Culture composition and culture method of stem cells

Abstract

The invention relates to the field of cell culture, in particular to a culture composition of stem cells and a culture method. The culture composition comprises NAD (Nicotinamide Adenine Dinucleotide) or a precursor thereof, flavonoid and coenzyme, and can be used for improving the proliferation capacity of stem cells and inhibiting the aging of the stem cells.

Description

Technical Field

[0001] The present invention relates to the field of cell culture, and particularly to a culture combination and a culture method for stem cells. Background Art

[0002] Numerous studies have shown that stem cells can not only self-renew, but also differentiate into other functional cells under appropriate conditions. Therefore, stem cells are expected to become an effective means for treating intractable human diseases. However, the content of stem cells in normal adult tissues is very low. How to rapidly expand and culture stem cells in vitro is an important technology for studying the mechanism of action of stem cells and exploring their treatment methods for human diseases.

[0003] Although the content of stem cells is very low, they are widely distributed in various tissues and organs of mammals, including but not limited to bone marrow, umbilical cord, adipose tissue, brain tissue, retina, heart, liver, lung, and skin, etc. A large number of studies have shown that these low-content stem cells can be further expanded in vitro by appropriate methods. The most conventional medium for culturing stem cells is a medium supplemented with fetal bovine serum. However, stem cells cultured with this medium not only grow relatively slowly, but also their potential to differentiate into functional cells is greatly reduced after more than 5 passages. The potential non-repeatability of bovine serum batches also greatly affects its large-scale production. On the other hand, bovine serum is heterologous to humans, which limits its clinical application.

[0004] NAD+ (nicotinamide adenine dinucleotide, abbreviated as coenzyme I) is a coenzyme of many dehydrogenases in the body, connecting the tricarboxylic acid cycle and the respiratory chain. Its function is to transfer the hydrogen removed in the metabolic process to flavoprotein. NAD+ can provide cell energy supply and promote cell regeneration. The metabolism of NAD+ in cells is as Figure 1 shown. Patent WO2021146505A1 provides the application of NAD+ in stem cell culture.

[0005] How to explore an optimized combination of culture conditions and medium composition for rapidly expanding stem cells without affecting their potential on this basis is still an urgent problem to be solved in this field. Summary of the Invention

[0006] The purpose of the present invention is to provide a culture combination for stem cells, which can promote the expansion of stem cells and improve the activity of stem cells.

[0007] The specific technical solution is as follows:

[0008] A culture combination for stem cells, the culture combination comprising NAD or its precursor, flavonoid, and coenzyme.

[0009] In some embodiments, the NAD precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food-grade or pharmaceutically acceptable salt, derivative, or prodrug thereof; preferably nicotinamide mononucleotide (NMN).

[0010] In some embodiments, the flavonoid is selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallocatechin, resveratrol, hesperetin, preferably quercetin and / or dihydroquercetin.

[0011] In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, S-adenosylmethionine or its derivatives, coenzyme A or its derivatives, coenzyme Qs or their derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

[0012] In some specific embodiments, the concentration of NAD+ or its precursor is 0.1 μM - 20 mM, such as 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 500 μM, 1 mM, 3 mM, 5 mM, 10 mM, 15 mM, 20 mM.

[0013] In some specific embodiments, the concentration of the flavonoid is 0.01 - 200 μM, such as 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, or 200 μM.

[0014] In some specific embodiments, the concentration of the coenzyme is 0.01 - 200 μM, such as 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, or 200 μM.

[0015] The present invention also provides a method for preparing stem cells, which uses the culture combination as described above.

[0016] In some embodiments, the preparation method includes the following steps:

[0017] 1) Obtaining stem cells from a subject;

[0018] 2) Treating the stem cells with the culture combination as described above;

[0019] wherein, when the culture combination is used, it is added to the stem cell medium.

[0020] In some specific embodiments, the stem cell culture medium comprises a xeno-free medium.

