WO2024251234A1 - Milieu de culture sans sérum pour cellules souches mésenchymateuses et son utilisation - Google Patents

Milieu de culture sans sérum pour cellules souches mésenchymateuses et son utilisation Download PDF

Info

Publication number
WO2024251234A1
WO2024251234A1 PCT/CN2024/097949 CN2024097949W WO2024251234A1 WO 2024251234 A1 WO2024251234 A1 WO 2024251234A1 CN 2024097949 W CN2024097949 W CN 2024097949W WO 2024251234 A1 WO2024251234 A1 WO 2024251234A1
Authority
WO
WIPO (PCT)
Prior art keywords
mesenchymal stem
culture
medium
cells
serum
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/097949
Other languages
English (en)
Chinese (zh)
Inventor
鄢晓君
刘伟
张元元
张昆
张志华
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Cytoniche Biotech Co Ltd
Original Assignee
Beijing Cytoniche Biotech Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Cytoniche Biotech Co Ltd filed Critical Beijing Cytoniche Biotech Co Ltd
Publication of WO2024251234A1 publication Critical patent/WO2024251234A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • the invention belongs to the technical field of cell culture, and particularly relates to a mesenchymal stem cell serum-free culture medium containing platelet lysate, and a preparation method and application thereof.
  • MSCs Mesenchymal stem cells
  • BM-MSCs bone marrow mesenchymal stem cells
  • mesenchymal stem cells have the characteristics of multidirectional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication. They have attracted widespread attention and are considered to be the stem cell products closest to clinical application. Since the separation and extraction of mesenchymal stem cells from umbilical cord tissue will not cause any damage, and the mesenchymal stem cells in the umbilical cord are large in number, high in quality, and pure in cells, they have become the stem cells with the most clinical application value and preservation value.
  • stem cell culture is mainly based on traditional two-dimensional culture, which is equivalent to supporting stem cell growth only on one plane.
  • the proliferation efficiency and space utilization are very low, which cannot meet the growing demand for large-dose application of clinical stem cells.
  • three-dimensional culture co-cultures the three-dimensional structure matrix with cells in vitro to form three-dimensional cell aggregates, allowing cells to grow, differentiate and migrate in vitro in a three-dimensional manner, more realistically simulating the living environment in the body, and more conducive to cell proliferation, survival and the maintenance of their own characteristics.
  • Cells have different nutritional requirements under different culture conditions. Therefore, suitable three-dimensional culture medium is required for three-dimensional culture technology to achieve the optimal large-scale production process.
  • HPLs Platelet lysate
  • HPLs can be collected from blood banks where expired platelets that are not suitable for transfusion are available.
  • HPLs contain a large number of cytokines, growth factors, proteins and other substances that can promote the growth of MSCs while maintaining their differentiation potential and immunomodulatory properties.
  • cytokines cytokines, growth factors, proteins and other substances that can promote the growth of MSCs while maintaining their differentiation potential and immunomodulatory properties.
  • most of the serum-free culture media for mesenchymal stem cells on the market are designed for two-dimensional culture.
  • the present invention aims to find a serum-free culture medium based on platelet lysate that is suitable for both two-dimensional and three-dimensional culture, so as to achieve good cell expansion quality and quantity and high safety.
  • the purpose of the present invention is to provide a novel serum-free culture medium for mesenchymal stem cells, which is serum-free and animal-derived, and can significantly improve the viability, proliferation and cell quality of mesenchymal stem cells in two-dimensional or three-dimensional culture, thereby solving the technical problem that the existing serum-free culture medium for mesenchymal stem cells in the prior art has poor effect when used for three-dimensional large-scale culture.
  • the present invention provides a mesenchymal stem cell serum-free culture medium, which comprises the following components and the contents thereof are as follows:
  • the mesenchymal stem cell serum-free culture medium further comprises one or more components selected from the following group, and the contents thereof are as follows:
  • the basal culture medium is selected from the group consisting of ⁇ -MEM medium, D/F12 medium and DMEM medium, and preferably the basal culture medium is ⁇ -MEM medium.
  • the content of the platelet lysate is 5% (v/v).
  • the content of L-glutamine is 4 mM.
  • the content of hydrocortisone is 500 ⁇ g/L.
  • the content of EGF is 5-20 ng/ml.
  • the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising:
  • the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising:
  • step 2) Adding the nutrient supplement preparation prepared in step 1) into the basal culture medium preparation.
  • the culture method is a two-dimensional culture method, comprising the following steps,
  • the culture method is a three-dimensional culture method, comprising the following steps,
  • Preparing microcarriers placing microcarriers in a culture container, optionally, transferring the microcarriers to the culture container after swelling with liquid in advance, adding complete medium of mesenchymal stem cells, and uniformly dispersing the microcarriers;
  • the mesenchymal stem cell complete culture medium is the mesenchymal stem cell serum-free culture medium of the present invention, and its composition is as described above.
  • the present invention provides a use of a mesenchymal stem cell serum-free medium in culturing mesenchymal stem cells.
  • the culture is a two-dimensional culture. In some embodiments, the culture is a three-dimensional culture.
  • the culture medium of the present invention does not contain components derived from serum, thus avoiding the risks of contamination of exogenous viruses and pathogenic factors caused by serum, poor reproducibility of products and experimental results, and easy to cause allergic reactions;
  • the culture medium of the present invention is also particularly suitable for three-dimensional culture of mesenchymal stem cells, achieving large-scale, high-quality expansion of mesenchymal stem cells and having the advantage of high safety.
  • FIG. 1 shows the cell viability during continuous culture in Example 5.
  • FIG. 3 shows the cell diameters of cells continuously cultured in Example 5.
  • FIG. 4 shows a microscopic image of continuous cell culture in Example 5.
  • FIG. 5 shows the cell staining results on the 1st and 4th days of three-dimensional culture of P7 in Example 7.
  • FIG6 shows the cell staining results of Example 8 on the 1st and 4th days of three-dimensional culture.
  • the present invention provides a serum-free culture medium for mesenchymal stem cells, which can be used for two-dimensional culture and three-dimensional culture of mesenchymal stem cells.
