Induced pluripotent stem cell culture medium and culture method of induced pluripotent stem cells

Abstract

The invention provides an induced pluripotent stem cell culture medium and a culture method of induced pluripotent stem cells. The induced pluripotent stem cell culture medium comprises a basic culture medium, a composite additive A and a composite additive B, the composite additive A comprises 5-30 [mu] g / mL of recombinant human insulin growth factors, 1-5 ng / mL of recombinant human transforming growth factors, 50-200 ng / mL of recombinant human basic fibroblast growth factors, 7-20 [mu] g / mL of sodium selenite, 0.5-2 mg / mL of albumin, 50-70 [mu] g / mL of ascorbic acid and 5-20 [mu] g / mL of transferrin; the composite additive B is prepared from 50 to 300mg / mL of a Stock B solution, 0.5 to 5 percent (v / v) of a non-essential amino acid solution, 50 to 200ng / mL of DL-piperidinecarboxylic acid, 50 to 200mu g / mL of gamma-aminobutyric acid, 0.1 to 1 percent (v / v) of a lipid concentrated solution, 0.5 to 5 percent (v / v) of an L-alanyl-L-glutamine solution and 0.1 to 0.1 percent of mercaptoethanol. The culture medium for the induced pluripotent stem cells provided by the invention can ensure the self-renewal capability of the iPSCs in a long-term passage process; and the genetic stability of the iPSCs cells is optimized according to the genome integrity and pluripotent state.

Description

Technical Field

[0001] This invention belongs to the field of culture medium technology, specifically relating to an induced pluripotent stem cell culture medium and a method for culturing induced pluripotent stem cells. Background Technology

[0002] Induced pluripotent stem cells (iPSCs), a promising discovery in stem cell research, have garnered widespread attention from the global academic and clinical medical communities in recent years. iPSCs are a class of cells that can be transformed from differentiated somatic cells (such as skin cells) into pluripotent cells similar to embryonic stem cells through gene reprogramming technology. This technological breakthrough not only offers new hope for regenerative medicine but also demonstrates enormous application potential in basic research, disease model development, drug screening, and personalized medicine.

[0003] Ensuring the quality and pluripotency of iPSCs to meet clinical needs during large-scale production presents numerous challenges. The pluripotency state of iPSCs is extremely sensitive and highly dependent on the culture medium (i.e., cell culture medium). Cell culture medium is crucial for maintaining healthy and proliferating cells by providing a good balance of nutrients, minerals, and pH. The cell culture medium, acting as a supporting structure for cell adhesion and proliferation, is typically encapsulated by feeder cells or growth support factors to further promote cell adhesion and growth.

[0004] However, most current iPSC cell culture media contain animal-derived serum components. Studies have shown that long-term use of animal-derived serum culture media may affect cell morphology, expansion potential, gene expression, and cytokine profile. Moreover, during long-term culture, current iPSC cell culture media are prone to spontaneous differentiation or gene mutations, leading to increased cell heterogeneity. In other words, after long-term passage culture, the cell morphology and pluripotency stability of iPSC cells are affected. Summary of the Invention

[0005] To address one of the problems existing in the prior art, the present invention provides an induced pluripotent stem cell culture medium and a method for culturing induced pluripotent stem cells.

[0006] According to a first aspect of the present invention, an induced pluripotent stem cell culture medium is provided, comprising a basal culture medium and compound additive A and compound additive B; compound additive A comprises recombinant human insulin-like growth factor 5-30 μg / mL, recombinant human transforming growth factor 1-5 ng / mL, recombinant human basic fibroblast growth factor 50-200 ng / mL, sodium selenite 7-20 ng / mL, albumin 0.5-2 mg / mL, ascorbic acid 50-70 μg / mL, and transferrin 5-20 μg / mL; compound additive B comprises Stock B solution 50-300 mg / mL, non-essential amino acid solution 0.5-5% (v / v), DL-piperidinecarboxylic acid 50-200 ng / mL, γ-aminobutyric acid 50-200 μg / mL, lipid concentrate 0.1-1% (v / v), L-alanyl-L-glutamine solution 0.5-5% (v / v), and mercaptoethanol 0.01-0.1% (v / v).

[0007] During long-term passaging, iPSCs face two major challenges: 1) loss of pluripotency (spontaneous differentiation); and 2) genomic instability (including genetic and epigenetic variations). Therefore, this invention constructs a microenvironment that mimics in vivo homeostasis and synergistically regulates multiple key signaling pathways by adding compound additives A and B, containing the specific materials described above, to the basal culture medium. This microenvironment enables iPSCs to maintain stable cell morphology and pluripotency gene expression and epigenetic stability during long-term passaging.

