In-vitro suspension culture medium and preparation method thereof

Abstract

The invention discloses an in-vitro suspension culture medium and a preparation method thereof, and relates to the technical field of cell culture, the in-vitro suspension culture medium comprises amino acid, vitamin, inorganic salt, biogenic amine, nucleotide, fatty acid, lipoid, hormone, saccharide, protein, polypeptide and water, and the polypeptide comprises recombinant human collagen peptide. According to the invention, in-vitro large-scale cell suspension culture without a microcarrier is realized.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to an in vitro suspension culture medium and a preparation method thereof. Background Art

[0002] Mesenchymal stem cells (MSCs), also known as multipotent stromal cells, are considered a valuable resource in the field of regenerative medicine due to their multidirectional differentiation potential and multiple biological properties. However, in practical applications, the in vitro expansion of mesenchymal stem cells and the maintenance of their differentiation ability have become key issues.

[0003] The conventional method for culturing mesenchymal stem cells in vitro is the two-dimensional monolayer adherent culture method, but this method is limited by the culture area and it is difficult to quickly obtain a large number of cells, which limits the development of mesenchymal stem cells in clinical applications. In order to solve this problem, the expansion method based on microcarrier bioreactors is widely used. By increasing the area to which mesenchymal stem cells can attach, higher density cell culture can be achieved. However, the microcarrier material may be internalized by mesenchymal stem cells, which will affect the safety of mesenchymal stem cell products. Summary of the invention

[0004] The main purpose of the present application is to provide an in vitro suspension culture medium and a preparation method thereof, so as to realize large-scale in vitro suspension culture of cells without microcarriers.

[0005] To achieve the above-mentioned purpose, an embodiment of the present application provides an in vitro suspension culture medium, which includes: amino acids, vitamins, inorganic salts, biogenic amines, nucleotides, fatty acids, lipids, hormones, carbohydrates, proteins, polypeptides and water, wherein the polypeptides include: recombinant human collagen peptides.

[0006] In one embodiment, the amino acids include: at least one of L-glycine, L-alanine, L-glutamine, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-cysteine ​​hydrochloride, L-cystine hydrochloride, L-glutamic acid, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

[0007] In one embodiment, the vitamins include at least one of biotin, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 and vitamin C.

[0008] In one embodiment, the inorganic salt includes at least one of calcium chloride, copper sulfate, ferric nitrate, ferrous sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, zinc sulfate and sodium selenite.

[0009] In one embodiment, the biogenic amine comprises: at least one of putrescine hydrochloride and ethanolamine;

[0010] And / or, the nucleotides include: at least one of hypoxanthine and thymine;

[0011] And / or, the fatty acid comprises: linoleic acid;

[0012] and / or, the lipids include: cholesterol;

[0013] and / or, the hormones include: hydrocortisone;

[0014] And / or, the sugar includes: glucose.

[0015] In one embodiment, the protein comprises at least one of plasma protein, enzyme and growth factor.

[0016] In one embodiment, the plasma protein includes: at least one of recombinant human transferrin, recombinant human serum albumin, recombinant human fibronectin and recombinant human insulin;

[0017] and / or, the enzyme comprises: at least one of catalase and superoxide dismutase;

[0018] And / or, the growth factor includes: at least one of recombinant human basic fibroblast growth factor, recombinant human epidermal growth factor, recombinant human insulin-like growth factor, recombinant human platelet-derived growth factor and recombinant human transforming growth factor.

[0019] In one embodiment, the in vitro suspension culture medium comprises: 430-2595 mg / L amino acids, 4.9-47.8 mg / L vitamins, 3030-15858 mg / L inorganic salts, 0.12-4 mg / L biogenic amines, 1.1-6 mg / L nucleotides, 0.01-1 mg / L fatty acids, 10-50 mg / L lipids, 0.001-0.01 mg / L hormones, 1000-8000 mg / L carbohydrates, 511-5080 mg / L plasma proteins, 20-100 mg / L enzymes, 4-80 ng / mL growth factors and 0.1-1 mg / L recombinant human collagen peptides, with the remainder being water.

[0020] In one embodiment, the in vitro suspension culture medium further comprises: at least one of choline chloride, inositol, lipoic acid and N-acetylcysteine.

[0021] In addition, to achieve the above-mentioned purpose, the present application also provides an in vitro suspension culture medium preparation method, which is used to prepare the above-mentioned in vitro suspension culture medium, comprising the following steps:

[0022] Amino acids, vitamins, inorganic salts, biogenic amines, nucleotides, fatty acids, lipids, hormones, sugars, proteins, polypeptides and water are mixed to prepare an in vitro suspension culture medium, wherein the polypeptides include recombinant human collagen peptides.

[0023] The embodiment of the present application provides an in vitro suspension culture medium, including: amino acids, vitamins, inorganic salts, biogenic amines, nucleotides, fatty acids, lipids, hormones, carbohydrates, proteins, polypeptides and water, wherein the polypeptides include: recombinant human collagen peptides. Cell proliferation is stimulated and cell viability is prolonged by adding amino acids; cell metabolism is regulated and controlled by adding vitamins, hormones and inorganic salts, and cells are helped to regulate cell membrane function; nucleotides are added to participate in the synthesis of genetic material, fatty acids and carbohydrates provide energy for cells; lipids are added to help maintain the fluidity and functionality of cell membranes; and proteins can provide nutrition, participate in cell signal transduction as a growth factor, and maintain the pH balance of the culture medium. In addition, recombinant human collagen peptides are added to the culture medium; human recombinant collagen peptides are peptides formed by enzymatic hydrolysis of recombinant human collagen. Collagen is insoluble in water, but after enzymatic hydrolysis to form peptides, these peptides are water-soluble and can be completely dissolved in water. Therefore, when mesenchymal stem cells meet human recombinant collagen peptides in the culture medium, the mesenchymal stem cells can bind to the peptides through the integrins of the cell membrane; at this time, the peptides, as matrix molecules combined with mesenchymal stem cells, competitively inhibit the adhesion of mesenchymal stem cells to the bottom of the culture flask. Therefore, when the peptide concentration reaches a certain level, the mesenchymal stem cells will no longer adhere to the culture flask, but will be suspended in the culture medium. When the mesenchymal stem cells suspended in the culture medium meet other mesenchymal stem cells, the mesenchymal stem cells bind through receptors on the surface of the cell membrane. In the cell cluster, the collagen peptides can serve as the extracellular matrix of the mesenchymal stem cells, further promoting the mutual binding of mesenchymal stem cells into spheres suspended in the culture medium; thereby realizing the in vitro large-scale culture of mesenchymal stem cells without microcarriers. This new in vitro culture system effectively reduces the culture cost and reduces the safety risk of mesenchymal stem cell products. And it enables mesenchymal stem cells to maintain good stem cell stemness after multiple generations of in vitro expansion. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a schematic diagram of the results of culturing mesenchymal stem cells in Example 1 of the present application;