  • stem cell refers to a type of multipotent cell with the ability to self-replicate. Under certain conditions, stem cells can differentiate into a variety of functional cells.
  • the term “mesenchymal stem cell” refers to an important member of the stem cell family, which is derived from the early mesoderm of development and belongs to multipotent stem cells. It was originally found in the bone marrow and has the characteristics of multidirectional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication.
  • Mesenchymal stem cells include bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, bone mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreatic mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord mesenchymal stem cells, etc.
  • the term “umbilical cord mesenchymal stem cells” refers to mesenchymal stem cells derived from the umbilical cord.
  • adipose mesenchymal stem cells refers to mesenchymal stem cells derived from adipose.
  • serum-free medium refers to a cell culture medium that is not supplemented with serum proteins such as fetal bovine serum. Serum-free medium is well known in the art.
  • the mesenchymal stem cell serum-free culture medium of the present invention comprises basal culture medium, about 88-99% (v/v); platelet lysate, about 1-12% (v/v); L-glutamine, about 2-4 mM; and hydrocortisone, about 0.05-5000 ⁇ g/L.
  • basal medium refers to the starting medium in which cells are added to start cultivation.
  • Basic medium is a solution of nutrients containing cells that nourish growth.
  • basal medium provides cells with essential and non-essential amino acids, vitamins, energy sources, lipids and trace elements required for minimal growth and/or survival.
  • basal medium is a commercially available culture medium or a culture medium known in the art that can maintain the growth or proliferation of mammalian cells.
  • Non-limiting examples of basal medium include, but are not limited to, DMEM, F12, MEM, ⁇ -MEM, MEGM, RPMI-1640 and combinations thereof.
  • the basal medium includes D/F12 (e.g., 1: 1 mixing ratio), ⁇ -MEM+DMEM (e.g., 1: 1 mixing ratio) and ⁇ -MEM.
  • the basal medium is ⁇ -MEM.
  • the basal medium accounts for about 88-99% (v/v) by volume ratio, for example, about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any value within the range of any of the above values as endpoints.
  • the basal medium accounts for about 88-97% (v/v) by volume ratio, for example, about 88-95% (v/v), about 88-93% (v/v), about 88-92% (v/v), about 88-91% (v/v), about 89-91% (v/v), about 90% (v/v), etc.
  • platelet lysate refers to a product that can be obtained from platelets.
  • Platelets can be obtained from a donor, preferably a human donor, and can be separated and pooled from multiple collection units of whole blood, or collected by platelet apheresis: blood is obtained from a donor and passed through a device that removes platelets. After separation, the platelets are typically lysed, for example, by one or more freeze/thaw cycles.
  • the platelet lysate used in the present invention can be a commercially available platelet lysate product that can be purchased commercially, such as the platelet lysate product EliteGro-adv purchased by EliteCell Biomedical Corp.
  • platelet lysate accounts for about 1-12% (v/v) by volume, for example, about 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, or the above.
  • the arbitrary numerical value is any value within the range of the endpoint.
  • the platelet lysate accounts for about 2-10% (v/v) by volume, for example, about 2.5-10% (v/v), about 2.5-7.5% (v/v), about 3.5-6.5% (v/v), about 4.5-5.5% (v/v), about 5% (v/v), etc.
  • the platelet lysate accounts for about 5% (v/v) by volume ratio calculated based on the total volume of the serum-free medium for mesenchymal stem cells.
  • the content of L-glutamine in the mesenchymal stem cell serum-free culture medium of the present invention is about 2-4mM, for example, about 2mM, 2.5mM, 3mM, 3.5mM, 4mM, or any value within the range of any of the above values as endpoints. In a preferred embodiment, the content of L-glutamine in the mesenchymal stem cell serum-free culture medium of the present invention is about 4mM.
  • cortisone is also referred to as cortisone, and refers to an adrenocortical hormone agent with a glucocorticoid effect.
  • cortisone is a commonly used additive that can affect the signal transduction pathway of cells, thereby affecting the function of cells.
  • the immune response and inflammatory response of cells can also be suppressed, thereby reducing the stress to which cells are subjected in culture, and improving the survival rate and growth rate of stem cells.
  • the content of cortisone is about 0.05-5000 ⁇ g/L, such as about 0.05 ⁇ g/L, 0.5 ⁇ g/L, 5 ⁇ g/L, 50 ⁇ g/L, 500 ⁇ g/L, 1000 ⁇ g/L, 2000 ⁇ g/L, 3000 ⁇ g/L, 4000 ⁇ g/L, 5000 ⁇ g/L, or any value within the range of the above arbitrary numerical value as endpoint.
  • the content of hydrocortisone is about 5-5000 ⁇ g/L, for example, about 100-1000 ⁇ g/L, for example, about 100 ⁇ g/L, 200 ⁇ g/L, 300 ⁇ g/L, 400 ⁇ g/L, 500 ⁇ g/L, 600 ⁇ g/L, 700 ⁇ g/L, 800 ⁇ g/L, 900 ⁇ g/L, 1000 ⁇ g/L.
  • the content of hydrocortisone is about 500 ⁇ g/L.
  • the mesenchymal stem cell serum-free culture medium of the present invention may also include growth factors.
  • Growth factors are supplementary factors necessary for maintaining the survival, proliferation and differentiation of cells in vitro. Growth factors are effective mitogens that can shorten the doubling time of cell populations. According to their chemical properties, they can be divided into polypeptide growth factors and steroid growth factors. Growth factors that can be added to the mesenchymal stem cell serum-free culture medium of the present invention include, but are not limited to, polypeptide growth factors and steroid growth factors, such as natural growth factors or corresponding recombinant growth factors obtained by genetic recombination.
  • the mesenchymal stem cell serum-free culture medium of the present invention includes one or more growth factors selected from the group consisting of epidermal growth factor (EGF); fibroblast growth factor (FGF), such as basic fibroblast growth factor (bFGF); and nerve growth factor (NGF).
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • NGF nerve growth factor
  • the mesenchymal stem cell serum-free culture medium of the present invention includes epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).
  • the content of EGF is about 0-20ng/ml, for example, about 0ng/ml, 1ng/ml, 2ng/ml, 3ng/ml, 4ng/ml, 5ng/ml, 6ng/ml, 7ng/ml, 8ng/ml, 9ng/ml, 10ng/ml, 11ng/ml, 12ng/ml, 13ng/ml, 14ng/ml, 15ng/ml, 16ng/ml, 17ng/ml, 18ng/ml, 19ng/ml, 20ng/ml, or any value in the range of endpoints of any of the above numerical values.
  • the content of EGF is about 5-20ng/ml. In a preferred embodiment, in the mesenchymal stem cell serum-free medium of the present invention, the content of EGF is about 20ng/ml.
  • the content of bFGF is about 5-20 ng/ml, for example, about 5 ng/ml, 6 ng/ml, 7 ng/ml, 8 ng/ml, 9 ng/ml, 10 ng/ml, 11 ng/ml, 12ng/ml, 13ng/ml, 14ng/ml, 15ng/ml, 16ng/ml, 17ng/ml, 18ng/ml, 19ng/ml, 20ng/ml, or any value within a range with any of the above values as endpoints.