[0008] Specifically, in compound additive A, recombinant human insulin-like growth factor, recombinant human transforming growth factor, and basic fibroblast growth factor jointly activate and balance signaling pathways related to stem cell pluripotency (such as PI3K / Akt, TGF-β / Smad, MAPK, etc.), promoting cell self-renewal while inhibiting differentiation tendency. This combination helps maintain typical iPSC morphology (such as high nucleocytoplasmic ratio and clear colony edges) and sustains the expression of pluripotency genes (such as OCT4, SOX2, Nanog). Sodium selenite (selenium source) and ascorbic acid (vitamin C) form a synergistic antioxidant combination, reducing the accumulation of reactive oxygen species, decreasing DNA oxidative damage and cellular stress, and mitigating oxidative damage from long-term passage, thereby protecting the stability of the cell genome and epigenetics. Ascorbic acid also acts as a cofactor in regulating epigenetic modifying enzymes such as histone demethylation, helping to maintain the open state of chromatin and the correct expression pattern of pluripotency-related genes. Transferrin efficiently transports iron ions essential for cell proliferation, while albumin acts as a carrier, binding and transporting fatty acids, hormones, and lipid-soluble substances. Simultaneously, albumin stabilizes the physicochemical environment of the culture medium, protecting various active ingredients (especially growth factors) and reducing their inactivation. Therefore, transferrin and albumin work synergistically to ensure the stable delivery and bioavailability of active ingredients such as growth factors and trace elements, providing balanced nutritional support for cells and improving the overall physicochemical stability and batch-to-batch consistency of the culture medium.

[0009] In compound additive B, Stock B solution (usually salt, buffer, etc.) and the basal culture medium together constitute a stable ionic and pH environment. L-alanyl-L-glutamine serves as a more stable source of glutamine. The non-essential amino acids and lipid concentrates together provide highly efficient and balanced energy and nutrient synthesis substrates for rapidly proliferating iPSCs, supporting their unique metabolic state. More importantly, γ-aminobutyric acid (GABA), as a signaling molecule, can participate in regulating cell proliferation, metabolism, and stress responses; DL-piperidinecarboxylic acid can act as a precursor or regulator affecting related metabolic pathways. Together, they finely regulate the intracellular signaling microenvironment, potentially contributing to improved iPSC cell state uniformity and stress resistance. Mercaptoethanol, as a reducing agent, helps maintain the necessary reducing environment in the culture medium, protecting cells from oxidative stress and potentially affecting the activity state of certain growth factors, ensuring their high activity.

[0010] Therefore, through the combined action of the aforementioned specific additives A and B, an advanced culture system is constructed that not only maintains pluripotency but also actively optimizes cell metabolism and signal homeostasis. Compared to traditional culture media that only provide growth factors, this system has greater advantages in terms of pluripotency quality, long-term stability, and functional adaptability. Consequently, iPSCs cells using this culture medium exhibit higher and longer-term epigenetic stability during long-term passage. In particular, this invention demonstrates through relevant experiments that when iPSCs cells are passaged using the induced pluripotent stem cell culture medium containing the aforementioned specific additives A and B, the resulting iPSCs cells, after long-term passage from P26 to P30, still possess a similar cell morphology to the iPSCs cells before passage. This indicates that the induced pluripotent stem cell culture medium of this invention is effective for passage culture of iPSCs cells, and the obtained iPSCs cells still exhibit good epigenetic stability even after long-term passage.

[0011] Furthermore, it should be noted that the induced pluripotent stem cell culture medium provided by this invention does not contain any animal-derived components and has a clearly defined chemical composition. This not only improves safety but also enables precise traceability and investigation in case of problems, which is crucial for Good Manufacturing Practices (GMP) in pharmaceutical manufacturing. Moreover, the compound additives A and B added in this invention have good interactions with the basal culture medium, which is more conducive to maintaining the stability of iPSCs during long-term passage.

[0012] Preferably, the Stock B solution comprises 0.33 mg / mL of L-ascorbic acid 2-phosphate sesquimagnesium hydrate, 41 μg / mL of vitamin B1, 10 μg / mL of reduced glutathione, and 66 μg / mL of benzoylthiamine.

[0013] Preferably, the non-essential amino acid solution is a Gibco™ MEM non-essential amino acid solution.

[0014] Preferably, the lipid concentrate is the Thermo Fisher Scientific product, catalog number 11905031, named Chemically Defined Lipid Concentrate.

[0015] Preferably, the L-alanyl-L-glutamine solution is Gibco™ GlutaMAX. TM Supplements.

