A human embryonic stem cell expansion medium

Abstract

This invention provides a human embryonic stem cell expansion culture medium, comprising DMEM / F12 medium, ITS-A, HEPES, magnesium ascorbate phosphate, non-essential amino acids, bFGF, FGF2-G3, GlutaMAX, LY-333531, gentamicin sulfate, Y27632, and CHIR99021. Y-27632 inhibits pluripotent stem cell apoptosis, improves adhesion efficiency, maintains pluripotency and colony formation ability; CHIR99021 enhances the self-renewal of pluripotent stem cells and inhibits their differentiation; LY-333531 inhibits spontaneous differentiation of pluripotent stem cells; and gentamicin sulfate reduces the risk of contamination during culture. The culture medium provided by this invention is serum-free, free of allogeneic substances, has a simple composition, and offers excellent expansion speed, effectively maintaining long-term stable passage of human embryonic stem cells, providing stable technical support for scientific research.

Description

Technical Field

[0001] This invention belongs to the field of cell culture and relates to a culture medium for expanding human embryonic stem cells. Background Technology

[0002] Human embryonic stem cells (ESCs, abbreviated as ES, EK or ESC cells) are a type of highly undifferentiated pluripotent stem cells isolated from early embryos (usually the inner cell mass of the blastocyst stage) or primitive gonads. These cells can proliferate indefinitely and maintain an undifferentiated state for a long time under suitable in vitro culture conditions. At the same time, under specific induction conditions, they can be directed to differentiate into almost all cell types derived from the ectoderm, mesoderm and endoderm, such as neurons, cardiomyocytes, pancreatic β cells, hepatocytes, hematopoietic cells and epidermal cells, showing great potential for reconstructing tissues and organs.

[0003] Since Sir Martin John Evans and Matthew Kaufman of the University of Cambridge, UK, first successfully established a mouse-human embryonic stem cell line on July 9, 1981, this field has rapidly become a cutting-edge hot topic in life sciences. Subsequently, scientists have successfully isolated and established human embryonic stem cells from humans and various other mammals, greatly expanding its research scope and application prospects. As one of the cornerstones of modern biomedical research, human embryonic stem cells, due to their unique biological characteristics, are considered an important bridge connecting developmental biology and regenerative medicine. They not only provide ideal in vitro models for studying early human development, cell fate determination mechanisms, and gene regulatory networks, but also demonstrate irreplaceable value in disease modeling, drug screening, toxicity assessment, and regenerative medicine. Especially in the exploration of treatments for degenerative diseases (such as Parkinson's disease and Alzheimer's disease), metabolic diseases (such as type 1 diabetes), cardiovascular diseases (such as myocardial infarction), retinopathy (such as age-related macular degeneration), and severe tissue damage, functional cell transplantation derived from ESCs is regarded as a revolutionary strategy that promises to achieve fundamental repair, bringing new hope to countless patients who currently have no effective cure.

[0004] However, the widespread application of human embryonic stem cells still faces numerous technical bottlenecks and challenges, among which the optimization of culture systems is particularly crucial. Pluripotent stem cell culture media are fundamental for maintaining their undifferentiated state, supporting efficient expansion, and achieving precise directed differentiation, directly impacting the reproducibility of research and the feasibility of clinical translation. Currently, various commercial culture systems exist globally. While they have made some progress in enhancing cell proliferation, maintaining pluripotency, and promoting specific lineage differentiation, significant limitations remain: some systems have only moderate effects on promoting cell proliferation and maintaining pluripotency; most systems rely on the addition of fetal bovine serum or animal-derived components, posing a risk of introducing exogenous pathogens and severely restricting their application in clinical-grade cell production; other systems, although serum-free formulations, have complex compositions, some components are not fully understood, and overall costs are high, significantly increasing research and industrialization costs. Therefore, developing a culture medium with fully defined components, no risk of foreign contamination, supporting efficient expansion and stable pluripotency maintenance, and with controllable costs has become a core technical bottleneck that urgently needs to be overcome in the current stem cell field. This is of great significance for promoting the large-scale clinical application of human embryonic stem cells from laboratory research. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a simple and efficient culture medium for expanding human embryonic stem cells.