[0021] In some more specific embodiments, the stem cell culture medium is selected from any one or more of RPMI (Roswell Park Memorial Institute)-1640 medium, CCM medium (Conditioned Culture Medium), MEM medium (Minimum Essential Medium), DMEM medium (Dulbecco's Modified Eagle Medium), IMDM medium (Iscove’s Modified Dulbecco Medium), EMEM medium (Eagle’s Minimum Essential Medium), CTS Essential 8 medium, Medium 199 medium, and StemFlex medium.

[0022] In some embodiments, the stem cell culture medium further contains any one or more of amino acids, growth factors, glutamic acid and its derivatives, and antibiotics.

[0023] In some embodiments, the stem cells include embryonic stem cells and adult stem cells.

[0024] In some specific embodiments, the adult stem cells are selected from one or more of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epidermal stem cells, skeletal muscle stem cells, adipose stem cells, pancreatic stem cells, corneal stem cells, liver stem cells, and intestinal epithelial stem cells.

[0025] In some specific embodiments, the preparation method has any one or more of the following features:

[0026] 1) The above culture combination is added to the stem cell culture medium after the stem cells are passaged (P) 1 (P1), P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20, P21, P22, P23, P24, P25, P26, P27, P28, P29, P30, P35, P40, P45, P50, P55, P60, P65, P70, P75, P80, P85, P90, P95 or P100.

[0027] 2) Add the above-mentioned culture combination to the stem cell medium at least once every 12, 24, 36, 48, 60, 72 hours, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 60 days;

[0028] 3) Add the above-mentioned culture combination to the stem cell medium at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times per week;

[0029] 4) Add the above-mentioned culture combination to the medium at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times each time the stem cells are passaged;

[0030] 5) Passage the stem cells at least once every 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162 or 168 hours.

[0031] The invention also provides the application of the above-mentioned culture combination and the preparation method in preventing, inhibiting and / or reducing stem cell senescence induced by cell culture.

[0032] The invention also provides the application of the above-mentioned culture combination and the preparation method in rescuing stem cells from senescence during the culture and / or amplification process.

[0033] The beneficial effects of the invention are as follows:

[0034] Adding NAD+ or its precursor, flavonoids and coenzymes to the stem cell medium can increase the intracellular NAD+ content, increase the mitochondrial content, provide sufficient energy to enhance cell function, improve cell metabolism and activity, thereby promoting cell proliferation, inhibiting cell senescence, and increasing the telomeres and longevity proteins of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the metabolism of NAD+ in cells. DETAILED DESCRIPTION OF THE INVENTION

[0036] DEFINITIONS

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0038] One of the objectives of the present invention is to provide a culture combination of stem cells, which can promote stem cell amplification and improve stem cell activity. The culture combination includes NAD or its precursor, flavonoids, and coenzymes.

[0039] The term "stem cell" refers to a type of cell derived from embryos, fetuses, or adult bodies that has the ability to self-renew and proliferate and differentiate without limitation under certain conditions. It can produce daughter cells with phenotypes and genotypes identical to its own, and can also produce specialized cells that make up the tissues and organs of the body. At the same time, it can also differentiate into progenitor cells. According to the differentiation potential, stem cells can be divided into three categories: totipotent stem cells, pluripotent stem cells, and unipotent stem cells. According to the developmental stage, stem cells can be further divided into: embryonic stem cells and adult stem cells.

[0040] In some specific embodiments, the adult stem cells include, but are not limited to, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epidermal stem cells, skeletal muscle stem cells, adipose stem cells, pancreatic stem cells, corneal stem cells, liver stem cells, and intestinal epithelial stem cells.

[0041] The term "NAD+ precursor" refers to any small molecule that causes an increase in NAD+. The small molecule can exist in a reduced or non-reduced form.

