A culture medium additive for improving the proliferation rate of pluripotent stem cells, a culture method, and a culture medium

Abstract

This invention belongs to the field of pluripotent stem cell culture, and relates to a culture medium additive, culture method, and culture medium for improving the proliferation rate of pluripotent stem cells. The additive is SUN11602; the additive is added to a basal culture medium with well-defined components. The technical solution of this application, while fully retaining and being compatible with existing culture medium formulations, significantly and safely improves the in vitro proliferation rate, colony formation rate, and population expansion efficiency of hPSCs by adding exogenous compounds without compromising hPSC pluripotency and genomic integrity.

Description

Technical Field

[0001] This invention belongs to the field of pluripotent stem cell culture, and relates to a culture medium additive, culture method and culture medium for improving the proliferation rate of pluripotent stem cells. Background Technology

[0002] Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are core tools for regenerative medicine, disease model building, and drug discovery. Their successful application relies on achieving efficient, stable, and homogeneous large-scale expansion in vitro. Currently, culture media with well-defined compositions and free of xenobiotic components, such as commercially available E8 medium, have become the gold standard for culturing hPSCs. E8 medium contains basal medium, insulin, selenite, transferrin, specific TGF-β family members (such as Noggin / GDF substitutes), and high concentrations of basic fibroblast growth factor (bFGF or FGF-2, typically used at 100 ng / mL). This formulation is designed to precisely regulate key signaling pathways such as SMAD and FGF / MAPK / ERK to maintain cell self-renewal and pluripotency.

[0003] Although E8 medium can support the basic growth and maintenance of hPSCs, the following core bottlenecks still exist in the large-scale culture and long-term expansion for industrialization. The fundamental problem lies not in the medium components themselves, but in the contradiction between proliferation rate and large-scale production: While the existing E8 medium system can maintain the pluripotency of hPSCs, the cell population doubling time it supports is relatively fixed, and the proliferation rate has not yet reached the theoretically optimal level. When conducting large-scale expansion for dozens or even hundreds of generations to obtain sufficient cells for treatment or screening, the current proliferation efficiency directly leads to long culture cycles, high equipment resource consumption, and high overall costs. Improving the proliferation rate within a single passage is key to shortening the production cycle and reducing costs. Summary of the Invention

[0004] This application provides a culture medium additive, culture method, and culture medium for improving the proliferation rate of pluripotent stem cells. While fully retaining and being compatible with existing culture medium formulations, the addition of exogenous compounds can safely, stably, and reproducibly significantly improve the in vitro proliferation rate, colony formation rate, and population expansion efficiency of hPSCs without compromising the pluripotency and genomic integrity of hPSCs.

[0005] To achieve the above technical objectives, the present application provides a culture medium additive for improving the proliferation rate of pluripotent stem cells, wherein the additive is SUN11602; the additive is added to a basal culture medium with clearly defined components for use.

[0006] As an improved technical solution of this application, the dosage of SUN11602 ranges from 0.1 to 3 μM.

[0007] As an improved technical solution of this application, the basal culture medium with clearly defined components is a serum-free culture medium containing FGF-2 and TGF-β family growth factors.

[0008] As an improved technical solution of this application, the basic culture medium with clearly defined components is E8 culture medium.

[0009] Another objective of this application is to provide a method for culturing pluripotent stem cells to improve their proliferation rate, comprising the following steps: Day 0: Culture pluripotent stem cells in E8 medium; Days 1-4: Discard the original culture medium and replace it with E8 medium containing SUN11602 for further culture.

[0010] As an improved technical solution in this application, the cell seeding amount per well is 5 × 10⁶. 4 indivual.

[0011] As an improved technical solution of this application, the dosage of SUN11602 ranges from 0.1 to 3 μM.

[0012] Another objective of this application is to provide a culture medium that enhances the proliferation rate of pluripotent stem cells, including the additive SUN11602.

[0013] As an improved technical solution of this application, E8 culture medium is also included.

