CN118421568A

CN118421568A - Culture medium of induced pluripotent stem cells and application thereof

Info

Publication number: CN118421568A
Application number: CN202410875287.6A
Authority: CN
Inventors: 刘佳
Original assignee: Shenzhen Zhongjia Biomedical Technology Co ltd
Current assignee: Shenzhen Zhongjia Biomedical Technology Co ltd
Priority date: 2024-07-02
Filing date: 2024-07-02
Publication date: 2024-08-02
Anticipated expiration: 2044-07-02

Abstract

The invention discloses a culture medium of induced pluripotent stem cells and application thereof, and relates to the technical field of cell culture media. The iPSCs culture medium provided by the invention does not contain serum, has definite components and good batch-to-batch repeatability; avoiding the rejection and conflict of the heteroplasms possibly caused by using serum, and having high safety. The small molecule additive has no toxic or side effect, and ensures the safety of subsequent application; the components are reasonably matched, the synergistic effect among the components can provide nutritional environment required by growth and proliferation of iPSCs from various cell sources, and the universality is good; the small molecular compound has low price, can replace most of expensive additives in the original culture medium, and greatly reduces the culture cost of the iPSCs. The culture medium is more suitable for proliferation and cloning clonality of iPSCs, and the cultured iPSCs also show more remarkable mitochondrial metabolism, have better cell activity and have considerable economic benefit and application prospect.

Description

Culture medium of induced pluripotent stem cells and application thereof

Technical Field

The invention relates to the technical field of cell culture media, in particular to a culture medium for induced pluripotent stem cells and application thereof.

Background

Induced pluripotent stem cells (induced pluripotent STEM CELLS, iPSCs) were initially obtained by reprogramming a combination of four transcription factors (OCT 4, KLF4, SOX2, c-Myc, OSKM) into differentiated somatic cells. Thereafter, iPSCs have been obtained from a variety of cell types and species, indicating that this is a common molecular mechanism. iPSCs not only have the multipotency of embryonic stem cells (ES cells) STEM CELLS, but are also abundant in source, with immeasurable potential value for regenerative medicine. Thus, more and more researchers use iPSCs to simulate the development of body tissues, organs, and other systems; furthermore, disease modeling can also be performed by reprogramming patient samples; in addition, human iPSCs have also become the basis for new cell therapies and drug discovery and have reached clinical use.

In the related art, most of the culture systems of Induced Pluripotent Stem Cells (iPSCs) contain serum, such as Fetal Bovine Serum (FBS). As the most commonly used additives in cell culture, serum provides proteins, nutrients, hormones, growth factors, and the like necessary for cell culture (proliferation, differentiation, etc.) in vitro. However, the composition of serum is complex, the overall composition is largely unknown, and there are significant differences between batches, component uncertainties, and batch-to-batch differences easily lead to reduced reproducibility. Furthermore, serum-containing media cannot avoid the xeno in animal serum, and human xeno rejection and collision can occur with cells cultured using animal serum; in addition, even though the serum is filtered, screened and the like, potential infectious pathogens such as tiny viruses or prions and the like still exist in the serum, so that the safety of the serum is reduced, and finally, the clinical application value and safety of the iPSCs are greatly reduced.

To circumvent these problems, serum-free media for iPSCs have been developed, such as serum-free media comprising serum substitutes (KOSRs). However, the components of the system are still not completely defined, and the system still has some disadvantages (such as variability among batches) of the culture medium containing the fetal bovine serum, and the problem that the proliferation rate of the cultured cells is slow is reported. Furthermore, serum substitutes such as Human Platelet Lysate (HPL) have the potential for inadequate availability and disease transmission between donors, and remain controversial. Another strategy is to culture iPSCs with a medium of complete chemical additives, which in most cases is free of heterologous cells, but the additive combinations required to meet the normal culture of iPSCs are very expensive, greatly limiting their potential value in clinical applications, and different additive combinations are generally only suitable for specific cell types.

Therefore, the provided iPSCs culture medium which has the advantages of no serum, definite components, small batch difference, good safety and universality with economic value and clinical application value is particularly important for researching the mechanism of iPSCs, screening medicines and applying the iPSCs.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a culture medium of induced pluripotent stem cells and application thereof, and aims to provide an iPSCs culture medium which is free of serum, definite in components, small in batch difference, good in safety and good in universality, so that the problems that the components of the existing iPSCs culture medium are undefined, the batch difference is obvious, rejection of human heteroplasmes exists and safety is insufficient in the serum-containing culture medium, and the problems that the chemical additive culture medium is high in price and poor in universality are solved, and powerful support is provided for transformation and clinical research of iPSCs.

An embodiment of the first aspect of the present invention provides a culture medium for induced pluripotent stem cells, the culture medium comprising a basal medium and an additive; the additive comprises flavanone, syringic acid, gastrodin, sesamin, loganin, glycitein, gentiopicroside, alpinetin, madecassoside and kaempferide; the medium does not contain serum.

The culture medium of the induced pluripotent stem cells according to the embodiment of the first aspect of the invention has at least the following beneficial effects: the invention provides a culture medium of Induced Pluripotent Stem Cells (iPSCs), which is characterized in that a small molecular compound combination is added into a basic culture medium as an additive. The culture medium does not contain serum, and the culture medium condition of the iPSCs is improved by adding a strategy of combining small molecular compounds, so that a new thought and strategy are provided for the culture of the iPSCs dependent on serum. The iPSCs culture medium has the following advantages:

(1) Serum-free, completely defined in components; the uncertainty of components in the serum culture medium and the difference between batches are avoided, and the repeatability of the culture medium between batches is good; avoiding rejection and conflict caused by using serum possibly caused by heterologous components, having high safety and greatly improving clinical application value and potential.

(2) Compared with the traditional serum culture medium, the serum-free iPSC culture medium is more suitable for iPSC cell proliferation and cell cloning, and simultaneously maintains microcosmic aspects and cell sizes which are approximately similar to those of the traditional culture medium; the cultured iPSCs also show more remarkable mitochondrial metabolism, have better cell activity and are also more beneficial to clinical transformation of various tissue engineering substitutes generated after the iPSCs are cultured.

(3) The additives are all naturally-occurring small molecular compounds, have no toxic or side effect, and ensure the safety of subsequent application; the components are reasonably matched, the synergistic effect among the components can provide nutritional environment required by growth and proliferation of iPSCs from various cell sources, and the universality is good; the small molecular compound has low price, can replace most of expensive additives in the original culture medium, greatly reduces the culture cost of the iPSCs, provides possibility for the subsequent industrialized application of the iPSCs, and has considerable economic benefit and application prospect.

Among the above additives of the present invention:

Flavanones, also known as 2, 3-dihydroflavones, 2-phenylbenzopyran-4-ones, 2-phenylchroman-4-ones, english name Flavanone (2, 3-Dihydroflavone, 4-Flavanone, 2-Phenyl-4-chromanone, 2-Phenylchroman-4-one), molecular formula C ₁₅H₁₂O₂, CAS number: 487-26-3. Flavanones have strong antioxidant and free radical scavenging activities, and can reduce the risk of certain chronic diseases, block certain cardiovascular diseases and certain cancers. The flavanone also has antiviral, antibacterial and antiinflammatory activities, and has effects of improving capillary fragility, inhibiting human platelet aggregation, resisting ulcer, and reducing anaphylaxis. The flavanone can act together with syringic acid to remove free radical and prevent oxidation of cells.

