CN116396930A - Mesenchymal stem cell serum-free medium and application thereof - Google Patents
Mesenchymal stem cell serum-free medium and application thereof Download PDFInfo
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- CN116396930A CN116396930A CN202310674350.5A CN202310674350A CN116396930A CN 116396930 A CN116396930 A CN 116396930A CN 202310674350 A CN202310674350 A CN 202310674350A CN 116396930 A CN116396930 A CN 116396930A
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Abstract
The present invention relates to a serum-free medium for mesenchymal stem cells, in particular, the serum-free medium comprises basal medium, platelet lysate, plant protein hydrolysate and other culture components such as but not limited to L-glutamine, hydrocortisone, growth factors and the like. The serum-free culture medium for the stem cells is particularly suitable for three-dimensional culture of the mesenchymal stem cells while being suitable for two-dimensional culture of the mesenchymal stem cells, and has the advantages of high stem cell expansion quality, large quantity, high safety and the like.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a mesenchymal stem cell serum-free medium containing a plant protein hydrolysate, and a preparation method and application thereof.
Background
Mesenchymal stem cells (Mesenchymal stem cells, MSCs), also known as pluripotent stromal cells, are a class of pluripotent stem cells belonging to the mesoderm, which are mainly present in connective tissue and organ interstitium, including bone marrow, umbilical cord, fat, mucosa, bone, muscle, lung, liver, pancreas and other tissues, amniotic fluid, amniotic membrane, placenta and the like. Can be differentiated into various tissue cells such as fat, bone, cartilage and the like under proper conditions. Research shows that mesenchymal stem cells have the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self replication and the like, and are widely paid attention to. The mesenchymal stem cells are isolated and extracted from the umbilical cord tissue without any damage, and the mesenchymal stem cells in the umbilical cord have the advantages of large quantity, high quality and pure cells, so the mesenchymal stem cells become the stem cells with the most clinical application value and preservation value at present.
At present, the stem cell culture mainly takes the traditional two-dimensional culture as a main part, which is equivalent to supporting the stem cell growth on only one plane, has low proliferation efficiency and space utilization rate, and cannot meet the requirement of increasing large-dose application of clinical stem cells. Compared with the two-dimensional plane cell culture technology, the three-dimensional culture co-cultures the three-dimensional structure matrix and the cells in vitro to form a three-dimensional cell aggregate, so that the cells can perform three-dimensional growth, differentiation and migration in vitro, simulate living environment in a organism more truly, and are more beneficial to proliferation, survival and self-property maintenance of the cells. The nutrient requirements of the cells in different culture conditions are different, so that the method is suitable for the three-dimensional culture technology and needs a proper three-dimensional culture medium to achieve the optimal mass production process.
The culture medium used for in vitro culture of mesenchymal stem cells is mostly supplemented with serum, which involves the risk of contaminating exogenous viruses and pathogenic agents during cell culture. Moreover, as the serum has a large number of unknown components, the biological active factors among different batches of serum are inconsistent, so that the reproducibility of products and experimental results is poor, the residual serum is easy to cause anaphylactic reaction of an inoculator to the serum, and the serum cannot be applied to clinical research. Therefore, in order to overcome various disadvantages caused by serum, it is necessary to find serum substitutes or to use serum-free media during cell culture.
The plant protein hydrolysate is prepared by hydrolyzing plant materials such as soybean, wheat, rice, etc. under the catalysis of acid or enzyme preparation to obtain amino acid and amino acid polypeptide intermediate mixed colloid, and processing. For example, soy protein hydrolysate is used as serum substitute, contains abundant dipeptide and polypeptide, and is more beneficial to MSCs proliferation and state maintenance relative to free amino acid. Furthermore, soy protein hydrolysates are rich in stabilized glutamate comprising polypeptides, which is considered as an important nucleic acid source in cell culture, as well as an excellent energy source. However, the application of the plant protein hydrolysate in MSCs culture is not reported, so the invention aims to find a serum-free culture medium which is based on the plant protein hydrolysate, has remarkable effect and high safety and is suitable for two-dimensional and three-dimensional culture.
Disclosure of Invention
The invention aims to provide a novel serum-free culture medium for mesenchymal stem cells, which has no serum and animal source components, and can remarkably improve the activity and proliferation capacity of the mesenchymal stem cells in two-dimensional culture or three-dimensional culture, thereby solving the technical problem that the existing serum-free culture medium for mesenchymal stem cells has poor effect when used for three-dimensional large-scale culture in the prior art.