[0025] Figure 2 This is a schematic diagram of the results of culturing mesenchymal stem cells in Comparative Example 1 of the present application;

[0026] Figure 3 This is a schematic diagram of the relative expression levels of pluripotency genes in mesenchymal stem cells cultured in Example 1 and Comparative Example 2 of the present application;

[0027] Figure 4 This is a schematic diagram of the CFU-F number results of mesenchymal stem cell culture in Example 1 and Comparative Example 2 of the present application.

[0028] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0030] Hereinafter, the in vitro suspension culture medium and the method for preparing the same are specifically disclosed with reference to the accompanying drawings in detail. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there may be cases where detailed descriptions of well-known matters and repeated descriptions of actually identical structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0031] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0032] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0033] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0034] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and understandable, the technical solution of the present application is further described below in conjunction with the accompanying drawings and embodiments. However, the present application is not limited to the listed embodiments, and should also include any other known changes within the scope of the rights claimed in the present application.

[0035] In order to better understand the technical solution of the present application, a detailed description will be given below in conjunction with the accompanying drawings and specific implementation methods.

[0036] In conventional techniques, the in vitro culture of mesenchymal stem cells mostly uses a two-dimensional monolayer adherent culture method, that is, the cells are cultured in vitro by combining receptors on their own membrane surfaces with extracellular matrix molecules. However, this method is limited by the culture area, and it is difficult to quickly obtain a large number of cells, which limits the development of mesenchymal stem cells in clinical applications. At the same time, when mesenchymal stem cells are cultured in a two-dimensional monolayer adherent culture, they lose the support and signals of the surrounding supporting cells in the physiological environment, and thus, as the culture time increases, the pluripotency of mesenchymal stem cells cannot be well maintained. It manifests as reduced expression of cell pluripotency genes, weakened in vitro differentiation ability, etc. Therefore, how to culture mesenchymal stem cells in large quantities in vitro while maintaining their good pluripotency has become a key issue in the application of mesenchymal stem cells. In order to solve this problem, the expansion method based on microcarrier bioreactors is widely used. Mesenchymal stem cells are attached to the porous microcarrier surface for division and proliferation, and the microcarrier greatly increases the area to which mesenchymal stem cells can be attached in a limited volume of culture fluid, thereby achieving higher density cell culture. However, the addition of microcarrier materials will increase the cost of the culture medium. At the same time, the microcarriers are likely to be internalized by mesenchymal stem cells, thus affecting the safety of mesenchymal stem cell products.

[0037] The embodiment of the present application provides an in vitro suspension culture medium, which stimulates cell proliferation and prolongs cell viability by adding amino acids; regulates and controls cell metabolism by adding vitamins, hormones and inorganic salts, and helps cells regulate cell membrane function; participates in the synthesis of genetic material by adding nucleotides, and fatty acids and sugars provide energy for cells; helps maintain the fluidity and functionality of cell membranes by adding lipids; and proteins can provide nutrition, participate in cell signal transduction as growth factors, and maintain the pH balance of the culture medium. In addition, recombinant human collagen peptides are added to the culture medium; human recombinant collagen peptides are peptides formed by enzymatic hydrolysis of recombinant human collagen. Collagen is insoluble in water, but after enzymatic hydrolysis to form peptides, these peptides are water-soluble and can be completely dissolved in water. Therefore, when mesenchymal stem cells and human recombinant collagen peptides meet in the culture medium, the mesenchymal stem cells can bind to the peptides through the integrins of the cell membrane; at this time, the peptides, as matrix molecules combined with mesenchymal stem cells, competitively inhibit the adhesion of mesenchymal stem cells to the bottom surface of the culture bottle. Therefore, when the peptide concentration reaches a certain level, the mesenchymal stem cells will no longer adhere to the culture flask, but will be suspended in the culture medium. When the mesenchymal stem cells suspended in the culture medium meet other mesenchymal stem cells, the mesenchymal stem cells bind through the receptors on the surface of the cell membrane. In the cell cluster, the collagen peptide can serve as the extracellular matrix of the mesenchymal stem cells, further promoting the mutual binding of the mesenchymal stem cells into spheres suspended in the culture medium; thus, the in vitro large-scale culture of mesenchymal stem cells can be achieved without microcarriers, effectively reducing the culture cost and the safety risk of mesenchymal stem cell products. And it enables the mesenchymal stem cells to maintain good stemness even after multiple generations of in vitro expansion.

[0038] The first aspect of an embodiment of the present application provides an in vitro suspension culture medium, which includes: amino acids, vitamins, inorganic salts, biogenic amines, nucleotides, fatty acids, lipids, hormones, carbohydrates, proteins, polypeptides and water, wherein the polypeptides include: recombinant human collagen peptides.

[0039] It should be understood that the in vitro suspension culture medium provided in the embodiments of the present application can be applied not only to mesenchymal stem cells, but also to other cell types that are difficult to spontaneously form suspension sphere cultures, such as cancer cell lines, etc., thereby establishing in vitro suspension culture spheres that are independent of carriers more quickly, economically and effectively.