[0006] To achieve the above objectives, the present invention discloses a human embryonic stem cell expansion culture medium, comprising a basal culture medium and culture medium additive A and culture medium additive B. Preferably, additive A of the present invention comprises DMEM / F12 medium, ITS-A, HEPES, magnesium ascorbate phosphate, MEM non-essential amino acids, bFGF, FGF2-G3, GlutaMAX, LY-333531, and gentamicin sulfate, and additive B comprises Y27632 and CHIR99021.

[0007] Preferably, the small molecule ROCK inhibitor Y-27632 can effectively reduce cell membrane damage and programmed cell death caused by mechanical stress during cryopreservation / thawing by blocking the ROCK-mediated cytoskeleton remodeling and contraction signaling pathway. In the early stage of thawing (initial culture stage), it can make it easier for single cells to form stable clones by inhibiting the cell cycle progress and cytokinesis in the G1-S phase, and can improve the adhesion efficiency of iPSCs and maintain pluripotency. The glycogen synthase kinase-3 inhibitor CHIR99021 can activate the Wnt / β-catenin signaling pathway by inhibiting the activity of GSK-3, promote the formation of high-quality, undifferentiated human embryonic stem cells and human embryonic stem cell populations, maintain their self-renewal and pluripotency and inhibit differentiation. The protein kinase C (PKC) β inhibitor LY-333531 can inhibit the spontaneous differentiation of pluripotent stem cells. Gentamicin sulfate can reduce the risk of contamination during the culture process.

[0008] Preferably, the basal culture medium in this invention is DMEM / F12 medium. In culture medium additive A, based on the final concentration of the complete culture medium, the concentration of ITS-A is 1% v / v, the concentration of HEPES is 25 mM, the concentration of magnesium ascorbate phosphate is 200 μg / ml, the concentration of MEM non-essential amino acids is 1% v / v, the concentration of bFGF is 10 ng / ml, the concentration of FGF2-G3 is 40 ng / ml, the concentration of GlutaMAX is 2 mM, the concentration of LY-333531 is 1 μM, and the concentration of gentamicin sulfate is 80 μg / ml. In culture medium additive B, the concentration of Y27632 is 10 mM, and the concentration of CHIR99021 is 1 mM.

[0009] This invention also provides a method for preparing a human embryonic stem cell expansion culture medium, wherein the preparation method includes: 1) Preparation of culture medium additive A: Dissolve ITS-A, HEPES, magnesium ascorbate phosphate, MEM non-essential amino acids, bFGF, FGF2-G3, GlutaMAX, LY-333531, and gentamicin sulfate in DMEM / F12 culture medium, adjust the pH to 7.2-7.7, and use a 0.2um PES filter membrane for sterilization; 2) Prepare culture medium additive B: Dissolve Y27632 and CHIR99021 in DMSO; 3) Thaw culture medium additives A and B at 2-8℃ before use. Mix additive A with the basal culture medium at a ratio of 1:50 to form a complete culture medium for human embryonic stem cell expansion. Add culture medium additive B is added to the complete culture medium at a ratio of 1:1000 on day 0 after inoculation.

[0010] Preferably, the pH value of the human embryonic stem cell expansion culture medium is 7.2-7.7, and the osmotic pressure of the human embryonic stem cell expansion culture medium is 260-320 mosm / L.

[0011] Preferably, the basal culture medium is stored at 2–8°C, the culture medium additives A and B are stored at -20–-80°C, and the complete culture medium is stored at 2–8°C and used within 3 weeks.

[0012] In this invention, preferably, the stem cells are embryonic stem cells; more preferably, they are human embryonic stem cells.

[0013] Compared with the prior art, the technical solution of the present invention has the following advantages: (1) This culture medium is free of serum and foreign substances, and is suitable for clinical and industrial production. (2) The small molecules such as Y-27632, CHR99021 and LY-333531 added to this culture medium can inhibit apoptosis of pluripotent stem cells, improve adhesion efficiency, enhance colony formation ability, maintain pluripotency and inhibit differentiation. Long-term culture can maintain good cell morphology and stable proliferation. (3) HEPES, GlutaMAX and FGF2-G3 in the culture medium can improve the stability of the culture medium. The composition is simple and the cost is low, which provides strong technical support for scientific research and industrial production. Attached Figure Description

[0014] Figure 1 This is a morphological image of human embryonic stem cell culture observed under a microscope in Example 2.