Syringic acid, also known as NSC 2129, SYRA, english name SYRINGIC ACID, molecular formula C ₉H₁₀O₅, CAS number: 530-57-4. Syringic acid is an effective antioxidant Chinese herbal medicine, and has potential anti-angiogenesis, anti-glycosylation, anti-hyperglycemia, neuroprotection and memory enhancing activities. Can act together with flavanone to remove free radical and prevent oxidation of cells.

Gastrodine, also known as gastrodine, english name Gastrodin (Gastrodine), molecular formula C ₁₃H₁₈O₇, CAS number: 62499-27-8. Gastrodin is a polyphenol (glucoside of 4-hydroxy benzyl alcohol), is the most important and effective component extracted from rhizoma Gastrodiae (Gastrodia elata), has antiinflammatory effect, and has long been used for research of dizziness, epilepsia, apoplexy and dementia. Can be used together with sesamin, loganin, glycitein, gentiopicroside, alpinetin, madecassoside and kaempferide to have antiinflammatory, antibacterial and antiviral effects and promote cell proliferation.

Sesamin, english name Sesamin (Fagarol), molecular formula C ₂₀H₁₈O₆, CAS no: 607-80-7. Sesamin is a lignan isolated from bark of Zanthoxylum (Fagara) plant and sesame oil, is a delta 5 desaturase inhibitor effective and selective in polyunsaturated fatty acid biosynthesis, and has effective neuroprotective effect on cerebral ischemia. Can be used together with gastrodin, loganin, glycitein, gentiopicroside, alpinetin, madecassoside and kaempferide to have antiinflammatory, antibacterial and antiviral effects and promote cell proliferation.

Loganin, also known as loganin, english name Loganin, molecular formula C ₁₇H₂₆O₁₀, CAS number: 18524-94-2. Loganin is the main iridoid glycoside compound in fructus Corni, and has antiinflammatory and antishock effects. Can be used together with gastrodin, sesamin, glycitein, gentiopicroside, alpinetin, madecassoside and kaempferide to have antiinflammatory, antibacterial and antiviral effects and promote cell proliferation.

Glycitein, also known as daidzin, huang Douhuang glycoside, daidzin, english name Glycitin (GLYCITEIN 7-O- β -glucoside), formula C ₂₂H₂₂O₁₀, CAS number: 40246-10-4. Glycitein is a natural isoflavone with antibacterial, antiviral, anticancer, anti-inflammatory, anti-aging and estrogenic effects, and may also modulate osteoblasts via TGF-beta or AKT signaling pathways in Bone Marrow Stem Cells (BMSCs). Can be used together with gastrodin, sesamin, loganin, gentiopicroside, alpinetin, madecassoside and kaempferide to have antiinflammatory, antibacterial and antiviral effects and promote cell proliferation.

Gentiopicroside, also known as gentiopicroside, english name Gentiopicroside, molecular formula C ₁₆H₂₀O₉, CAS number: 20831-76-9. Gentiopicroside is a natural iridoid glycoside with anti-inflammatory and antioxidant activities. Can be used together with gastrodin, sesamin, loganin, glycitein, alpinetin, madecassoside and kaempferide to have antiinflammatory, antibacterial and antiviral effects and promote cell proliferation.

Alpinetin, english name ALPINETIN, molecular formula C ₁₆H₁₄O₄, CAS no: 36052-37-6. Alpinetin is a flavonoid compound separated from semen Alpiniae, can activate PPAR-gamma, and has anti-inflammatory activity. Can be used together with gastrodin, sesamin, loganin, glycitein, gentiopicroside, madecassoside and kaempferide to have antiinflammatory, antibacterial and antiviral effects and promote cell proliferation.

Madecassoside, english name Madecassoside (Asiaticoside A), molecular formula C ₄₈H₇₈O₂₀, CAS no: 34540-22-2. Madecassoside is a pentacyclic triterpene isolated from centella asiatica and has anti-inflammatory, antioxidant, anti-apoptotic and anti-autophagic properties. Can be used together with gastrodin, sesamin, loganin, glycitein, gentiopicroside, alpinetin and kaempferide to have antiinflammatory, antibacterial and antiviral effects and promote cell proliferation.

Kaempferide, english name KAEMPFERIDE (4 ' -Methylkaempferol, 4' -O-Methylkaempferol, kaempferol 4' -methylether), molecular formula C ₁₆H₁₂O₆, CAS number: 491-54-3. Kaempferide is a natural product extracted from rhizoma Kaempferiae root, and has various biological activities including anticancer, antiinflammatory, antioxidant, antibacterial and antiviral activities. Can be combined with gastrodin, sesamin, loganin, glycitein, gentiopicroside, alpinetin, madecassoside to play the roles of anti-inflammatory, antibacterial, antiviral activity and promoting cell proliferation.

In the serum-free iPSCs culture medium provided by the invention, the additives are reasonably matched, the components are synergistic, a nutritional environment required by iPSCs growth and proliferation can be provided, iPSCs cell proliferation can be promoted, the culture medium is more suitable for cell cloning, and the cultured iPSCs cells also show more remarkable mitochondrial metabolism and have better cell activity, so that powerful support is provided for clinical application of the iPSCs cells.

In some embodiments of the invention, the final concentration of the medium: the concentration of the flavanone is 0.05-1 mu M; the concentration of the eugenol acid is 0.1-1 mu M; the concentration of the gastrodin is 0.05-1 mu M; the concentration of sesamin is 0.1-2 mu M; the concentration of the loganin is 0.05-1 mu M; the concentration of the glycitein is 0.05-1 mu M; the concentration of gentiopicroside is 0.05-1 mu M; the concentration of the alpinetin is 0.05-1 mu M; the concentration of the madecassoside is 0.05-1 mu M; the concentration of kaempferide is 0.05-1 mu M.

In some embodiments of the invention, the final concentration of the medium: the concentration of the flavanone is 0.05-0.2 mu M; the concentration of the eugenol acid is 0.1-0.4 mu M; the concentration of the gastrodin is 0.05-0.2 mu M; the concentration of sesamin is 0.1-1 mu M; the concentration of the loganin is 0.05-0.2 mu M; the concentration of the glycitein is 0.05-0.2 mu M; the concentration of gentiopicroside is 0.05-0.2 mu M; the concentration of the alpinetin is 0.05-0.2 mu M; the concentration of the madecassoside is 0.05-0.2 mu M; the concentration of kaempferide is 0.05-0.2 mu M.

In some embodiments of the present invention, the flavanone concentration is 0.05 to 1 μm, preferably 0.05 to 0.2 μm, more preferably about 0.1 μm (about 22.425 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the flavanone is dissolved in DMSO to make up 44mg/ml (196.2 mM) of mother liquor, which is diluted to a final concentration when used.

In some embodiments of the invention, the concentration of the eugenoic acid is 0.1-1 [ mu ] M, preferably 0.1-0.4 [ mu ] M, more preferably about 0.2 [ mu ] M (about 39.6 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the syringic acid is dissolved using ethanol to prepare a 39mg/ml (196.8 mM) mother liquor, which is diluted to a final concentration when used.

In some embodiments of the present invention, the gastrodin concentration is 0.05 to 1 μm, preferably 0.05 to 0.2 μm, more preferably about 0.1 μm (about 28.6 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the gastrodin is dissolved in DMSO to prepare a 50mg/ml (174.65 mM) stock solution, which is diluted to a final concentration when used.