[0040] In one feasible embodiment, the amino acids include: at least one of L-glycine, L-alanine, L-glutamine, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-cysteine ​​hydrochloride, L-cystine hydrochloride, L-glutamic acid, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

[0041] Amino acids are the basic units that make up proteins. They provide cells with the nitrogen elements needed to build nucleotides, amino acids and vitamins. They also stimulate cell proliferation and prolong the viability of cultured cells. They also help maintain the genomic stability and low mutation rate of stem cells, prevent genetic mutations in stem cells during the culture process, and ensure the reliability and safety of stem cells.

[0042] Exemplarily, the concentration of amino acids in the in vitro suspension medium is 430-2595 mg / L; for example, 430 mg / L, 500 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L, 2500 mg / L, 2595 mg / L, etc.

[0043] Exemplarily, the concentration of L-glycine in the in vitro suspension medium is 6 to 30 mg / L; for example, 6 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, etc.

[0044] Exemplarily, the concentration of L-alanine in the in vitro suspension medium is 2 to 15 mg / L; for example, 2 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, etc.

[0045] Exemplarily, the concentration of L-glutamine in the in vitro suspension medium is 200-600 mg / L; for example, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, 600 mg / L, etc.

[0046] Exemplarily, the concentration of L-arginine hydrochloride in the in vitro suspension medium is 40-300 mg / L; for example, 40 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, etc.

[0047] Exemplarily, the concentration of L-asparagine in the in vitro suspension medium is 3 to 20 mg / L; for example, 3 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, etc.

[0048] Exemplarily, the concentration of L-aspartic acid in the in vitro suspension medium is 3 to 20 mg / L; for example, 3 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, etc.

[0049] Exemplarily, the concentration of L-cysteine ​​hydrochloride in the in vitro suspension medium is 5 to 50 mg / L; for example, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, etc.

[0050] Exemplarily, the concentration of L-cystine hydrochloride in the in vitro suspension medium is 8-40 mg / L; for example, 8 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, etc.

[0051] Exemplarily, the concentration of L-glutamic acid in the in vitro suspension medium is 3 to 20 mg / L; for example, 3 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, etc.

[0052] Exemplarily, the concentration of L-histidine hydrochloride in the in vitro suspension medium is 10 to 90 mg / L; for example, 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 90 mg / L, etc.

[0053] Exemplarily, the concentration of L-isoleucine in the in vitro suspension medium is 15 to 150 mg / L; for example, 15 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L, 150 mg / L, etc.

[0054] Exemplarily, the concentration of L-leucine in the in vitro suspension medium is 20-150 mg / L; for example, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L, 150 mg / L, etc.

[0055] Exemplarily, the concentration of L-lysine hydrochloride in the in vitro suspension medium is 30-250 mg / L; for example, 30 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, 250 mg / L, etc.

[0056] Exemplarily, the concentration of L-methionine in the in vitro suspension medium is 5 to 50 mg / L; for example, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, etc.

[0057] Exemplarily, the concentration of L-phenylalanine in the in vitro suspension medium is 10-100 mg / L; for example, 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, etc.

[0058] Exemplarily, the concentration of L-proline in the in vitro suspension medium is 5 to 50 mg / L; for example, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, etc.

[0059] Exemplarily, the concentration of L-serine in the in vitro suspension medium is 8 to 80 mg / L; for example, 8 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, etc.

[0060] Exemplarily, the concentration of L-threonine in the in vitro suspension medium is 15 to 150 mg / L; for example, 15 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L, 150 mg / L, etc.

[0061] Exemplarily, the concentration of L-tryptophan in the in vitro suspension medium is 3 to 30 mg / L; for example, 3 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, etc.

[0062] Exemplarily, the concentration of L-tyrosine in the in vitro suspension medium is 20-200 mg / L; for example, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L, 160 mg / L, 180 mg / L, 200 mg / L, etc.

[0063] Exemplarily, the concentration of L-valine in the in vitro suspension medium is 20-200 mg / L; for example, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 140 mg / L, 160 mg / L, 180 mg / L, 200 mg / L, etc.

[0064] In a feasible embodiment, the vitamins include at least one of biotin, D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 and vitamin C.

[0065] Vitamins are trace organic compounds necessary for maintaining cell growth and function. They can participate in a variety of metabolic pathways in cells. For example, D-pantothenic acid can be converted into coenzyme A and participate in catalytic reactions in metabolism; vitamin B12 participates in the synthesis of folic acid and fatty acids. They not only promote cell growth and maintain cell function, but also enhance the proliferation ability of cells. For example, vitamin C can enhance the proliferation ability of bone marrow mesenchymal stem cells from aging individuals. In addition, vitamins help maintain the morphological and phenotypic stability of cells, ensuring that stem cells maintain their original morphology and molecular characteristics during the culture process. They are also involved in maintaining cell gene expression and stability, preventing stem cells from genetic mutations during the culture process, and ensuring the reliability and safety of stem cells. Therefore, vitamins are indispensable in the culture medium of mesenchymal stem cells, and they play a key role in maintaining the normal growth, differentiation and genetic stability of mesenchymal stem cells.

[0066] Exemplarily, the concentration of the vitamin in the in vitro suspension medium is 4.9-47.8 mg / L; for example, 4.9 mg / L, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 45 mg / L, 47 mg / L, 47.8 mg / L, etc.

[0067] Illustratively, the concentration of biotin in the in vitro suspension culture medium is 0.001-0.01 mg / L; for example, 0.001 mg / L, 0.002 mg / L, 0.004 mg / L, 0.006 mg / L, 0.008 mg / L, 0.01 mg / L, etc.

[0068] Illustratively, the concentration of D-calcium pantothenate in the in vitro suspension medium is 1 to 10 mg / L; for example, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, etc.

[0069] Illustratively, the concentration of folic acid in the in vitro suspension medium is 1 to 10 mg / L; for example, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, etc.

[0070] Illustratively, the concentration of nicotinamide in the in vitro suspension medium is 0.5-5 mg / L; for example, 0.5 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, etc.

[0071] Exemplarily, the concentration of pyridoxine hydrochloride in the in vitro suspension medium is 1 to 9 mg / L; for example, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, 9 mg / L, etc.

[0072] Illustratively, the concentration of riboflavin in the in vitro suspension culture medium is 0.1-1 mg / L; for example, 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, etc.

