A serum-free medium for mesenchymal stem cells with determined components and its application

Abstract

The present invention discloses a serum-free medium with defined components for culturing mesenchymal stem cells and its applications. The present invention discloses a serum-free medium with defined components for culturing mesenchymal stem cells. The components of the serum-free medium include a basal medium and L-glutamine, transferrin, L-ascorbic acid, lipid mixture, putrescine, human serum albumin, heparin sodium, progesterone, HEPES, fetuin, hydrocortisone, trimipramine, and insulin-like growth factor. The present invention provides the use of the aforementioned serum-free medium in culturing mesenchymal stem cells and its application in a system, device, or equipment for preparing a serum-free medium for automated production of mesenchymal stem cells. The serum-free medium of the present invention has defined components, can avoid the interference of serum on cell culture, and can effectively promote the in vitro expansion of mesenchymal stem cells, highlighting the outstanding culture advantages of the serum-free medium described in the present invention.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a serum-free medium for mesenchymal stem cells with determined components and its application. Background Art

[0002] As a kind of adult stem cells, mesenchymal stem cells have many advantages, such as easy availability of materials, no ethical issues, high proliferation, multi-directional differentiation and immunomodulation. They have become potential therapeutic materials for various major diseases, such as neurodegenerative diseases, diabetes, stroke, pulmonary fibrosis, rheumatoid arthritis and graft-versus-host disease, etc. At home and abroad, numerous clinical trials of MSCs have been carried out in recent years and achieved positive therapeutic effects.

[0003] Traditional mesenchymal stem cell culture usually uses basal media such as DMEM / F12, MEM-alpha, and adds fetal bovine serum for cell culture. Such as the commercial serum-containing mesenchymal stem cell medium (STEMCELL, MesenCult TM Amplification Kit (human), #05411), and another example is that Chinese Patent Application CN103146647A discloses a mesenchymal stem cell medium, which uses MEM-alpha as the basal medium and adds 10% fetal bovine serum. Such serum-containing media have many disadvantages: the components are uncertain, restricting the application; containing heterologous proteins, the yield of amplified mesenchymal stem cells is low, the number of passages is limited, and the cells carry bovine serum proteins by endocytosis, which can cause receptor immune reactions, and at the same time there is a risk of introducing bacteria and viruses carried by heterologous serum, greatly restricting the application of mesenchymal stem cells.

[0004] Currently, there are also commercially available serum-free media for mesenchymal stem cells, including serum-free media containing platelet lysate (hPL) and serum-free media with defined components. The medium containing platelet lysate (such as Helios) is added to MEM-α or DMEM / F12 medium for use, and the cells grow rapidly. However, the components of this medium are not clear, the safety and batch-to-batch consistency in clinical applications are poor, and more stringent quality control release requirements are needed for downstream applications such as stem cell preparations. Serum-free media with defined components (such as: STEMCELL, MesenCult TM -ACF Plus Culture Kit, product number: 05448; Gibco, StemPro TMSerum-free and xenogeneic-free medium for mesenchymal stem cells, product number: A1067501; ExcellBIOS, serum-free medium for MSC proliferation, product number: ME000-N023), and a serum-free medium for mesenchymal stem cells and its use disclosed in Chinese Patent Application CN110331130A. These media are all media with defined components and can be used for the culture of mesenchymal stem cells. However, the cell culture effects of these media, especially in the isolation of primary seed cells, still have a large gap compared with the medium containing platelet lysate. Summary of the Invention

[0005] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0006] The first aspect of the present invention provides a serum-free medium for mesenchymal stem cells with defined components. The serum-free medium for mesenchymal stem cells with defined components does not contain serum, and the serum-free medium for mesenchymal stem cells with defined components includes a basal medium and L-glutamine, transferrin, L-ascorbic acid, lipid mixture, putrescine, human albumin, sodium heparin, progesterone, HEPES, fetuin, hydrocortisone, trimipramine, insulin-like growth factor.

[0007] Further, the insulin-like growth factor includes IGF-1LR3.

[0008] Further, not containing serum includes not containing any animal serum such as horse serum, fetal bovine serum, bovine serum, human serum, etc.

[0009] In some embodiments, not containing serum means not containing serum with undefined components directly separated from animal blood, including serum separated from the blood of any mammal.

[0010] Further, the basal medium is selected from one of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, F12 medium, IMDM medium.

[0011] Further, the basal medium is selected from multiple or all of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, F12 medium, IMDM medium.

[0012] Further, the basal medium is MEM medium or DMEM / F12 medium.

[0013] Further, the basal medium is α-MEM medium or DMEM / F12 medium.

[0014] Further, the pH value of the serum-free medium is 7.1 - 7.5.

[0015] In some embodiments, the pH value of the serum-free medium is 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5.

[0016] Furthermore, the osmotic pressure of the serum-free medium is 290 mOSM / kg to 340 mOSM / kg.