In some embodiments of the present invention, the sesamin concentration is 0.1-2 μm, preferably 0.1-1 μm, more preferably about 0.5 μm (about 177 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, sesamin is dissolved in DMSO to prepare a 70mg/ml (197.54 mM) mother liquor, which is diluted to a final concentration when used.

In some embodiments of the present invention, the concentration of loganin is 0.05 to 1 μm, preferably 0.05 to 0.2 μm, more preferably about 0.1 μm (about 39 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the loganin is dissolved in DMSO to make up 78mg/ml (199.8 mM) of mother liquor, which is diluted to a final concentration when used.

In some embodiments of the invention, the glycitein concentration is 0.05 to 1. Mu.M, preferably 0.05 to 0.2. Mu.M, more preferably about 0.1. Mu.M (about 44.64 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the glycitein is dissolved in DMSO to make up 89mg/ml (199.37 mM) of mother liquor, which is diluted to final concentration when used.

In some embodiments of the present invention, the gentiopicroside concentration is 0.05 to 1 μm, preferably 0.05 to 0.2 μm, more preferably about 0.1 μm (about 35.6 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the gentiopicroside is dissolved using DMSO to make up 71mg/ml (199.25 mM) of mother liquor, which is diluted to a final concentration when used.

In some embodiments of the present invention, the concentration of alpinetin is 0.05 to 1 μm, preferably 0.05 to 0.2 μm, more preferably about 0.1 μm (about 27 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the alpinetin is dissolved in DMSO to prepare a 54mg/ml (199.79 mM) stock solution, which is diluted to a final concentration when used.

In some embodiments of the invention, the concentration of madecassoside, based on the final concentration of the medium, is between 0.05 and 1. Mu.M, preferably between 0.05 and 0.2. Mu.M, more preferably about 0.1. Mu.M (about 97.5 ng/ml).

In some embodiments of the invention, the madecassoside is dissolved in DMSO to prepare a mother liquor of 100mg/ml (102.55 mM) and diluted to a final concentration when used.

In some embodiments of the present invention, the kaempferide concentration is 0.05-1 μm, preferably 0.05-0.2 μm, more preferably about 0.1 μm (about 30 ng/ml), based on the final concentration of the culture medium.

In some embodiments of the invention, the kaempferide is dissolved in DMSO to prepare a 60mg/ml (199.82 mM) mother liquor, which is diluted to a final concentration when used.

In some embodiments of the invention, the additive further comprises the following components that support the growth of induced pluripotent stem cells: one or more lipids, one or more transferrin and transferrin substitutes, one or more insulin and insulin substitutes, one or more trace elements, one or more vitamins, one or more amino acids, one or more receptor tyrosine kinases, one or more hormones, and one or more hormone-like compounds.

Specifically, the receptor tyrosine kinase includes at least one of basic fibroblast growth factor (bFGF), epidermal Growth Factor (EGF), vascular Endothelial Growth Factor (VEGF), platelet growth factor (PDGF), insulin-like growth factor (IGF), and liver growth factor (HGF). Preferably an epidermal growth factor.

In some embodiments of the invention, the additive comprises the following components: lipid concentrate, transferrin, crystalline bovine insulin, selenium, glucocorticoid, isoproterenol hydrochloride and epidermal cell growth factor.

In some embodiments of the invention, the lipid concentrate (Lipid Concentrate) is a concentrated lipid emulsion, whose chemical composition is defined, free of protein additives, useful in a variety of applications, including CHO, hybridoma and growth and maintenance of insect cell cultures, production of monoclonal antibodies by the hybridoma, and expression of viruses in insect cells. Commercially available lipid concentrates, for example 10 x or 20 x or 50 x, may be used with dilution to 1 x.

In some embodiments of the present invention, the concentration of transferrin (transferrin) is 1-10 μg/ml, preferably 5-6 μg/ml, more preferably about 5.5 μg/ml, based on the final concentration of the culture medium. Transferrin is a glycoprotein comprising iron-containing transferrin and iron-free transferrin, wherein the preferred transferrin is iron-containing transferrin capable of regulating the metabolism of elemental iron in vivo, transferring elemental iron into cells via cell surface transferrin receptors; in addition, it can be combined with other trace elements to regulate the growth proliferation and functional expression of iPSCs.

In some embodiments of the present invention, the concentration of the crystalline bovine insulin (crystallized bovine insulin) is 1 to 20 μg/ml, preferably 5 to 15 μg/ml, more preferably about 10 μg/ml, based on the final concentration of the culture medium.

In some embodiments of the invention, the concentration of selenium (selenium) is 5 to 10ng/ml, preferably 6 to 7ng/ml, more preferably about 6.7ng/ml, based on the final concentration of the medium.

In some embodiments of the present invention, the glucocorticoid concentration is 0.5 to 1.5 μm, preferably 0.9 to 1.2 μm, and more preferably about 1.1 μm, based on the final concentration of the culture medium.

In some embodiments of the invention, the glucocorticoid is hydrocortisone (hydrocortisone), also known as cortisol, having the chemical formula C ₂₁H₃₀O₅, and is an organic compound extracted from the adrenal cortex and has the strongest effect on carbohydrate metabolism.

In some embodiments of the present invention, the concentration of isoprenaline hydrochloride (isoproterenol hydrochloride) is 0.1-0.5 μg/ml, preferably 0.2-0.3 μg/ml, more preferably about 0.212 μg/ml, based on the final concentration of the culture medium.

In some embodiments of the invention, the concentration of the epidermal growth factor (EPIDERMAL GROWTH FACTOR, EGF) is 1 to 20ng/ml, preferably 5 to 15ng/ml, more preferably about 10ng/ml, based on the final concentration of the medium.

In some embodiments of the invention, the additive further comprises the following components: penicillin and gentamicin.

In some embodiments of the invention, the penicillin (penicillin) is at a concentration of about 100U/ml based on the final concentration of the medium.

In some embodiments of the invention, the concentration of gentamicin (gentamicin) is about 25mg/ml, based on the final concentration of the medium.

In some embodiments of the invention, the basal medium comprises at least one of DMEM (Dulbecco's Modified Eagle's Medium)、MEM (Minimal Essential Medium)、BME (Basal Medium Eagle)、F-12、RPMI 1640、GMEM (Glasgow's Minimal Essential Medium). Preferably, the basal medium is DMEM. The basal medium refers to artificially prepared culture containing nutrients required by cell growth such as saccharides, amino acids, inorganic salts, vitamins, lipids and the like, and can provide survival and minimum physiological activities of iPSCs.

In some embodiments of the invention, it is known to those skilled in the art that other components may also be added to the medium in order to facilitate cell growth. It is known to those skilled in the art to select other components such as L-glutamine, NEAA MEM, 2-mercaptoethanol, etc., which need to be added to a specific medium depending on the cells being cultured and other conditions.

In a specific embodiment of the invention, the culture medium of the induced pluripotent stem cells comprises a basic culture medium DMEM and additives of flavanone, syringic acid, gastrodin, sesamin, loganin, glycitein, gentiopicroside, alpinetin, madecassoside and kaempferide. More specifically, the culture medium of the induced pluripotent stem cells comprises the following formula: dmem+0.1 μM flavanone+0.2 μM syringic acid+0.1 μM gastrodin+0.5 μM sesamin+0.1 μM loganin+0.1 μM glycitein+0.1 μM gentiopicroside+0.1 μM alpinetin+0.1 μM madecassoside+0.1 μM kaempferide.

