Composition and its application in stem cell culture

Abstract

This invention relates to the field of stem cell culture technology, and more particularly to compositions and their applications in stem cell culture. The invention utilizes a composition formed by combining acetate, a pyruvate dehydrogenase kinase (PDK) specific inhibitor, and an HMG-CoA reductase inhibitor to enhance the ability of mesenchymal stem cells to maintain stemness and prevent differentiation during passage. Based on this composition, further incorporating various nutrients, the resulting composition provides ample nutrition to mesenchymal stem cells, maintaining their good proliferative capacity and stemness during passage, even in the absence of serum and animal-derived components, with batch-to-batch stability.

Description

Technical Field

[0001] This invention relates to the field of stem cell culture technology, and more particularly to compositions and their application in stem cell culture. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cell derived from the mesoderm, possessing the potential for self-renewal, multi-lineage differentiation, and low immunogenicity. In vitro, MSCs can be induced to differentiate into various tissue cells, including epithelial, bone, cartilage, adipose, neural, and cardiac cells, and exhibit functions such as promoting angiogenesis, protecting nerves, and providing cell replacement therapy. MSCs also show potential clinical applications in hematopoietic support, promoting stem cell implantation, and immune regulation.

[0003] Although mesenchymal stem cells are relatively abundant in sources, such as adipose tissue, umbilical cord blood, umbilical cord, and placenta, they are adult stem cells with very low initial concentrations in vivo. To meet the needs of clinical treatment, they need to be rapidly expanded and cultured in vitro. At the same time, they also need to maintain multi-lineage differentiation potential and undifferentiated state, i.e., undifferentiated proliferation. This is an urgent problem to be solved for mesenchymal stem cells to enter the clinical stage.

[0004] However, long-term in vitro culture of MSCs has shown a continuous decrease in their differentiation potential, and their phenotypic and immunomodulatory properties also change. To improve the efficiency of in vitro culture, it is essential to maintain the physiological characteristics and features of MSCs throughout the entire culture process. Therefore, optimizing the culture conditions and protocols for MSCs is a significant research focus.

[0005] Currently, in in vitro expansion culture, traditional mesenchymal stem cell culture media consist of a basal medium supplemented with 5% to 20% fetal bovine serum (FBS). Serum is an indispensable component for cell growth, but animal serum, as a xenogeneic serum, is not only complex in composition, containing a large number of amino acids, nucleosides, proteins, hormones, lipids, and other trace components, the content and specific functions of which are not yet fully determined. Furthermore, since FBS originates from different individuals, batch-to-batch variability is inevitable, and there is a risk of introducing exogenous pathogens, such as prions associated with mad cow disease, in human treatments. Subsequently, human serum, platelet lysate, or umbilical cord serum have been used as alternatives to FBS. However, blood-derived components, whether animal or human, are of unknown composition, and batch-to-batch variability is unavoidable. Therefore, adding blood-derived components to the culture medium leads to reduced reproducibility of results or increased cell population heterogeneity. In addition, the high price of serum or human serum and human platelet lysate in actual production greatly limits the widespread promotion of stem cell therapy. Furthermore, residual serum may cause allergic reactions in patients during the clinical application of stem cells.

[0006] Scientists have attempted to develop specific culture media for the growth of MSCs in animals or humans, such as StemPro™ MSC SFM and StemPro™ MSC SFM XenoFree™ (Thermo Fisher Scientific, Waltham, MA, USA), StemXVico (R&D Systems, Minneapolis, MN, USA), PowerStem MSC1 (Pan Biotech, Aidenbach, Germany), MSC NutriStem® XF (Biological Industries, Kibbutz Beit-Haemek, Israel), PRIME-XV MSC Expansion SFM (Irvine Scientific, Santa Ana, CA, USA), MesenCult™-ACF Plus (Stem Cell Technologies, Vancouver, Canada), and MSCGM-CD (Lonza, Basel, Switzerland). However, these media have also shown limited performance, such as supporting only a single passage culture or slow, multiple passages for cell expansion. Furthermore, all these studies used cells that had previously been exposed to serum during the initial isolation / expansion phase. When cells are exposed to serum and then placed under serum-free conditions, serum-derived contaminants may be carried by the cells, and this serum-exposed culture process may ultimately limit its therapeutic applications. Furthermore, existing serum-free culture media are expensive, resulting in the cost of the required materials being several times higher than that of animal serum, which greatly limits the widespread adoption of stem cell therapy.