[0017] In some embodiments, the osmotic pressure of the serum-free medium is 290 mOSM / kg, 295 mOSM / kg, 300 mOSM / kg, 305 mOSM / kg, 310 mOSM / kg, 315 mOSM / kg, 320 mOSM / kg, 325 mOSM / kg, 330 mOSM / kg, 335 mOSM / kg, 340 mOSM / kg.

[0018] Furthermore, the pH value of the serum-free medium is 7.1, 7.2, 7.4.

[0019] Furthermore, the pH value of the serum-free medium is adjusted using dilute hydrochloric acid and sodium hydroxide.

[0020] Furthermore, the osmotic pressure of the serum-free medium is 340 mOsm / kg.

[0021] Furthermore, the osmotic pressure of the serum-free medium is adjusted using sodium chloride.

[0022] Furthermore, the concentration of each component in the serum-free medium in the serum-free medium is:

[0023] The concentration of L-glutamine is 1 - 10 mM,

[0024] The concentration of transferrin is 100 - 500 nM,

[0025] The concentration of L-ascorbic acid is 150 - 300 μM,

[0026] The concentration of the lipid mixture is 0.1 - 1% v / v,

[0027] The concentration of putrescine is 30 - 80 μM,

[0028] The concentration of human serum albumin is 1 - 10 mg / mL,

[0029] The concentration of sodium heparin is 100 - 300 ng / mL,

[0030] The concentration of progesterone is 10 - 30 μM,

[0031] The concentration of HEPES is 10 - 25 mM,

[0032] The fetuin concentration is 1 - 5 mg / mL,

[0033] The hydrocortisone concentration is 50 - 150 nM,

[0034] The trimipramine concentration is 5 - 20 μM,

[0035] The insulin-like growth factor concentration is 10 - 100 ng / mL.

[0036] In some embodiments, the L-glutamine concentration is 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM.

[0037] In some embodiments, the transferrin concentration is 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM.

[0038] In some embodiments, the L-ascorbic acid concentration is 150 μM, 200 μM, 250 μM, 300 μM.

[0039] In some embodiments, the lipid mixture concentration is 0.1% v / v, 0.2% v / v, 0.3% v / v, 0.4% v / v, 0.5% v / v, 0.6% v / v, 0.7% v / v, 0.8% v / v, 0.9% v / v, 1% v / v.

[0040] In some embodiments, the putrescine concentration is 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM.

[0041] In some embodiments, the human serum albumin concentration is 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL.

[0042] In some embodiments, the heparin sodium concentration is 100 ng / mL, 150 ng / mL, 200 ng / mL, 250 ng / mL, 300 ng / mL.

[0043] In some embodiments, the progesterone concentration is 10 μM, 15 μM, 20 μM, 25 μM, 30 μM.

[0044] In some embodiments, the HEPES concentration is 10 mM, 15 mM, 20 mM, 25 mM.

[0045] In some embodiments, the clomipramine concentration is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, 20 μM.

[0046] In some embodiments, the insulin-like growth factor concentration is 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL.

[0047] In some embodiments, transferrin, L-ascorbic acid, lipid mixture, putrescine, human serum albumin, sodium heparin, progesterone can maintain the survival of mesenchymal stem cells.

[0048] In some embodiments, after the addition of clomipramine, it can effectively promote the proliferation of mesenchymal stem cells.

[0049] In some embodiments, hydrocortisone and fetuin can enhance the survival and proliferation of primary cells. In some embodiments, the lipid mixture can enhance the transport of cellular proteins and enhance cell survival.

[0050] In some embodiments, the synergistic effect of various components in the technical solution endows the cultured mesenchymal stem cells with excellent self-renewal ability.

[0051] In some embodiments, the culture medium does not use animal-derived components, thereby effectively avoiding the uncertainties brought by animal-derived substances to the self-renewal and differentiation processes of cultured cells and the occurrence of contamination of cultured cells, and can completely avoid the induction of immune responses during the process of cell therapy.

[0052] Further, the L-glutamine concentration is 2 mM, 4 mM, 10 mM.

[0053] Further, the transferrin concentration is 100 nM, 300 nM, 500 nM.

[0054] Further, the L-ascorbic acid concentration is 150 μM, 200 μM, 300 μM.

[0055] Further, the lipid mixture concentration is 0.1% v / v, 0.3% v / v, 1% v / v.

[0056] Further, the putrescine concentration is 30 μM, 60 μM, 80 μM.

[0057] Further, the human albumin concentration is 1 mg / ml, 4 mg / ml, or 10 mg / ml.

[0058] Further, the heparin sodium concentration is 100 ng / ml, 150 ng / ml, or 300 ng / ml.

[0059] Further, the progesterone concentration is 10 μM, 20 μM, or 30 μM.

[0060] Further, the HEPES concentration is 10 mM, 15 mM, or 25 mM.

[0061] Further, the fetuin concentration is 1 mg / mL, 2 mg / mL, or 5 mg / mL.