[0016] Preferably, compound additive A comprises recombinant human insulin-like growth factor 20 μg / mL, recombinant human transforming growth factor 2 ng / mL, recombinant human basic fibroblast growth factor 100 ng / mL, sodium selenite 14 ng / mL, albumin 1 mg / mL, ascorbic acid 66 μg / mL, and transferrin 11 μg / mL; compound additive B comprises Stock B solution 200 mg / mL, non-essential amino acid solution 1% (v / v), DL-piperidinecarboxylic acid 129.8 ng / mL, γ-aminobutyric acid 103.1 μg / mL, lipid concentrate 0.2% (v / v), L-alanyl-L-glutamine solution 1% (v / v), and mercaptoethanol 0.07% (v / v).

[0017] Furthermore, the concentrations of each component in compound additive A and compound additive B are as described above. The components in compound additive A and compound additive B can exert a better synergistic effect without affecting the performance of each component itself. Therefore, it is more conducive to ensuring the self-renewal capacity, genome integrity and pluripotency of iPSCs during long-term passage.

[0018] Preferably, the basal culture medium is DMEM / F-12 medium. The selected DMEM / F-12 medium can synergistically interact with compound additive B, providing more comprehensive nutrition and facilitating the efficient utilization of growth factor signals from compound additive A by cells for protein synthesis, energy metabolism, and proliferation. Furthermore, the basic components of DMEM / F-12 (such as some salt ions), together with the antioxidant network of compound additive A and the reducing environment (mercaptoethanol) of compound additive B, construct a multi-layered oxidative stress defense system. Therefore, DMEM / F-12 medium can produce a better synergistic effect with the added compound additives A and B, forming a specific culture system that more effectively maintains the long-term passage pluripotency, genomic stability, and culture consistency of iPSCs.

[0019] Preferably, the volume ratio of compound additive A, compound additive B, and basal culture medium is 1:1.5~6:45~105. This volume ratio ensures that the final concentrations of key active ingredients in compound additives A and B are precisely controlled within the optimal window, thus guaranteeing the best synergistic effect of each functional component in the complex system and maintaining the pluripotency and genomic stability of iPSCs during long-term passage.

[0020] Preferably, the volume ratio of compound additive A, compound additive B and basal culture medium is 1:3:100.

[0021] Preferably, the basal culture medium is stored at 2~8℃, and the additives are stored at -15℃~-25℃.

[0022] Preferably, compound additive A and / or compound additive B are thawed at 2-8°C before being used to prepare induced pluripotent stem cell culture medium.

[0023] According to a second aspect of the present invention, a method for culturing induced pluripotent stem cells (iPSCs) is provided, comprising the following steps: (a) resuscitation: iPSCs are seeded into a first culture dish coated with matrix gel for initial culture, the first culture dish containing a first culture medium comprising any one of the aforementioned iPSC culture medium and a first ROCK inhibitor; (b) passage: when the iPSCs resuscitated in step (a) have adhered and grown to a confluence of 85% or more, they are passaged in a second culture dish coated with matrix gel, the second culture dish containing a second culture medium comprising iPSC culture medium and a second ROCK inhibitor. In the iPSC culture method provided by the present invention, the present invention-specific culture medium (i.e., the iPSC culture medium provided by the present invention) with the same composition is always used from the iPSC resuscitation, expansion and subsequent passage. This eliminates the component fluctuations, osmotic pressure changes and signal differences caused by changing different brands or formulations of culture medium. iPSCs are extremely sensitive to changes in the culture environment, and the method of the present invention ensures that the cells are in a microenvironment with clear signals, stable composition and consistent physicochemical properties from the moment of thawing until all subsequent passages. This high degree of uniformity minimizes the interference of environmental stress on cells, providing a fundamental guarantee for maintaining the homogeneity of the cell population and the stability of the genome.

[0024] Furthermore, during both step (a) thawing and step (b) passaging, ROCK inhibitors were actively added to the culture medium. During thawing, the inhibitors protected the most vulnerable cells, which had just recovered from cryopreservation, ensuring successful cell adhesion and laying a high-activity foundation for the entire culture process. During passaging, the inhibitors protected cells dispersed due to digestion, ensuring high viability for re-adhesion and the formation of new clones. Therefore, this process ensured high cell viability in each expansion cycle, effectively preventing clonal selective drift and genomic instability caused by repeated apoptosis stress over the long term.

[0025] Preferably, the first and second ROCK inhibitors are Y-27632. Using Y-27632 as a ROCK inhibitor allows for a more significant synergistic effect with the induced pluripotent stem cell culture medium system provided by this invention, which is more conducive to ensuring a smooth transition of cells during the resuscitation and passage process, thereby fully demonstrating the optimization effect of the culture medium on the long-term passage stability of iPSCs.