[0014] As an improved technical solution of this application, the dosage of SUN11602 ranges from 0.1 to 3 μM. Beneficial effects

[0015] Significantly improves hPSC proliferation efficiency, directly reducing time and cost: This invention, without altering the core components and formulation of existing mature culture media, can safely and significantly enhance the in vitro proliferation capacity of human pluripotent stem cells (hPSCs) by adding specific compounds. Experiments have confirmed that using the culture medium composition of this invention can increase the proliferation rate of iPSCs by 10%-25%. This means that more cells can be obtained in the same culture time, or the culture cycle required to obtain the same number of cells can be significantly shortened, directly reducing the time and overall economic costs associated with equipment usage, manual operation, and culture medium consumption. This lays a key technological foundation for the large-scale production of hPSCs.

[0016] Fully compatible with existing standardized processes, easy to use and risk-free: The additive of this invention is fully compatible with currently used internationally recognized culture media with well-defined compositions (such as E8 medium). Users do not need to change their existing, validated basal culture medium formulations, extracellular matrix, or standard operating procedures; it can be added directly to existing culture media for it to take effect. This greatly reduces the technology transfer threshold and risk, avoiding the risks of uncertainty in cell adaptability and pluripotency that may arise from changing to a completely new culture medium system, and facilitating rapid adoption and application by laboratories and manufacturing companies.

[0017] With clearly defined components and controllable safety, it has the potential for industrialization. The additives used in this invention are chemically defined small molecule compounds or recombinant proteins, without introducing animal-derived components or extracts of unknown composition, thus meeting the regulatory requirements for raw materials in cell therapy products. This approach improves efficiency while maintaining the clarity and safety of the culture system's composition, clearing obstacles for its application in clinical-grade cell production that complies with Good Manufacturing Practice (GMP) standards, and demonstrating broad prospects for industrialization. Attached Figure Description

[0018] Figure 1 Proliferation at 96 hours in Example 1; Figure 2 SSEA4 flow cytometry detection plot in Example 2; Figure 3 Oct4 flow cytometry detection plot in Example 2; Figure 4 Nanog flow cytometry detection plot in Example 2; Figure 5 The effect on the mesoblast differentiation capacity of iPSCs in Example 3; Figure 6 The effect on the endoderm differentiation ability of iPSCs in Example 3; Figure 7 The effect on the ectoderm differentiation ability of iPSCs in Example 3. Detailed Implementation

[0019] The technical solution of this application will be clearly and completely described below with reference to the accompanying drawings and specific embodiments.

[0020] Definition of noun In this article, the small molecule compound and the small molecule S refer to SUN11602.

[0021] In this article, E8 medium is referred to as Essential 8, which is E8 complete medium (containing 2 ng / mL TGF-β1 and 100 ng / mL FGF-2).

[0022] In this article, pluripotent stem cells include induced pluripotent stem cells (iPSCs).

[0023] Manufacturers and models of reagents used in the experiments in the examples III. The technical solution of this application will be verified under different experimental conditions below. Example 1

[0024] The effect of adding small molecule SUN11602 to E8 complete medium on iPSC proliferation. Experimental groups: Group A: E8 complete culture medium; Group B: E8 complete culture medium + 10% FGF; Group C: E8 complete medium + 30% FGF; Group D: E8 complete medium + 100% FGF; Group E: E8 complete culture medium + 0.01 μM small molecule S; Group F: E8 complete medium + 0.1 μM small molecule S; Group G: E8 complete culture medium + 1 μM small molecule S; Group H: E8 complete medium + 3 μM small molecule S; Group I: E8 complete culture medium + 10 μM small molecule S.

[0025] Experimental steps: Day 0: iPSCs were seeded as single cells in Matrigel-coated 12-well plates, with a seeding density of 5 × 10⁶ cells per well. 4 Cells. Cultured using E8 complete medium. Triple replicates per condition.

[0026] Days 1-4: Discard the original culture medium and replace it with the corresponding culture medium for group A, group B, group C, group D, group E, group F, group G, group H, and group I, respectively. Change the medium daily.