[0062] Further, the hydrocortisone concentration is 50 nM, 100 nM, or 150 nM.

[0063] Further, the trimipramine concentration is 5 μM, 10 μM, or 15 μM.

[0064] Further, the insulin-like growth factor concentration is 10 ng / mL, 50 ng / mL, or 100 ng / mL.

[0065] In a second aspect of the present invention, there is provided the use of the aforementioned serum-free medium in culturing mesenchymal stem cells, wherein the mesenchymal stem cells include mesenchymal stem cells in humans or non-human mammals.

[0066] In a third aspect of the present invention, there is provided a preparation method based on the aforementioned serum-free medium, the method comprising the steps of:

[0067] A1. Using a basal medium as the base solution, adding the components other than the basal medium in the aforementioned serum-free medium into the basal medium according to the content, and mixing evenly;

[0068] A2. Adjusting the pH value and osmotic pressure;

[0069] A3. Sterilizing the mixed solution obtained in A2 to obtain a serum-free medium.

[0070] Further, the sterilization is carried out by filtration sterilization using a microporous membrane.

[0071] Further, the obtained serum-free medium is stored at 4°C.

[0072] In some embodiments, the preparation method of the serum-free medium has simple steps, easily available raw materials, does not contain animal serum, can be specifically used for the culture and expansion of mesenchymal stem cells, can maximize the preservation of the undifferentiated state of mesenchymal stem cells, and maintain their cell pluripotency.

[0073] Further, the preparation method includes:

[0074] A1. Using the basal medium as the base solution, adding L-glutamine, transferrin, L-ascorbic acid, lipid mixture, putrescine, human serum albumin, sodium heparin, progesterone, HEPES, fetuin, hydrocortisone, trimipramine, and insulin-like growth factor into the basal medium according to the contents in the aforementioned serum-free medium, and mixing evenly until there is no obvious precipitation;

[0075] A2. Using dilute hydrochloric acid and sodium hydroxide to adjust the pH value of the mixed liquid to 7.1 - 7.5, and using sodium chloride to adjust the osmotic pressure of the mixed liquid after adjusting the pH value to 290 mOSM / kg - 340 mOSM / kg;

[0076] A3. Performing a sterilization operation on the final mixed liquid obtained in A2 to obtain a serum-free medium.

[0077] Further, the sterilization operation is carried out by filtration sterilization through a microporous membrane.

[0078] The fourth aspect of the present invention provides the application of the aforementioned serum-free medium and the aforementioned preparation method in preparing a system, device, or equipment for automatically producing mesenchymal stem cell serum-free medium.

[0079] The fifth aspect of the present invention provides a product, which includes a processor that executes the aforementioned preparation method, and a computer program is stored in the processor, and the computer program can execute the aforementioned preparation method.

[0080] Further, the product includes a system, device, or equipment.

[0081] Further, the computer program includes programmed program instructions.

[0082] Further, the processor retrieves the program instructions.

[0083] Further, the program instructions control the processor to execute the step of using the basal medium as the base solution, adding the components other than the basal medium in the aforementioned serum-free medium into the basal medium, and mixing evenly.

[0084] Further, the program instructions control the processor to execute the steps of adjusting the pH value and osmotic pressure.

[0085] Further, the program instructions control the processor to execute the step of filtration sterilization.

[0086] Further, the computer program is stored in a computer-readable storage medium.

[0087] As used herein, the terms "computer program" and "program" refer to one or more application programs, software components, instruction sets, procedures, functions, objects, classes, instances, associated data, or portions thereof adapted to be implemented in a suitable computer-readable program code.

[0088] The term "computer-readable storage medium" shall be regarded as including a single medium or multiple media (e.g., a centralized or distributed database, and / or associated cache memories and servers) that store a set or more sets of computer-executable instructions or data. The term "computer-readable storage medium" shall also be regarded as including any medium that is capable of storing or encoding a set of instructions for execution by a processor and causing the processor to perform any one or more of the methods in the present disclosure. Thus, the term "computer-readable storage medium" shall be regarded as including, but not limited to, solid-state memories, optical media, and magnetic media. For example, a computer-readable storage medium can be one or more volatile, non-transitory, or non-volatile tangible computer-readable media.

[0089] As used herein, the term "processor" refers to any type of processor, such as a microprocessor, an embedded processor, a digital signal processor (DSP), a network processor, or other device for executing code, and may include more than one processor, such as a multi-core design or multiple processors each having a multi-core design. The processor can be configured to execute a sequence of computer program instructions, such as those stored in a memory, to perform various operations, processes, and methods according to exemplary embodiments of the present invention.

[0090] As used herein, the term "L-glutamine" refers to 2-aminopentanedioic acid (l’acide 2-aminoglutaramique). L-glutamine is a semi-essential, polar uncharged, and hydrophilic amino acid.