[0042] NAD+ is a cofactor that plays a central role in metabolism, and the level of NAD+ decreases with age. NAD+ can help repair DNA, regulate immune cell conduction, provide cell energy, and reduce aging, etc. During the in vitro preparation process of immune cells, adding NAD+ treatment can provide sufficient energy for immune cell amplification, improve immune cell amplification, and reduce cell aging indicators. NAD+ precursors are compounds that are chemically converted into NAD+.

[0043] Flavonoids have anti-free radical and antioxidant effects and can delay cell apoptosis. In some embodiments, the flavonoids are selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallocatechin, resveratrol, hesperetin, preferably quercetin and / or dihydroquercetin. Among them, quercetin is a plant flavonol from the flavonoid group of polyphenols. It exists in many fruits, vegetables, leaves, seeds, and grains and is a naturally occurring polar auxin transport inhibitor. Dihydroquercetin is a dihydroflavonol compound belonging to the vitamin P group and is a widely used bioactive agent with various biological activities in the human body, including antioxidant and free radical scavenging effects.

[0044] Coenzymes are a general term for a large class of organic cofactors and are essential factors for enzymes to catalyze oxidation-reduction reactions, group transfer, and isomerization reactions. In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, S-adenosylmethionine or its derivatives, coenzyme A or its derivatives, coenzyme Qs or their derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10. Among them, pyrroloquinoline quinone (PQQ) is a redox enzyme cofactor with the chemical formula C14H6N2O8, which participates in a variety of redox reactions and mediates a series of cellular and molecular biochemical reactions in the form of a coenzyme, thus affecting the physiological and metabolic processes of the human body.

[0045] In some embodiments, the NAD+ precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a pharmaceutically or food acceptable salt, derivative, or prodrug thereof; preferably nicotinamide mononucleotide (NMN). As a direct precursor of NAD+, NMN can be easily and directly converted into NAD+ by NMNAT enzymes and is easily absorbed by cells.

[0046] In some specific embodiments, the concentration of the NAD+ or its precursor is 0.1 μM - 20 mM, such as 0.1 μM, 1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 50 μM, 80 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 500 μM, 1 mM, 3 mM, 5 mM, 10 mM, 15 mM, 20 mM.

[0047] In some embodiments, the flavonoid is selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallocatechin, resveratrol, hesperetin, preferably quercetin and / or dihydroquercetin.

[0048] In some embodiments, the concentration of the flavonoid is 0.01 - 200 μM, such as 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, or 200 μM.

[0049] In some embodiments, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, S-adenosylmethionine or its derivatives, coenzyme A or its derivatives, coenzyme Qs or their derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

[0050] In some embodiments, the concentration of the coenzyme is 0.01 - 200 μM, such as 10 nM, 20 nM, 50 nM, 100 nM, 200 nM, 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM or 200 μM.

[0051] The invention also provides a method for preparing stem cells, the preparation method comprising the following steps:

[0052] 1) Obtaining stem cells from a subject;

[0053] 2) Treating the stem cells with the culture combination of the invention;

[0054] Wherein, when the culture combination is used, it is added to the stem cell medium.

[0055] In some embodiments, the subject is a human or a mammal, and the mammals include but are not limited to pigs, cows, sheep, camels, alpacas, etc. The obtained stem cells are freshly collected or cells after cryopreservation and resuscitation.

[0056] In some embodiments, the stem cell medium further comprises a xeno - free medium.

[0057] The term "xeno - free medium" refers to a medium that does not contain components of xenogeneic animal origin (such as fetal bovine serum, etc.) and only contains human - derived or recombinant human proteins.

[0058] In some specific embodiments, the stem cell medium includes but is not limited to RPMI - 1640 medium, CCM medium, MEM medium, DMEM medium, IMDM medium, EMEM medium, CTS Essential 8 medium, Medium 199 medium, StemFlex medium.

[0059] The medium may also contain other commonly used culture components, such as amino acids, growth factors, glutamic acid and its derivatives, antibiotics, etc.