  • the content of bFGF in the serum-free medium for mesenchymal stem cells of the present invention is about 20ng/ml.
  • the mesenchymal stem cell serum-free culture medium of the present invention may also include other supplementary factors.
  • the supplementary factors that can be added to the mesenchymal stem cell serum-free culture medium of the present invention include, but are not limited to, hormones, binding proteins, adhesion factors, and other added factors.
  • the serum-free medium for mesenchymal stem cells of the present invention includes hormones, such as insulin, growth hormone, glucagon, etc.
  • hormones such as insulin, growth hormone, glucagon, etc.
  • insulin is a polypeptide that can bind to the insulin receptor on cells to form a complex, promote the synthesis of RNA, protein and fatty acids, and induce cell apoptosis, and is an important cell survival factor.
  • the mesenchymal stem cell serum-free medium of the present invention includes binding proteins, such as transferrin and albumin.
  • transferrin and albumin There are specific transferrin receptors on most mammalian cells. The complex binding of the receptor with transferrin and iron ions is the main source of the necessary trace element iron for cells.
  • transferrin also has the properties of a growth factor and can bind to other trace elements such as vanadium. Different cells also have different requirements for transferrin.
  • Albumin is also a commonly used additive factor in serum-free medium. It stabilizes and regulates the activity of the above substances in serum-free medium by binding with vitamins, lipids, hormones, metal ions and growth factors, and also has the effect of binding toxins and reducing the effects of proteases on cells.
  • the content of albumin is about 0.5-1.0g/L, for example, about 0.5g/L, 0.6g/L, 0.7g/L, 0.8g/L, 0.9g/L, 1.0g/L, or any value within the range of the above arbitrary numerical values as endpoints.
  • a component complex when adding components to the mesenchymal stem cell serum-free medium of the present invention, a component complex may be added, such as a complex comprising a hormone such as insulin and a binding protein such as transferrin.
  • the component complex is ITS, for example, including but not limited to ITS-X, ITS-G and ITS-E, etc.
  • ITS-X is included in the mesenchymal stem cell serum-free medium of the present invention.
  • the ITS-X that can be added to the mesenchymal stem cell serum-free medium of the present invention can be a commercially available ITS-X product that can be purchased commercially.
  • the content of ITS-X is about 0.8-1.2% (v/v), for example, about 0.8% (v/v), 0.9% (v/v), 1.0% (v/v), 1.1% (v/v), 1.2% (v/v), or any value within the range of any of the above values as endpoints.
  • the serum-free medium for mesenchymal stem cells of the present invention further comprises other added factors, such as amino acids, vitamins, glucose, and salts including inorganic salts and organic salts.
  • Amino acids are the basic units for synthesizing proteins.
  • the mesenchymal stem cell serum-free medium of the present invention may include essential amino acids and non-essential amino acids that the cells themselves cannot synthesize and can synthesize, for the needs of cell growth. Those skilled in the art are familiar with the relevant knowledge of non-essential amino acids used in cell culture.
  • the non-essential amino acids that can be added to the mesenchymal stem cell serum-free medium of the present invention can be commercially available non-essential amino acid products that can be purchased commercially, such as non-essential amino acid products (article number: M7145) that can be provided by Sigma.
  • the content of non-essential amino acids is about 0.1-1.0mM, for example, about 0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1.0mM, or any value within the range of any of the above numerical values as endpoints.
  • Vitamins are a class of biologically active substances that maintain cell growth and play a major role in cell metabolism. Most of them form the cofactors or coenzymes of enzymes in cells. Without vitamins, enzymes will be inactive and metabolic activities will not be able to proceed.
  • the vitamins added to the mesenchymal stem cell serum-free medium of the present invention can be complex vitamins, such as commercially available complex vitamin products that can be purchased commercially, such as the MEM vitamin solution product (Cat. No.: S420JV) provided by Shanghai Yuanpei Biotechnology Co., Ltd.
  • the content of vitamins is about 0.8-1.2% (v/v), for example, about 0.8% (v/v), 0.9% (v/v), 1.0% (v/v), 1.1% (v/v), 1.2% (v/v), or any value within the range of any of the above values as endpoints, based on the total volume of the mesenchymal stem cell serum-free medium.
  • Glucose provides an energy source for the growth of mesenchymal stem cells in the culture medium.
  • the content of glucose is about 11-27mM, such as about 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, or any value within the range of any of the above values as endpoints.
  • Salts such as inorganic salts or organic salts can maintain the osmotic pressure balance of the culture medium and participate in the metabolic activities of the cells.
  • the main ones are Na + , K + , Ca2 + , Mg2 + , etc.
  • Na + is the most important cation in the extracellular fluid and plays a decisive role in maintaining the constant osmotic pressure.
  • K + is mainly distributed in the intracellular fluid. Intracellular K + is necessary for activating certain enzymes and is also extremely important in regulating the acid-base balance of the intracellular environment.
  • the role of Ca2+ in the extracellular fluid is to adhere cells inside the tissue to each other, and participate in many important cell physiological activities in the cell, such as conduction, participating in muscle cell contraction, etc.
  • Mg2+ is an important component of the intercellular matrix and is of great significance for the stable binding between cells.
  • Phosphorus compounds play an important role in the metabolism of cell substances and the regulation of physiological functions.
  • sodium pyruvate is included in the mesenchymal stem cell serum-free medium of the present invention.
  • the content of sodium pyruvate in the mesenchymal stem cell serum-free medium of the present invention is about 1-2 mM, such as about 1 mM, 2 mM, or any value within the range of any of the above values as endpoints.
  • Hepes is included in the mesenchymal stem cell serum-free medium of the present invention.
  • the content of Hepes is about 10-25mM, for example, about 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, or any value in the range of any of the above values as endpoints.
  • the mesenchymal stem cell serum-free culture medium of the present invention further comprises one or more components selected from the following group: sodium pyruvate, 1-2 mM; EGF, 0-20 ng/ml or 5-20 ng/ml; bFGF, 5-20 ng/ml; ITS-X, 0.8-1.2% (v/v); glucose, 11-27 mM; albumin, 0.5-1.0 g/L; vitamins, 0.8-1.2% (v/v); non-essential amino acids, 0.1-1.0 mM; and Hepes, 10-25 mM.