[0091] As used herein, the term "transferrin" generally refers to a glycoprotein that can bind and transport multivalent ions. For example, transferrin can be a single-chain glycoprotein. For example, transferrin can have a molecular weight of about 77,000 D. For example, transferrin can have polysaccharides. For example, transferrin can have two ion-binding sites. For example, the ion-binding sites can have different affinities for iron ions. For example, the multivalent ion can be an iron ion, a chromium ion, a manganese ion, a cadmium ion, or a nickel ion. For example, each transferrin molecule can bind two ferric iron atoms. For example, transferrin can be iron-saturated transferrin containing iron or apo-transferrin without iron. For example, transferrin can be mouse transferrin.

[0092] The terms "L-ascorbic acid" and "vitamin C" used in the present invention are used interchangeably herein and may be any chemical form of L-ascorbic acid found in an aqueous solution, such as undissociated, present in its free acid form, or dissociated into an anion. The dissolved salt form of L-ascorbic acid is an anion, along with any kind of cation commonly found in a fermentation supernatant, such as potassium, sodium, ammonium, or calcium. Also included may be isolated crystals of the free acid form of L-ascorbic acid. On the other hand, isolated crystals of the salt form of L-ascorbic acid are named with the name of its corresponding salt, i.e., sodium ascorbate, potassium ascorbate, calcium ascorbate, etc.

[0093] The term "lipid mixture" used in the present invention refers to a mixture of monoacylphospholipids and / or diacylphospholipids (collectively referred to as phospholipids) and cholesterol used in a liposome formulation. Phospholipids having different head groups (attached to glycerol) or different acyl chains (esterified to glycerol) can be combined with cholesterol in different ratios to produce liposomes having desired characteristics, such as effective and stable ITE loading. The lipid components in the lipid mixture can be characterized by the molar ratio of lipid species expressed in percentage terms (i.e., where the total lipid components of the liposome are 100%).

[0094] The term "putrescine" used in the present invention is a substance produced by the decarboxylation of ornithine or the hydrolysis of spermine. Putrescine can be found in putrefaction, but is also often found in normal components in organisms. Putrescine is a polyamine and functions to constitute ribosomes and promote cell growth or RNA synthesis.

[0095] The term "human serum albumin" used in the present invention, also known as "albumin", "serum albumin", "plasma albumin", is synthesized by the liver and is the main protein component in normal human serum total protein. Albumin is a single-chain polypeptide with a molecular weight of 66,458 daltons, composed of 585 amino acids, and an isoelectric point of 4.6. Under physiological conditions, human serum albumin mainly exists in a reduced form with one free sulfhydryl group (HSA-SH), also known as sulfhydryl albumin. HSA-SH contains many charged residues, such as positively charged lysine and arginine, and negatively charged carboxylates. The spatial structure of albumin is a heart-shaped tertiary structure: α-helix: composed of amino acids; substructure: composed of 6 α-helices; domain: composed of 2 substructures.

[0096] The term "sodium heparin" used in the present invention is an anticoagulant that can interfere with many links in the blood coagulation process and has anticoagulant effects both in vivo and in vitro. Its mechanism of action is relatively complex and mainly enhances the inhibitory effect of antithrombin III (AT-III) on activated coagulation factors II, IX, X, XI, and XII by binding to it.

[0097] The term "progesterone" used in the present invention is also known as progesterone, pregnenolone, luteosterone, corpus luteum hormone, progestin, gestagen or progestone, and is a steroid involved in the female menstrual cycle, pregnancy and has an impact on embryos of humans and other animals, with the chemical formula C 21 H 30 O2. It includes, for example, compounds having progesterone activity, such as progesterone, norethynodrel, norgestrel, megestrol acetate, norethisterone, progesterone-based agents, etc.

[0098] The term "HEPES" used in the present invention refers to 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

[0099] The term "fetuin" used in the present invention is a blood protein produced in the liver and secreted into the blood. They belong to a large class of binding proteins that mediate the transport and availability of various cargo substances in the blood. "Fetuin" includes fetuin-A and fetuin-B.

[0100] The term "hydrocortisone" used in the present invention is also known as cortisone, and refers to an adrenocortical hormone agent having glucocorticoid effects. In the culture of mesenchymal stem cells, hydrocortisone is a commonly used additive that can affect the cell signaling pathway, thereby affecting cell function. It can also inhibit the immune response and inflammatory response of cells, thereby reducing the stress on cells in culture and increasing the survival rate and growth rate of stem cells.

[0101] The term "Trimipramine" used in the present invention, also known as trimipramine maleate, is a chemical in the form of a white or off-white crystalline powder. The chemical name is trimipramine, and the molecular formula is C 20 H 26 N2, and the molecular weight is 294.43400.

[0102] The term "insulin-like growth factors" (abbreviated as IGFs) used in the present invention is a protein hormone highly similar to the insulin sequence. It can regulate a complex system of the physiological environment, and this system consists of two cell surface receptors (IGF1R and IGF2R) and two ligands (IGF-1 and IGF-2).