[0060] The term "Amino acid" is an organic compound containing a basic amino group and an acidic carboxyl group. A compound formed by replacing the hydrogen atom on the carboxylic acid carbon atom with an amino group. The amino acids of the invention refer to non - essential amino acids (NEAA), including but not limited to glutamic acid, alanine, glycine, aspartic acid, cystine, proline, serine and tyrosine, etc., which can be used to stimulate cell proliferation and prolong cell viability, and preferably glutamic acid.

[0061] In some embodiments, the concentration of the amino acid is 100 - 300 ng / ml, such as 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, and preferably 200 ng / ml.

[0062] The term "growth factor" refers to a class of organic compounds that are essential for regulating the normal growth and metabolism of microorganisms but cannot be synthesized by simple carbon and nitrogen sources. In a broad sense, growth factors include, in addition to vitamins, bases, purines, pyrimidines, biotin, nicotinic acid, etc., and sometimes also include the amino acids required by amino acid auxotrophic mutants; while in a narrow sense, growth factors generally refer only to vitamins. A class of polypeptide substances that regulate multiple effects such as cell growth and other cell functions by binding to specific and high-affinity cell membrane receptors. The growth factors of the invention include but are not limited to epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGFα, TGFβ), fibroblast growth factor (FGF), insulin-like growth factor (IGF), nerve growth factor (NGF), interleukin growth factor (IL), erythropoietin (EPO), colony-stimulating factor (CSF), and preferably epidermal growth factor (EGF).

[0063] In some embodiments, the concentration of the growth factor is 100 - 300 ng / ml, such as 100 ng / ml, 150 ng / ml, 200 ng / ml, 250 ng / ml, 300 ng / ml, and preferably 200 ng / ml.

[0064] The glutamate derivative is selected from any one of L-glutamine and GlutaMAX, and preferably GlutaMAX.

[0065] The term "GlutaMAX" refers to L-alanyl-L-glutamine (Alanyl-glutamine, Ala-Glu), also known as alanyl glutamine, alanyl glutamic acid dipeptide, which is very stable in aqueous solution and will not spontaneously degrade.

[0066] In some embodiments, the dosage of the glutamate and its derivative is 0.5x - 1.5x, such as 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, and preferably 1x.

[0067] The term "antibiotic" is also known as antibacterial, which refers to a class of secondary metabolites produced by microorganisms (including bacteria, fungi, actinomycetes) or higher plants and animals during their life processes, which have anti-pathogenic or other activities and can interfere with the development functions of other living cells. The antibiotic of the invention refers to Penicillin-Streptomycin mixture (Pen / Strep).

[0068] The dosage of the penicillin-streptomycin mixture is 0.5x - 1.5x, such as 0.5x, 0.6x, 0.7x, 0.8x, 0.9x, 1x, 1.1x, 1.2x, 1.3x, 1.4x, 1.5x, and preferably 1x.

[0069] In some embodiments, the preparation method includes placing the cells or cell line in a culture medium containing a culture combination (including NAD or its precursor, flavonoid, and coenzyme). The culture medium does not need to contain the culture combination, but the culture combination can be added to the culture medium before or after adding the cells or cell line. Specifically, the culture combination can be added to the culture medium before adding the cells or cell line for culture and / or expansion. Or the culture combination can be added after or simultaneously with adding the cells or cell line for culture and / or expansion, but before any possibility of culture-induced senescence. For example, the culture combination can be added near passage, can be added during any passage during the initial culture or subsequent passages, including but not limited to passage (P)1 (P1), P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20, P21, P22, P23, P24, P25, P26, P27, P28, P29, P30, P35, P40, P45, P50, P55, P60, P65, P70, P75, P80, P85, P90, P95 or P100.

[0070] In addition, the addition of the culture combination has an effect of inhibiting, reducing, and / or preventing culture-induced senescence on cells, and can also have a therapeutic effect on cell cultures that have shown signs of senescence or have already senesced. Therefore, the invention discloses a method for rescuing cells or cell lines from senescence during culture and expansion, the method including placing the cells or cell line in a culture medium containing a culture combination or adding the culture combination to the culture medium before or after adding the cells or cell line.