In one aspect, the invention provides a serum-free medium for mesenchymal stem cells, which comprises the following components in the following content:
and optionally one or more cell culture components selected from the group consisting of: glutamine, growth factors, hormones, binding proteins, glutathione, hydrocortisone, and other additive factors.
In some embodiments, the basal medium is selected from the group consisting of an alpha-MEM medium, a D/F12 medium, and a DMEM medium, preferably the basal medium is an alpha-MEM medium.
In some embodiments, the platelet lysate is present in an amount of 1-5% (v/v), preferably 2% (v/v).
In some embodiments, the plant protein hydrolysate is present in an amount of 5-10g/L, preferably 5g/L.
In some embodiments, the glutamine is L-glutamine in an amount of 2 to 4mM, preferably 4mM.
In some embodiments, the glutathione is reduced glutathione in an amount of 80-120 μg/L, preferably 100 μg/L.
In some embodiments, hydrocortisone is present in an amount of 0.5 to 1.5mg/L, preferably 1.0mg/L.
In some embodiments, the growth factor comprises one or more selected from the group consisting of: epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta), and platelet-derived growth factor-BB (PDGF-BB); the content of Epidermal Growth Factor (EGF) is 5-20ng/ml, preferably 20ng/ml; the content of basic fibroblast growth factor (bFGF) is 5-20ng/ml, preferably 20ng/ml; the content of transforming growth factor-beta (TGF-beta) is 5-15ng/ml, preferably 10ng/ml: and the platelet-derived growth factor-BB (PDGF-BB) is present in an amount of 5-15ng/ml, preferably 10ng/ml.
In some embodiments, the hormone comprises one or more selected from the group consisting of: insulin such as recombinant human insulin, and progesterone; the content of recombinant human insulin is 5-15mg/L, preferably 10mg/L; the content of progesterone is 15-25ng/ml, preferably 20ng/L.
In some embodiments, the binding protein comprises transferrin, e.g., recombinant human transferrin, in an amount of 15-25mg/L, preferably 20mg/L.
In some embodiments, the other additive factors include one or more selected from the group consisting of: vitamins such as vitamin C, glucose and salts such as sodium pyruvate and sodium selenite; the content of vitamin C is 15-25mg/L, preferably 20mg/L; glucose content is 20-30mM, preferably 25mM; the content of sodium pyruvate is 1-2mM, preferably 1mM; the content of sodium selenite is 25-35 mg/L, preferably 30-mg/L.
In some embodiments, the present invention provides a mesenchymal stem cell serum-free medium comprising the following components and in the amounts shown below:
in a preferred embodiment, the invention provides a mesenchymal stem cell serum-free medium comprising the following components in the following amounts:
In another aspect, the present invention provides a method for preparing a serum-free medium of mesenchymal stem cells, comprising:
the components are added into the basal medium in proportion.
In some alternative embodiments, the invention provides a method of preparing a serum-free medium of mesenchymal stem cells, comprising:
1) Mixing other components except the basic culture medium in proportion to prepare a nutrition additive; and
2) And (3) adding the nutritional additive prepared in the step (1) into the basic culture medium.
In yet another aspect, the present invention provides a method of culturing mesenchymal stem cells.
The two-dimensional culture method comprises the following steps,
1) Uniformly mixing a cell suspension containing cells with a mesenchymal stem cell complete culture medium;
2) Collecting cells, and re-suspending the cells with a mesenchymal stem cell complete medium;
3) Inoculating cells into a cell culture container, adding a mesenchymal stem cell complete culture medium, and placing the cells in an incubator for culture; and
4) The cells were harvested.
The mesenchymal stem cell complete medium is the mesenchymal stem cell serum-free medium of the present invention, and the composition thereof is as described above.
The three-dimensional culture method comprises the following steps,
1) Preparing a microcarrier: placing the microcarrier in a culture container, and adding a mesenchymal stem cell complete culture medium to uniformly disperse the microcarrier;
2) Cell inoculation and culture: adding the cell suspension into a culture container, and placing the culture container into an incubator for culture; and
3) The cells were harvested.
The mesenchymal stem cell complete medium is the mesenchymal stem cell serum-free medium of the present invention, and the composition thereof is as described above.
In yet another aspect, the invention provides the use of a serum-free medium of mesenchymal stem cells for culturing mesenchymal stem cells. In some embodiments, the culturing is two-dimensional. In some embodiments, the culturing is three-dimensional.