  • the mesenchymal stem cell serum-free medium of the present invention optionally includes a surplus of water. It should be understood by those skilled in the art that, during the preparation of the serum-free medium, due to factors such as the mixing and miscibility of liquids and the dissolution of solid components in liquid components, the volume obtained after preparation may be less than the sum of the volumes of the components. In this case, the mesenchymal stem cell serum-free medium with an expected total volume can be obtained by supplementing the surplus of water.
  • the components contained in the serum-free medium for mesenchymal stem cells of the present invention may be commercially available products.
  • the mesenchymal stem cell serum-free culture medium of the present invention has the following composition:
  • the mesenchymal stem cell serum-free culture medium of the present invention has the following composition:
  • the mesenchymal stem cell serum-free medium of the present invention is a two-component formulation.
  • the two components are stored separately and then mixed evenly before use, ready for use.
  • the mesenchymal stem cell serum-free medium of the present invention is a two-component formulation, wherein the first component is a basal medium component, including a basal medium; the second component is a nutrient addition component, including other components other than the basal medium.
  • the content of the components in each component is as defined herein.
  • the two components are stored separately, wherein the basal medium component is stored at 2-8°C, and the nutrient supplement component is stored at -18 to -22°C, such as -20°C.
  • the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising the following steps:
  • the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising:
  • step 2) Adding the nutrient supplement preparation prepared in step 1) into the basal culture medium preparation.
  • the components before adding, can be dissolved in water or an organic solvent, such as DMSO.
  • DMSO an organic solvent
  • raw materials such as EGF, bFGF, glucose, albumin, glutamine, and/or sodium pyruvate are dissolved with cell culture water.
  • hydrocortisone is dissolved with DMSO and/or alcohol.
  • stirring or other means well known in the art are optionally performed during the process of adding components to promote uniform mixing of the components.
  • filtration can be optionally performed using a filtration method known in the art, such as filtering using a filter membrane.
  • filtering is performed using a filter membrane with a pore size of no more than 0.22 ⁇ m.
  • filtering is performed using a filter membrane with a pore size of 0.22 ⁇ m, and in some embodiments, filtering is performed using a filter membrane with a pore size greater than 0.22 ⁇ m.
  • the mesenchymal stem cell serum-free medium of the present invention can be used immediately after preparation, or alternatively, can be stored in a low temperature environment for subsequent use.
  • the two components of the mesenchymal stem cell serum-free medium of the present invention are stored separately during storage, wherein the basal medium component is stored at a temperature of 2-8°C, and the nutrient supplement component is stored at a temperature of -18 to -22°C, for example, -20°C.
  • the present invention provides a method for culturing mesenchymal stem cells, wherein the mesenchymal stem cell serum-free culture medium of the present invention is used for culturing.
  • the mesenchymal stem cell serum-free culture medium of the present invention is not only suitable for two-dimensional culture of mesenchymal stem cells, but also can be applied to three-dimensional culture of mesenchymal stem cells, especially large-scale three-dimensional culture.
  • two-dimensional culture refers to a culture in which cells are exposed to conditions that are compatible with cell growth and allow cells to grow in a monolayer.
  • Devices suitable for such growth are referred to as “two-dimensional culture devices.” Such devices typically have a flat growth surface.
  • Non-limiting examples of devices for two-dimensional culture are cell culture dishes and cell culture plates.
  • three-dimensional culture refers to the in vitro co-culturing of carriers with three-dimensional structures and different materials with various types of cells, so that the cells can migrate and grow in the three-dimensional spatial structure of the carrier, forming a three-dimensional cell-carrier complex, changing or reducing the cell's adhesion characteristics during the culture process, allowing the cells to obtain more living space in space and reducing cell contact inhibition.
  • the cells to be cultured are stem cells, for example, mesenchymal stem cells, including but not limited to bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, bone mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreatic mesenchymal stem cells, amniotic fluid mesenchymal stem cells, umbilical cord mesenchymal stem cells, etc.
  • mesenchymal stem cells including but not limited to bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, bone mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreatic mesenchymal stem cells
  • the two-dimensional culture method comprises the following steps,
  • the mesenchymal stem cell complete culture medium is the mesenchymal stem cell serum-free culture medium of the present invention, and its composition is as described in the above aspects.
  • the cells to be cultured may be cryopreserved cells. Before culturing, the cells are thawed according to procedures well known to those skilled in the art, such as placing the frozen cells in a 37°C water bath and shaking to thaw, and taking out the cells when the ice crystals in the cell suspension are about to completely disappear by naked eye observation, thereby obtaining a cell suspension containing cells that can continue to be cultured.
  • the mesenchymal stem cell complete medium is used immediately after being prepared at room temperature. In some embodiments, the mesenchymal stem cell complete medium is stored at low temperature and has been restored to room temperature before use.
  • the mesenchymal stem cell complete culture medium is added dropwise and mixed evenly with the cell suspension.
  • cells are collected by centrifugation, for example, at 200 ⁇ g for 5 min.
  • the cells are accurately counted after resuspending them in complete mesenchymal stem cell culture medium.
  • the seeding density may be 6000-10000/ cm2 , such as 6000/ cm2 , 7000/ cm2 , 8000/ cm2 , 9000/ cm2 , 10000/ cm2 , or any value in the range of any of the above values.
  • the seeding density is 8000/ cm2 .
  • the culture is performed using conventional incubation conditions in the art, such as placing in an incubator at 37° C., 5% CO 2 concentration, and saturated humidity.
  • the continuous culture time varies with the time required for the cell confluence to reach an appropriate standard, such as 75-90% or 80-85%.
  • the continuous culture can be 2 days, 3 days, 4 days, etc.
  • the cells are harvested according to conventional methods in the art, or subsequent steps such as selection and passaging can be performed.
  • the two-dimensional culture method of mesenchymal stem cells of the present invention comprises the following steps.