[0027] Day 5: Perform endpoint testing (cell count / viability testing).

[0028] Cell counting / viability assay: After digesting the cells into single cells, they were stained with AO / PI staining solution and the number and viability of cells in each well were analyzed using a Countstar automated cell counter.

[0029] Combination Figure 1It can be seen that the small molecule SUN11602 can enhance the proliferation capacity of iPSCs and cannot be replaced by FGF protein. The 96-hour proliferation rate of iPSCs after adding 1 μM SUN11602 to the E8 complete medium (group G) was significantly higher than that of the E8 culture group (group A), while the addition of 0.01 μM, 0.1 μM, 3 μM, and 10 μM SUN11602 (groups E, F, H, and I) did not have the same effect as group G. Furthermore, the 96-hour proliferation rate of iPSCs after adding 10%–100% FGF (group BD) did not show a significant difference compared to the E8 culture group (group A). Example 2

[0030] To verify the effect on iPSC dryness, the experimental group was: Control group: E8 complete culture medium; Preferred group: E8 complete culture medium + 1 μM small molecule S.

[0031] Experimental steps: Day 0: iPSCs were seeded as single cells in Matrigel-coated 12-well plates, with a seeding density of 5 × 10⁶ cells per well. 4 Each well was cultured using E8 complete medium. Three replicates were performed for each condition.

[0032] Days 1-4: Discard the original culture medium and replace it with the corresponding culture medium for the control group and the optimized group, changing the medium daily.

[0033] Day 5: Digest cells with TrypLE and passage them, repeating the steps from Day 0 to Day 4.

[0034] After 10 passages of cell culture, endpoint assays (SSEA4, Oct4, Nanog assays) were performed using the following flow cytometry method: SSEA4: Take 1.0 × 10 6 Cell suspension was centrifuged at 300g for 5 min, and the supernatant was removed. Cell blocking buffer was applied and incubated at 4°C for 10 min. DPBS was added and mixed thoroughly, then divided into two tubes (SSEA4 tube and SSEA4 isotype control tube). Cells were washed by centrifugation at 300g for 5 min. After removing the supernatant, 100 μL of SSEA4 antibody working solution was added to the SSEA4 tube, and 100 μL of SSEA4 isotype control working solution was added to the SSEA4 isotype control tube. The mixture was incubated at room temperature in the dark for 30 min. After incubation, cells were washed with DPBS, centrifuged at 300g for 5 min, and the supernatant was removed. Cells were then resuspended in 200 μL of DPBS, and the resuspended solution was sent to a flow cytometer for analysis.

[0035] Oct4, Nanog: Take 3.0 × 10 6Cell suspension was centrifuged at 300g for 5 min, and the supernatant was removed. Cell blocking buffer was applied and incubated at 4°C for 10 min. DPBS was added, mixed, and centrifuged at 300g for 5 min to wash the cells. After removing the supernatant, fixation and permeabilization buffer was added, mixed, and incubated at room temperature in the dark for 15 min. 1% BSA was added, mixed, and divided into three tubes (isotype control, Oct4, and Nanog), and each tube was centrifuged at 300g for 5 min to wash the cells. After removing the supernatant, 100 μL of 1% BSA was added to the isotype control tube, 100 μL of Oct4 antibody working solution to the Oct4 tube, and 100 μL of Nanog working solution to the Nanog tube. The tubes were incubated at room temperature in the dark for 30 min. After incubation, cells were washed with DPBS, centrifuged at 300g for 5 min, and the supernatant was removed. The cells were then resuspended in 200 μL of DPBS, and the resuspended solution was sent to a flow cytometer for analysis.

[0036] Combination Figures 1-4 show: After long-term culture, under the condition of adding 1 μM SUN11602 (preferred group) to E8 complete medium, the pluripotency of iPSCs was not significantly different from that of the E8 culture group (control group). Example 3

[0037] To verify the effect on the differentiation ability of iPSCs into three germ layers, the experimental group was: Control group: E8 complete culture medium; Preferred group: E8 complete culture medium + 1 μM small molecule S.