[0103] The term "IGF-1LR3" used in the present invention refers to long-chain LR3 insulin-like growth factor (LR3 IGF-1), which belongs to the insulin gene family and is structurally similar to the insulin precursor protein.

[0104] The term "transforming growth factor beta-1 (TGF-β1)" used in the present invention refers to the protein encoded by the human gene TGFB1 or its homologous sequences. The exemplary amino acid sequence of human TGF-β1 is provided by Swiss-Prot accession number P01137.

[0105] The terms "fibroblast growth factor 2 (FGF-2)", "basic FGF", "FGF-b", "FGFB", "BFGF", "heparin-binding growth factor 2 (HBGF-2)" or "prostatropin" used in the present invention are used interchangeably and refer to known members of the fibroblast growth factor family.

[0106] Advantages and beneficial effects of the present invention:

[0107] The present invention provides a serum-free medium and a method for preparing mesenchymal stem cells. The components of the serum-free medium are determined, which can avoid the interference of serum on cell culture and effectively promote the in vitro expansion of mesenchymal stem cells, highlighting the outstanding culture advantages of the serum-free medium described in the present invention. Description of the Drawings

[0108] Figure 1 It is a cell state diagram of mesenchymal stem cells in each group. Detailed Embodiments

[0109] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. For the operations and methods involved in the examples, unless otherwise specified, they are all conventional methods; for the reagents involved, unless otherwise specified, they are all conventional reagents. The technical solutions of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the examples are only for helping to understand the present invention and should not be regarded as specific limitations of the present invention.

[0110] Example 1 Components and Preparation Method of Serum-Free Medium

[0111] 1. Experimental Materials

[0112] Putrescine·2HCl (Sigma, P7505), sodium selenite (SIGMA, 214485-5G), heparin sodium (Wanbang Medicine, GSNZSY), insulin (Wanbang Medical, YIDAOSUZHUSHEYE), transferrin (Wanbang Medical, YIDAOSUZHUSHEYE), L-glutamine (Wanbang Medical, YIDAOSUZHUSHEYE), L-ascorbic acid (Sigma, A8960-5G), lipid mixture (GIBCO, 11905031), human albumin (Takeda, S20180020), progesterone (Sigma, P-069-1ML), HEPES (Yeasen, 60110ES76), fetuin (Zhihe Tongchuang, RP1005-100μg), hydrocortisone (Sinopharm, H20023069), trimipramine (MCE, HY-B1213), insulin-like growth factor (Tongli Haiyuan, GMP-TL510), α-MEM (GIBCO, 12561049), DMEM / F-12 (GIBCO, 12-719F).

[0113] 2. Experimental methods

[0114] 1) Experimental example 1:

[0115] 1.1 The formulation of the serum-free medium for mesenchymal stem cells in Experimental example 1 is as follows:

[0116] The concentration of L-glutamine is 4 mM, the concentration of the transferrin is 300 nM, the concentration of the L-ascorbic acid is 200 μM, the concentration of the lipid mixture is 0.1% v / v, the concentration of the putrescine is 60 μM, the concentration of the human albumin is 4 mg / mL, the concentration of the heparin sodium is 100 ng / mL, the concentration of the progesterone is 20 μM, the concentration of the HEPES is 10 mM, the concentration of the fetuin is 1 mg / mL, the concentration of the hydrocortisone is 100 nM, the concentration of the trimipramine is 5 μM, and the concentration of the insulin-like growth factor is 50 ng / mL. The basal medium is DMEM / F12.

[0117] 1.2 The preparation steps of the serum-free medium for mesenchymal stem cells in Experimental example 1 are as follows:

[0118] 1.2.1 Use DMEM-F12 as the basal medium; weigh each component in turn according to the formulation of Experimental example 1 and add them to the DMEM-F12 medium, and mix evenly until there is no obvious precipitate;

[0119] 1.2.2 Use dilute hydrochloric acid and sodium hydroxide to adjust the pH value to 7.1, and use sodium chloride to adjust the osmotic pressure to 340 mOsm / kg;

[0120] 1.2.3 Filter the mixture obtained in step 1.2.2 through a microporous membrane to sterilize it, and then the serum-free medium can be obtained and stored at 4°C.

[0121] 2) Experimental Example 2:

[0122] 2.1 The formulation of the serum-free medium for mesenchymal stem cells in Experimental Example 2 is as follows:

[0123] The concentration of L-glutamine is 10 mM, the concentration of the transferrin is 500 nM, the concentration of L-ascorbic acid is 150 μM, the concentration of the lipid mixture is 0.3% v / v, the concentration of putrescine is 30 μM, the concentration of human serum albumin is 1 mg / mL, the concentration of sodium heparin is 150 ng / mL, the concentration of progesterone is 30 μM, the concentration of HEPES is 15 mM, the concentration of fetuin is 2 mg / mL, the concentration of hydrocortisone is 150 nM, the concentration of trimipramine is 10 μM, and the concentration of insulin-like growth factor is 100 ng / mL. The basal medium is α-MEM.