[0073] Illustratively, the concentration of thiamine hydrochloride in the in vitro suspension medium is 1 to 10 mg / L; for example, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, etc.

[0074] Illustratively, the concentration of vitamin B12 in the in vitro suspension culture medium is 0.2 to 2 mg / L; for example, 0.2 mg / L, 0.5 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L, etc.

[0075] Exemplarily, the concentration of vitamin C in the in vitro suspension culture medium is 0.176 to 0.704 mg / L; for example, 0.176 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.704 mg / L, etc.

[0076] In one feasible embodiment, the inorganic salt includes at least one of calcium chloride, copper sulfate, ferric nitrate, ferrous sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, zinc sulfate and sodium selenite.

[0077] Inorganic salts are essential elements for maintaining normal physiological functions and metabolic activities of cells, including sodium, potassium, calcium, magnesium, chloride ions, phosphates, sulfates and bicarbonates, which work together to maintain the osmotic pressure balance of cell culture fluid. For example, sodium and potassium ions are the main cations in extracellular fluid, and they play a core role in maintaining the constant osmotic pressure inside and outside the cell, ensuring the reasonable distribution of water inside and outside the cell. Magnesium ions, as conjugate ions of ATP, play a key role in the energy metabolism process. In addition, inorganic salts also help regulate the function of cell membranes, which is very important for the electrophysiological properties and signal transduction of cells. Some trace elements such as zinc, copper, manganese and selenium are essential for the activity of various enzymes, and they are involved in the energy production and antioxidant defense of cells. Bicarbonate participates in maintaining the stability of the pH value of the culture medium, which is essential for the survival and metabolic activities of cells. Therefore, the role of inorganic salts in the culture medium of mesenchymal stem cells is multifaceted, from maintaining basic cell physiological functions to participating in complex metabolic and signal transduction processes, they are indispensable, and they provide cells with a suitable growth environment to ensure the normal growth and differentiation of cells.

[0078] Exemplarily, the concentration of inorganic salts in the in vitro suspension culture medium is 3030-15858 mg / L; for example, 3030 mg / L, 4000 mg / L, 5000 mg / L, 6000 mg / L, 7000 mg / L, 8000 mg / L, 9000 mg / L, 10000 mg / L, 11000 mg / L, 12000 mg / L, 13000 mg / L, 14000 mg / L, 15000 mg / L, etc.

[0079] Exemplarily, the concentration of calcium chloride in the in vitro suspension medium is 30-200 mg / L; for example, 30 mg / L, 50 mg / L, 100 mg / L, 150 mg / L, 200 mg / L, etc.

[0080] Illustratively, the concentration of copper sulfate in the in vitro suspension culture medium is 0.001-0.01 mg / L; for example, 0.001 mg / L, 0.002 mg / L, 0.004 mg / L, 0.008 mg / L, 0.01 mg / L, etc.

[0081] Illustratively, the concentration of ferric nitrate in the in vitro suspension medium is 0.01-0.1 mg / L; for example, 0.01 mg / L, 0.02 mg / L, 0.04 mg / L, 0.06 mg / L, 0.08 mg / L, 0.1 mg / L, etc.

[0082] Illustratively, the concentration of ferrous sulfate in the in vitro suspension medium is 0.1-1 mg / L; for example, 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, etc.

[0083] Exemplarily, the concentration of magnesium chloride in the in vitro suspension medium is 8 to 40 mg / L; for example, 8 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, etc.

[0084] Exemplarily, the concentration of magnesium sulfate in the in vitro suspension medium is 15-100 mg / L; for example, 15 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, etc.

[0085] Illustratively, the concentration of potassium chloride in the in vitro suspension medium is 100-500 mg / L; for example, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, 500 mg / L, etc.

[0086] Exemplarily, the concentration of sodium bicarbonate in the in vitro suspension medium is 800-5000 mg / L; for example, 800 mg / L, 1000 mg / L, 2000 mg / L, 3000 mg / L, 4000 mg / L, 5000 mg / L, etc.

[0087] Illustratively, the concentration of sodium chloride in the in vitro suspension medium is 2000-10000 mg / L; for example, 2000 mg / L, 4000 mg / L, 6000 mg / L, 8000 mg / L, 10000 mg / L, etc.

[0088] Exemplarily, the concentration of disodium hydrogen phosphate in the in vitro suspension medium is 50-400 mg / L; for example, 50 mg / L, 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, etc.

[0089] Exemplarily, the concentration of sodium dihydrogen phosphate in the in vitro suspension medium is 10 to 80 mg / L; for example, 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, etc.

[0090] Illustratively, the concentration of zinc sulfate in the in vitro suspension medium is 0.1-1 mg / L; for example, 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, etc.

[0091] Illustratively, the concentration of sodium selenite in the in vitro suspension medium is 17.3-35 mg / L; for example, 17.3 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, etc.

[0092] In one feasible embodiment, the biogenic amine includes at least one of putrescine hydrochloride and ethanolamine.

[0093] Putrescine hydrochloride can promote the synthesis of proteins and nucleic acids, regulate intracellular pH, help maintain the stability of the intracellular environment, and protect cells from damage by oxidative stress.

[0094] Ethanolamine is an important component of cell membrane phospholipids. It can maintain the structure and function of the cell membrane, participate in the synthesis and metabolism of cell membrane phospholipids, and affect the fluidity and receptor function of the cell membrane.

[0095] Illustratively, the concentration of the biogenic amine in the in vitro suspension culture medium is 0.12-4 mg / L; for example, 0.12 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, etc.

[0096] Exemplarily, the concentration of putrescine hydrochloride in the in vitro suspension medium is 0.02-2 mg / L; for example, 0.02 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L, etc.

[0097] Exemplarily, the concentration of ethanolamine in the in vitro suspension medium is 0.1-2 mg / L; for example, 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, 1.5 mg / L, 2 mg / L, etc.

[0098] In a feasible embodiment, the nucleotide includes at least one of hypoxanthine and thymine.

[0099] Hypoxanthine and thymine are important precursors for nucleic acid synthesis and can support DNA synthesis and cell proliferation.