[0015] Figure 2 The image shows the flow cytometry results of human embryonic stem cell markers Nanog, OCT3 / 4, and Sox17 in Example 3. Detailed Implementation

[0016] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples, so that those skilled in the art can understand the present invention and its advantages.

[0017] Those skilled in the art should understand that the present invention is not limited to these specific embodiments. The embodiments are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0018] Unless otherwise specified, the materials, reagents, human embryonic stem cells, etc. used in the following examples can be obtained commercially.

[0019] Example 1: Preparation of human embryonic stem cell expansion culture medium Preparation of culture medium additive A: Based on the final concentration of the complete culture medium, under aseptic conditions, ITS-A 1% v / v, HEPES 25mM, magnesium ascorbate phosphate 200ug / ml, MEM non-essential amino acids 1% v / v, bFGF 10ng / ml, FGF2-G3 40ng / ml, GlutaMAX 2mM, LY-333531 1uM, and gentamicin sulfate 80ug / ml were dissolved in DMEM / F12 medium according to their respective properties. The pH was adjusted to 7.2-7.7 using NaOH and hydrochloric acid. The mixture was then sterilized by filtration through a 0.2um PES filter membrane. The osmotic pressure was adjusted to 320mosm / L with sodium chloride. After preparation, the solution was aliquoted and stored at -20 to -80℃.

[0020] Preparation of culture medium additive B: Under aseptic conditions, dissolve 10 mM Y27632 and 1 mM CHIR99021 in DMSO, and after preparation, dispense into containers and store at -20 to -80°C.

[0021] In the human embryonic stem cell expansion culture medium of the embodiment, culture medium additives A and B are thawed at 2-8°C before use. Additive A is mixed with the basal culture medium at a ratio of 1:50 to prepare the complete culture medium for human embryonic stem cell expansion. The culture medium additive B is added to the complete culture medium at a ratio of 1:1000 on day 0 of inoculation.

[0022] Example 2: Vitronectin-coated culture plates 1. Thaw Vitronectin at room temperature (15-25℃). 2. Coat at 1 μg / cm. Take a small amount of VTN-coated protein and add it to DMEM / F12. Gently mix and dilute, without vortexing.

[0023] 3. Add the coating solution to the six-well plate and gently shake to mix. Ensure the diluted Vitronectin solution is evenly spread on the bottom surface of the plate.

[0024] 4. Let stand at room temperature (15-25℃) for 1 hour, and discard the coating solution before use.

[0025] iPSC successor 5. Passage is performed when the confluence of human embryonic stem cell clones reaches about 85%.

[0026] 6. Mix additive A and basal culture medium at a ratio of 1:50 to prepare complete culture medium for the expansion of adult embryonic stem cells, and add culture medium additive B at a ratio of 1:1000, and allow to return to room temperature (~25℃).

[0027] 7. Discard the culture medium in the iPSC wells, add 2 mL / well of DPBS (calcium and magnesium-free), gently shake and discard.

[0028] 8. Add 2 mL of Accutase per well to completely cover the bottom of the well.

[0029] 9. Incubate in a 37℃ incubator for 7-8 minutes.

[0030] 10. Add 2 mL of pre-warmed human embryonic stem cell expansion complete culture medium per well, horizontally cross-shake the 6-well plate to detach the cells from the substrate, and gently pipette 1-2 times.

[0031] 11. Centrifuge 200g of cell suspension for 5 minutes, discard the supernatant, and resuspend the cells in human embryonic stem cell expansion complete culture medium.

[0032] 12. Discard the VTN coating solution and seed the cell suspension into the coated 6-well plate at a ratio of 1:10.

[0033] 13. Shake the 6-well plate horizontally three times, place it in an incubator at 37°C, 5% CO2 concentration, and saturated humidity, shake the 6-well plate horizontally three times again, and incubate overnight.

[0034] 14. Replace with fresh human embryonic stem cell expansion complete culture medium (without culture medium additive B) after 18-24 hours. Change the medium daily thereafter, and continue passage after 3-4 days.

[0035] Example 3: In this example, the markers Nanog, OCT3 / 4, and Sox17 of the cells harvested in Example 2 were analyzed by flow cytometry.

[0036] 1. Digestion and cell collection: Discard the culture medium in the well plate, add 2 mL / well of DPBS (without calcium and magnesium) to wash, and then discard.

[0037] 2. Add 2 mL of Accutase and incubate at 37°C for 7-8 min.