[0007] Therefore, serum-free culture media with defined components, batch-stable quality control, and strong cell proliferation and passage capacity remain the focus of current research. Summary of the Invention

[0008] In view of this, the technical problem to be solved by the present invention is to provide a composition for promoting undifferentiated proliferation and its application in stem cell culture.

[0009] The present invention provides a composition comprising two or more of acetate, pyruvate dehydrogenase kinase inhibitor and HMG-CoA reductase inhibitor.

[0010] In this invention, the acetate includes at least one of sodium acetate, potassium acetate, ammonium acetate, lead acetate, and zinc acetate. In some embodiments, the acetate is sodium acetate.

[0011] In this invention, the pyruvate dehydrogenase kinase inhibitor includes at least one of dichloroacetic acid, dichloro acetate, or dichloroacetate. In some embodiments, the pyruvate dehydrogenase kinase inhibitor is sodium dichloroacetate.

[0012] In this invention, the HMG-CoA reductase inhibitors include simvastatin, lovastatin, rosuvastatin calcium, fluvastatin sodium, and pravastatin sodium. In some embodiments, the HMG-CoA reductase inhibitor is lovastatin.

[0013] In this embodiment of the invention, the composition consists of sodium acetate, sodium dichloroacetate, and lovastatin.

[0014] In some embodiments, the molar ratio of sodium acetate, sodium dichloroacetate, and lovastatin in the composition is (1~10):(0.5~5):(0.005~0.02).

[0015] In some specific embodiments, the composition comprises sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 5:1:0.01.

[0016] In some specific embodiments, the composition comprises sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 10:5:0.02.

[0017] In some specific embodiments, the composition contains sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 10:0.5:0.02.

[0018] In some specific embodiments, the composition comprises sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 1:0.5:0.02.

[0019] In some specific embodiments, the composition comprises sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 1:5:0.02.

[0020] In some specific embodiments, the composition contains sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 10:5:0.005.

[0021] In some specific embodiments, the composition contains sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 10:0.5:0.005.

[0022] In some specific embodiments, the composition comprises sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 1:0.5:0.005.

[0023] In some specific embodiments, the composition contains sodium acetate, sodium dichloroacetate and lovastatin in a molar ratio of 1:5:0.005.

[0024] The composition provided by the present invention also includes human serum albumin, growth factors, laminin, hormones, antioxidants, transport proteins, ethanolamine, 4-hydroxyethylpiperazine ethanesulfonic acid, inorganic salts, amino acids, lipids, vitamins and pH adjusters.

[0025] In this invention, the growth factor includes at least one selected from PDGF, EGF, bFGF, IGF-1, and IGF-2. In some embodiments of this invention, the growth factor includes at least one selected from bFGF or EGF. In some embodiments, the growth factor is both bFGF and EGF.

[0026] In this invention, the hormone includes at least one of insulin or a trypsin inhibitor. In some embodiments, the hormone is both insulin and a trypsin inhibitor.

[0027] In this invention, the antioxidant includes at least one of glutathione or L-ascorbic acid-2-phosphate. In some embodiments, the antioxidant is glutathione and L-ascorbic acid-2-phosphate.

[0028] In this invention, the transport protein includes at least one of transferrin or human lipid transporter 2. In some embodiments, the transport protein is both transferrin and human lipid transporter 2.

[0029] In this invention, the inorganic salt includes at least one selected from sodium pyruvate, sodium selenite, or ferrous sulfate. In some embodiments, the inorganic salt is sodium pyruvate, sodium selenite, and ferrous sulfate.

[0030] In this invention, the amino acids include a non-essential amino acid composition and glutamine. In some embodiments, the non-essential amino acid composition includes glycine, alanine, serine, aspartic acid, glutamic acid (and its amines), proline, arginine, histidine, tyrosine, and cysteine. The non-essential amino acid composition used in the culture medium of this invention is named MEM Non-essential Amino Acid Solution (100×) (Thermo Fisher Scientific, #11140050).

[0031] In this invention, the lipids include at least one selected from linoleic acid, linolenic acid, lecithin, or choline. In some embodiments, the lipids are linoleic acid, linolenic acid, lecithin, and choline.

[0032] In this invention, the vitamin includes at least one of vitamin E or vitamin B12. In some embodiments, the vitamin is both vitamin E and vitamin B12.

[0033] In this invention, the pH adjuster is sodium bicarbonate.

[0034] The composition of the present invention comprises the following components: sodium acetate, sodium dichloroacetate, lovastatin, human serum albumin, basic fibroblast growth factor, epidermal growth factor, laminin, insulin, trypsin inhibitor, glutathione, L-ascorbic acid-2-phosphate, transferrin, human lipid transporter 2, ethanolamine, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium pyruvate, sodium selenite, ferrous sulfate, L-glutamine, non-essential amino acids, linoleic acid, linolenic acid, lecithin, choline, vitamin E, vitamin B12, and sodium bicarbonate.

[0035] The components in the composition of this invention can exist independently or in mixtures of two or more; this invention does not limit this. The composition is preferably in solution form. In the solution, the concentration of each component in the composition can be 1 to 100 times the working concentration, for example, 2, 10, 20, 40, 50, or 100 times the working concentration. The working concentration referred to in this invention refers to the concentration of each component in the culture medium during the culture of mesenchymal stem cells.

[0036] In some embodiments, the working concentrations of the components in the composition are:

[0037]

[0038] In some embodiments, the working concentration of sodium acetate is 3-7 mmol / L, the working concentration of sodium dichloroacetate is 0.7-2 mmol / L, and the working concentration of lovastatin is 7-15 μmol / L.

[0039] In some embodiments, the working concentration of sodium acetate is 4-6 mmol / L, the working concentration of sodium dichloroacetate is 0.8-1.2 mmol / L, and the working concentration of lovastatin is 8-12 μmol / L.

[0040] In some specific embodiments, the working concentration of sodium acetate is 5 mmol / L, the working concentration of sodium dichloroacetate is 1 mmol / L, and the working concentration of lovastatin is 10 μmol / L.

[0041] In some specific embodiments, the working concentration of sodium acetate is 10 mmol / L, the working concentration of sodium dichloroacetate is 5 mmol / L, and the working concentration of lovastatin is 20 μmol / L.

[0042] In some specific embodiments, the working concentration of sodium acetate is 10 mmol / L, the working concentration of sodium dichloroacetate is 0.5 mmol / L, and the working concentration of lovastatin is 20 μmol / L.

[0043] In some specific embodiments, the working concentration of sodium acetate is 1 mmol / L, the working concentration of sodium dichloroacetate is 0.5 mmol / L, and the working concentration of lovastatin is 20 μmol / L.

[0044] In some specific embodiments, the working concentration of sodium acetate is 1 mmol / L, the working concentration of sodium dichloroacetate is 5 mmol / L, and the working concentration of lovastatin is 20 μmol / L.

[0045] In some specific embodiments, the working concentration of sodium acetate is 10 mmol / L, the working concentration of sodium dichloroacetate is 5 mmol / L, and the working concentration of lovastatin is 5 μmol / L.

[0046] In some specific embodiments, the working concentration of sodium acetate is 10 mmol / L, the working concentration of sodium dichloroacetate is 0.5 mmol / L, and the working concentration of lovastatin is 5 μmol / L.

[0047] In some specific embodiments, the working concentration of sodium acetate is 1 mmol / L, the working concentration of sodium dichloroacetate is 0.5 mmol / L, and the working concentration of lovastatin is 5 μmol / L.

[0048] In some specific embodiments, the working concentration of sodium acetate is 1 mmol / L, the working concentration of sodium dichloroacetate is 5 mmol / L, and the working concentration of lovastatin is 5 μmol / L.

[0049] In this invention, the concentration of human lipid transporter 2 has no significant effect on the culture effect. The concentration of human lipid transporter 2 used in the embodiments of this invention is 20 mg / L.

[0050] The present invention also provides a culture medium comprising a basal culture medium and the composition described herein.

[0051] In this invention, the basal culture medium is DMEM / F12 medium, DMEM-LG medium, or α-MEM. The three media have similar culture effects, with DMEM / F12 medium providing the best culture effect, followed by DMEM-LG medium.

[0052] The present invention also provides the use of the composition or culture medium in the culture of mesenchymal stem cells.

[0053] The composition or culture medium described in this invention is suitable for culturing various types of mesenchymal stem cells, such as mesenchymal stem cells derived from tissues such as adipose tissue, umbilical cord blood, umbilical cord, and placenta, as well as mesenchymal stem cells differentiated from induced pluripotent stem cells or embryonic stem cells. In embodiments of this invention, the mesenchymal stem cells include umbilical cord mesenchymal stem cells.

[0054] The present invention also provides a method for culturing mesenchymal stem cells, which includes seeding mesenchymal stem cells into the culture medium described in the present invention, culturing and passage.

[0055] The present invention also provides a method for differentiating mesenchymal stem cells, which involves culturing mesenchymal stem cells in the culture medium described in the present invention and then inducing differentiation.

[0056] The differentiation described in this invention includes epithelial cell differentiation, osteogenic differentiation, chondrogenic differentiation, adipogenic differentiation, neurogenic differentiation, or cardiac cell differentiation.

[0057] This invention utilizes a composition consisting of acetate, a pyruvate dehydrogenase kinase (PDK) specific inhibitor, and an HMG-CoA reductase inhibitor to enhance the passage capacity of mesenchymal stem cells and prevent their differentiation during passage. Furthermore, by incorporating various nutrients into this composition, a comprehensive solution is created that provides ample nutrition to mesenchymal stem cells, maintaining their excellent passage capacity and stemness during passage even in the absence of blood-derived components, while ensuring batch-to-batch stability. Attached Figure Description

[0058] Figure 1 The cell density of each group after 8 days of culture in the culture medium is shown.

[0059] Figure 2 The results of P15 generation cell morphology and flow cytometry analysis are shown.

[0060] Figure 3 The diagram shows the cell culture conditions in Group 1 using the same culture medium as those in commercially available culture media. In Group A, cells cultured in the ordinary culture medium showed significant morphological changes at passage P15, losing the inherent morphology of mesenchymal stem cells (40×). Group B shows the growth curves of mesenchymal stem cells (MSCs) cultured in the culture medium of this invention and the ordinary culture medium. It is evident that the growth rate of MSCs cultured in the culture medium of this invention is significantly better than that in the ordinary culture medium. Detailed Implementation

[0061] This invention provides compositions and their applications in stem cell culture. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0062] In the composition of this invention, acetate is an exogenous source of acetyl-CoA, which can be converted into acetyl-CoA in the cytoplasm, thereby increasing the level of histone acetylation and reversing stem cell senescence. When combined with a pyruvate dehydrogenase kinase (PDK) specific inhibitor and an HMG-CoA reductase inhibitor, it can more significantly promote stem cell proliferation while maintaining stem cell stemness, resulting in a significant synergistic effect among the three.

[0063] This invention combines a composition consisting of acetate, a pyruvate dehydrogenase kinase (PDK) specific inhibitor, and an HMG-CoA reductase inhibitor with the aforementioned nutrients to provide mesenchymal stem cells with sufficient nutrition. Even without blood-derived components, the mesenchymal stem cells maintain good passage capacity and stemness during passage, and are stable between batches.

[0064] The reagents and consumables used in this invention are all commercially available products. The invention is further illustrated below with reference to specific examples:

[0065] Example 1

[0066] I. Design an experiment according to the formula in the table below:

[0067] Table 1. Culture medium formulations for each group

[0068]

[0069]

[0070] The preparation methods for each group of culture media include:

[0071] Under the premise of using ultrapure water, the culture medium can be prepared by three methods, and the preparation method does not affect the results.

[0072] The first method: Dissolve the above substances separately in ultrapure water, stir evenly, leaving no precipitate or suspension, filter through a 0.22μm filter membrane for sterilization, and store at 4℃;

[0073] The second method: Prepare concentrated solutions of the above components using ultrapure water, filter them through a 0.22μm filter membrane for sterilization, store them at 4℃, and finally mix them according to a certain ratio before use.

[0074] The third method involves first dissolving easily soluble and stable components in ultrapure water to prepare a solution, then filtering it through a 0.22μm filter membrane for sterilization and storing it at 4℃. Next, dissolve the components that are difficult to dissolve and store separately, and prepare concentrated solutions of different concentrations according to different proportions. Filter the solutions through a 0.22μm filter for sterilization, freeze them to protect them from light, and finally mix them together in a certain proportion before use.

[0075] After the culture medium is prepared, a small amount should be drawn and placed in a culture flask at 37°C for 24-48 hours to check for contamination. Only use the culture medium in experiments after confirming it is free of contamination.

[0076] II. Resuscitation and Culture of Mesenchymal Stem Cells

[0077] Remove the cryopreservation tubes of human umbilical cord mesenchymal stem cells (MSCs) or human induced mesenchymal stem cells (iMSCs) from the liquid nitrogen container and immerse them directly in a 37°C water bath, shaking occasionally to thaw them quickly. Then transfer them to centrifuge tubes and add at least four times the volume of serum-free culture medium to each tube, mixing well. Centrifuge, discard the supernatant, add serum-free mesenchymal stem cell culture medium, count the cells, and then inoculate each group of cells at 1.0 × 10⁻⁶ cells / mL. 4 cells / cm 2 The cells were densely inoculated into 24-well culture plates and incubated statically at 37°C in a CO2 incubator.

[0078] When MSCs reached 90% confluence, the culture medium was aspirated, washed with PBS, and digested with 0.25% trypsin for 2-5 minutes. The trypsin was discarded, and the serum-free mesenchymal stem cell culture medium (groups 1-7) was added. The cells were dispersed, centrifuged, and the supernatant was removed. The cells were then resuspended in the serum-free mesenchymal stem cell culture medium (groups 1-7), counted, and the cell density was adjusted. The cells were then seeded into cell culture dishes or flasks and incubated at 37°C in a CO2 incubator. A control group was also established under the same culture conditions, culturing MSCs in conventional serum-containing culture medium.

[0079] III. Evaluation of Cultivation Effect:

[0080] 3.1 After culturing for 8 days, the cell density of each group was measured, and the results are as follows: Figure 1 And Table 2:

[0081] Table 2 Cell density of each group (10) 4 cells / cm 2 )

[0082]

[0083] The results showed that the cell density in groups 1-7 was significantly higher than that in the control group, and the difference was statistically significant (p<0.05). Among them, treatment 1 had the highest cell density, which was significantly better than the control group and the other treatment groups (groups 2-7).

[0084] 3.2 Further detection of relevant indicators: After culturing umbilical cord mesenchymal stem cells in serum-free medium to the 20th generation of umbilical cord mesenchymal stem cells in Group 1, the surface markers of the 20th generation of umbilical cord mesenchymal stem cells were measured.

[0085] 3.2.1 Marker detection (flow cytometry data)

[0086] Figure 2 Mesenchymal stem cells cultured in the culture medium of Group 1 of this invention up to the 15th generation, as shown in the bright-field plot and flow cytometry analysis, still maintained stem cell characteristics. Bright-field was achieved under 10× light microscopy; flow cytometry showed that CD73 and CD90 were positive in over 90% of cells, while negative markers included a mixture of CD34 and CD45 (Miltenyi, #130-125-285).

[0087] 3.2.2 Cell morphology and growth rate detection

[0088] The growth curves of umbilical cord mesenchymal stem cells cultured in Group 1 medium and those cultured in fetal bovine serum medium using existing MSCs were compared. The growth curves show that the growth efficiency of the treatment group was significantly better than that of the control group.

[0089] Figure 3 Mesenchymal stem cell (MSC) growth morphology and cell growth curves under different culture medium conditions. A. Cells cultured in ordinary culture medium showed significant morphological changes at passage P15, losing the inherent morphology of MSCs (40×). B. Growth curves of MSCs cultured in the culture medium of this invention and ordinary culture medium. It can be seen that the growth rate of MSCs cultured in the culture medium of this invention is significantly better than that in ordinary culture medium.

[0090] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Composition, characterized in that, Sodium acetate, sodium dichloroacetate, lovastatin, human serum albumin, basic fibroblast growth factor, epidermal growth factor, laminin, insulin, trypsin inhibitor, glutathione, L-ascorbic acid-2 phosphate, transferrin, human lipocalin 2, ethanolamine, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium pyruvate, sodium selenite, ferrous sulfate, L-glutamine, non-essential amino acids, linoleic acid, linolenic acid, lecithin, choline, vitamin E, vitamin B12 and sodium bicarbonate; The molar ratio of sodium acetate, sodium dichloroacetate and lovastatin is 5:1:0.01, The working concentration of each component is as follows: sodium acetate 1mmol / L-10mmol / L, sodium dichloroacetate 0.5μmol / L-5mmol / L, lovastatin 5-20μmol / L, human serum albumin 10mg / L, bFGF 20μg / L, EGF 5μg / L, laminin 1mg / L, insulin 10mg / L, trypsin inhibitor 0.5mg / L, glutathione 5mg / L, L-ascorbic acid-2 phosphate 58mg / L, transferrin 10mg / L, human lipocalin 2 20mg / L-100mg / L, ethanolamine 0.4mg / L, 4-hydroxyethylpiperazine ethanesulfonic acid 1000mg / L, sodium pyruvate 150mg / L, sodium selenite 14μg / L, ferrous sulfate 0.8mg / L, L-glutamine 150mg / L, non-essential amino acids 1mmol / L, linoleic acid 0.4mg / L, linolenic acid 0.5mg / L, lecithin 0.5mg / L, choline 30nmol / L, vitamin E 30mg / L, vitamin B12 20μmol / L and sodium bicarbonate 2000mg / L.

2. The composition of claim 1, wherein, The working concentration of sodium acetate is 5mmol / L, the working concentration of sodium dichloroacetate is 1mmol / L, and the working concentration of lovastatin is 10μmol / L.

3. A culture medium, characterized by, The composition of claim 1 or 2 or the medium of claim 3 or 4.

4. The medium according to claim 3, characterized in that, The medium is DMEM / F12 medium, DMEM-LG medium or α-MEM.

5. The use of the composition of claim 1 or 2 or the medium of claim 3 or 4 in mesenchymal stem cell culture.

6. Use according to claim 5, characterized in that, The mesenchymal stem cell is umbilical cord mesenchymal stem cell, bone marrow mesenchymal stem cell, adipose and dental pulp derived mesenchymal stem cell, mesenchymal stem cell differentiated from induced pluripotent stem cell.

7. A method for culturing mesenchymal stem cells, characterized by, The mesenchymal stem cell is seeded into the medium of claim 3 or 4, cultured and subcultured.

8. A method for differentiation of mesenchymal stem cells, characterized by, After the mesenchymal stem cell is cultured in the medium of claim 3 or 4, differentiation induction is performed.

Images