[0100] Illustratively, the concentration of nucleotides in the in vitro suspension medium is 1.1 to 6 mg / L; for example, 1.1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, 6 mg / L, etc.

[0101] Illustratively, the concentration of hypoxanthine in the in vitro suspension medium is 1 to 5 mg / L; for example, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, etc.

[0102] Illustratively, the concentration of thymine in the in vitro suspension culture medium is 0.1-1 mg / L; for example, 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, etc.

[0103] In one possible embodiment, the fatty acid includes: linoleic acid.

[0104] Linoleic acid can promote cell growth and the secretion of angiogenesis mediators, and as an energy storage and molecular precursor, it can also participate in the formation of cell structure.

[0105] Illustratively, the concentration of linoleic acid in the in vitro suspension medium is 0.01-1 mg / L; for example, 0.01 mg / L, 0.05 mg / L, 0.1 mg / L, 0.2 mg / L, 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1 mg / L, etc.

[0106] In one possible embodiment, the lipid comprises: cholesterol.

[0107] Cholesterol is a key component of cell membrane structure and function, can regulate cell signaling, and is essential for normal cell growth and function.

[0108] Illustratively, the concentration of cholesterol in the in vitro suspension medium is 10-15 mg / L; for example, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, etc.

[0109] In one possible embodiment, the hormone includes: hydrocortisone.

[0110] Hydrocortisone (also known as cortisol) can promote cell attachment and proliferation, and at the same time improve the purity of the desired stem cell population by having a certain inhibitory effect on fibroblasts in primary culture.

[0111] Illustratively, the concentration of hydrocortisone in the in vitro suspension culture medium is 0.001-0.01 mg / L; for example, 0.001 mg / L, 0.002 mg / L, 0.003 mg / L, 0.004 mg / L, 0.006 mg / L, 0.008 mg / L, 0.01 mg / L, etc.

[0112] In one possible embodiment, the sugar includes: glucose.

[0113] Glucose is the main energy source for cell life activities. It can provide energy, promote cell growth and proliferation, participate in cell metabolism, maintain the stability of the intracellular environment, and serve as a key factor in cell survival and function.

[0114] Illustratively, the concentration of glucose in the in vitro suspension medium is 1000-8000 mg / L; for example, 1000 mg / L, 2000 mg / L, 4000 mg / L, 6000 mg / L, 8000 mg / L, etc.

[0115] In a feasible embodiment, the protein includes at least one of plasma protein, enzyme and growth factor.

[0116] It is to be understood that the plasma protein used in the present application is plasma protein produced by in vitro recombinant production.

[0117] Exemplarily, the concentration of plasma protein in the in vitro suspension culture medium is 511-5080 mg / L; for example, 511 mg / L, 1000 mg / L, 2000 mg / L, 3000 mg / L, 4000 mg / L, 5000 mg / L, 5080 mg / L, etc.

[0118] Illustratively, the concentration of the enzyme in the in vitro suspension medium is 20-100 mg / L; for example, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, etc.

[0119] Exemplarily, the concentration of the growth factor in the in vitro suspension culture medium is 4 to 80 ng / mL; for example, 4 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL, 60 ng / mL, 80 ng / mL, etc.

[0120] Exemplarily, the concentration of recombinant human collagen peptide in the in vitro suspension culture medium is 0.1-1 mg / L; for example, 0.1 mg / L, 0.2 mg / L, 0.3 mg / L, 0.4 mg / L, 0.5 mg / L, 0.6 mg / L, 0.7 mg / L, 0.8 mg / L, 0.9 mg / L, 1 mg / L, etc.

[0121] Exemplarily, the plasma protein includes at least one of recombinant human transferrin, recombinant human serum albumin, recombinant human fibronectin and recombinant human insulin.

[0122] In serum-free or low-serum cell culture systems, transferrin is the preferred delivery form of iron.

[0123] Recombinant human serum albumin can provide nitrogen and essential amino acids that are important for cell growth and help maintain osmotic pressure balance in the cell culture environment. It can also serve as a substrate for cell attachment, help stabilize pH, and even protect cells from stress and damage.

[0124] Recombinant human fibronectin can be used as a cell culture matrix to replace rat tail collagen, blood-derived fibronectin, etc., to increase cell adhesion rate, enhance cell metabolism level, and shorten cell growth cycle.

[0125] Recombinant human insulin can regulate the uptake and utilization of glucose and affect cell growth and differentiation.

[0126] Exemplarily, the concentration of recombinant human transferrin in the in vitro suspension culture medium is 5 to 50 mg / L; for example, 5 mg / L, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, etc.

[0127] Exemplarily, the concentration of recombinant human serum albumin in the in vitro suspension culture medium is 500-5000 mg / L; for example, 500 mg / L, 1000 mg / L, 2000 mg / L, 3000 mg / L, 4000 mg / L, 5000 mg / L, etc.

[0128] Illustratively, the concentration of recombinant human fibronectin in the in vitro suspension culture medium is 1 to 10 mg / L; for example, 1 mg / L, 2 mg / L, 4 mg / L, 6 mg / L, 8 mg / L, 10 mg / L, etc.

[0129] Exemplarily, the concentration of recombinant human insulin in the in vitro suspension culture medium is 5 to 20 mg / L; for example, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, etc.

[0130] Exemplarily, the enzyme includes at least one of catalase and superoxide dismutase.

[0131] Superoxide dismutase is an antioxidant metalloenzyme whose main function is to catalyze the dismutation reaction of superoxide anion free radicals, converting them into hydrogen peroxide and oxygen, thereby removing free radicals and protecting cells from oxidative damage. Catalase further decomposes hydrogen peroxide into water and oxygen, further reducing the damage of reactive oxygen free radicals to cells.

[0132] Exemplarily, the concentration of catalase in the in vitro suspension culture medium is 10-50 mg / L; for example, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, etc.

[0133] Illustratively, the concentration of superoxide dismutase in the in vitro suspension culture medium is 20-100 mg / L; for example, 10 mg / L, 20 mg / L, 30 mg / L, 40 mg / L, 50 mg / L, etc.