  • the three-dimensional culture method comprises the following steps,
  • Preparing microcarriers placing microcarriers in a culture container, optionally, transferring the microcarriers to the culture container after swelling with liquid in advance, adding complete medium of mesenchymal stem cells, and uniformly dispersing the microcarriers;
  • the mesenchymal stem cell complete culture medium is the mesenchymal stem cell serum-free culture medium of the present invention, and its composition is as described above.
  • the cells to be cultured may be cryopreserved cells. Before culturing, the cells are thawed according to procedures well known to those skilled in the art, such as placing the frozen cells in a 37°C water bath and shaking to thaw, and taking out the cells when the ice crystals in the cell suspension are about to completely disappear by naked eye observation, thereby obtaining a cell suspension containing cells that can continue to be cultured.
  • the mesenchymal stem cell complete medium is used immediately after being prepared at room temperature. In some embodiments, the mesenchymal stem cell complete medium is stored at low temperature and has been restored to room temperature before use.
  • conventional reactors in the art are used to culture according to conventional conditions for three-dimensional culture.
  • the parameters of the reactor are set to a combination of variable speed culture and constant speed culture, such as 40 rpm, 5 min, 1 rpm, 2 h, for 1 day.
  • the reactor is adjusted to a constant speed of 40 rpm, and culture is continued for several days, such as 1 day, 2 days, 3 days, 4 days, and the time to stop culture is determined according to the results of cell sampling and detection.
  • conventional procedures in the art may be used to perform cell sampling and detection. For example, a small amount of microcarrier suspension is drawn with a sterile pipette and placed in a microplate, such as a 96-well plate, and cells are observed by fluorescent labeling and staining.
  • the cells can be harvested using conventional steps in the art.
  • the microcarriers used are microcarriers that can be lysed with a lysate. After the microcarriers are completely degraded, the cell suspension is collected, centrifuged, and the supernatant is discarded to harvest the cells. Cells can also be resuspended with PBS, centrifuged again, and the supernatant is discarded. The cells can be harvested after optionally repeating multiple times. After harvesting the cells, they can be resuspended to a suitable density for standby use.
  • cell counting is optionally included to detect relevant parameters such as the number of live cells, cell viability, cell diameter, etc.
  • a small amount of cell suspension that has been lysed and resuspended once is taken and placed in a cell counter for counting, and finally the number of live cells, cell viability, cell diameter, etc. are detected.
  • the three-dimensional culture method of mesenchymal stem cells of the present invention comprises the following steps:
  • Microslide and cell preparation Place the microslide into a culture bottle, add complete mesenchymal stem cell culture medium, and shake the culture bottle to evenly disperse the microslide;
  • Cell inoculation and culture Add the cell suspension into the culture bottle, add the complete medium of mesenchymal stem cells, and culture in the incubator. During the culture, the complete medium of mesenchymal stem cells can be supplemented optionally.
  • Optional cell sampling and observation use a sterile pipette to draw a small amount of microcarrier suspension and place it in a microplate, and observe the cells by fluorescent labeling and staining;
  • Cell harvesting stop stirring the bioreactor, discard the supernatant after the microcarriers settle, add lysis buffer for lysis and digestion, collect the cell suspension after the microcarriers are completely degraded, and optionally perform the steps of centrifugation, discard the supernatant, resuspend the cells in PBS, centrifuge again, and discard the supernatant. Optionally, resuspend the cells to a suitable density according to needs for later use;
  • Optional cell counting Take a small amount of cell suspension that has been lysed and resuspended once, place it on a cell counter for counting, and finally detect relevant parameters such as the number of living cells, cell viability, and cell diameter.
  • three-dimensional cell culture Compared with two-dimensional cell culture, three-dimensional cell culture has many advantages: 1. Closer to the in vivo environment, cells can form more complex tissue structures in three-dimensional structures, which are closer to the internal environment of the human body, which makes three-dimensional cultured cells more biologically meaningful and clinically valuable. 2. Better cell-cell and cell-matrix interactions: Three-dimensional culture can enable cells to better interact with each other, thereby better simulating the biological environment in the body, increasing communication between cells, and facilitating the plasticity of growth and differentiation. 3. Better drug screening, three-dimensional culture can better simulate the biological environment in the body, so it is more suitable for drug screening. This method can help better predict the efficacy and safety of new drugs, thereby better improving the efficiency of drug research and development. 4.
  • three-dimensional culture can provide better models and methods for tissue engineering and regenerative medicine. Through three-dimensional culture, multiple cell types can be combined into complex tissue structures, which is of great help in studying cell-to-cell interactions and cell differentiation. In addition, three-dimensional culture can also provide better methods for tissue repair and regeneration, such as tissue engineering using stem cells and pluripotent cells.
  • microcarrier refers to a supporting matrix particle that allows adherent cells such as mesenchymal stem cells to grow in a bioreactor.
  • the microcarrier can be a sphere, a cylinder or a flat carrier.
  • the microcarrier can be solid or porous.
  • the microcarrier size can be 10-1000 microns, for example 20-1000 microns, 50-500 microns of porous microspheres, wherein there are a number of interconnected pores greater than 10 micron diameters that form a porous permeable structure to wrap the cells, just like the relationship between a honeycomb and a bee, the porous structure of the microcarrier provides a nest for the cells to protect them from the influence of adverse external factors.
  • Microcarrier can be made of multiple different materials.
  • microcarrier is made of non-biodegradable materials, such as but not limited to cellulose, DEAE-dextran, hydroxylated methacrylate, polyacrylamide, polystyrene, plastics, glass, pottery and silicone.
  • microcarrier is made of biodegradable materials, such as but not limited to collagen, alginate, dextran, gellan gum and gelatin. These microcarrier materials and different surface chemistry can affect cell behavior, including form and proliferation.