In another specific embodiment of the invention, the culture medium of the induced pluripotent stem cells comprises a basal culture medium DMEM, and additives of flavanone, syringic acid, gastrodin, sesamin, loganin, glycitein, gentiopicroside, alpinetin, madecassoside, kaempferin, lipid concentrate, transferrin, crystalline bovine insulin, selenium, hydrocortisone, isoprenaline hydrochloride, epidermal growth factor, penicillin, gentamicin. More specifically, the culture medium of the induced pluripotent stem cells comprises the following formula: dmem+0.1 μM flavanone+0.2 μM syringic acid+0.1 μM gastrodin+0.5 μM sesamin+0.1 μM loganin+0.1 μM glycitein+0.1 μM gentiopicroside+0.1 μM alpinetin+0.1 μM madecassoside+0.1 μM kaempferide+1×lipid concentrate+5.5 μg/ml transferrin+10 μg/ml crystalline bovine insulin 6.7ng/ml selenium+1.1 μM M hydrocortisone hydrocortisone+0.212 μg/ml isoprenaline hydrochloride+10 ng/ml epidermal cell growth factor+100U/ml penicillin+25 mg/ml gentamycin.

In a second aspect of the present invention, there is provided a use of the medium for the culture of induced pluripotent stem cells described above for non-diagnostic or therapeutic purposes.

The application of the embodiment according to the second aspect of the invention has at least the following advantages: the culture medium of the Induced Pluripotent Stem Cells (iPSCs) is applied to iPSCs culture, so that the culture cost of the cells is reduced, and the possibility is provided for the subsequent industrial application of the iPSCs. The culture medium does not contain serum, the components are completely clear, the uncertainty of the components and the difference between batches of the serum culture medium are avoided, the repeatability between batches of the culture medium is good, the rejection and the conflict caused by the use of the serum possibly caused by the heterologous components are avoided, the safety is high, higher safety is provided for transformation and clinical research, and a foundation is laid for the clinical application of iPSCs. Moreover, the culture medium is applied to iPSCs culture, can promote iPSCs cell proliferation, is more suitable for cell cloning, and the cultured iPSCs cells also show more remarkable mitochondrial metabolism, have better cell activity and are more beneficial to clinical transformation of various tissue engineering substitutes generated after iPSCs cell culture. The natural small molecule additive strategy also provides another thought for improving the conditions of in vitro iPSCs culture: the amplification and function of iPSCs are improved by different culture conditions including natural small molecule additives, cytokine combinations, co-stimulatory molecules, various genetically engineered cells, and the like.

An embodiment of the third aspect of the present invention provides an induced pluripotent stem cell, which is obtained by culturing the induced pluripotent stem cell in a medium as described above.

According to the embodiment of the third aspect of the invention, the induced pluripotent stem cells have at least the following beneficial effects: the Induced Pluripotent Stem Cells (iPSCs) provided by the invention have the advantages that the culture medium does not contain serum, the components are completely clear, rejection and conflict caused by the use of the serum possibly caused by the heterologous components are avoided, the safety is high, and a foundation is laid for the subsequent clinical application of the iPSCs. And the iPSCs have faster cell proliferation, better cell clonality, more remarkable mitochondrial metabolism and better cell activity, and are more suitable for producing cell or tissue substitutes for clinical application after the iPSCs are cultured.

In a fourth aspect of the present invention, there is provided a method for culturing induced pluripotent stem cells, comprising the steps of:

(i) Introducing one or more stem cell multipotential factors into a somatic cell;

(ii) Inducing and culturing somatic cells with stem cell multipotential factors introduced into the culture medium of the induced multipotential stem cells to obtain induced cells;

(iii) Detecting and analyzing the pluripotency of the induced cells in (ii);

(iv) Selecting a monoclonal of the induced cells having pluripotency;

(v) And (3) culturing the monoclonal selected in (iv) by adopting the culture medium of the induced pluripotent stem cells to obtain the induced pluripotent stem cells.

The method for culturing induced pluripotent stem cells according to the embodiment of the fourth aspect of the invention has at least the following advantageous effects: the culture method of the induced pluripotent stem cells provided by the invention is simple and convenient, and has lower dependence on technicians; the cell culture cost is lower, and the method provides possibility for the subsequent industrialized application of the iPSCs. Moreover, the iPSCs cultured by the method have faster cell proliferation and better cell clonality, and meanwhile, the cells can keep microscopic aspects, cell sizes and metabolism which are approximately similar to those of the traditional culture method; the cultured iPSCs also show more remarkable mitochondrial metabolism, have better cell activity and are also more beneficial to clinical transformation of various tissue engineering substitutes generated after the iPSCs are cultured.

In some embodiments of the invention, the term "somatic cell" is a concept relative to "germ cell" and "embryonic stem cell", which is a cell that is not multipotent any more resulting from differentiation of "embryonic stem cell" but has a specific function, which is not multipotent more resulting from differentiation of "embryonic stem cell" or continued development of an inner cell mass, which is generally a cell with a specific function, which is generally obtained from a fetal mouse or a adult mouse at a stage of development that is located after blastocyst stage (in particular 3.5 days after fertilization in a mouse), and from which germ cells and sources thereof (e.g., spermatogenic stem cells, germ crest stem cells, etc.) that may have a multipotency are generally avoided. The somatic cells used in the present invention are preferably derived from mammals, more preferably from humans, monkeys, dogs, cats, rats or mice. The somatic cells in the present invention may be any type of somatic cells in the body, preferably fibroblasts or epithelial cells.

In some embodiments of the invention, the term "inducing" refers to the process of dedifferentiating somatic cells into pluripotent stem cells. Preferably, somatic cell dedifferentiation into pluripotent stem cells is induced by introducing a stem cell-maintaining pluripotent factor cDNA into the somatic cells. Wherein, the stem cell multipotency factor refers to a factor capable of reverting differentiated cells to a multipotency state, and is mostly a nuclear transcription factor, including but not limited to ：SOX2、OCT3/4、KIF4、NONOG、LIN28、c-Myc、lin28、esrrb、tbx3、VPA、CHIR99021、616452、Tranylcypromine、Forskolin、DZNep、TTNPB or a combination thereof.

In some embodiments of the invention, the stem cell pluripotency factor comprises a combination of VPA, CHIR99021, 616452, tranylcypromine, forskolin, DZNep, TTNPB.

In some embodiments of the invention, the method of introducing a stem cell pluripotent factor into a somatic cell may be a variety of techniques well known to those skilled in the art, including viral infection, liposome transfection, transposon mediated insertion expression, transmembrane proteins, drug induction, electrotransfection, particle bombardment, and the like, and methods of transferring DNA into a cell.

In some embodiments of the invention, methods for detecting and analyzing the pluripotency of cells are well known to those of skill in the art, including identifying expression of a pluripotency molecular marker, detection of methylation status of a cell, formation of embryonal body EB, formation of teratomas, and administration of induced pluripotent stem cell forming chimeric mice, and the like.

The embodiment of the sixth aspect of the invention provides an application of the induced pluripotent stem cells or the induced pluripotent stem cells obtained by culturing by the culture method in constructing a disease model, screening new drugs and preparing drugs for treating nervous system diseases or cardiovascular system diseases, wherein the application is for non-diagnosis or treatment purposes.