[0134] Exemplarily, the growth factor includes at least one of recombinant human basic fibroblast growth factor, recombinant human epidermal growth factor, recombinant human insulin-like growth factor, recombinant human platelet-derived growth factor and recombinant human transforming growth factor.

[0135] Recombinant human basic fibroblast growth factor can stimulate cell proliferation and migration, and has a supporting effect on maintaining the stem cell characteristics of undifferentiated human mesenchymal stem cells.

[0136] Recombinant human epidermal growth factor regulates the cell growth cycle by regulating the expression of multiple cytokines related to cell growth, thereby affecting cell proliferation and migration, differentiation and survival.

[0137] Recombinant human insulin-like growth factor can promote cell proliferation and differentiation, promote growth, and inhibit cell apoptosis.

[0138] Recombinant human platelet-derived growth factor is a potent mitogen that regulates cell growth and division and plays an important role in cell proliferation, migration, survival and chemotaxis.

[0139] Recombinant human transforming growth factor can regulate cell proliferation, growth, differentiation and cell migration.

[0140] Exemplarily, the concentration of recombinant human basic fibroblast growth factor in the in vitro suspension culture medium is 1 to 20 ng / mL; for example, 1 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, etc.

[0141] Exemplarily, the concentration of recombinant human epidermal growth factor in the in vitro suspension culture medium is 1 to 12 ng / mL; for example, 1 ng / mL, 5 ng / mL, 10 ng / mL, 12 ng / mL, etc.

[0142] Exemplarily, the concentration of recombinant human insulin-like growth factor in the in vitro suspension culture medium is 1 to 20 ng / mL; for example, 1 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, etc.

[0143] Exemplarily, the concentration of recombinant human platelet-derived growth factor in the in vitro suspension culture medium is 1 to 20 ng / mL; for example, 1 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, etc.

[0144] Exemplarily, the concentration of recombinant human transforming growth factor in the in vitro suspension culture medium is 0.1 to 5 ng / mL; for example, 0.1 ng / mL, 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, etc.

[0145] In a feasible embodiment, the concentration of each component in the in vitro suspension culture medium includes: 430-2595 mg / L amino acids, 4.9-47.8 mg / L vitamins, 3030-15858 mg / L inorganic salts, 0.12-4 mg / L biogenic amines, 1.1-6 mg / L nucleotides, 0.01-1 mg / L fatty acids, 10-50 mg / L lipids, 0.001-0.01 mg / L hormones, 1000-8000 mg / L carbohydrates, 511-5080 mg / L plasma proteins, 20-100 mg / L enzymes, 4-80 ng / mL growth factors and 0.1-1 mg / L recombinant human collagen peptides, with the remainder being water.

[0146] In a feasible embodiment, the in vitro suspension medium further comprises: at least one of choline chloride, inositol, lipoic acid and N-acetylcysteine.

[0147] Illustratively, the concentration of choline chloride in the in vitro suspension medium is 3-30 mg / L; for example, 3 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, etc.

[0148] Illustratively, the concentration of inositol in the in vitro suspension medium is 3 to 20 mg / L; for example, 3 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, etc.

[0149] Illustratively, the concentration of lipoic acid in the in vitro suspension medium is 0.05-5 mg / L; for example, 0.05 mg / L, 0.1 mg / L, 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, 5 mg / L, etc.

[0150] Exemplarily, the concentration of N-acetylcysteine ​​in the in vitro suspension medium is 10-100 mg / L; for example, 10 mg / L, 20 mg / L, 40 mg / L, 60 mg / L, 80 mg / L, 100 mg / L, etc.

[0151] Exemplarily, the in vitro suspension medium includes: 6-30 mg / L L-glycine, 2-15 mg / L L-alanine, 200-600 mg / L L-glutamine, 40-300 mg / L L-arginine hydrochloride, 3-20 mg / L L-asparagine, 3-20 mg / L L-aspartic acid, 5-50 mg / L L-cysteine ​​hydrochloride, 8-40 mg / L L-cystine hydrochloride, 3-20 mg / L L-glutamic acid, 10-90 mg / L L-histidine hydrochloride, 15-150 mg / L L-isoleucine, 20-150 mg / L L-leucine, 30-250 mg / L L-lysine hydrochloride, 5-50 mg / L L-methionine, 10-100 mg / L L-phenylalanine, 5-50 mg / L L-proline, 8-80 mg / L L-serine, 15-150mg / L L-threonine, 3-30mg / L L-tryptophan, 20-200mg / L L-tyrosine, 20-200mg / L L-valine, 0.001-0.01mg / L biotin, 3-30mg / L choline chloride, 1-10mg / L calcium pantothenate, 1-10mg / L folic acid, 0.5-5mg / L nicotinamide, 1-9mg / L pyridoxine hydrochloride, 0.1-1mg / L riboflavin, 1-10mg / L thiamine hydrochloride, 0.2-2mg / L vitamin B12, 3-20mg / L inositol, 30-200mg / L calcium chloride, 0.001-0.01mg / L copper sulfate, 0.01-0.1mg / L ferric nitrate, 0.1-1mg / L ferrous sulfate, 8-40mg / L magnesium chloride, 15-100mg / L magnesium sulfate , 100-500mg / L potassium chloride, 800-5000mg / L sodium bicarbonate, 2000-10000mg / L sodium chloride, 50-400mg / L disodium hydrogen phosphate, 10-80mg / L sodium dihydrogen phosphate, 0.1-1mg / L zinc sulfate, 17.3-35.0mg / L sodium selenite, 1000-8000mg / L glucose, 1-5mg / L hypoxanthine, 0.01-1mg / L linoleic acid, 0.05-5mg / L lipoic acid, 0.176-0.704mg / L vitamin C, 0.02-2mg / L putrescine hydrochloride, 0.1-1mg / L thymine, 10-100mg / L N-acetylcysteine, 0.1-1mg / L recombinant human collagen peptide, 0.1-2mg / L ethanolamine, 10-50mg / L cholesterol, 0.001-0.01mg / L hydrocortisone, 5-50mg / L recombinant human transferrin, 500-5000mg / L recombinant human serum albumin, 1-10mg / L recombinant human fibronectin, 5-20mg / L recombinant human insulin, 10-50mg / L catalase, 10-50mg / L superoxide dismutase, 1-20ng / ml recombinant human basic fibroblast growth factor, 1-12ng / ml recombinant human epidermal growth factor, 1-20ng / ml recombinant human insulin-like growth factor, 1-20ng / ml recombinant human platelet-derived growth factor and 0.1-5ng / ml recombinant human transforming growth factor, the balance is water.