  • Microcarriers that can be used for three-dimensional cell culture can be purchased commercially from manufacturers such as Global Cell Solutions, GE Healthcare, Cultispher Percell, SoloHill Engineering, Cytiva, and Beijing Huakan.
  • the microcarrier used in three-dimensional culture is a three-dimensional porous microcarrier, preferably, the three-dimensional porous microcarrier has a particle size of 80-400 ⁇ m and a pore size of 30-50 ⁇ m.
  • the mesenchymal stem cell serum-free culture medium of the present invention can be applied to the three-dimensional culture of mesenchymal stem cells using various commercially available three-dimensional culture microcarriers.
  • mesenchymal stem cell serum-free medium of the present invention should not be limited to the above-described embodiments. All mesenchymal stem cell culturing methods using the mesenchymal stem cell serum-free medium of the present invention should fall within the scope of the present invention.
  • mesenchymal stem cell serum-free medium Use of mesenchymal stem cell serum-free medium in culturing mesenchymal stem cells
  • the present invention provides a use of a mesenchymal stem cell serum-free medium in culturing mesenchymal stem cells.
  • the culture is a two-dimensional culture. In some embodiments, the culture is a three-dimensional culture.
  • the platelet lysate used in the following examples was EliteGro-adv purchased from EliteCell Biomedical Corp.
  • culture media A, B, C, and D are as follows:
  • Human umbilical cord mesenchymal stem cells P5 were revived and inoculated (inoculation volume 2 ⁇ 10 5 ) in T25 culture flasks for 3 days, and the cell number, proliferation times, cell viability and cell diameter were detected.
  • the experimental results showed that the proliferation of group D using ⁇ -MEM cells was 14.3 times, which was significantly better than the other groups (see Table 1), but the proliferation times of groups B and C were more than 10 times, so D/F12, ⁇ -MEM+DMEM (1:1) and ⁇ -MEM can be selected as the basic culture medium, and the basic culture medium ⁇ -MEM is preferred.
  • Human umbilical cord mesenchymal stem cells P5 were revived and inoculated (inoculation amount 2 ⁇ 10 5 ) in T25 culture flasks, cultured for 3 days, and inoculated (inoculation amount 50 ⁇ 10 5/ well) in 6-well plates without cell attachment coating (microcarrier 20 mg/well) for three-dimensional culture.
  • the culture medium volume was 8 ml and the speed was cultured (40 rpm, 5 min; 1 rpm, 2 h). After 1 day (day 1), the speed was adjusted to a constant 40 rpm. Culture for another 3 days. Detect cell number, proliferation times and cell viability.
  • Human umbilical cord mesenchymal stem cells P3 were revived and inoculated (inoculation volume 2 ⁇ 10 5 ) in T25 culture flasks for two-dimensional culture, and J-1 and J culture medium were selected for culture for 3 days.
  • Inoculation (inoculation volume 2.5 ⁇ 10 6 ) was inoculated in 125ml spinner flasks (microcarrier 100mg) for three-dimensional culture, and J-1 and J culture medium volume was 50ml, and the speed was cultured (40rpm, 5min; 1rpm, 2h). After 1 day (day 1), 25ml of culture medium was supplemented, samples were counted and stained, and the speed was adjusted to a constant 40rpm, and cultured for another 3 days. The cell number, proliferation times, cell viability and cell diameter were detected. .
  • the experimental results confirmed that the proliferation times of two-dimensional and three-dimensional cells were not affected by the concentration change when hydrocortisone was 400 and 500 ⁇ g/L (see Table 3-2).
  • Example 4 Effects of different concentrations of platelet lysate on two-dimensional and three-dimensional culture of MSCs
  • the components and concentrations of medium L and M are as follows:
  • Human umbilical cord mesenchymal stem cells P5 were revived and inoculated (inoculation amount 2 ⁇ 10 5 ) in T25 culture flasks for two-dimensional culture for 3 days. Inoculated (inoculation amount 2.5 ⁇ 10 6 ) in 125ml spinner bottles (microcarrier 100mg) for three-dimensional culture, each culture medium volume 50ml, inter-speed culture (40rpm, 5min; 1rpm, 2h). One day later (day 1), 25ml of culture medium was supplemented, samples were counted and stained, the speed was adjusted to a constant speed of 40rpm, and cultured for another 3 days. The number of cells, proliferation times, cell viability and cell diameter were detected.
  • the experimental results showed that when the platelet lysate concentration was 5% in two-dimensional culture, the cell proliferation reached more than 10 times. When the platelet lysate concentration was 5% in three-dimensional culture, the proliferation was more than 12 times (see Table 4), and the platelet lysate concentration was preferably 5%.
  • culture media M, M1, M2, and M3 are as follows:
  • Human umbilical cord mesenchymal stem cells P5 were revived and inoculated (inoculation amount 6 ⁇ 10 5 ) in T75 culture flasks for two-dimensional culture for 3 days. Inoculated (inoculation amount 5 ⁇ 10 5 ) in non-TC6 well plates (microcarrier 20 mg/well) for three-dimensional culture, each culture medium volume 8 ml, and cultured at different speeds (40 rpm, 5 min; 1 rpm, 2 h). After 1 day (day 1), the speed was adjusted to a constant speed of 40 rpm and cultured for another 3 days. The number of cells, proliferation times, cell viability and cell diameter were detected.
  • the experimental results showed that when the EGF dosage was gradually reduced to 0 in two-dimensional culture, the cell proliferation reached about 10 times, and the proliferation was not significantly affected.
  • the EGF dosage was gradually reduced to 0 in three-dimensional culture, the proliferation was more than 9 times, and when the platelet lysate was reduced to 2%, the two-dimensional and three-dimensional proliferation decreased insignificantly (see Table 5). Therefore, the optimal range of EGF dosage is 0-20 ng/ml.
  • Human umbilical cord mesenchymal stem cells P4 were revived, cultured for 3 days, digested, counted, and continued to be cultured in two-dimensional and three-dimensional cultures.
  • Two-dimensional culture 2 ⁇ 10 50,000 cells were inoculated in a T25 culture flask containing culture medium L and N and cultured for 3 days.
  • Three-dimensional culture 2.5 ⁇ 10 6 cells were inoculated in a 125ml spinner bottle, microcarrier 100mg, culture medium volume 50ml, and medium speed culture (40rpm, 5min; 1rpm, 2h). After 1 day (day 1), 25ml of culture medium was supplemented, the speed was adjusted to a constant speed of 40rpm, and culture was continued for 3 days.
  • culture medium N is a commercial culture medium from Biological Industries (Cat. No.: 05-200-1A+PLTGOLD010R). The experimental results are shown in Table 6.
  • Culture medium L has good effects in both two-dimensional and three-dimensional culture, but culture medium N is only effective in two-dimensional culture and poor in three-dimensional culture.
  • Human umbilical cord mesenchymal stem cells P3 were revived and inoculated (inoculation volume 6 ⁇ 10 5 ) in a T75 culture flask containing medium L. After 3 days of culture, they were inoculated in a 125ml spinner flask with a cell inoculation volume of 2.5 ⁇ 10 6 , a microcarrier of 100 mg, a medium volume of 50 ml N, and medium speed culture (40 rpm, 5 min; 1 rpm, 2 h).