[0071] Before transferring the culture to a new culture container (i.e., passage), passage can be performed at a rate sufficient to enable the cells to reach the desired confluence or amplification level in the culture medium (including appropriate attachment time). The time required for the cells to obtain an appropriate degree of amplification depends on the cell type, culture medium, culture container size, and culture conditions. In some embodiments, the cells are passaged at least once every 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162 or 168 hours.

[0072] In addition, the time between passages can render a single administration of the culture combination insufficient to maintain an effective amount or concentration. Thus, in some embodiments, the culture combination of the invention is added to the culture medium at least once every 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96 hours, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 60 days. In some embodiments, the culture combination of the invention is added to the culture medium at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times per week. In some embodiments, the culture combination of the invention is added to the culture medium at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times each time the cells or cell line is passaged.

[0073] The method disclosed in the present invention relates to stem cell culture and needs to be carried out under specific culture conditions, such as the presence of serum, culture medium supplements, CO 2 percentage, temperature, O 2 percentage, etc., which can vary according to the specific cell type.

[0074] The present invention also provides a pharmaceutical composition comprising stem cells prepared by the preparation method of the present invention and pharmaceutically acceptable excipients.

[0075] In some embodiments, the pharmaceutical composition is a hematopoietic stem cell preparation.

[0076] In some embodiments, the pharmaceutical composition is a mesenchymal stem cell preparation.

[0077] In some embodiments, the pharmaceutical composition is a neural stem cell preparation.

[0078] In some embodiments, the pharmaceutical composition is an epidermal stem cell preparation.

[0079] In some embodiments, the pharmaceutical composition is a skeletal muscle stem cell preparation.

[0080] In some embodiments, the pharmaceutical composition is an adipose stem cell preparation.

[0081] In some embodiments, the pharmaceutical composition is a pancreatic stem cell preparation.

[0082] In some embodiments, the pharmaceutical composition is a corneal stem cell preparation.

[0083] In some embodiments, the pharmaceutical composition is a preparation of liver stem cells.

[0084] In some embodiments, the pharmaceutical composition is a preparation of intestinal epithelial stem cells.

[0085] The stem cells prepared by the culture combinations or preparation methods disclosed by the present invention can be used to study the development and differentiation of stem cells, including differentiation into various other cell types. That is, the stem cells can be used to generate a variety of more differentiated cell types, which can be used for both research purposes and clinical applications.

[0086] The stem cells prepared by the culture combinations or preparation methods disclosed by the present invention can be cultured in vitro and used as a source for obtaining other reagents. For example, mesenchymal stem cells can be used as a source for secreting immunomodulators (such as IL-10), or mesenchymal stem cells can be used as a source for extracellular vesicles (EVs) (such as exosomes). Such reagents can be purified from stem cells or the culture medium in which the stem cells grow by standard methods known in the art.

[0087] In addition, the stem cells prepared by the culture combinations or preparation methods disclosed by the present invention can be used for therapeutic purposes. The stem cells for therapeutic purposes can be unmodified cells or stem cells that have been modified (such as expressing therapeutic agents, secreting recombinant proteins or mRNAs, etc.). Indications for using stem cells for treatment include, but are not limited to, heart injury (such as ischemic cardiomyopathy) and autoimmune diseases (such as Crohn's disease, multiple sclerosis, systemic lupus erythematosus, systemic sclerosis).

[0088] The present invention will be illustrated below in the form of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. The methods and materials used in the examples are conventional materials and methods in the art unless otherwise specified.

[0089] I. Experimental setup

[0090] Primary cryopreserved mesenchymal stem cells (MSCs) were selected and isolated from alpaca umbilical cord blood. The screening criteria were as follows: the mesenchymal stem cell surface markers CD34, CD45, and HLA-DR were negative (all less than 2%), and CD73, CD90, and CD105 were positive (all greater than 95%). Cells at passage 3 (P3), passage 6 (P6), and passage 9 (P9) were taken for standby.