[0038] 3. Add 2 mL of pre-warmed human embryonic stem cell expansion complete culture medium and pipette to single cells. 4. Transfer the cell suspension to a 15ml centrifuge tube, centrifuge at 200g for 5 minutes, discard the supernatant, and keep the cell pellet.

[0039] 5. Resuspend the cell pellet in 1 ml of DPBS and count the cells. Add Fixation Buffer to fix for 20 min, centrifuge at 400 g for 5 min, and discard the supernatant.

[0040] 6. Add the nuclear membrane-breaking agent, incubate on ice in the dark for 30 minutes, centrifuge at 400g for 5 minutes, and discard the supernatant. 7. Add antibody for staining, incubate on ice in the dark for 30 min, centrifuge at 400g for 5 min, discard the supernatant, and keep the cell pellet.

[0041] 8. Wash twice: Resuspend the cell pellet in 1 ml of DPBS, centrifuge at 400 g for 5 min, and discard the supernatant.

[0042] 9. Resuspend the cells in 400 μL of DPBS and analyze Nanog, OCT3 / 4, and Sox17 using flow cytometry.

[0043] As attached Figures 1-2 As shown, the human embryonic stem cell expansion culture medium described in this invention exhibits excellent performance in terms of cell morphology and activity, and can effectively maintain the pluripotency of human embryonic stem cells and inhibit differentiation.

Claims

1. A human embryonic stem cell expansion culture medium, characterized in that, The human embryonic stem cell expansion culture medium includes: a basal culture medium and culture medium additive A and culture medium additive B; culture medium additive A includes DMEM / F12 medium, ITS-A, HEPES, magnesium ascorbate phosphate, MEM non-essential amino acids, bFGF, FGF2-G3, GlutaMAX, LY-333531, and gentamicin sulfate; and additive B includes Y27632 and CHIR99021.

2. The human embryonic stem cell expansion culture medium according to claim 1, characterized in that, The basal culture medium is DMEM / F12 medium.

3. The human embryonic stem cell expansion culture medium according to claim 1, characterized in that: In the culture medium additive A, based on the final concentration of the complete culture medium, the concentration of ITS-A is 1% v / v, the concentration of HEPES is 25 mM, the concentration of magnesium ascorbate phosphate is 200 ug / ml, the concentration of MEM non-essential amino acids is 1% v / v, the concentration of bFGF is 10 ng / ml, the concentration of FGF2-G3 is 40 ng / ml, the concentration of GlutaMAX is 2 mM, the concentration of LY-333531 is 1 uM, and the concentration of gentamicin sulfate is 80 ug / ml.

4. The human embryonic stem cell expansion culture medium according to claim 1, characterized in that: In the culture medium additive B, the concentration of Y27632 is 10 mM and the concentration of CHIR99021 is 1 mM.

5. The human embryonic stem cell expansion culture medium according to claim 1, characterized in that: The pH value of the human embryonic stem cell expansion medium is 7.2-7.7, and the osmotic pressure of the human embryonic stem cell expansion medium is 260-320 mosm / L.

6. The human embryonic stem cell expansion culture medium according to claim 3, characterized in that, The preparation method of the culture medium additive A is to dissolve ITS-A, HEPES, magnesium ascorbate phosphate, MEM non-essential amino acids, bFGF, FGF2-G3, GlutaMAX, LY-333531, and gentamicin sulfate in DMEM / F12 culture medium, adjust the pH value to 7.2-7.7, and use a 0.2um PES filter membrane for sterilization.

7. The human embryonic stem cell expansion culture medium according to claim 4, characterized in that, The culture medium additive B is prepared by dissolving Y27632 and CHIR99021 in DMSO.

8. The human embryonic stem cell expansion culture medium according to claim 1, characterized in that, The basal culture medium is stored at 2–8°C, and the culture medium additives A and B are stored at -20–-80°C.

9. A method for preparing the human embryonic stem cell expansion culture medium as described in claim 1, characterized in that... Includes the following steps: Thaw culture medium additives A and B at 2–8°C, and mix additive A with the basal culture medium at a ratio of 1:50 to form a complete culture medium for expanding human embryonic stem cells. Additive B is added to the complete culture medium at a ratio of 1:1000 on day 0 after inoculation.

10. The preparation method according to claim 9, characterized in that, Once the complete culture medium is prepared, it should be stored at 2–8°C and used within 4 weeks.

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