[0152] In this embodiment, amino acids are added to stimulate cell proliferation and prolong cell viability; vitamins, hormones and inorganic salts are added to regulate and control cell metabolism and help cells regulate cell membrane function; nucleotides are added to participate in the synthesis of genetic material, and fatty acids and carbohydrates provide energy for cells; lipids are added to help maintain the fluidity and functionality of cell membranes; and proteins can provide nutrition, participate in cell signal transduction as growth factors, and maintain the pH balance of the culture medium. In addition, recombinant human collagen peptides are added to the culture medium; human recombinant collagen peptides are peptides formed by enzymatic hydrolysis of recombinant human collagen. Collagen is insoluble in water, but after enzymatic hydrolysis to form peptides, these peptides are water-soluble and can be completely dissolved in water. Therefore, when mesenchymal stem cells meet human recombinant collagen peptides in the culture medium, the mesenchymal stem cells can bind to the peptides through the integrins of the cell membrane; at this time, the peptides, as matrix molecules combined with mesenchymal stem cells, competitively inhibit the adhesion of mesenchymal stem cells to the bottom of the culture bottle. Therefore, when the peptide concentration reaches a certain level, the mesenchymal stem cells will no longer adhere to the culture flask, but will be suspended in the culture medium. When the mesenchymal stem cells suspended in the culture medium meet other mesenchymal stem cells, the mesenchymal stem cells bind through the receptors on the surface of the cell membrane. In the cell cluster, the collagen peptide can serve as the extracellular matrix of the mesenchymal stem cells, further promoting the mutual binding of the mesenchymal stem cells into spheres suspended in the culture medium; thus, the in vitro large-scale culture of mesenchymal stem cells can be achieved without microcarriers, effectively reducing the cost of the culture medium and the safety risk of mesenchymal stem cell products. And it enables the mesenchymal stem cells to maintain good stem cell stemness even after multiple generations of in vitro expansion.

[0153] A second aspect of the present application provides a method for preparing an in vitro suspension culture medium, comprising the following steps:

[0154] Step S10, mixing amino acids, vitamins, inorganic salts, biogenic amines, nucleotides, fatty acids, lipids, hormones, carbohydrates, proteins, polypeptides and water to prepare an in vitro suspension culture medium, wherein the polypeptides include: recombinant human collagen peptides.

[0155] In a feasible embodiment, the above substances are accurately weighed and mixed, and the pH value is adjusted to 7.3-7.4 to prepare an in vitro suspension culture medium.

[0156] In this embodiment, a serum-free medium for in vitro suspension culture of mesenchymal stem cells is prepared, which does not contain animal-derived components, and can achieve in vitro serum-free suspension culture of mesenchymal stem cells independent of a carrier. The suspension culture system can maintain good stemness of stem cells during in vitro expansion.

[0157] In order to enable the details and operations of the above embodiments of the present application to be clearly understood by those skilled in the art, and to significantly demonstrate the improved performance of the embodiments of the present application, the above technical solutions are illustrated by means of multiple embodiments below.

[0158] Example 1

[0159] The in vitro suspension medium provided includes: 18.75 mg / L L-glycine, 4.45 mg / L L-alanine, 300 mg / L L-glutamine, 147.5 mg / L L-arginine hydrochloride, 7.5 mg / L L-asparagine, 6.65 mg / L L-aspartic acid, 17.56 mg / L L-cysteine ​​hydrochloride, 31.29 mg / L L-cystine hydrochloride, 7.35 mg / L L-glutamic acid, 31.48 mg / L L-histidine hydrochloride, 54.47 mg / L L-isoleucine, 59 mg / L L-leucine, 91.25 mg / L L-lysine hydrochloride, 17.24 mg / L L-methionine, 35.48 mg / L L-phenylalanine, 17.25 mg / L L-proline, 26.25 mg / L L-serine, 53.45mg / L L-threonine, 9mg / L L-tryptophan, 55.79mg / L L-tyrosine, 52.85mg / L L-valine, 0.0035mg / L biotin, 8.98mg / L choline chloride, 2.24mg / LD calcium pantothenate, 2.65mg / L folic acid, 2.02mg / L nicotinamide, 2mg / L pyridoxine hydrochloride, 0.219mg / L riboflavin, 2.17mg / L thiamine hydrochloride, 0.68mg / L vitamin B12, 12.6mg / L inositol, 116.6mg / L calcium chloride, 0.0013mg / L copper sulfate, 0.05mg / L ferric nitrate, 0.417mg / L ferrous sulfate, 28.64mg / L magnesium chloride, 48.84mg / L magnesium sulfate, 311.8mg / L potassium chloride, 2438mg / L sodium bicarbonate, 6999.5mg / L sodium chloride, 71.02mg / L disodium hydrogen phosphate, 62.5mg / L sodium dihydrogen phosphate, 0.32mg / L zinc sulfate, 0.01mg / L sodium selenite, 3151mg / L glucose, 2.39 mg / L hypoxanthine, 0.042mg / L linoleic acid, 0.105mg / L lipoic acid, 0.5mg / L vitamin C, 0.15mg / L putrescine hydrochloride, 0.365mg / L thymine, 65mg / L N-acetylcysteine, 0.5mg / L recombinant human collagen peptide, 1mg / L ethanolamine, 20mg / L cholesterol, 0.005mg / L hydrocortisone, 5.5mg / L recombinant human transferrin, 1000mg / L recombinant human serum albumin, 3mg / L recombinant human fibronectin, 10mg / L recombinant human insulin, 20mg / L catalase, 20mg / L superoxide dismutase, 10ng / ml recombinant human basic fibroblast growth factor, 10ng / ml recombinant human epidermal growth factor, 10ng / ml recombinant human insulin-like growth factor, 10ng / ml recombinant human platelet-derived growth factor and 0.5ng / ml recombinant human transforming growth factor, the balance is water.