[0026] Preferably, the volume ratio of the first ROCK inhibitor to the first culture medium is 0.7~1.5:1000; the volume ratio of the second ROCK inhibitor to the second culture medium is 0.7~1.5:1000. By controlling the proportion of ROCK inhibitor in the culture medium within the above ranges during the resuscitation and passage of iPSCs, sufficient concentrations of Y-27632 can be ensured to maximize the inhibition of apoptosis and improve cell survival and colony formation rates after resuscitation and passage. Simultaneously, the reduced efficacy due to excessively low concentrations (insufficient protection) or excessively high concentrations (potential side effects and cost waste) can be avoided.

[0027] Preferably, step (a) revival includes the following steps: (a1) Spreading the matrix gel into the first culture dish and placing it in an incubator for at least 30 minutes; (a2) Taking the cryovial containing induced pluripotent stem cells and gently shaking it in a 37±1℃ water bath until no ice crystals remain; (a3) ​​Adding the cell suspension from the cryovial to the basal culture medium, centrifuging, and discarding the supernatant; (a4) Resuspending the cells in induced pluripotent stem cell culture medium, staining and counting, replenishing the induced pluripotent stem cell culture medium, and adding the first ROCK inhibitor, mixing well; (a5) Discarding the matrix gel from the first culture dish in (a1), then transferring the cell suspension obtained in (a4) to the first culture dish, shaking well, and placing it in an incubator for initial culture. The culture medium needs to be changed once a day. Here, the culture medium refers to induced pluripotent stem cell culture medium. The first ROCK inhibitor only needs to be added on the first day when seeding the flask, and does not need to be added after changing the medium daily. The above specific revival steps can maximize the survival and functional recovery of iPSCs, laying an important foundation for the subsequent passage stability of iPSCs.

[0028] Preferably, in (a1), the culture conditions in the incubator are: 5±0.5% CO2, 37±1℃.

[0029] Preferably, in (a3), the centrifugation conditions are: 280~320g, 4~6min.

[0030] Preferably, in (a4), the cell suspension is counted using trypan blue staining, and the volume ratio of trypan blue to cell suspension is 1:1.

[0031] Preferably, in (a5), the initial culture conditions are: 5±0.5% CO2, 37±1℃.

[0032] Preferably, step (b) passage includes the following steps: (b1) Spread the matrix gel into the second culture dish and place it in an incubator for more than 30 minutes for later use; (b2) When the cell adhesion and confluence of the cells revived in step (a) reaches more than 85%, discard the old culture medium and add basal culture medium for washing 2-3 times; (b3) Discard the washing solution and add digestion solution to digest the cells. When 50% or more of the cells become rounded, stop the digestion; (b4) Transfer the cell suspension obtained in (b3) to a centrifuge tube containing basal culture medium, centrifuge, and discard the supernatant; (b5) Resuspend the cells in induced pluripotent stem cell culture medium, stain and count, take the viable cells, add the second ROCK inhibitor, and mix well; (b6) Discard the matrix gel in the second culture dish in (b1), then transfer the cell suspension obtained in (b5) to the second culture dish, shake well, and place it in an incubator for passage culture. The culture medium needs to be changed once a day. The culture medium mentioned here refers to induced pluripotent stem cell culture medium. The second ROCK inhibitor only needs to be added on the first day of seeding; it does not need to be added after daily medium changes. The specific steps of passage described above optimize the entire process from iPSC digestion to reseeding. In particular, it achieves gentle and efficient cell digestion and effectively and systematically eliminates digestion stress, ensuring that cells enter the next round of expansion in optimal condition, which is beneficial for maintaining the consistency and genetic stability of the iPSC cell population.

[0033] Preferably, in (b1), the culture conditions in the incubator are: 5±0.5% CO2, 37±1℃.

[0034] Preferably, in (b3), the digestion solution is Tryple Express digestion solution. Preferably, cell morphology is observed using a microscope. Preferably, after adding the digestion solution, the culture dish is placed in an incubator for 6-8 minutes under the following culture conditions: 5±0.5% CO2, 37±1℃.

[0035] Preferably, in (b4), the centrifugation conditions are: 280~320g, 4~6min.

[0036] Preferably, in (b5), the cell suspension is counted using trypan blue staining, and the volume ratio of trypan blue to cell suspension is 1:1.

[0037] Preferably, in (b6), the subculture conditions are: 5±0.5% CO2, 37±1℃.

[0038] In summary, the present invention provides an induced pluripotent stem cell culture medium prepared by a specific composition of compound additives A and B, which can effectively provide a stable and uniform culture medium environment for the resuscitation and passage of iPSCs. Therefore, it can ensure the self-renewal capacity, genomic integrity and pluripotency of iPSCs during long-term passage, and optimize the genetic stability of iPSCs. Attached Figure Description

[0039] Figure 1 This is a microscope image of iPSCs passaged to P26 in Example 1 of the present invention.

[0040] Figure 2 This is a microscope image of iPSCs passaged to P27 in Example 1 of the present invention.

[0041] Figure 3 This is a microscope image of iPSCs passaged to P28 cells in Example 1 of this invention.

[0042] Figure 4 This is a microscope image of iPSCs passaged to P29 in Example 1 of the present invention.

[0043] Figure 5 This is a microscope image of iPSCs passaged to P30 in Example 1 of the present invention.

[0044] Figure 6 This is a microscope image of iPSCs passaged to P5 in Example 1 of this invention. Detailed Implementation

[0045] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0046] Example 1 In this embodiment, the induced pluripotent stem cell culture medium used in the iPSC cell resuscitation and passage process consists of DMEM / F-12 medium (basal medium) and compound additive A and compound additive B, with a volume ratio of compound additive A, compound additive B, and DMEM / F-12 medium of 1:3:100. Compound additive A includes the following components: recombinant human insulin-like growth factor 20 μg / mL, recombinant human transforming growth factor 2 ng / mL, recombinant human basic fibroblast growth factor 100 ng / mL, sodium selenite 14 ng / mL, albumin 1 mg / mL, ascorbic acid 66 μg / mL, and transferrin 11 μg / mL; compound additive B includes Stock... Stock B solution contains 200 mg / mL of Stock B, 1% (v / v) of non-essential amino acid solution, 129.8 ng / mL of DL-piperidinecarboxylic acid, 103.1 μg / mL of γ-aminobutyric acid, 0.2% (v / v) of lipid concentrate, 1% (v / v) of L-alanyl-L-glutamine solution, and 0.07% (v / v) of mercaptoethanol. Specifically, Stock B solution includes 0.33 mg / mL of L-ascorbic acid 2-phosphate sesquimagnesium hydrate, 41 μg / mL of vitamin B1, 10 μg / mL of reduced glutathione, and 66 μg / mL of benzoylthiamine; the non-essential amino acid solution is Gibco™ MEM non-essential amino acid solution; the lipid concentrate is a product from Thermo Fisher Scientific, catalog number 11905031, product name Chemically Defined Lipid Concentrate; and the L-alanyl-L-glutamine solution is Gibco™ GlutaMAX™ supplement.

[0047] The preparation method is as follows: dissolve each of the above components according to their respective solubility characteristics, filter and sterilize using a filter membrane, and then prepare compound additive A and compound additive B in the specified amounts. Store in aliquots at -20℃. Under aseptic conditions, add compound additive A and compound additive B to DMEM / F-12 basal medium in the specified proportions, mix well, adjust the pH to 7.0-7.4, and store the prepared medium (induced pluripotent stem cell medium) at 2-8℃. The shelf life is two weeks.

[0048] The iPSCs cells of this embodiment were revived and passaged according to the following steps: (a) Recovery: (a1) Take 5 mL of the bottle-laying gel (matrix gel) into T75 and place it in the incubator for more than 30 min, then set aside for use; the incubation conditions in the incubator are: 5% CO2, 37℃; (a2) Remove the cryopreservation tube containing induced pluripotent stem cells from the liquid nitrogen tank and quickly place it in a 37°C water bath and gently shake until no ice crystals remain; (a3) Wipe the outside of the cryovial with an alcohol swab, add the cell suspension in the cryovial to a 15 mL centrifuge tube containing 8 mL of DMEM / F-12 medium and mix well, then rinse the cryovial once with 1 mL of DMEM / F-12 medium, and combine the washings into a 15 mL centrifuge tube; centrifuge (300 g, 5 min) and discard the supernatant; (a4) Resuspend the cells in 1 mL of complete culture medium, stain with trypan blue at a 1:1 ratio and count them, then add 7 mL of complete culture medium (i.e., induced pluripotent stem cell culture medium), and add 8 μL of Y-27632 (ROCK inhibitor) to the cell suspension and mix well. (a5) Discard the gelling agent in T75 from (a1), transfer the cell suspension to a T75 culture flask, shake well and place in an incubator. The culture conditions are 5% CO2, 37℃, and the culture medium needs to be changed once a day. (b) Transmission: (b1) Take 5 mL of the bottle-laying gel (matrix gel) into T75 and place it in the incubator for more than 30 min before use; the incubation conditions in the incubator are: 5% CO2, 37℃; (b2) When the cell adhesion and confluence of the cells revived in step (a) reaches more than 85%, discard the old culture medium and add 5 ml of LDM / 12 culture medium for washing twice; (b3) Discard the washing solution and add 5 mL of Tryple Express digestion solution to digest the cells. Stop digestion when 50% or more of the cells become round. In this step, the culture flask is placed in the incubator and left to stand for 6-8 minutes. The specific judgment for stopping digestion is still based on when 50% or more of the cells become round. The conditions for static culture are: 5% CO2, 37℃. (b4) Add 15 mL of DMEM / F12 medium to a 50 mL centrifuge tube, remove the digested iPSCs and pipette the adherent cells off the wall, transfer the cell suspension to a 50 mL centrifuge tube containing DMEM / F12 medium, centrifuge (300 g, 5 min), and discard the supernatant. (b5) Take 1 mL of complete culture medium (i.e. induced pluripotent stem cell culture medium) to resuspend the cells, stain with trypan blue 1:1 and count them, take 1.5 million live cells; add 8 μL of Y-27632 to the cell suspension and mix well; (b6) Add the complete culture medium to 8 mL, discard the matrix gel in the (b1) T75 culture flask, and seed the cells into the T75 culture flask for frequent subculturing. The culture medium needs to be changed once a day.