[0038] Experimental steps: iPSCs were seeded as single cells in Matrigel-lined 12-well plates at a density of 5 × 10⁶ cells per well. 4 Cells. Cultured using E8 complete medium. Triple replicates per condition.

[0039] Discard the original culture medium and replace it with the corresponding culture medium for the control group and the optimized group, changing the medium daily.

[0040] When the cell fusion rate reaches 30-50%, trigerm layer differentiation is performed (differentiation methods include: Xu F, Deng C, Ren Z, Sun L, Meng Y, Liu W, Wan J, Chen G. Lysophosphatidic acid shiftsmetabolic and transcriptional landscapes to induce a distinct cellular state in human pluripotent stem cells. Cell Rep. 2021 Nov 30;37(9):110063. doi:10.1016 / j.celrep.2021.110063. PMID: 34852227.).

[0041] Forty-eight hours after differentiation, iPSCs differentiated into mesoderm were collected for qPCR detection of mesoderm-specific markers (TBXT, MIXL1).

[0042] Ninety-six hours after differentiation, iPSCs differentiating into endoderm and ectoderm were collected for qPCR detection of endoderm-specific markers (SOX17, GATA4) and ectoderm-specific markers (PAX6, SOX1). The qPCR method was as follows: RNA was extracted from the cells to be tested, and the RNA concentration was measured. Then, reverse transcription was performed to obtain cDNA solution from the cells to be tested. The reagent kit reaction solution, forward and reverse primers (10 μM) were thawed at room temperature, vortexed and mixed, centrifuged at 2680 g, and placed on ice to prepare the amplification system for each marker. After thoroughly mixing all components, a short centrifugation was performed, and then 6 μL of the amplification system was added to each reaction tube of a 384-well plate on ice. 4 μL of cDNA solution from the cells to be tested was added to each of the above reaction tubes, the plates were sealed, and centrifuged at 500 g for 80 s in a microplate centrifuge before qPCR detection.

[0043] Combination Figures 5-7 The results showed that there was no significant difference in the trigerm layer differentiation ability between iPSCs cultured in E8 complete medium with an additional 1 μM SUN11602 (preferred group) and those cultured in E8 complete medium (control group).

Claims

1. A culture medium additive for improving the proliferation rate of pluripotent stem cells, characterized in that, The additive is SUN11602; the additive is added to a well-defined basal culture medium for use.

2. The culture medium additive for improving the proliferation rate of pluripotent stem cells according to claim 1, characterized in that, The dosage range of SUN11602 is 0.1-3 μM.

3. The culture medium additive for improving the proliferation rate of pluripotent stem cells according to claim 1, characterized in that, The basal culture medium with clearly defined components is a serum-free medium containing FGF-2 and TGF-β family growth factors.

4. The culture medium additive for improving the proliferation rate of pluripotent stem cells according to claim 3, characterized in that, The basal culture medium with clearly defined components is E8 medium.

5. A method for culturing pluripotent stem cells to improve their proliferation rate, characterized in that: Includes the following steps: Day 0: Culture pluripotent stem cells in E8 medium; Days 1-4: Discard the original culture medium and replace it with E8 medium containing SUN11602 for further culture.

6. The method for improving the proliferation rate of pluripotent stem cells according to claim 5, characterized in that: The cell seeding density per well is 5 × 10⁶ cells. 4 indivual.

7. The method for improving the proliferation rate of pluripotent stem cells according to claim 5, characterized in that: The dosage range of SUN11602 is 0.1-3 μM.

8. A culture medium for improving the proliferation rate of pluripotent stem cells, characterized in that: Includes additive SUN11602.

9. The culture medium for improving the proliferation rate of pluripotent stem cells according to claim 8, characterized in that: It also includes E8 culture medium.

10. The culture medium for improving the proliferation rate of pluripotent stem cells according to claim 8, characterized in that: The dosage range of SUN11602 is 0.1-3 μM.

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