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Rheumatology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Milieu de culture sans sérum pour cellules souches mésenchymateuses. Plus particulièrement, le milieu de culture sans sérum comprend un milieu de culture basique, un lysat plaquettaire et d'autres composants de culture tels que, mais sans y être limités, la L-glutamine, l'hydrocortisone, un facteur de croissance, etc. Le milieu de culture sans sérum pour cellules souches est approprié pour une culture bidimensionnelle de cellules souches mésenchymateuses tout en étant particulièrement approprié pour une culture tridimensionnelle de cellules souches mésenchymateuses, et présente les avantages d'une qualité d'amplification de cellules souches élevée, d'une grande quantité, d'une sécurité élevée, etc.
PCT/CN2024/097949 2023-06-08 2024-06-07 Milieu de culture sans sérum pour cellules souches mésenchymateuses et son utilisation Pending WO2024251234A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202310674353.9A CN116426470B (zh) 2023-06-08 2023-06-08 间充质干细胞无血清培养基及其应用
CN202310674353.9 2023-06-08

Publications (1)

Publication Number Publication Date
WO2024251234A1 true WO2024251234A1 (fr) 2024-12-12

Family

ID=87092993

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/097949 Pending WO2024251234A1 (fr) 2023-06-08 2024-06-07 Milieu de culture sans sérum pour cellules souches mésenchymateuses et son utilisation