[0091] A blank group (MEM medium) and a model group (MEM medium, MSCs in the presence of 200 μM H 2 O 2cultured in the medium for 2 hours), MNM group (taking the cells of the model group and culturing them in MEM medium supplemented with NMN for 24 hours), NMN+PQQ group (taking the cells of the model group and culturing them in MEM medium supplemented with NMN+PQQ for 24 hours), NMN+PQQ+dihydroquercetin group (taking the cells of the model group and culturing them in MEM medium supplemented with NMN+PQQ+dihydroquercetin for 24 hours), pretreatment group (culturing MSC in MEM medium supplemented with NMN+PQQ+dihydroquercetin for 24 hours, and then culturing in the basal medium containing 200 μM H 2 O 2 for 2 hours)

[0092] II. Detection methods

[0093] 2.1 Mitochondrial morphology: Incubate MSC with 100 nM MitoTracker Red CMXRos (Thermo) in the medium at 37 °C for 30 minutes. After washing, incubate the cells with 3.7% formaldehyde at 37 °C for 15 minutes. Image using an Olympus 1X70 microscope. The indicators for evaluating mitochondrial shape include roundness (area / perimeter), length-to-width ratio (maximum diameter / minimum diameter), and nucleus-to-cytoplasm ratio.

[0094] 2.2 Mitochondrial membrane potential: Resuspend MSC with the basal medium to prepare a cell suspension. Add the mitochondrial membrane potential probe MitoTracker Red CMXRos (Invitrogen), and incubate in a 37 °C incubator for 30 min. After incubation, centrifuge at 400 g for 5 min to discard the supernatant, and resuspend and centrifuge again with pre-warmed DPBS for washing. After centrifugation, discard the supernatant, add DPBS and mix well, and detect on a CytoFLEX S flow cytometer (Beckman).

[0095] 2.3 Reactive oxygen species (ROS): Resuspend MSC with the basal medium to prepare a cell suspension. Add the probe DCFH-DA (2,7-dichlorofluorescein diacetate), mix well, and incubate in a 37 °C incubator for 20 min. Invert and mix every 3 - 5 min to ensure full contact of the probe with the cells. Wash twice with DPBS at 500 g for 5 min to fully remove the DCFH-DA that has not entered the cells, and resuspend with DPBS. Detect on a CytoFLEX S flow cytometer (Beckman).

[0096] 2.4 Stem cell migration: A scratch assay was performed to evaluate the migration activity of MSCs. Cells in a 12-well plate were scratched using a pipette. Images were taken at 0, 24, and 36 hours after scratching under an inverted fluorescence microscope to evaluate cell migration into the wound area. The cell migration rate was calculated by analysis using an image processing program (NIS Elements) on the images taken at 0, 24, and 36 hours after scratching. The following is the calculation formula:

[0097]

[0098] RS: Relative area ratio

[0099] St36 / St0: Cell growth area at 36 hours or 0 hours in the experimental group

[0100] Sn36 / Sn0: Cell growth area at 36 hours or 0 hours in the model group

[0101] Efficacy verification of the culture combination of the examples

[0102] Using P3, P6, and P9 cells respectively, the following functional verifications were carried out: 1) Proliferation ability: The cell numbers before and after culture were detected using a flow cytometer, and the amplification fold was calculated; 2) Mitochondrial function: The mitochondrial morphology and mitochondrial membrane potential of MSCs in each group before and after culture were detected; 3) Antioxidant ability: The levels of reactive oxygen species (ROS) in MSCs in each group before and after culture were detected; 4) Stem cell migration ability: The stem cell migration ability of MSCs in each group before and after culture was detected.

Claims

1. A combination of stem cell cultures, characterized in that, the combination of cultures comprises NAD or its precursors, flavonoids and coenzymes.