The application of the embodiment according to the sixth aspect of the invention has at least the following advantages: the iPSCs obtained by the invention have the technical effects described above, and the application of the iPSCs in various aspects such as construction of disease models, new drug screening, preparation of drugs for treating nervous system diseases or cardiovascular system diseases and the like has the advantages of the iPSCs (such as pluripotency of embryonic stem cells, rich sources, no human ethics problems and the like), does not introduce heterologous substances, has better safety, and has immeasurable potential value for the fields of clinical application, regenerative medicine and the like. In addition, the cell culture cost is lower, the proliferation is faster, the activity is better, and the method has extremely obvious advantages in the aspects of economic benefit and industrial application.

Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.

Drawings

Fig. 1 (a) is a schematic diagram of microscopic cell morphology of iPSCs cells derived from Urine (UE) cells provided in an embodiment of the present invention;

Fig. 1 (B) is a schematic diagram of microscopic cell morphology of iPSCs cells derived from Hair Follicle (HF) cells provided in an embodiment of the invention;

Fig. 1 (C) is a schematic diagram of microscopic cell morphology of iPSCs cells derived from Skin (SF) cells provided in the examples of the present invention;

FIG. 2 is a schematic diagram of the measurement results of the size diameter of iPSCs provided in the examples of the present invention;

FIG. 3 is a graph showing the results of doubling time measurement of iPSCs provided in the examples of the present invention;

FIG. 4 is a schematic diagram showing the result of evaluating clonality of iPSCs provided in the examples of the present invention;

FIG. 5 is a schematic diagram showing the result of evaluation of extracellular acidification rate (ECAR) of iPSCs provided by the examples of the present invention;

Fig. 6 is a schematic diagram of the results of evaluation of Oxygen Consumption Rate (OCR) of mitochondrial respiration of iPSCs cells provided in the examples of the present invention.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

In the description of the present invention, unless otherwise indicated, the numerical ranges "a-b" represent shorthand representations of any combination of real numbers between a and b, where a and b are both real numbers. Unless otherwise indicated, the various reactions or operational steps may or may not be performed sequentially. Preferably, the reaction process in the present invention is carried out sequentially.

The following examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the product specifications. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, immunology and recombinant DNA, which are within the skill of the art. All reagents or equipment were commercially available as conventional products without the manufacturer's knowledge.

In the following examples, all cellular materials were taken from Shenzhen second people hospital, skin tissue was taken from skin tissue samples obtained from tumor surgery or circumcision, hair pull was taken from staff in stem cell laboratory, urine was taken from staff in stem cell laboratory, all donors signed body fluid, tissue, cell donation informed consent, and the process of obtaining materials was in compliance with the relevant ethics regulations.

Example 1 serum-free iPSCs Medium

The serum-free iPSCs medium of this example included basal medium and additives.

Basal medium was DMEM, purchased from GIBCO brand.

The components of the additive are calculated according to the final concentration, and the additive comprises the following components: 0.1 [ mu ] M flavanone+0.2 [ mu ] M syringic acid+0.1 [ mu ] M gastrodin+0.5 [ mu ] M sesamin+0.1 [ mu ] M loganin+0.1 [ mu ] M glycitein+0.1 [ mu ] M gentiopicroside+0.1 [ mu ] M alpinetin+0.1 [ mu ] M madecassoside+0.1 [ mu ] M kaempferide+1X lipid concentrate+5.5 [ mu ] g/ml transferrin+10 [ mu ] g/ml crystalline bovine insulin 6.7ng/ml selenium+1.1 [ mu ] M hydrocortisone (hydrocortisone) +) +0.212 [ mu ] g/ml isoprenaline hydrochloride+10 ng/ml epidermal cell growth factor+100U/ml penicillin+25 mg/ml gentamycin.

The additive purchase information is as follows (unless otherwise specified: flavanones were purchased from MCE brand, cat# CAS No.: 487-26-3; syringic acid was purchased from MCE brand, cat No. CAS No. 530-57-4; gastrodine is purchased from MCE brand, cat# CAS No.: 62499-27-8; sesamin is purchased from MCE brand, cat No. CAS No. 607-80-7; loganin is available from MCE brand under the accession number CAS No.: 18524-94-2; glycitein is purchased from MCE brand, cat# CAS No.: 40957-83-3; gentiopicroside is available from MCE brand under the CAS number 20831-76-9; mountain Jiang Su from MCE brand, cat# CAS No.: 36052-37-6; madecassoside is available from MCE brand under the CAS No.: 34540-22-2; kaempferide is available from MCE brand under the CAS number 491-54-3;1 Xlipid concentrate was purchased from GIBCO brand, cat: 11905031; transferrin is purchased from Aladin brand under the accession number T302200-10mg; crystalline bovine insulin is available from YuanYe under the trade designation S12033-1g; selenium is purchased from Aladin brand, cat# S105199-5g; hydrocortisone available from Aladin brand under the designation H657409-5g; isoprenaline hydrochloride is available from Aladin brand under the product number I129810-5g; epidermal growth factor was purchased from Sigma-Aldrich under the trade designation E9644-.5MG; penicillin is purchased from Aladin brand, cat# P113151-20ml; gentamicin was purchased from YuanYe brand under the trade designation R20013-10ml.

EXAMPLE 2 serum-free iPSCs Medium

Basal medium was DMEM, purchased from GIBCO brand.

The components of the additive are calculated according to the final concentration, and the additive comprises the following components: 0.05 [ mu ] M flavanone+0.1 [ mu ] M syringic acid+0.05 [ mu ] M gastrodin+0.1 [ mu ] M sesamin+0.05 [ mu ] M loganin+0.05 [ mu ] M glycitein+0.05 [ mu ] M gentiopicroside+0.05 [ mu ] M alpinetin+0.05 [ mu ] M madecassoside+0.05 [ mu ] M kaempferide+1 Xlipid concentrate+5 [ mu ] g/ml transferrin+5 [ mu ] g/ml crystalline bovine insulin 6ng/ml selenium+0.9 [ mu ] M hydrocortisone (hydrocortisone) +0.2 [ mu ] g/ml isoprenaline hydrochloride+5 ng/ml epidermal cell growth factor+100U/ml penicillin+25 mg/ml gentamicin.

Example 3 serum-free iPSCs Medium

Basal medium was DMEM, purchased from GIBCO brand.

The components of the additive are calculated according to the final concentration, and the additive comprises the following components: 0.2 [ mu ] M flavanone+0.4 [ mu ] M syringic acid+0.2 [ mu ] M gastrodin+1 [ mu ] M sesamin+0.2 [ mu ] M loganin+0.2 [ mu ] M glycitein+0.2 [ mu ] M gentiopicroside+0.2 [ mu ] M alpinetin+0.2 [ mu ] M madecassoside+0.2 [ mu ] M kaempferide+1 Xlipid concentrate+6 [ mu ] g/ml transferrin+15 [ mu ] g/ml crystalline bovine insulin+7 ng/ml selenium+1.2 [ mu ] M hydrocortisone (hydrocortisone) +0.3 [ mu ] g/ml isoprenaline hydrochloride+15 ng/ml epidermal cell growth factor+100U/ml penicillin+25 mg/ml gentamicin.