The beneficial effects of the invention are as follows:
1. the culture medium does not contain components derived from serum, so that the disadvantages of risk of foreign virus and pathogenic factors pollution, poor reproducibility of products and experimental results, easiness in causing anaphylactic reaction and the like caused by the serum are avoided;
2. according to the invention, the plant protein hydrolysate is added to the culture medium on the basis of adding the platelet lysate, so that the usage amount of the platelet lysate is effectively reduced, and the three-dimensional culture effect of the mesenchymal stem cells is obviously improved;
3. The culture medium is particularly suitable for three-dimensional culture of the mesenchymal stem cells while being suitable for two-dimensional culture of the mesenchymal stem cells, realizes large-scale high-quality expansion of the mesenchymal stem cells, and has the advantage of high safety.
Drawings
FIG. 1 shows a morphology of P0 cells in example 3.
FIG. 2 shows a cell morphology (P8) in example 4.
FIG. 3 shows the cell continuous culture activity in example 4.
FIG. 4 shows the multiplication factor of the cell continuous culture in example 4.
FIG. 5 shows the cell diameter size of the continuous culture of cells in example 4.
FIG. 6 shows the result of cell staining at day 4 of example 6 (P6).
FIG. 7 shows the cell continuous culture activity at day 4 of example 6.
FIG. 8 shows the cell proliferation fold of the continuous culture at day 4 of example 6.
FIG. 9 shows the cell diameter of the cell culture at day 4 of example 6.
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.
As used herein, the term "comprising" is intended to specify the presence of stated features, integers, components or steps, but does not preclude 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 invention provides a serum-free 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 class of pluripotent cells that have 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 early-stage mesoderm, belongs to multipotent stem cells, and is originally found in bone marrow and has the characteristics of multipotent differentiation, hematopoietic support, promotion of stem cell implantation, immune regulation, self replication and the like. The mesenchymal stem cells include bone marrow mesenchymal stem cells, dental pulp mesenchymal stem cells, adipose mesenchymal stem cells, synovial mesenchymal stem cells, skeletal mesenchymal stem cells, muscle mesenchymal stem cells, lung mesenchymal stem cells, liver mesenchymal stem cells, pancreatic mesenchymal stem cells, amniotic mesenchymal stem cells, umbilical cord mesenchymal stem cells and the like. The term "umbilical cord mesenchymal stem cells" refers to mesenchymal stem cells derived from umbilical cord. The term "adipose mesenchymal stem cells" refers to adipose-derived mesenchymal stem cells.
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 media are well known in the art.
The mesenchymal stem cell serum-free medium of the present invention comprises a basal medium of about 90-99% (v/v); 1-10 g/L of vegetable protein hydrolysate; and one or more cell culture components selected from the group consisting of: glutamine, growth factors, hormones, binding proteins, glutathione, hydrocortisone, and other additive factors.
As used herein, the term "basal medium" refers to the starting medium into which cells are added to begin culturing. The basal medium is a solution containing nutrients that nourish the growing cells. Typically, the basal medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements that are required by the cells for minimal growth and/or survival. In some embodiments, the basal medium is a commercially available medium or a medium known in the art to be capable of maintaining growth or proliferation of mammalian cells. Non-limiting examples of basal media include, but are not limited to, DMEM, F12, MEM, alpha-MEM, MEGM, RPMI-1640, and combinations thereof. In some embodiments, the basal medium comprises D/F12 (e.g., 1:1 mix ratio), alpha-MEM+DMEM (e.g., 1:1 mix ratio), and alpha-MEM. In a preferred embodiment, the basal medium is alpha-MEM. In some embodiments, the mesenchymal stem cells of the present invention comprise about 90-99% (v/v) of the basal medium by volume ratio, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any value within a range ending with any of the above. In some embodiments, the mesenchymal stem cells of the present invention comprise about 90-97% (v/v), such as about 92-97% (v/v), about 93-96% (v/v), about 94-95% (v/v), about 95% (v/v), etc., of the basal medium in terms of volume ratio.