[0049] After passaged to P26, the iPSCs cells of this embodiment were tested in T25 culture flasks for cell concentration, viability, total number of viable cells, and total number of cells. Simultaneously, based on P26, the cell concentration, viability, total number of viable cells, and total number of cells were tested in T25 culture flasks for passages of P27, P28, P29, and P30. The cell growth fold at harvest for P27, P28, P29, and P30, as well as the expansion fold after three consecutive passages of P27, P28, and P29, were also calculated. The relevant results are shown in Table 1. Furthermore, microscopic images of the iPSCs cells from P26, P27, P28, P29, and P30 were taken, and the images are shown below. Figure 1 , 2 As shown in Figures 3, 4, and 5.

[0050] Table 1 Summary of relevant parameters for iPSC cell passage to P26-P30

[0051] In Table 1 above, T25*1 represents one T25 culture flask, T25*2 represents two T25 culture flasks, and so on; survival rate = total number of live cells / total number of cells; growth fold at harvest = total number of live cells / number of inoculated cells; the growth fold after three consecutive passages (P26~P29) is the product of the growth folds at harvest of P27, P28, and P29.

[0052] Depend on Figures 1-6 As can be seen, using the induced pluripotent stem cell culture medium and method provided in this embodiment, iPSCs cells can be passaged to P26~P30 (corresponding to P26~P30 respectively). Figures 1-5 After that, its cell morphology remained relatively stable, similar to that of the early stages of passage, such as passage to P5 (corresponding to P5). Figure 6 The fact that there was no significant change compared to the previous data suggests that iPSCs cells still possess good genetic stability after long-term passage.

[0053] As can be seen from Table 1, using the induced pluripotent stem cell culture medium and culture method provided in this embodiment, iPSCs cells still have a high cell survival rate and a high growth rate after being passaged to P26~P30.

[0054] Example 2 In this embodiment, the induced pluripotent stem cell culture medium used during iPSC resuscitation and passage differs from that in Example 1 in that the compound additive A contains 50 ng / mL of recombinant human basic fibroblast growth factor. The rest is the same as in Example 1.

[0055] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0056] Example 3 In this embodiment, the induced pluripotent stem cell culture medium used during iPSC resuscitation and passage differs from that in Example 1 in that the compound additive A contains 1 ng / mL of recombinant human transforming growth factor. The rest is the same as in Example 1.

[0057] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0058] Example 4 In this embodiment, the induced pluripotent stem cell culture medium used during iPSC resuscitation and passage differs from that in Example 1 in that the compound additive A contains 0.5 mg / mL albumin. The rest is the same as in Example 1.

[0059] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0060] Comparative Example 1 The difference between the induced pluripotent stem cell culture medium used in this comparative example for iPSC cell resuscitation and passage and that in Example 1 is that the compound additive A does not contain albumin. Otherwise, it is the same as in Example 1.

[0061] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0062] Comparative Example 2 The difference between the induced pluripotent stem cell culture medium used in the iPSC resuscitation and passage process in this comparative example and Example 1 is that the compound additive A does not contain recombinant human basic fibroblast growth factor. Otherwise, it is the same as in Example 1.

[0063] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0064] Comparative Example 3 The difference between the induced pluripotent stem cell culture medium used in this comparative example for iPSC cell resuscitation and passage and that in Example 1 is that sodium selenite was not included in compound additive A. Otherwise, the contents were the same as in Example 1.

[0065] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0066] Comparative Example 4 The difference between the induced pluripotent stem cell culture medium used in the iPSC resuscitation and passage process in this comparative example and Example 1 is that the compound additive B does not contain a non-essential amino acid solution. Otherwise, it is the same as in Example 1.

[0067] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0068] Comparative Example 5 The difference between the induced pluripotent stem cell culture medium used in the iPSC resuscitation and passage process in this comparative example and Example 1 is that the compound additive B does not contain DL-piperidinecarboxylic acid. Otherwise, it is the same as in Example 1.