Example 4 Urine (UE) cell-derived iPSCs

1. Urine (UE) derived cell preparation

Collecting urine of normal adult, transferring the urine into a 50ml centrifuge tube, centrifuging 400g for 10 minutes, absorbing and discarding supernatant, leaving 1-5 ml of supernatant in each tube, mixing into one centrifuge tube, adding PBS containing blue/streptomycin (PBS is added into 95ml for uniformly mixing with 5ml of blue/streptomycin), and slightly and uniformly mixing. Centrifuging 400g for 10 minutes, absorbing and removing the supernatant until 0.5-1 ml of liquid remains, adding DMEM/F12, re-suspending uniformly, transferring to a six-hole plate placed in a 5% CO ₂ incubator at 37 ℃ for 20 minutes (the six-hole plate is coated with 0.1% gelatin), placing the six-hole plate in the 37 ℃ and 5% CO ₂ incubator, standing for 3 days, taking out for observation, discarding if the culture medium is polluted, adding 1ml of culture medium if the culture medium is normal, and continuing to perform standing culture.

2. IPSCs culture

The UE cells were induced to reprogram iPSCs by electrotransfer of the seven compounds VPA, CHIR99021, 616452, tranylcypromine, forskolin, DZNep, TTNPB in combination, as described in the art literature, using the serum-free iPSCs medium of example 1. The culture plate is coated by Matrigel (the culture plate is placed in a 37 ℃ and 5% CO ₂ carbon dioxide incubator for at least 30 minutes), cells are uniformly shaken and then are placed in the 37 ℃ and 5% CO ₂ carbon dioxide incubator for culture, daily observation and liquid replacement are carried out, the cells can be passaged after about 70% of common passaging for 3-4 days, the cells are too large in passaging after the evening, and the cells are too large in cloning and are easy to differentiate, so that the cells are passaged for 5-7 days even if the cell density is not high. Detecting and analyzing the pluripotency of the obtained cells, picking out the monoclone with the pluripotency, and continuing to culture to obtain the reprogrammed iPSCs.

The UE cells induced by the method of the embodiment are reprogrammed to iPSCs, the whole process does not use feeder cells, does not use viral vectors, and is cultured by adopting Serum-free medium (SFM) with definite components, and the human iPSCs have no exogenous gene integration and multiple functions.

EXAMPLE 5 iPSCs derived from Hair Follicle (HF) cells

1. Preparation of Hair Follicle (HF) source cells

Selecting normal adult brain back hair or eyebrow as hair source, cutting off hair root until only about 1cm of hair root is left, sterilizing with 75% alcohol for 10 min, rapidly pulling out hair root with clean eyebrow forceps, rinsing in PBS, and inoculating into culture dish coated with matrigel, wherein the culture solution is DMEM/F12 containing 1% B27, 10ng/ml bFGF and 50ng/ml EGF. After 1 week hair growth was observed. The liquid is changed every 3-4 days later.

2. IPSCs culture

Induction of HF cells to reprogram iPSCs cells by electrotransfer of seven compound combinations of VPA, CHIR99021, 616452, tranylcypromine, forskolin, DZNep, TTNPB, induction methods see the literature in the art, induction culture was performed using serum-free iPSCs medium of example 2. The culture plate is coated by Matrigel (the culture plate is placed in a 37 ℃ and 5% CO ₂ carbon dioxide incubator for at least 30 minutes), cells are uniformly shaken and then are placed in the 37 ℃ and 5% CO ₂ carbon dioxide incubator for culture, daily observation and liquid replacement are carried out, the cells can be passaged after about 70% of common passaging for 3-4 days, the cells are too large in passaging after the evening, and the cells are too large in cloning and are easy to differentiate, so that the cells are passaged for 5-7 days even if the cell density is not high. Detecting and analyzing the pluripotency of the obtained cells, picking out the monoclone with the pluripotency, and continuing to culture to obtain the reprogrammed iPSCs.

The HF cells induced by the method of this example were reprogrammed to iPSCs without feeder cells, without using viral vectors, and cultured in Serum-free medium (SFM) with defined composition of the present invention, and such human iPSCs had no foreign gene integration and were pluripotent.

Example 6 Skin (SF) cell-derived iPSCs

1. Preparation of Skin (SF) derived cells

The fibroblasts used in this example were derived from surgical skin fibroblasts. In the operation process, a piece of tissue with the size of 2cm of abdomen is taken, the tissue is placed in PBS (phosphate buffer solution) or physiological saline with double antibody at 4 ℃ for transportation, after the PBS with double antibody is fully washed to remove blood, subcutaneous tissues are removed as much as possible, a leather strip is completely covered by 0.2% collagenase and sealed by a preservative film, the tissue is digested at 4 ℃ overnight (12-16 hours), the digestion condition is observed every hour from 12 hours, and the digestion time is recorded according to the detection standard so that the epidermis can be gently torn off by using an ophthalmic forceps. The digested specimens were discarded in a sterile room, rinsed 2 times with DMEM containing 10% fetal bovine serum, and stopped. The epidermis was gently removed with ophthalmic forceps and the dermis was cut into 1mm x 1mm size pieces with ophthalmic scissors. Adding 0.2% collagenase to submerge the tissue, and digesting for 1-4 hours or more at 37 ℃ until the tissue blocks are basically dispersed, wherein no obvious tissue blocks are visible; digestion was stopped by adding DMEM containing 10% fetal bovine serum, residual tissue mass was removed by filtration through a 100 μm filter, and the filtrate was eluted twice by centrifugation at 1000rpm for 5 minutes and inoculated with a culture medium at a set density of 2 x 10 ⁵, incubated at 37 ℃ in a 5% CO ₂ humidity incubator. And changing the liquid every 2-3 days later, observing the cell wall-attached growth condition, and carrying out passage by a pancreatin digestion method when the cells are about 80-90% confluent.

2. IPSCs culture

SF cells were induced to reprogram iPSCs cells by electrotransfer of a combination of seven compounds VPA, CHIR99021, 616452, tranylcypromine, forskolin, DZNep, TTNPB, induction methods were described in the art literature, induction culture was performed using serum-free iPSCs medium of example 3. The culture plate is coated by Matrigel (the culture plate is placed in a 37 ℃ and 5% CO ₂ carbon dioxide incubator for at least 30 minutes), cells are uniformly shaken and then are placed in the 37 ℃ and 5% CO ₂ carbon dioxide incubator for culture, daily observation and liquid replacement are carried out, the cells can be passaged after about 70% of common passaging for 3-4 days, the cells are too large in passaging after the evening, and the cells are too large in cloning and are easy to differentiate, so that the cells are passaged for 5-7 days even if the cell density is not high. Detecting and analyzing the pluripotency of the obtained cells, picking out the monoclone with the pluripotency, and continuing to culture to obtain the reprogrammed iPSCs.

The SF cells induced by the method of the present example were reprogrammed to iPSCs without feeder cells, without using viral vectors, and cultured in Serum-free medium (SFM) with the composition of the present invention, and such human iPSCs had no foreign gene integration and were pluripotent.

Comparative example 1

The difference between comparative example 1 and example 4 is: different culture mediums, comparative example 1 used classical fetal bovine serum culture medium (FBSCM, abbreviated FCM) to induce culture of UE-derived iPSCs cells. The remainder is the same as in example 4 and will not be described in detail here.