2. The combination of cultures according to claim 1, characterized in that, the NAD precursor is selected from one or more of tryptophan, quinolinic acid, nicotinic acid (NA), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or a food or pharmaceutically acceptable salt, derivative or prodrug thereof; preferably nicotinamide mononucleotide (NMN).

3. The combination of cultures according to claim 1 or 2, characterized in that, the flavonoids are selected from one or more of quercetin, dihydroquercetin, fisetin, catechin, gallocatechin, resveratrol, hesperetin, preferably quercetin and / or dihydroquercetin.

4. The combination of cultures according to any one of claims 1-3, characterized in that, the coenzyme is selected from at least one of glutathione or its derivatives, adenosine triphosphate and its derivatives, pyrroloquinoline quinone or its derivatives, S-adenosylmethionine or its derivatives, coenzyme A or its derivatives, coenzyme Qs or their derivatives, preferably pyrroloquinoline quinone and / or coenzyme Q10.

5. The combination of cultures according to any one of claims 1-4, characterized in that, the concentration of NAD+ or its precursor is 0.1 μM - 20 mM.

6. The combination of cultures according to any one of claims 1-4, characterized in that, the concentration of the flavonoids is 0.01 - 200 μM.

7. The combination of cultures according to any one of claims 1-4, characterized in that, the concentration of the coenzyme is 0.01 - 200 μM.

8. A method for preparing stem cells, characterized in that, the preparation method uses the combination of cultures according to any one of claims 1-7.

9. The preparation method according to claim 8, characterized in that, the preparation method comprises the following steps: 1) Obtaining stem cells from a subject; 2) Treating the stem cells with the combination of cultures according to any one of claims 1-7; wherein, the combination of cultures is added to the stem cell medium when used.

10. The preparation method according to claim 9, characterized in that, the stem cell medium comprises a xeno-free medium.

11. The preparation method according to claim 10, characterized in that, the stem cell medium is selected from any one or more of RPMI-1640 medium, CCM medium, MEM medium, DMEM medium, IMDM medium, EMEM medium, CTS Essential 8 medium, Medium 199 medium, StemFlex medium.

12. The preparation method according to any one of claims 9-11, characterized in that, the stem cell medium further contains any one or more of amino acids, growth factors, glutamic acid and its derivatives, antibiotics.

13. The preparation method according to any one of claims 8-12, characterized in that, the stem cells include embryonic stem cells, adult stem cells.

14. The preparation method according to claim 13, characterized in that, The adult stem cells are selected from one or more of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epidermal stem cells, skeletal muscle stem cells, adipose stem cells, pancreatic stem cells, corneal stem cells, liver stem cells, and intestinal epithelial stem cells.

15. The preparation method according to any one of claims 8-14, characterized in that it has any one or more of the following characteristics: 1) After the stem cells are passaged (P) 1 (P1), P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, P13, P14, P15, P16, P17, P18, P19, P20, P21, P22, P23, P24, P25, P26, P27, P28, P29, P30, P35, P40, P45, P50, P55, P60, P65, P70, P75, P80, P85, P90, P95 or P100, the culture combination according to any one of claims 1-7 is added to the stem cell medium; 2) Every 12, 24, 36, 48, 60, 72 hours, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 60 days, the culture combination according to any one of claims 1-7 is added to the stem cell medium at least once; 3) The culture combination according to any one of claims 1-7 is added to the stem cell medium at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times per week; 4) Each time the stem cells are passaged, the culture combination according to any one of claims 1-7 is added to the medium at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 times; 5) The stem cells are passaged at least once every 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78, 84, 90, 96, 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162 or 168 hours.

16. Use of the culture combination according to any one of claims 1-7 and the preparation method according to any one of claims 8-15 in preventing, inhibiting and / or reducing cell culture-induced stem cell senescence.

17. Use of the culture combination according to any one of claims 1-7 and the preparation method according to any one of claims 8-15 in rescuing stem cells from senescence during the culture and / or amplification process.

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