[0069] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0070] Comparative Example 6 The difference between the induced pluripotent stem cell culture medium used in the iPSC resuscitation and passage process in this comparative example and that in Example 1 is that compound additive B does not contain γ-aminobutyric acid (GABA). Otherwise, it is the same as in Example 1.

[0071] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0072] Comparative Example 7 The difference between the induced pluripotent stem cell culture medium used in the iPSC resuscitation and passage process in this comparative example and Example 1 is that the compound additive B does not contain mercaptoethanol. Otherwise, it is the same as in Example 1.

[0073] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0074] Comparative Example 8 The difference between the induced pluripotent stem cell culture medium used in this comparative example for iPSC cell resuscitation and passage and that in Example 1 is that the compound additive A contains 0.2 ng / mL of recombinant human transforming growth factor. The rest is the same as in Example 1.

[0075] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0076] Comparative Example 9 The difference between the induced pluripotent stem cell culture medium used in this comparative example for iPSC cell resuscitation and passage and that in Example 1 is that the compound additive A contains 5 ng / mL of recombinant human basic fibroblast growth factor. The rest is the same as in Example 1.

[0077] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0078] Comparative Example 10 The difference between the induced pluripotent stem cell culture medium used in this comparative example for iPSC cell resuscitation and passage and that in Example 1 is that the compound additive A contains 25 μg / mL of ascorbic acid. The rest is the same as in Example 1.

[0079] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0080] Comparative Example 11 The difference between the induced pluripotent stem cell culture medium used in this comparative example for iPSC cell resuscitation and passage and that in Example 1 is that the compound additive A contains 2 μg / mL of transferrin. The rest is the same as in Example 1.

[0081] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0082] Comparative Example 12 The difference between the induced pluripotent stem cell culture medium used in this comparative example for iPSC cell resuscitation and passage and that in Example 1 is the presence of Stock B solution at a concentration of 40 mg / mL in compound additive B. The rest of the contents are the same as in Example 1.

[0083] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0084] Comparative Example 13 The difference between the induced pluripotent stem cell culture medium used in the iPSC resuscitation and passage process in this comparative example and Example 1 is that the compound additive B contains 0.1% (v / v) L-alanyl-L-glutamine solution. The rest is the same as in Example 1.

[0085] The preparation methods for compound additive A and compound additive B are as described in Example 1.

[0086] Test Analysis (1) Growth and amplification performance test The amplification fold (calculated by passage 3 times as shown in Table 1 of Example 1) and the average survival rate (average survival rate over 3 passages) of Examples 1-4 and Comparative Examples 1-13 were tested. The specific testing methods are the same as in Example 1, and the results are shown in Table 2.

[0087] Table 2 summarizes the growth and amplification performance test results of the examples and comparative examples.

[0088] As shown in Table 2, in the three-generation expansion cultures, the culture medium in Examples 1-4 had a better effect on promoting the proliferation of pluripotent stem cells, with the expansion fold of all three generations exceeding 160. In contrast, the expansion fold of Comparative Examples 1-13 was below 160, and the cell survival rate was also lower than that in Examples 1-4. These results indicate that the culture medium involved in this invention effectively maintains the survival of pluripotent stem cells (iPSCs) and significantly promotes their proliferation.

[0089] (2) Cell flow cytometry detection In Examples 1-4 and Comparative Examples 1-13 above, after three consecutive passages (refer to Table 1 in Example 1 for three passages), the same number of cells from the last generation were taken for flow cytometry detection to detect the expression (positive rate) of SSEA4, TRA-1-81, Oct4, etc. The results are shown in Table 3 below.

[0090] Table 3 Summary of Flow Cytometry Marker Performance Test Results

[0091] As shown in Table 3, the pluripotency genes SSEA4, TRA-1-81, and Oct4 in Examples 1-4 were all highly expressed, and the differences between the groups were not significant. This indicates that the culture medium involved in this invention can maintain the pluripotency of iPS cells. However, in Comparative Examples 1-13, the expression of pluripotency genes SSEA4, TRA-1-81, or Oct4 was below 70% to varying degrees, indicating that differentiation had occurred and the pluripotency of iPSCs cells could not be maintained well.

[0092] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A culture medium for induced pluripotent stem cells, characterized in that, Includes basal culture medium and compound additive A and compound additive B; The compound additive A includes recombinant human insulin-like growth factor 5-30 μg / mL, recombinant human transforming growth factor 1-5 ng / mL, recombinant human basic fibroblast growth factor 50-200 ng / mL, sodium selenite 7-20 ng / mL, albumin 0.5-2 mg / mL, ascorbic acid 50-70 μg / mL, and transferrin 5-20 μg / mL; The composite additive B comprises Stock B solution 50~300mg / mL, non-essential amino acid solution 0.5~5% (v / v), DL-piperidinecarboxylic acid 50~200ng / mL, γ-aminobutyric acid 50~200μg / mL, lipid concentrate 0.1~1% (v / v), L-alanyl-L-glutamine solution 0.5~5% (v / v), and mercaptoethanol 0.01~0.1% (v / v).

2. The induced pluripotent stem cell culture medium as described in claim 1, characterized in that: The compound additive A comprises 20 μg / mL of recombinant human insulin-like growth factor, 2 ng / mL of recombinant human transforming growth factor, 100 ng / mL of recombinant human basic fibroblast growth factor, 14 ng / mL of sodium selenite, 1 mg / mL of albumin, 66 μg / mL of ascorbic acid, and 11 μg / mL of transferrin. The compound additive B comprises 200 mg / mL of the Stock B solution, 1% (v / v) of the non-essential amino acid solution, 129.8 ng / mL of DL-piperidinecarboxylic acid, 103.1 μg / mL of γ-aminobutyric acid, 0.2% (v / v) of the lipid concentrate, 1% (v / v) of the L-alanyl-L-glutamine solution, and 0.07% (v / v) of mercaptoethanol.

3. The induced pluripotent stem cell culture medium as described in claim 1, characterized in that: The basal culture medium is DMEM / F-12 medium.

4. The induced pluripotent stem cell culture medium as described in claim 1, characterized in that: The volume ratio of the composite additive A, the composite additive B and the basal culture medium is 1:1.5~6:45~105.

5. The induced pluripotent stem cell culture medium as described in claim 4, characterized in that: The volume ratio of the composite additive A, the composite additive B, and the basal culture medium is 1:3:

100.

6. A method for culturing induced pluripotent stem cells, characterized in that, Includes the following steps: (a) Resuscitation: Induced pluripotent stem cells are seeded into a first culture dish lined with matrix gel for initial culture, the first culture dish containing a first culture medium, the first culture medium comprising the induced pluripotent stem cell culture medium as described in any one of claims 1 to 7 and a first ROCK inhibitor; (b) Passaging: When the induced pluripotent stem cells revived in step (a) adhere to the culture vessel and grow to a confluence of 85% or more, they are passaged in a second culture dish covered with matrix gel. The second culture dish contains a second culture medium, which includes the induced pluripotent stem cell culture medium and the second ROCK inhibitor.

7. The method for culturing induced pluripotent stem cells as described in claim 6, characterized in that: The first ROCK inhibitor and the second ROCK inhibitor are Y-27632.

8. The method for culturing induced pluripotent stem cells as described in claim 6, characterized in that: The volume ratio of the first ROCK inhibitor to the first culture medium is 0.7~1.5:1000; The volume ratio of the second ROCK inhibitor to the second culture medium is 0.7~1.5:1000.

9. The method for culturing induced pluripotent stem cells as described in claim 6, characterized in that, Step (a) of recovery includes the following steps: (a1) Spread the matrix gel into the first petri dish and place it in an incubator for more than 30 minutes before use; (a2) Take the cryopreservation tube containing induced pluripotent stem cells and place it in a water bath at 37±1℃ and gently shake until no ice crystals remain; (a3) Add the cell suspension in the cryovial to the basal culture medium, centrifuge, and discard the supernatant; (a4) Resuspend the cells in the induced pluripotent stem cell culture medium, stain and count them, replenish the induced pluripotent stem cell culture medium, add the first ROCK inhibitor, and mix well; (a5) Discard the matrix gel in the first culture dish in (a1), then transfer the cell suspension obtained in (a4) to the culture dish, shake well and place it in an incubator for initial culture. The culture medium needs to be changed once a day.

10. The method for culturing induced pluripotent stem cells as described in claim 6, characterized in that, Step (b) of the progeny includes the following steps: (b1) Spread the substrate gel into the second culture dish and place it in an incubator for at least 30 minutes before use; (b2) When the cell adhesion and confluence of the cells revived in step (a) reaches more than 85%, discard the old culture medium and add basal culture medium to wash 2-3 times; (b3) Discard the washing solution and add digestion solution to digest the cells. When 50% or more of the cells become rounded, stop digestion. (b4) Transfer the cell suspension obtained in (b3) to a centrifuge tube containing the basal culture medium, centrifuge, and discard the supernatant; (b5) Resuspend the cells in the induced pluripotent stem cell culture medium, stain and count them, take the live cells, add the second ROCK inhibitor and mix well; (b6) Discard the matrix gel in the second culture dish in (b1), then transfer the cell suspension obtained in (b5) to the second culture dish, shake well and place in an incubator for subculture. The culture medium needs to be changed once a day.

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