Wherein, the formula of the fetal bovine serum culture medium (FBSCM, abbreviated as FCM) is as follows: dmem+5% Fetal Bovine Serum (FBS) +24.3 μg/ml adenine (adenine) +5 μg/ml crystalline bovine insulin (crystallized bovine insulin) +1.1 μg M hydrocortisone (hydrocortisone) +0.212 μg/ml isoprenaline hydrochloride (isoproterenol hydrochloride) +10ng/ml epidermal cell growth factor (EPIDERMAL GROWTH FACTOR) +100U/ml penicillin (penicillin) +25mg/ml gentamicin (gentamicin).

Comparative example 2

The difference between comparative example 2 and example 5 is: different culture mediums, comparative example 2 used classical fetal bovine serum culture medium (FBSCM, abbreviated FCM) to induce culture of HF-derived iPSCs cells. The remainder is the same as in example 5 and will not be described in detail here.

Comparative example 3

The difference between comparative example 3 and example 6 is: different culture mediums, comparative example 3 used classical fetal bovine serum culture medium (FBSCM, abbreviated FCM) to induce SF-derived iPSCs cells. The remainder is the same as in example 6 and will not be described in detail here.

Cell microscopic appearance evaluation of test example 1 iPSCs

The morphology of iPSCs cells in examples 4-6 and comparative examples 1-3 was observed under a microscope, and the results are shown in fig. 1 (a) -1 (C), wherein fig. 1 (a) is iPSCs derived from Urine (UE) cells, UE (SFM) represents iPSCs cells obtained in example 4, and UE (FCM) represents iPSCs cells obtained in comparative example 1; FIG. 1 (B) is an iPSC derived from Hair Follicle (HF) cells, HF (SFM) represents the iPSC cells obtained in example 5, and HF (FCM) represents the iPSC cells obtained in comparative example 2; FIG. 1 (C) is a Skin (SF) cell-derived iPSCs, SF (SFM) represents the iPSCs cells obtained in example 6, and HF (FCM) represents the iPSCs cells obtained in comparative example 3. From FIGS. 1 (A) -1 (C), it can be seen that there was no significant difference between the morphology of the iPSCs cells of different origins, either in fetal bovine serum medium (FCM) or in serum-free medium (SFM).

Test example 2 iPSCs proliferation potential evaluation

Gelatin coating was used overnight before seeding cells in a petri dish or flask. The iPSCs of examples 4-6 and comparative examples 1-3 were then thawed and seeded into 12-well plates at 10% confluency. For 4 consecutive days, daily replacement of the medium (SFM) of examples 1 to 3 or the medium FCM of the comparative example was performed. On day 4 of passage 1 (P1) and passage 2 (P2), the cells were trypsinized and inoculated for subsequent passages. Cells were collected from wells daily and counted using the coulter beckman Z2 system, respectively, and the average cell size was calculated. Statistical analysis was performed using GRAPHPADPRI-smv.9.2 software. The results are expressed as means with standard deviation. In addition, two-way analysis of variance (ANOVA) was used to interpret the data.

Theoretically, the proliferative potential of a cell is inversely proportional to its size. Thus, the cell sizes in examples 4 to 6 and comparative examples 1 to 3 were measured daily, and the results are shown as a graph. The results are shown in FIG. 2, wherein UE, HF, SF represent iPSCs cells derived from UE, HF, SF, respectively, and SFM, FCM represent cells cultured using corresponding serum-free medium (SFM) or fetal bovine serum medium (FCM), respectively; a represents the iPSC cell size in example 5 (SFM), comparative example 2 (FCM), B represents the iPSC cell size in example 4 (SFM), comparative example 1 (FCM), and C represents the iPSC cell size in example 6 (SFM), comparative example 3 (FCM). No significant difference in the change in the size diameter of the iPSCs cells of the P1, P2, and P3 generations was observed, and the curves approximately overlapped.

Test example 3 iPSCs doubling time measurement

Cell culture the doubling time was calculated using the proliferation curve as in test example 2. Statistical analysis was performed using GRAPHPADPRI-smv.9.2 software. The results are expressed as means with standard deviation. In addition, two-way analysis of variance (ANOVA) was used to interpret the data.

The results are shown in FIG. 3, wherein UE, HF, SF represent iPSCs cells derived from UE, HF, SF, respectively, and SFM, FCM represent cells cultured using corresponding serum-free medium (SFM) or fetal bovine serum medium (FCM), respectively; a represents the iPSC cell doubling time in example 5 (SFM), comparative example 2 (FCM), B represents the iPSC cell doubling time in example 4 (SFM), comparative example 1 (FCM), and C represents the iPSC cell doubling time in example 6 (SFM), comparative example 3 (FCM). Compared with the SFM in the comparative example, the doubling time of the P1 generation iPSCs cultured by the HF and UE source iPSCs cells in the FCM is relatively higher, and the time of the P3 generation is prolonged under the SFM culture condition; whereas SF-derived iPSCs cells were cultured in SFM for a consistently higher doubling time. For iPSCs cells, FCM was compared to SFM culture, with 3 cell cultures, no high variability was observed between generations (n=3).

Test example 4 iPSCs clonality evaluation

The iPSCs cells from UE and HF in examples 4 and 5, respectively, were seeded at a density of 1.3×10 ⁵ in 25cm ² flasks. The medium was changed once on day 5. On day 10, cells were fixed with 3.7% formaldehyde, after which the cells were stained with the Nile blue a/rhodoamine mixture for 15min, then rinsed 3 times with tap water and the flasks were air dried at room temperature. Colony Forming Units (CFU) were then carefully calculated directly in the flask. Next, to enhance the assessment and eliminate observer bias, the assessment used a scanner, the flasks were scanned using a Typhoon trio+ scanner, and the stained areas were assessed using ImageJ software. Statistical analysis was performed using GRAPHPADPRI-smv.9.2 software. The results are expressed as means with standard deviation. In addition, two-way analysis of variance (ANOVA) was used to interpret the data. Data are presented as counts of CFU/25cm ² and scanned stain/surface (AU).

The results are shown in FIG. 4, wherein UE and HF represent iPSCs cells derived from UE and HF, respectively, and SFM and FCM represent cells cultured using corresponding serum-free medium (SFM) or fetal bovine serum medium (FCM), respectively; A. b represents the clone number (A) and staining result (B) of iPSCs in example 5 (SFM) and comparative example 2 (FCM), respectively, and C, D represents the clone number (C) and staining result (D) of iPSCs in example 4 (SFM) and comparative example 1 (FCM), respectively. As can be seen from fig. 4, SFM produced more CFU than FCM culture during 3 generation iPSCs cell culture (fig. 4).

Test example 5 iPSCs metabolic evaluation

IPSCs cells (200,000 cells/cm ² cultured in 100 μl cell culture medium/well) were seeded in XFe96 well plates. Cells were incubated with the corresponding media (FCM or SFM) for three days before analysis was performed. Seahorse XFe96 sensor cartridge plates were hydrated with XF calibration the day prior to analysis and incubated overnight at 37 ℃. Cells were washed and incubated with medium for 1 hour before measuring energy metabolism. Extracellular acidification rates (Extracellular Acidification Rate, ECAR) representing glycolytic metabolism and oxygen consumption rates (Oxygen Consumption Rate, OCR) representing mitochondrial respiration were determined using XFe extracellular flux analyzer. The concentration of each injection represents the final concentration in the well.

Mitochondrial respiration is achieved by sequential injection 1.5µM ATP synthase inhibitor oligomycin, 0.5µM FCCP, 0.5µM of a combination of the mitochondrial complex I inhibitor rotenone, 0.5µM mitochondrial complex III inhibitor antimycin A.

Glycolytic metabolism is by sequential injection 10mM D-(+)-glucose, 1.5µM of the ATP synthase inhibitor oligomycin (67.5% oligomycin A complex), 50mM 2-deoxy-D-glucose (2-DG).

At least three measurement cycles (3 min mixed +3 min measured) were completed before and after each injection. Oxygen Consumption Rate (OCR) and extracellular acidification (ECAR) were calculated using Wave software v 2.6. Normalization was performed according to cell number using CyQuant cell proliferation assay kit. Fluorescence was measured at 485 nm/535 nm for 0.1 seconds using a Victor2 1420 multi-tag counter plate reader and Wallac1420 software. The normalized values were calculated from fluorescence measurements by Microsoft Excel software and applied to metabolic values.

Cell metabolism was assessed using Seahorse XFe96 extracellular flux analyzer. The energy metabolism value is normalized according to the cell number using CyQuant cell proliferation assay kit.

Statistical analysis was performed using GRAPHPADPRI-smv.9.2 software. The results are expressed as means with standard deviation. In addition, two-way analysis of variance (ANOVA) was used to interpret the data.

The results are shown in FIGS. 5-6, wherein UE, HF, SF represent iPSCs cells derived from UE, HF, SF, respectively, and SFM, FCM represent culturing cells using corresponding serum-free medium (SFM) or fetal bovine serum medium (FCM), respectively; FIG. 5A shows the extracellular acidification rates of iPSCs in example 5 (SFM) and comparative example 2 (FCM), FIG. 5B shows the extracellular acidification rates of iPSCs in example 4 (SFM) and comparative example 1 (FCM), and FIG. 5C shows the extracellular acidification rates of iPSCs in example 6 (SFM) and comparative example 3 (FCM); fig. 6 a shows the oxygen consumption rate of mitochondrial respiration of iPSCs cells in example 5 (SFM) and comparative example 2 (FCM), fig. 6B shows the oxygen consumption rate of mitochondrial respiration of iPSCs cells in example 4 (SFM) and comparative example 1 (FCM), and fig. 6C shows the oxygen consumption rate of mitochondrial respiration of iPSCs cells in example 6 (SFM) and comparative example 3 (FCM).

As can be seen from fig. 5-6, in contrast, when iPSCs cells were cultured in SFM, their maximum Oxygen Consumption Rate (OCR) was significantly higher than FCM (fig. 5). There is a trend of increasing ECAR base and ECAR maximum values of SFM compared to FCM (fig. 6).

In summary, the present invention evaluates the applicability of serum-free media (SFM) in the culture of iPSCs cells, and by comparison with conditions using classical fetal bovine serum media (FCM), the characteristics of iPSCs cells, including microscopic morphology, cell size, doubling time, clonogenic, glycolysis, and mitochondrial metabolism, were evaluated. The results indicate that SFM culture is more suitable for iPSCs cell proliferation and cloning while maintaining morphology and cell size similar to FCM culture. Furthermore, iPSCs cells under SFM conditions also showed greater mitochondrial respiration, exhibited more pronounced mitochondrial metabolism, and better cellular activity than FCM conditions. Finally, the SFM of the invention overcomes the problems and the barriers encountered when serum is used in cell culture, is more suitable for producing cell or tissue substitutes for clinical application, and the safety and the definite components of the SFM can be helpful for clinical transformation of various tissue engineering substitutes produced after iPSC cell culture.

While the preferred embodiment of the present application has been described in detail, the present application is not limited to the above embodiment, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.

Claims

1. A culture medium for induced pluripotent stem cells, wherein the culture medium comprises a basal medium and an additive; the additive comprises flavanone, syringic acid, gastrodin, sesamin, loganin, glycitein, gentiopicroside, alpinetin, madecassoside and kaempferide; the medium does not contain serum.

2. The culture medium of induced pluripotent stem cells according to claim 1, wherein the culture medium comprises, based on the final concentration of the culture medium: the concentration of the flavanone is 0.05-1 mu M; the concentration of the eugenol acid is 0.1-1 mu M; the concentration of the gastrodin is 0.05-1 mu M; the concentration of sesamin is 0.1-2 mu M; the concentration of the loganin is 0.05-1 mu M; the concentration of the glycitein is 0.05-1 mu M; the concentration of gentiopicroside is 0.05-1 mu M; the concentration of the alpinetin is 0.05-1 mu M; the concentration of the madecassoside is 0.05-1 mu M; the concentration of kaempferide is 0.05-1 mu M.

3. The culture medium of induced pluripotent stem cells according to claim 2, wherein the culture medium comprises, based on the final concentration of the culture medium: the concentration of the flavanone is 0.05-0.2 mu M; the concentration of the eugenol acid is 0.1-0.4 mu M; the concentration of the gastrodin is 0.05-0.2 mu M; the concentration of sesamin is 0.1-1 mu M; the concentration of the loganin is 0.05-0.2 mu M; the concentration of the glycitein is 0.05-0.2 mu M; the concentration of gentiopicroside is 0.05-0.2 mu M; the concentration of the alpinetin is 0.05-0.2 mu M; the concentration of the madecassoside is 0.05-0.2 mu M; the concentration of kaempferide is 0.05-0.2 mu M.

4. The culture medium of induced pluripotent stem cells according to claim 1, wherein the additive further comprises the following components supporting the growth of induced pluripotent stem cells: one or more lipids, one or more transferrin and transferrin substitutes, one or more insulin and insulin substitutes, one or more trace elements, one or more vitamins, one or more amino acids, one or more receptor tyrosine kinases, one or more hormones, and one or more hormone-like compounds.

5. The culture medium of induced pluripotent stem cells according to claim 4, wherein the additive comprises the following components: lipid concentrate, transferrin, crystalline bovine insulin, selenium, glucocorticoid, isoproterenol hydrochloride and epidermal cell growth factor.

6. The culture medium of induced pluripotent stem cells of claim 1, wherein the basal medium comprises at least one of DMEM, MEM, BME, F-12, RPMI 1640, GMEM.

7. Use of a culture medium of induced pluripotent stem cells according to any one of claims 1 to 6 for culturing induced pluripotent stem cells, for non-diagnostic or therapeutic purposes.

8. An induced pluripotent stem cell obtained by culturing the induced pluripotent stem cell according to any one of claims 1 to 6 in a medium.

9. A method of culturing induced pluripotent stem cells, comprising the steps of:

(ii) Inducing and culturing somatic cells having stem cell multipotential factors introduced into (i) using the culture medium for induced pluripotent stem cells according to any one of claims 1 to 6, to obtain induced cells;

(iii) Detecting and analyzing the pluripotency of the induced cells in (ii);

(iv) Selecting a monoclonal of the induced cells having pluripotency;

(v) Culturing the selected monoclonal in (iv) with the medium of the induced pluripotent stem cells according to any one of claims 1 to 6, to obtain the induced pluripotent stem cells.

10. Use of the induced pluripotent stem cells according to claim 8 or the induced pluripotent stem cells obtained by culturing according to the culturing method of claim 9 for constructing a disease model, screening for new drugs, preparing a medicament for treating a neurological disease or a cardiovascular disease, the use being for non-diagnostic or therapeutic purposes.