve the anchorage rate of the mesenchymal stem cells when the mesenchymal stem cells are recovered.

Example 3

DMEM/F12 complete medium containing 10% FBS (control 1), MSC from BI company

XF Medium (control group 2), of national cell Co

and (3) taking out the frozen UC-MSCs from the liquid nitrogen, rapidly thawing in a water bath at 37 ℃, transferring to an ultra-clean workbench after complete thawing, diluting the cell suspension by 10 times of volume of DPBS, and taking a proper amount of suspension for cell counting and calculating the survival rate. Then, the cells were resuspended at 3X 10 in the medium corresponding to each group as a result of viable cell count by centrifugation at 1200rpm for 5min and discarding the supernatant⁴The suspension/mL, in an amount of 0.1 mL/well, was seeded in 96-well plates in 3 replicates per set. Then put in 5% CO₂The cells were cultured in an incubator at 37 ℃ and the cell viability of each group was measured using CCK-8 kit 24 hours after inoculation.

The results for CCK-8 for each group are shown in Table 3 and FIG. 7.

TABLE 3

Denotes p <0.01, denotes p < 0.05.

As can be seen from table 3 and fig. 7, the cell viability of the mesenchymal stem cells of the experimental groups 3 to 6 was higher than that of the control group 1, the control group 2, the control group 3, the experimental group 1 and the experimental group 2. Compared with control groups 1-2, experiment group 1 and experiment group 2, the cell survival rates of the mesenchymal stem cells of the experiment groups 3-6 are respectively very significant (p is less than 0.01), and compared with the control group 1 and control group 2, the cell survival rates of the mesenchymal stem cells of the experiment group 1, experiment group 2 and control group 3 are respectively significant (p is less than 0.05).

The results show that the mesenchymal stem cell culture medium of the experimental group 3-6 can obviously improve the survival rate of the mesenchymal stem cells during recovery culture.

Example 4

DMEM/F12 complete medium containing 10% FBS (control group 1), from national cell Co

The mesenchymal stem cell culture media of hMSC-SFM (control 3) and experimental group 4 and experimental group 5 prepared in example 1 were used as resuscitation media to resuscitate mesenchymal stem cells (UC-MSCs), respectively. The operation of each group is as follows:

and (3) taking out the frozen UC-MSCs from the liquid nitrogen, rapidly thawing in a water bath at 37 ℃, transferring to an ultra-clean workbench after complete thawing, diluting the cell suspension by 10 times of volume of DPBS, and taking a proper amount of suspension for cell counting and calculating the survival rate. Then, the mixture was centrifuged at 1200rpm for 5min, the supernatant was discarded, the mixture was resuspended in DPBS as a result of viable cell count, and the suspension was then resuspended at 1X 10⁴/cm²Was inoculated into T25 flasks containing groups of medium, each group being set up in 3 replicates. Then put in 5% CO₂After culturing in an incubator at 37 ℃ for 3 days, digesting with 0.05% trypsin solution to collect UC-MSCs in each group, and detecting the expression of surface markers such as CD105, CD73, CD90, CD34, CD45, HLA-DR and the like by a flow cytometer, the results are shown in Table 4 and FIGS. 8-11. Fig. 8 is a result of surface markers of mesenchymal stem cells of the control group 1; fig. 9 is a result of surface markers of mesenchymal stem cells of the control group 3; FIG. 10 is the mesenchymal stem of Experimental group 4Results of surface markers of cells; fig. 1 is a result of surface markers of mesenchymal stem cells of experimental group 5.

TABLE 4

Group of

CD73

CD90

CD105

CD34

CD45

HLA-DR

Control group

1

100.00％

99.64％

0.02％

0.33％

1.14％

Control group 3

100.00％

99.96％

99.53％

0.00％

0.12％

0.06％

Experimental group 4

99.98％

99.91％

99.45％

0.30％

0.05％

0.29％

Experimental group 5

99.98％

99.36％

0.29％

0.06％

0.72％

As can be seen from Table 4 and FIGS. 8 to 11, the mesenchymal stem cells of the experimental group 4, the experimental group 5, the control group 1 and the control group 3 had positive expression (not less than 95%) of the surface markers CD105, CD73 and CD90, but negative expression (not more than 2%) of CD34, CD45 and HLA-DR, and there was no significant difference between the groups. As can be seen from the above, the mesenchymal stem cell culture media of examples 4 and 5 did not affect the expression of cell surface markers of the revived mesenchymal stem cells.

Example 5

taking out the frozen UC-MSCs from the liquid nitrogen, rapidly thawing in 37 deg.C water bath, transferring to a superclean bench after completely thawing, diluting with 10 times volume of DPBSAnd (4) taking a proper amount of suspension to count the cell amount and calculate the survival rate. Then, the mixture was centrifuged at 1200rpm for 5min, the supernatant was discarded, the mixture was resuspended in DPBS as a result of viable cell count, and the suspension was then resuspended at 1X 10⁴Per cm²Was inoculated into T25 flasks containing groups of medium, each group being set up in 3 replicates. Then put in 5% CO₂After culturing at 37 ℃ for 72 hours in an incubator, total RNA extraction from cells was carried out according to the instructions of TRIzol kit, followed by obtaining cDNA using M-MLV reverse transcription kit. GAPDH is used as an internal reference gene, expression levels of UC-MSCs dry genes Oct4, Nanog and Sox2 of each group are detected according to SYBR Green quantitative PCR kit instructions (sequences of primers used in PCR are shown in Table 5), 3 parallel samples are designed for each group, and 2 parallel samples are used^-△△CtThe relative expression level of the target gene was quantitatively analyzed by the method, and the results are shown in FIG. 12.

TABLE 5

As can be seen from fig. 12, there was no significant difference in the expression amounts of the three sternness genes of experimental group 4 and experimental group 5 from control group 1 and control group 3 (p >0.05), which indicates that the mesenchymal stem cell culture medium of experimental group 4 and experimental group 5 had no effect on the sternness of MSCs.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

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Claims

1. A composition comprising progesterone, estradiol, vitronectin, collagen and L-ascorbic acid.

2. The composition according to claim 1, wherein the composition is a powder or a solution; and/or the estradiol is β-estradiol, and the collagen is type IV collagen protein.

3. Use of the composition of claim 1 or 2 in a cell culture medium.

4. The application according to claim 3, wherein the estradiol is beta-estradiol, the collagen is type IV collagen, and the working concentration of the composition is in the following range: corpus luteum Ketone 1nM～10nM, β-estradiol 0.4nM～4nM, vitronectin 20μg/L～200μg/L, type IV collagen 20μg/L～200μg/L, L-ascorbic acid 10mg/L～100mg/L;

Preferably, the working concentration of the composition is in the following range: the working concentration of progesterone is 2nM～5nM; the working concentration of β-estradiol is 1nM～2nM; the working concentration of vitronectin is 50μg/L～100μg /L; the working concentration of type IV collagen is 50μg/L～100μg/L; the working concentration of L-ascorbic acid is 25mg/L～50mg/L;

Preferably, the working concentration of the composition when culturing cells is in the following range: the working concentration of progesterone is 5nM, the working concentration of β-estradiol is 2nM, the working concentration of vitronectin is 100μg/L, IV The working concentration of collagen type was 100 μg/L, and the working concentration of L-ascorbic acid was 50 mg/L.

5. A cell culture medium, comprising the composition according to any one of claims 1 to 2 and a serum-free medium.

6. The cell culture medium according to claim 5, wherein the serum-free culture medium is a serum-free mesenchymal stem cell culture medium.

7. A method for resuscitating mesenchymal stem cells, comprising the following steps:

After the frozen mesenchymal stem cells are thawed, they are inoculated into the cell culture medium according to any one of claims 5 to 6.

8 . The method for resuscitating mesenchymal stem cells according to claim 7 , wherein the seeding density is 0.6×10 ⁴ cells/cm ² to 1×10 ⁴ cells/cm ² .

9 . The method for resuscitating mesenchymal stem cells according to claim 7 , wherein the mesenchymal stem cells are umbilical cord-derived mesenchymal stem cells. 10 .

10. The method for resuscitating mesenchymal stem cells according to any one of claims 7 to 9, wherein the rejuvenation is performed under a water bath condition of 37°C.