Country Status (2)

Country Link
CN (1) CN116426470B (fr)
WO (1) WO2024251234A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119709611A (zh) * 2025-02-28 2025-03-28 山东丽山生物科技有限公司 一种促间充质干细胞增殖的无血清多肽培养基及其制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116426470B (zh) * 2023-06-08 2023-09-22 北京华龛生物科技有限公司 间充质干细胞无血清培养基及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011101834A1 (fr) * 2010-02-22 2011-08-25 Advanced Neuro-Science Allies Private Limited Procédé d'obtention de cellules souches mésenchymateuses, milieux utilisés, procédés et composition associés
CN112522191A (zh) * 2020-12-18 2021-03-19 云南中科灵长类生物医学重点实验室 一种间充质干细胞的培养方法
CN112608894A (zh) * 2020-12-31 2021-04-06 任建华 一种间充质干细胞培养基
WO2021227573A1 (fr) * 2020-05-14 2021-11-18 梦芊科技知识产权有限公司 Milieu de culture exempt de xeno et procédé de multiplication de cellules souches mésenchymateuses au moyen de celui-ci
CN114540298A (zh) * 2022-03-25 2022-05-27 北京瑷格干细胞科技有限公司 一种干细胞无血清培养基及其制备方法
CN116426470A (zh) * 2023-06-08 2023-07-14 北京华龛生物科技有限公司 间充质干细胞无血清培养基及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011101834A1 (fr) * 2010-02-22 2011-08-25 Advanced Neuro-Science Allies Private Limited Procédé d'obtention de cellules souches mésenchymateuses, milieux utilisés, procédés et composition associés
WO2021227573A1 (fr) * 2020-05-14 2021-11-18 梦芊科技知识产权有限公司 Milieu de culture exempt de xeno et procédé de multiplication de cellules souches mésenchymateuses au moyen de celui-ci
CN112522191A (zh) * 2020-12-18 2021-03-19 云南中科灵长类生物医学重点实验室 一种间充质干细胞的培养方法
CN112608894A (zh) * 2020-12-31 2021-04-06 任建华 一种间充质干细胞培养基
CN114540298A (zh) * 2022-03-25 2022-05-27 北京瑷格干细胞科技有限公司 一种干细胞无血清培养基及其制备方法
CN116426470A (zh) * 2023-06-08 2023-07-14 北京华龛生物科技有限公司 间充质干细胞无血清培养基及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HU YOUJIN, LIU XIONGHAO, LONG PANPAN, XIAO DI, CUN JINTAO, LI ZHUO, XUE JINFENG, WU YONG, LUO SHA, WU LINGQIAN, LIANG DESHENG: "Nonviral Gene Targeting at rDNA Locus of Human Mesenchymal Stem Cells", BIOMED RESEARCH INTERNATIONAL, vol. 2013, 1 January 2013 (2013-01-01), pages 135189 - 135189-10, XP093247374, ISSN: 2314-6133, DOI: 10.1155/2013/135189 *
NIKOLITS ILIAS, NEBEL SABRINA, EGGER DOMINIK, KREß SEBASTIAN, KASPER CORNELIA: "Towards Physiologic Culture Approaches to Improve Standard Cultivation of Mesenchymal Stem Cells", CELLS, vol. 10, no. 4, pages 886, XP055854993, DOI: 10.3390/cells10040886 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119709611A (zh) * 2025-02-28 2025-03-28 山东丽山生物科技有限公司 一种促间充质干细胞增殖的无血清多肽培养基及其制备方法

Also Published As

Publication number Publication date
CN116426470B (zh) 2023-09-22
CN116426470A (zh) 2023-07-14

Similar Documents

Publication Publication Date Title
US20230383245A1 (en) Culture medium composition
CN111117946B (zh) 一种鼻粘膜类器官培养基及培养方法
JP5670053B2 (ja) マイクロキャリアを使用した、産褥由来の細胞の生体外での拡大
JP2023175783A (ja) ナノファイバーを用いた細胞培養
WO2024251234A1 (fr) Milieu de culture sans sérum pour cellules souches mésenchymateuses et son utilisation
CN106414722B (zh) 红系细胞的体外扩增
TWI698528B (zh) 血管平滑肌細胞的培養方法
CN114790446B (zh) 一种宫颈腺癌类器官培养基及其构建方法
US20220135947A1 (en) Methods for culturing mesenchymal stem cells, products thereof, and applications thereof
US20150329826A1 (en) Materials and methods for cell culture
CN119855898A (zh) iPSC在生物反应器中的分化
CN110951686A (zh) 一种造血干细胞体外扩增培养体系和方法
US10370696B2 (en) Method for cell recovery
CN112852709B (zh) 小鼠肺类器官培养方法
CN120082503B (zh) 一种肝祖类器官培养基及培养方法
CN118703430A (zh) 一种基于可溶性微载体的干细胞球大规模制备方法
US12480093B2 (en) Production of megakaryocytes in bioreactors
CN116396930B (zh) 间充质干细胞无血清培养基及其应用
Tasto et al. and Scalable Expansion Process
TWI303278B (en) A method of forming spheroids from the cultured cells
JP4186043B2 (ja) 細胞分化誘導方法および細胞培養物
CN120384048A (zh) 一种化学成分限定的间充质干细胞培养基及其应用
CN118256435A (zh) 一种人源诱导多能干细胞来源的血小板裂解物用于自然杀伤细胞的培养和冻存
Zhang et al. Timed morphological changes of human hepatocytes L-02 cultured at high density by the support of spherical porous chitosan microcarriers
CN107805624A (zh) 一种体外诱导胰岛内分泌细胞形成岛样结构的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24818764

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE