WO2024251234A1 - Serum-free culture medium for mesenchymal stem cells and use thereof - Google Patents

Abstract

A serum-free culture medium for mesenchymal stem cells. Specifically, the serum-free culture medium comprises a basic culture medium, a platelet lysate, and other culture components such as but not limited to L-glutamine, hydrocortisone, a growth factor, etc. The serum-free culture medium for stem cells is suitable for two-dimensional culture of mesenchymal stem cells while being especially suitable for three-dimensional culture of mesenchymal stem cells, and has the advantages of high stem cell amplification quality, large quantity, high safety, etc.

Description

Serum-free culture medium for mesenchymal stem cells and its application

This application claims priority to Chinese patent application No. 202310674353.9, filed on June 8, 2023, entitled “Serum-free culture medium for mesenchymal stem cells and its application”, the entire text of which is hereby incorporated by reference.

Technical Field

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.

Background Art

Mesenchymal stem cells (MSCs), also known as multipotent stromal cells, are a type of multipotent stem cells belonging to the mesoderm. They are mainly found in connective tissue and organ stroma, including bone marrow, umbilical cord, fat, mucosa, bone, muscle, lung, liver, pancreas and other tissues, as well as amniotic fluid, amniotic membrane, placenta, etc. Under appropriate conditions, they can differentiate into a variety of tissue cells such as fat, bone, and cartilage. In 1976, Freidenstein first discovered bone marrow mesenchymal stem cells (BM-MSCs). Later studies found that 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.

At present, 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. Compared with two-dimensional planar cell culture technology, 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.

Most of the culture media used for in vitro culture of mesenchymal stem cells need to be supplemented with serum, which poses a risk of contamination with exogenous viruses and pathogenic factors during the cell culture process. Moreover, due to the large number of unknown components in serum, the bioactive factors between different batches of serum are inconsistent, resulting in poor reproducibility of products and experimental results. Residual serum can also easily cause allergic reactions to serum in recipients, posing a huge challenge to clinical research. In order to overcome the various disadvantages brought by serum, it is very necessary to use serum-free culture medium in the cell culture process.

Platelet lysate (HPLs) is a xeno-free, animal serum-free cell culture medium supplement derived from human platelets. 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. Currently, 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.

Summary of the invention

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.

In one aspect, the present invention provides a mesenchymal stem cell serum-free culture medium, which comprises the following components and the contents thereof are as follows:

In some embodiments, 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:

In some embodiments, the basal culture medium is selected from the group consisting of α-MEM medium, D/F12 medium and DMEM medium, and preferably the basal culture medium is α-MEM medium.

In some embodiments, the content of the platelet lysate is 5% (v/v).

In some embodiments, the content of L-glutamine is 4 mM.

In some embodiments, the content of hydrocortisone is 500 μg/L.

In some embodiments, the content of EGF is 5-20 ng/ml.

In another aspect, the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising:

1) adding the other components except the platelet lysate into the basal medium in proportion; and

2) After filtration, add platelet lysate according to the proportion.

In some alternative embodiments, the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising:

1) mixing the other components except the basic culture medium in proportion to prepare a nutrient supplement composition; and

2) Adding the nutrient supplement preparation prepared in step 1) into the basal culture medium preparation.

In yet another aspect, 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.

In some embodiments, the culture method is a two-dimensional culture method, comprising the following steps,

1) Mixing the cell suspension containing cells and the complete medium of mesenchymal stem cells evenly;

2) Collect cells and resuspend them in mesenchymal stem cell complete medium;

3) inoculating the cells into a cell culture container, adding a complete medium for mesenchymal stem cells, and culturing the container in an incubator; and

4) Harvest cells.

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.

In some embodiments, the culture method is a three-dimensional culture method, comprising the following steps,

1) 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;

2) Cell inoculation and culture: adding the cell suspension into a culture container and culturing it in an incubator; and

3) Harvest cells.

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.

In another aspect, the present invention provides a use of a mesenchymal stem cell serum-free medium in culturing mesenchymal stem cells. In some embodiments, the culture is a two-dimensional culture. In some embodiments, the culture is a three-dimensional culture.

The beneficial effects of the present invention are:

1. 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;

2. While being suitable for two-dimensional culture of mesenchymal stem cells, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the cell viability during continuous culture in Example 5.

FIG. 2 shows the proliferation multiples of cells in 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.

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.

DETAILED DESCRIPTION

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

When used herein, the terms “comprising” are intended to specify the presence of stated features, integers, components or steps, but they do not exclude the presence or addition of one or more other features, integers, components, steps or groups thereof.

Mesenchymal Stem Cell Serum-Free Medium

In one aspect, 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.

As used herein, the term "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. The term "adipose mesenchymal stem cells" refers to mesenchymal stem cells derived from adipose.

As used herein, the term "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 μg/L.

When used in this article, the term "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. Generally, 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. In some embodiments, 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, α-MEM, MEGM, RPMI-1640 and combinations thereof. In some embodiments, the basal medium includes D/F12 (e.g., 1: 1 mixing ratio), α-MEM+DMEM (e.g., 1: 1 mixing ratio) and α-MEM. In a preferred embodiment, the basal medium is α-MEM. In some embodiments, in the serum-free medium for mesenchymal stem cells of the present invention, 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. In some embodiments, in the serum-free medium for mesenchymal stem cells of the present invention, 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.

As used herein, the term "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. In some embodiments, in the serum-free medium for mesenchymal stem cells of the present invention, 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. In some embodiments, in the serum-free medium for mesenchymal stem cells of the present invention, 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. In a preferred embodiment, in the serum-free medium for mesenchymal stem cells of the present invention, 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.

During cell culture, most cells have a high requirement for glutamine, because glutamine is used by cells as both an energy source and a carbon source, and is an essential amino acid for cells to synthesize nucleic acids and proteins. In the absence of glutamine, cells grow poorly and die. The glutamine that cells can use is the L-isomer, and D-glutamine cannot be used. In some embodiments, 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.

When used in this article, the term "cortisone" is also referred to as cortisone, and refers to an adrenocortical hormone agent with a glucocorticoid effect. In mesenchymal stem cell culture, 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. In some embodiments, in the mesenchymal stem cell serum-free culture medium of the present invention, the content of cortisone is about 0.05-5000 μg/L, such as about 0.05 μg/L, 0.5 μg/L, 5 μg/L, 50 μg/L, 500 μg/L, 1000 μg/L, 2000 μg/L, 3000 μg/L, 4000 μg/L, 5000 μg/L, or any value within the range of the above arbitrary numerical value as endpoint. In some embodiments, in the serum-free medium for mesenchymal stem cells of the present invention, the content of hydrocortisone is about 5-5000 μg/L, for example, about 100-1000 μg/L, for example, about 100 μg/L, 200 μg/L, 300 μg/L, 400 μg/L, 500 μg/L, 600 μg/L, 700 μg/L, 800 μg/L, 900 μg/L, 1000 μg/L. In a preferred embodiment, in the serum-free medium for mesenchymal stem cells of the present invention, the content of hydrocortisone is about 500 μg/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. In some embodiments, 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). In some embodiments, the mesenchymal stem cell serum-free culture medium of the present invention includes epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). In some embodiments, in the mesenchymal stem cell serum-free medium of the present invention, 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. In some embodiments, in the mesenchymal stem cell serum-free medium of the present invention, 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. In some embodiments, in the serum-free medium for mesenchymal stem cells of the present invention, 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. In a preferred embodiment, 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. For example, 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.

In some embodiments, the serum-free medium for mesenchymal stem cells of the present invention includes hormones, such as insulin, growth hormone, glucagon, etc. Almost all cell lines require insulin, which 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.

In some embodiments, the mesenchymal stem cell serum-free medium of the present invention includes binding proteins, such as 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. In addition, 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. In some embodiments, in the mesenchymal stem cell serum-free medium of the present invention, 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.

In some embodiments, 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. In some specific embodiments, the component complex is ITS, for example, including but not limited to ITS-X, ITS-G and ITS-E, etc. In some embodiments, 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. In some embodiments, in the mesenchymal stem cell serum-free medium of the present invention, 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.

In some embodiments, 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. In some embodiments, in the mesenchymal stem cell serum-free medium of the present invention, 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. In some embodiments, in the mesenchymal stem cell serum-free medium of the present invention, 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. In some embodiments, in the mesenchymal stem cell serum-free culture medium of the present invention, 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.

In some embodiments, sodium pyruvate is included in the mesenchymal stem cell serum-free medium of the present invention. In some embodiments, 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.

In some embodiments, Hepes is included in the mesenchymal stem cell serum-free medium of the present invention. In some embodiments, 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.

In some embodiments, 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.

In some embodiments, 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.

It should be understood by those skilled in the art that the components contained in the serum-free medium for mesenchymal stem cells of the present invention may be commercially available products.

In some embodiments, the mesenchymal stem cell serum-free culture medium of the present invention has the following composition:

In some embodiments, the mesenchymal stem cell serum-free culture medium of the present invention has the following composition:

In some embodiments, 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. In some embodiments, 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.

In some embodiments, 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.

Preparation of serum-free culture medium for mesenchymal stem cells

In another aspect, the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising the following steps:

1) adding the other components except the platelet lysate into the basal medium in proportion; and

2) Add platelet lysate according to the proportion.

In some alternative embodiments, the present invention provides a method for preparing a serum-free medium for mesenchymal stem cells, comprising:

1) mixing the other components except the basic culture medium in proportion to prepare a nutrient supplement composition; and

2) Adding the nutrient supplement preparation prepared in step 1) into the basal culture medium preparation.

In some embodiments, before adding, the components can be dissolved in water or an organic solvent, such as DMSO. In some embodiments, before adding, raw materials such as EGF, bFGF, glucose, albumin, glutamine, and/or sodium pyruvate are dissolved with cell culture water. In some embodiments, before adding, hydrocortisone is dissolved with DMSO and/or alcohol.

In some embodiments, in each step of the above method, 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.

In some embodiments, in each step of the above method, after the components are mixed, filtration can be optionally performed using a filtration method known in the art, such as filtering using a filter membrane. In some embodiments, filtering is performed using a filter membrane with a pore size of no more than 0.22 μm. In some embodiments, filtering is performed using a filter membrane with a pore size of 0.22 μm, and in some embodiments, filtering is performed using a filter membrane with a pore size greater than 0.22 μm.

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. In some embodiments, 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.

Methods for culturing mesenchymal stem cells

In yet another aspect, 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.

As used herein, the term "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.

As used in this article, the term "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.

In the present invention, 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.

The two-dimensional culture method comprises the following steps,

1) Mixing the cell suspension containing cells and the complete medium of mesenchymal stem cells evenly;

2) Collect cells and resuspend them in mesenchymal stem cell complete medium;

3) inoculating the cells into a cell culture container, adding a complete medium for mesenchymal stem cells, and culturing the container in an incubator; and

4) Harvest cells.

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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the mesenchymal stem cell complete culture medium is added dropwise and mixed evenly with the cell suspension.

In some embodiments, cells are collected by centrifugation, for example, at 200×g for 5 min.

In some embodiments, the cells are accurately counted after resuspending them in complete mesenchymal stem cell culture medium.

In some embodiments, 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. In a preferred embodiment, the seeding density is 8000/ ^cm2 .

In some embodiments, 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.

In some embodiments, 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%. For example, the continuous culture can be 2 days, 3 days, 4 days, etc. After the cell confluence reaches an appropriate standard, such as 75-90% or 80-85%, the cells are harvested according to conventional methods in the art, or subsequent steps such as selection and passaging can be performed.

In a specific embodiment, the two-dimensional culture method of mesenchymal stem cells of the present invention comprises the following steps.

Pipette the cell suspension into a centrifuge tube, add mesenchymal stem cell complete medium restored to room temperature, and mix gently. Collect the cells by centrifugation, then remove the supernatant, and add mesenchymal stem cell complete medium to resuspend the cells. Then inoculate the cells into a cell culture container, add an appropriate amount of fresh mesenchymal stem cell complete medium restored to room temperature, and culture in an incubator. Continuously culture until the appropriate cell confluence can be selected for subculture.

The three-dimensional culture method comprises the following steps,

1) 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;

2) Cell inoculation and culture: adding the cell suspension into a culture container and culturing it in an incubator; and

3) Harvest cells.

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.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, conventional reactors in the art are used to culture according to conventional conditions for three-dimensional culture. In some embodiments, 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. After optionally supplementing with complete medium for mesenchymal stem cells, 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.

In some embodiments, 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.

In some embodiments, the cells can be harvested using conventional steps in the art. In some specific embodiments, 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.

In some embodiments, cell counting is optionally included to detect relevant parameters such as the number of live cells, cell viability, cell diameter, etc. In a specific embodiment, 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.

In a specific embodiment, the three-dimensional culture method of mesenchymal stem cells of the present invention comprises the following steps:

1. 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;

2. 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.

3. 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;

4. 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;

5. 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.

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. Better tissue engineering and regenerative medicine applications, 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.

A three-dimensional culture system comprising microcarriers can be used when carrying out three-dimensional cell culture. When used in this article, the term "microcarrier" refers to a supporting matrix particle that allows adherent cells such as mesenchymal stem cells to grow in a bioreactor. In the present invention, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 μm and a pore size of 30-50 μm. 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.

It should be understood that the method of culturing mesenchymal stem cells using the 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.

Use of mesenchymal stem cell serum-free medium in culturing mesenchymal stem cells

In another aspect, the present invention provides a use of a mesenchymal stem cell serum-free medium in culturing mesenchymal stem cells. In some embodiments, the culture is a two-dimensional culture. In some embodiments, the culture is a three-dimensional culture.

Example

The present invention will be described in further detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are conventional products that can be purchased from the market. It will be appreciated by those skilled in the art that the present invention belongs to that the methods and materials described below are exemplary only and should not be construed as limiting the scope of the present invention.

The platelet lysate used in the following examples was EliteGro-adv purchased from EliteCell Biomedical Corp.

Example 1. Effects of different basal culture media on 2D culture of MSCs

The components and concentrations of 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 α-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, α-MEM+DMEM (1:1) and α-MEM can be selected as the basic culture medium, and the basic culture medium α-MEM is preferred.

Table 1 Cell culture conditions on the third day of two-dimensional culture in Example 1

Example 2. Effects of different concentrations of glutamine on two-dimensional and three-dimensional culture of MSCs

The components and concentrations of medium E and F are as follows:

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. The experimental results show that the two-dimensional cell proliferation effect of group F using 4mM L-glutamine is significantly better than that of group E (see Table 2), but the cell proliferation of groups E and F is more than 10 times, and the three-dimensional culture effect of group F using 4mM L-glutamine is equivalent to that of group E. Therefore, L-glutamine 2-4mM can be selected, and L-glutamine 4mM is preferred to ensure both two-dimensional and three-dimensional culture effects.

Table 2: Cultivation of two-dimensional and three-dimensional cells on day 3 in Example 2

Example 3. Effects of different concentrations of hydrocortisone on two-dimensional and three-dimensional culture of MSCs

The components and concentrations of medium G, H, I, J, and K are as follows:

Human umbilical cord mesenchymal stem cells P5 were revived and inoculated (inoculation volume 2×10 ⁵ ) in T25 culture flasks, cultured for 3 days, and the cell number, proliferation times and cell diameter were detected. The experimental results showed that the cell proliferation times reached the highest point when hydrocortisone was 500μg/L, and the cell proliferation did not increase with further increase in concentration (see Table 3-1). When hydrocortisone was 5-5000μg/L, the cell proliferation could reach more than 10 times, so hydrocortisone 5-5000μg/L can be selected, and hydrocortisone 500μg/L is preferred.

Table 3-1 Cell culture conditions on the third day of two-dimensional culture in Example 3

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 500μg/L (see Table 3-2).

Table 3-2 Two-dimensional and three-dimensional cell culture conditions in Example 3

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%.

Table 4 Cell culture conditions on the third day of two-dimensional culture in Example 4

Example 5. Effects of different concentrations of EGF on two-dimensional and three-dimensional culture of MSCs

The components and concentrations of 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. When 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.

Table 5 Two-dimensional and three-dimensional cell culture conditions in Example 5

Example 6. Effect of continuous passage of MSCs in two-dimensional culture

Experimental method: Human umbilical cord mesenchymal stem cells P5 were revived and inoculated (inoculation amount 2×10 ⁵ cells) in T25 culture flasks containing medium L. After 3 days of culture, they were subcultured in T25 culture flasks and cultured for another 3 days. The cells were subcultured again and this process was repeated. The cells were cultured to P10, and the cell viability, proliferation times and cell diameter were tested at each generation. The experimental results are shown in Figures 1-4. The proliferation times of cells from P6 to P10 were relatively stable, all above 10 times, the cell viability was high, and the cell diameter increased slightly with the increase of the passage. This shows that the medium L can stably culture MSC cells in two dimensions.

Example 7. Comparison of the effects of two-dimensional and three-dimensional culture of MSCs

Experimental methods: 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. Among them, 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.