[0124] 2.2 The preparation steps of the serum-free medium for mesenchymal stem cells in Experimental Example 2 are as follows:

[0125] 2.2.1 Use α-MEM as the basal medium; weigh each component successively according to the formulation of Experimental Example 2, add them to the α-MEM medium, and mix evenly until there is no obvious precipitate;

[0126] 2.2.2 Adjust the pH value to 7.2 using dilute hydrochloric acid and sodium hydroxide, and adjust the osmotic pressure to 340 mOsm / kg using sodium chloride;

[0127] 2.2.3 Filter the mixture obtained in step 2.2.2 through a microporous membrane to sterilize it, and then the serum-free medium can be obtained and stored at 4°C.

[0128] 3) Experimental Example 3:

[0129] 3.1 The formulation of the serum-free medium for mesenchymal stem cells in Experimental Example 3 is as follows:

[0130] The concentration of L-glutamine is 2 mM, the concentration of the transferrin is 100 nM, the concentration of L-ascorbic acid is 300 μM, the concentration of the lipid mixture is 1% v / v, the concentration of putrescine is 80 μM, the concentration of human serum albumin is 10 mg / mL, the concentration of sodium heparin is 300 ng / mL, the concentration of progesterone is 10 μM, the concentration of HEPES is 25 mM, the concentration of fetuin is 5 mg / mL, the concentration of hydrocortisone is 50 nM, the concentration of trimipramine is 15 μM, and the concentration of insulin-like growth factor is 10 ng / mL. The basal medium is α-MEM.

[0131] 3.2 Experimental Example 3 Preparation steps of mesenchymal stem cell serum-free medium are as follows:

[0132] 3.2.1 Use α-MEM as the basal medium; Weigh each component successively according to the formula of Experimental Example 3, add them to the α-MEM medium, and mix evenly until there is no obvious precipitate;

[0133] 3.2.2 Adjust the pH value to 7.4 using dilute hydrochloric acid and sodium hydroxide, and adjust the osmotic pressure to 340 mOsm / kg using sodium chloride;

[0134] 3.2.3 Filter and sterilize the mixture obtained in step 3.2.2 through a microporous filter membrane to obtain the serum-free medium, and store it at 4°C.

[0135] 4) Comparative Example 1:

[0136] 4.1 The formula of the medium for mesenchymal stem cells in Comparative Example 1 is as follows:

[0137] The concentration of L-glutamine is 2 mM, the concentration of the transferrin is 100 nM, the concentration of L-ascorbic acid is 300 μM, the concentration of the lipid mixture is 1% v / v, the concentration of putrescine is 80 μM, the concentration of human serum albumin is 10 mg / mL, the concentration of heparin sodium is 300 ng / mL, the concentration of progesterone is 10 μM, the concentration of HEPES is 25 mM, the concentration of fetuin is 5 mg / mL, the concentration of hydrocortisone is 50 nM, and the concentration of insulin-like growth factor is 10 ng / mL. The basal medium is α-MEM.

[0138] 4.2 Preparation steps of the medium for mesenchymal stem cells in Comparative Example 1 are as follows:

[0139] 4.2.1 Use α-MEM as the basal medium; Weigh each component successively according to the formula of Comparative Example 1, add them to the α-MEM medium, and mix evenly until there is no obvious precipitate;

[0140] 4.2.2 Adjust the pH value to 7.4 using dilute hydrochloric acid and sodium hydroxide, and adjust the osmotic pressure to 340 mOsm / kg using sodium chloride;

[0141] 4.2.3 Filter and sterilize the mixture obtained in step 4.2.2 through a microporous filter membrane to obtain the mesenchymal stem cell medium, and store it at 4°C.

[0142] 5) Comparative Example 2:

[0143] 5.1 Components of the medium for mesenchymal stem cells in Comparative Example 2 are as follows:

[0144] The concentration of L-glutamine is 2mM, the concentration of transferrin is 100nM, the concentration of L-ascorbic acid is 300μM, the concentration of lipid mixture is 1% v / v, the concentration of putrescine is 80μM, the concentration of human albumin is 10mg / mL, the concentration of heparin sodium is 300ng / mL, the concentration of progesterone is 10μM, the concentration of HEPES is 25mM, the concentration of fetuin is 5mg / mL, the concentration of hydrocortisone is 50nM, the concentration of insulin-like growth factor is 10ng / mL, the concentration of transforming growth factor β1 is 2.0ng / mL, and the concentration of fibroblast growth factor 2 is 20ng / ml. α-MEM is selected as the basic culture medium.

[0145] 5.2 Comparative Example 2 The steps for preparing the culture medium for mesenchymal stem cells are as follows:

[0146] 5.2.1 Use α-MEM as the basic culture medium; weigh each component in accordance with the formula of Comparative Example 2, add it to the α-MEM culture medium, and mix it evenly until there is no obvious precipitation;

[0147] 5.2.2 Use dilute hydrochloric acid and sodium hydroxide to adjust the pH value to 7.4, and use sodium chloride to adjust the osmotic pressure to 340mOsm / kg;

[0148] 5.2.3 Filter the mixed solution obtained in step 5.2.2 through a microporous filter membrane for sterilization to obtain the mesenchymal stem cell culture medium, and store it at 4°C.

[0149] Example 2 Observation of mesenchymal stem cell morphology

[0150] 1. Experimental methods

[0151] Umbilical cord mesenchyme was added at 4.4×10 3 cell / cm 2 The density of the samples was inoculated in 6-well plates, and the experimental examples were added

[0152] 1. Experimental Example 2, Experimental Example 3, Comparative Example 1, and Comparative Example 2 were cultured in each culture medium, and cell morphology was observed and images were collected every day starting from the second day.

[0153] 2. Experimental results

[0154] The results are as follows Figure 1 As shown, normal growth of cells can be observed in the culture media of Experimental Example 1, Experimental Example 2, Experimental Example 3 and Comparative Example 1, Comparative Example 2, and the cells appear to be elongated fibrous under a microscope.

[0155] Example 3 Detection of cell proliferation activity of mesenchymal stem cells

[0156] 1. Experimental methods

[0157] The umbilical cord mesenchyme was inoculated into a 6-well plate at a density of 5×10 3 cells / cm 2 . The culture media of Experimental Example 1, Experimental Example 2, Experimental Example 3, Comparative Example 1, and Comparative Example 2 were added respectively for culture. On the 4th day of culture, the cells in each group were collected to calculate the cell amplification multiple and cell viability rate.

[0158] The calculation formula for the cell amplification multiple is:

[0159] The number of cells collected on the 4th day / the initial number of seeded plates.

[0160] The calculation formula for the cell viability rate is:

[0161] (The number of live cells / the total number of cells) × 100%.

[0162] 2. Experimental results

[0163] The experimental results are shown in Table 1. The results show that the cell amplification multiples of the cells in Experimental Example 1, Experimental Example 2, and Experimental Example 3 are higher than those of the cells in Comparative Example 1 and Comparative Example 2, indicating that the proliferation ability of the cells in Experimental Example 1, Experimental Example 2, and Experimental Example 3 of the present invention is better.

[0164] Table 1

[0165] Group Cell amplification multiple Cell viability Experimental Example 1 24.21 96.12％ Experimental Example 2 23.32 95.22％ Experimental Example 3 23.89 97.03％ Comparative Example 1 9.32 89.44％ Comparative Example 2 16.11 93.03％

[0166] Detection of cell surface markers of mesenchymal stem cells in Example 4

[0167] 1. Experimental method

[0168] The mesenchymal stem cells were cultured starting from the primary generation using the mesenchymal stem cell culture media of Experimental Example 1, Experimental Example 2, Experimental Example 3, Comparative Example 1, and Comparative Example 2 respectively, and continuously cultured to the P6 passage. The cells were collected. Take 5×10 5 cell suspension, label the antibodies of CD90, CD105, CD73, CD19, CD14, CD34, CD45, HLA-DR, incubate in the dark at 4°C for 30 min, and wash away the unbound antibodies with PBS. Use the corresponding isotype control antibody as the negative control group, and perform marker analysis by flow cytometry.

[0169] Among them, for the positive markers: the positive rates of CD90, CD105, and CD73 need to reach more than 95% to meet the standard; for the negative markers: the positive rates of CD19, CD14, CD34, CD45, and HLA-DR need to be below 2% to meet the standard.

[0170] 2. Experimental results

[0171] Flow cytometry analysis was performed using a CytoFLEX flow cytometer, and the results are shown in Table 2. In the present invention, the mesenchymal stem cells cultured in the culture media of Experimental Example 1, Experimental Example 2, and Experimental Example 3 can maintain their stem cell characteristics without change, and the positive rates of CD90, CD105, and CD73 are all above 95%, while the positive rates of CD19, CD14, CD34, CD45, and HLA-DR are all below 2%.

[0172] Table 2 Expression levels of pluripotent genes in mesenchymal stem cells of each group

[0173]

[0174]

[0175] All documents mentioned in the present invention are incorporated herein by reference as if each individual document was specifically and individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A serum-free medium for mesenchymal stem cells with determined components, characterized in that, The serum-free medium for the determined mesenchymal stem cells comprises a basal medium and L-glutamine, transferrin, L-ascorbic acid, lipid mixture, putrescine, human albumin, sodium heparin, progesterone, HEPES, fetuin, hydrocortisone, trimipramine, insulin-like growth factor; The insulin-like growth factor is IGF-1 LR3; The basal medium is selected from one of DMEM medium, MEM medium, DMEM / F12 medium, F10 medium, F12 medium, IMDM medium; The concentrations of the components in the serum-free medium in the serum-free medium are as follows: The concentration of L-glutamine is 1-10 mM, The concentration of transferrin is 100-500 nM, The concentration of L-ascorbic acid is 150-300 μM, The concentration of the lipid mixture is 0.1-1% v / v, and the lipid mixture is the product with the catalog number 11905031 of GIBCO company, The concentration of putrescine is 30-80 μM, The concentration of human albumin is 1-10 mg / mL, The concentration of sodium heparin is 100-300 ng / mL, The concentration of progesterone is 10-30 μM, The concentration of HEPES is 10-25 mM, The concentration of fetuin is 1-5 mg / mL, The concentration of hydrocortisone is 50-150 nM, The concentration of trimipramine is 5-20 μM, The concentration of insulin-like growth factor is 10-100 ng / mL.

2. The serum-free medium according to claim 1, characterized in that, The basal medium is MEM medium or DMEM / F12 medium.

3. The serum-free medium according to claim 1, characterized in that, The basal medium is α-MEM medium or DMEM / F12 medium.

4. The serum-free medium according to claim 1, wherein The pH value of the serum-free medium is 7.1-7.

5.

5. The serum-free medium according to claim 1, wherein The osmotic pressure of the serum-free medium is 290 mOSM / kg to 340 mOSM / kg.

6. The serum-free medium according to claim 1, characterized in that, The pH value of the serum-free medium is 7.1, 7.2, 7.

4.

7. The serum-free medium according to claim 1, wherein The pH value of the serum-free medium is adjusted using dilute hydrochloric acid and sodium hydroxide.

8. The serum-free medium according to claim 1, wherein The osmotic pressure of the serum-free medium is 340 mOsm / kg.

9. The serum-free medium according to claim 1, characterized in that, The osmotic pressure of the serum-free medium is adjusted using sodium chloride.

10. The serum-free medium according to claim 1, wherein, The concentration of the L-glutamine is 2 mM, 4 mM, 10 mM.

11. The serum-free medium according to claim 1, wherein, The concentration of the transferrin is 100 nM, 300 nM, 500 nM.

12. The serum-free medium according to claim 1, characterized in that, The concentration of the L-ascorbic acid is 150 μM, 200 μM, 300 μM.

13. The serum-free medium according to claim 1, wherein, The concentration of the lipid mixture is 0.1% v / v, 0.3% v / v, 1% v / v.

14. The serum-free medium according to claim 1, wherein The concentration of the putrescine is 30 μM, 60 μM, 80 μM.

15. The serum-free medium according to claim 1, characterized in that, The concentration of the human albumin is 1 mg / ml, 4 mg / ml, 10 mg / ml.

16. The serum-free medium according to claim 1, characterized in that, The concentration of the sodium heparin is 100 ng / ml, 150 ng / ml, 300 ng / ml.

17. The serum-free medium according to claim 1, wherein The concentration of the progesterone is 10 μM, 20 μM, 30 μM.

18. The serum-free medium according to claim 1, characterized in that, The concentration of the HEPES is 10 mM, 15 mM, 25 mM.

19. The serum-free medium according to claim 1, wherein The concentration of the fetuin is 1 mg / mL, 2 mg / mL, 5 mg / mL.

20. The serum-free medium according to claim 1, wherein The concentration of the hydrocortisone is 50 nM, 100 nM, 150 nM.

21. The serum-free medium according to claim 1, wherein, The concentration of the trimipramine is 5 μM, 10 μM, 15 μM.

22. The serum-free medium according to claim 1, wherein The insulin-like growth factor concentration is 10 ng / mL, 50 ng / mL, or 100 ng / mL.

23. Use of the serum-free medium according to any one of claims 1-22 for culturing mesenchymal stem cells, characterized in that, The mesenchymal stem cells include mesenchymal stem cells in humans or non-human mammals.

24. A preparation method of the serum-free medium according to any one of claims 1-22, characterized in that, The method includes the steps of: A1. Using a basal medium as the base solution, adding the components other than the basal medium in the serum-free medium according to any one of claims 1-22 into the basal medium, and mixing well; A2. Adjusting the pH value and osmotic pressure; A3. Sterilizing the mixed solution obtained in A2 to obtain a serum-free medium.

25. The preparation method according to claim 24, characterized in that, The sterilization is performed by filtration through a microporous membrane.

26. The preparation method according to claim 24, characterized in that, The obtained serum-free medium is stored at 4°C.

27. Use of the serum-free medium according to any one of claims 1-22 in the preparation of a system or device for automated production of a serum-free medium for mesenchymal stem cells.

28. Use of the preparation method according to any one of claims 24-26 in the preparation of a system or device for automated production of a serum-free medium for mesenchymal stem cells.

29. A product, characterized in that, The product includes a processor that executes the preparation method according to any one of claims 24-26. A computer program is stored in the processor, and the computer program can execute the preparation method according to any one of claims 24-26.

30. The product according to claim 29, wherein, The product includes a system or device.

Images