[0160] Comparative Example 1

[0161] The components are the same as those in Example 1, except that no recombinant human collagen peptide is added.

[0162] Comparative Example 2

[0163] The cells were provided with DMEM / F12 medium supplemented with 10 wt.% fetal bovine serum.

[0164] In vitro suspension culture was performed using the in vitro suspension culture medium in Example 1 and Comparative Example 1. The frozen P3 human umbilical cord mesenchymal stem cells were revived and cultured at 1×10 4 cells / cm 2 The revived cells were inoculated into a T25 culture flask treated with cell culture at a density of 100 μg / cm2 and cultured for 3 days before observation under a phase contrast microscope. The experimental results of Example 1 are shown in FIG. Figure 1 , it can be seen that a large number of cell balls are suspended in the culture medium. The experimental results of comparative example 1 are as follows Figure 2 , it can be seen that a large number of cells grow attached to the wall. Therefore, the in vitro suspension culture medium provided in the embodiment of the present application can successfully establish in vitro suspension culture of mesenchymal stem cells.

[0165] The P0 human umbilical cord mesenchymal stem cells were revived and cultured at 1×10 4 cells / cm 2 The revived cells were inoculated into a conventional T25 culture flask at a density of . The in vitro suspension culture was performed using the in vitro suspension culture medium of Example 1, and the conventional two-dimensional adherent culture was performed using the culture medium of Comparative Example 2. After each amplification for 5 generations, the cultured cells were harvested.

[0166] Real-time quantitative PCR was used to detect the expression of pluripotency genes. Figure 3 As shown, the horizontal axis is the pluripotency genes Oct4, Sox2, Nanog, Klf4 and c-myc of mesenchymal stem cells, and the vertical axis is the relative level of expression of each gene. Figure 3 It can be seen that the expression levels of pluripotency genes Oct4, Sox2, Nanog, Klf4 and c-myc expressed by mesenchymal stem cells cultured in suspension in Example 1 are significantly higher than those in Comparative Example 2 cultured in adherent culture.

[0167] P5 cells cultured in the two media were plated at a density of 50 to 100 cells / cm 2 The density of the cells was inoculated into a 10 cm culture dish. After 10 to 12 days of culture, the cells were fixed, stained and counted. The results are shown in Figure 4 .according to Figure 4 It can be seen that the number of CFU-F colonies formed by mesenchymal stem cells cultured in suspension was significantly higher than that in the control group cultured in conventional adherent culture.

[0168] The above are only preferred embodiments of the present application, and do not limit the scope of the present application. For those skilled in the art, the present application may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of patent protection of the present application.

Claims

1. An in vitro suspension culture medium, characterized in that The in vitro suspension culture medium comprises: amino acids, vitamins, inorganic salts, biogenic amines, nucleotides, fatty acids, lipids, hormones, sugars, proteins, polypeptides and water, wherein the polypeptides comprise: recombinant human collagen peptides.

2. The in vitro suspension culture medium according to claim 1, characterized in that The amino acids include at least one of L-glycine, L-alanine, L-glutamine, L-arginine hydrochloride, L-asparagine, L-aspartic acid, L-cysteine ​​hydrochloride, L-cystine hydrochloride, L-glutamic acid, L-histidine hydrochloride, L-isoleucine, L-leucine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine.

3. The in vitro suspension culture medium according to claim 1, characterized in that The vitamins include: at least one of biotin, D-calcium pantothenate, folic acid, nicotinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 and vitamin C.

4. The in vitro suspension culture medium according to claim 1, characterized in that The inorganic salt comprises at least one of calcium chloride, copper sulfate, ferric nitrate, ferrous sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, zinc sulfate and sodium selenite.

5. The in vitro suspension culture medium according to claim 1, characterized in that The biogenic amine comprises: at least one of putrescine hydrochloride and ethanolamine; And / or, the nucleotides include: at least one of hypoxanthine and thymine; And / or, the fatty acid comprises: linoleic acid; and / or, the lipids include: cholesterol; and / or, the hormones include: hydrocortisone; And / or, the sugar includes: glucose.

6. The in vitro suspension culture medium according to claim 1, characterized in that The protein comprises at least one of plasma protein, enzyme and growth factor.

7. The in vitro suspension culture medium according to claim 6, characterized in that The plasma protein comprises: at least one of recombinant human transferrin, recombinant human serum albumin, recombinant human fibronectin and recombinant human insulin; and / or, the enzyme comprises: at least one of catalase and superoxide dismutase; And / or, the growth factor includes: at least one of recombinant human basic fibroblast growth factor, recombinant human epidermal growth factor, recombinant human insulin-like growth factor, recombinant human platelet-derived growth factor and recombinant human transforming growth factor.

8. The in vitro suspension culture medium according to claim 6, characterized in that The in vitro suspension culture medium comprises: 430-2595 mg / L amino acids, 4.9-47.8 mg / L vitamins, 3030-15858 mg / L inorganic salts, 0.12-4 mg / L biogenic amines, 1.1-6 mg / L nucleotides, 0.01-1 mg / L fatty acids, 10-50 mg / L lipids, 0.001-0.01 mg / L hormones, 1000-8000 mg / L sugars, 511-5080 mg / L plasma proteins, 20-100 mg / L enzymes, 4-80 ng / mL growth factors and 0.1-1 mg / L recombinant human collagen peptides, with the remainder being water.

9. The in vitro suspension culture medium according to any one of claims 1 to 8, characterized in that The in vitro suspension culture medium further comprises: at least one of choline chloride, inositol, lipoic acid and N-acetylcysteine.

10. A method for preparing an in vitro suspension culture medium, characterized in that: For preparing an in vitro suspension culture medium according to any one of claims 1 to 9, the method comprises the following steps: Amino acids, vitamins, inorganic salts, biogenic amines, nucleotides, fatty acids, lipids, hormones, sugars, proteins, polypeptides and water are mixed to prepare an in vitro suspension culture medium, wherein the polypeptides include recombinant human collagen peptides.

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