Table 6 Comparison of 2D and 3D MSCs culture effects in Example 6

Example 8. Effect of three-dimensional continuous passage of MSCs

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). One day later (day 1), 25 ml of medium was added, samples were counted and stained, the speed was adjusted to a constant 40 rpm, and after another 3 days of culture, staining and counting were performed, and the cells were continuously passaged to P7. The experimental results showed that using L medium, after 3 consecutive generations, the proliferation multiples still reached more than 10 times, the cell activity was high, and the cells maintained a good state (see Table 7 and Figure 5).

Table 7 Cell culture conditions on the 1st and 4th day of three-dimensional culture P7 in Example 7

Example 9. Culture effects of MSCs from different sources

Experimental method: Resuscitation of P4 generation human umbilical cord mesenchymal stem cells UCMSC, human adipose mesenchymal stem cells ADMSC and human dental pulp mesenchymal stem cells DPMSC, after 3 days of culture, digestion, counting, and continued two-dimensional and three-dimensional culture. Two-dimensional culture: 2×10 ^50,000 MSCs from different sources were inoculated in a T25 culture bottle containing medium L and cultured for 3 days. Three-dimensional culture: 2.5×10 ⁶ MSCs from different sources 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. The experimental results are shown in Table 8 and Figure 6. Each cell has a good culture effect in both two-dimensional and three-dimensional. Therefore, medium L is suitable for two-dimensional and three-dimensional culture of UCMSC, ADMSC, and DPMSC.

Table 8 Two-dimensional and three-dimensional culture of MSCs from different sources

The embodiments of the present invention are not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and detail, and these are considered to fall within the scope of protection of the present invention.

Claims (12)

A mesenchymal stem cell serum-free medium comprises the following components and the contents thereof are as follows: based on the total volume of the mesenchymal stem cell serum-free medium,
The mesenchymal stem cell serum-free medium according to claim 1, wherein the basal medium is selected from the group consisting of α-MEM medium, D/F12 medium and DMEM medium. The mesenchymal stem cell serum-free culture medium according to claim 1 or 2, wherein the content of the platelet lysate is 5% (v/v). The mesenchymal stem cell serum-free medium according to claim 1 or 2, wherein the content of L-glutamine is 4 mM. The mesenchymal stem cell serum-free culture medium according to claim 1 or 2, wherein the content of hydrocortisone is 500 μg/L. The mesenchymal stem cell serum-free medium according to claim 1 or 2, comprising the following components and the contents thereof are as follows: based on the total volume of the mesenchymal stem cell serum-free medium,

A method for preparing a serum-free medium for mesenchymal stem cells according to any one of claims 1 to 6, comprising: 1) adding the other components except the platelet lysate into the basal medium in proportion; and 2) Add platelet lysate according to the proportion. A method for preparing a serum-free medium for mesenchymal stem cells according to any one of claims 1 to 6, comprising: 1) mixing the other components except the basic culture medium in proportion to prepare a nutrient supplement composition; and 2) Adding the nutrient supplement preparation prepared in step 1) into the basal culture medium preparation. A method for culturing mesenchymal stem cells, wherein the mesenchymal stem cell serum-free culture medium according to any one of claims 1 to 6 is used for culturing. The method for culturing mesenchymal stem cells according to claim 9, wherein the culturing method is a two-dimensional culturing method, comprising the following steps: 1) Mixing the cell suspension containing cells and the complete medium of mesenchymal stem cells evenly; 2) Collect cells and resuspend them in mesenchymal stem cell complete medium; 3) inoculating the cells into a cell culture container, adding a complete medium for mesenchymal stem cells, and culturing the container in an incubator; and 4) harvesting cells; Wherein, the mesenchymal stem cell complete culture medium is a mesenchymal stem cell serum-free culture medium according to any one of claims 1 to 6. The method for culturing mesenchymal stem cells according to claim 9, wherein the culturing method is a three-dimensional culturing method, comprising the following steps: 1) 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; 2) Cell inoculation and culture: adding the cell suspension into a culture container and culturing it in an incubator; and 3) harvesting cells; Wherein, the mesenchymal stem cell complete culture medium is a mesenchymal stem cell serum-free culture medium according to any one of claims 1 to 6. Use of the mesenchymal stem cell serum-free medium according to any one of claims 1 to 6 in culturing mesenchymal stem cells.