As used herein, the term "platelet lysate" refers to a preparation obtainable from platelets. Platelets can be obtained from a donor, preferably a human donor, can be isolated and pooled from whole blood from multiple collection units, or collected by platelet apheresis: blood is obtained from a donor and passed through a platelet removal device. After separation, the platelets are typically lysed, for example, by one or more freeze/thaw cycles. The platelet lysate used in the present invention may be a commercially available platelet lysate product commercially available, for example, the platelet lysate product EliteGro-adv purchased for EliteCell Biomedical Corp. In some embodiments, the mesenchymal stem cell serum-free medium of the present invention comprises about 1-10% (v/v) of platelet lysate, e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any value within a range ending with any of the above values, by volume. In some embodiments, the mesenchymal stem cells of the present invention comprise about 1-10% (v/v), such as about 1-5% (v/v), about 1-4% (v/v), about 1-3% (v/v), about 1-2% (v/v), etc., of platelet lysate by volume ratio in a serum-free medium. In a preferred embodiment, the mesenchymal stem cell serum-free medium of the present invention comprises about 2% (v/v) of platelet lysate based on the total volume of the mesenchymal stem cell serum-free medium.
As used herein, the term "plant protein hydrolysate" has a meaning recognized in the art and generally refers to a product obtained by hydrolyzing a plant material under the catalysis of an acid or enzyme preparation to obtain an intermediate mixed colloid of amino acids and amino acid polypeptides, and then processing the resulting product. In certain embodiments, the plant protein hydrolysate is a hydrolysate of beans such as soybeans, peas, chickpeas, or grains such as wheat, rice, or cotton, and the like. The hydrolysates used in the serum-free medium of mesenchymal stem cells of the present invention are commercially available plant protein hydrolysate products, such as those available from Sigma company (cat# 51841). In some embodiments, the amount of plant protein hydrolysate in the serum-free medium of mesenchymal stem cells of the present invention is about 1-10g/L, for example about 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 10g/L, or any value within the range ending with any of the above values. In preferred embodiments, the plant protein hydrolysate is present in an amount of about 5 to about 10g/L, such as about 5 to about 9g/L, about 5 to about 8g/L, about 5 to about 7g/L, about 5 to about 6g/L, about 5g/L, etc.
In some embodiments, the mesenchymal stem cell serum-free medium of the present invention comprises a binding protein, such as transferrin. The presence of specific transferrin receptors on most mammalian cells, the binding of the receptor to the complex of transferrin and ferric ions is a major source of the trace element iron necessary for cell acquisition, and transferrin has the properties of growth factors and is able to bind to other trace elements such as vanadium and the like. The amount of transferrin required varies from cell to cell. The transferrin that can be added to the serum-free medium of mesenchymal stem cells of the present invention may be recombinant human transferrin, for example a commercially available recombinant human transferrin product such as that available from the division of Wohan Biotechnology, inc. (cat# HYC044M 01). In some embodiments, the amount of recombinant human transferrin in the serum-free medium of mesenchymal stem cells of the present invention is about 15-25mg/L, for example about 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L, 25mg/L, or any value within the range ending with any of the above values. In a preferred embodiment, the recombinant human transferrin is present in an amount of about 20mg/L.
In some embodiments, the mesenchymal stem cell serum-free medium of the present invention comprises a hormone, such as insulin, growth hormone, glucagon, progesterone, and the like. Almost all cell lines require insulin, a polypeptide that binds to insulin receptors on cells to form complexes, promote synthesis of RNA, proteins and fatty acids, and consistent apoptosis is an important cell survival factor. The insulin that can be added to the serum-free medium of mesenchymal stem cells of the present invention may be recombinant human insulin, for example, commercially available recombinant human insulin products such as those available from the division of the Wohan Biotechnology Co., ltd (cat# S454 RV). In some embodiments, the amount of recombinant human insulin in the mesenchymal stem cell serum-free medium of the present invention is about 5-15mg/L, for example about 5mg/L, 6mg/L, 7mg/L, 8mg/L, 9mg/L, 10mg/L, 11mg/L, 12mg/L, 13mg/L, 14mg/L, 15mg/L, or any value within the range ending with any of the above values. In a preferred embodiment, the recombinant human insulin is present in an amount of about 10mg/L. Progesterone, also known as progesterone, luteinizing hormone, is the major bioactive progestogen secreted by the ovaries. In some embodiments, in the mesenchymal stem cell serum-free medium of the present invention, the content of progesterone is about 15-25ng/ml, for example about 15ng/ml, 16ng/ml, 17ng/ml, 18ng/ml, 19ng/ml, 20ng/ml, 21ng/ml, 22ng/ml, 23ng/ml, 24ng/ml, 25ng/ml, or any value within the range ending with any of the above values. In a preferred embodiment, the progestin is present in an amount of about 20 ng/ml.
In cell culture, most cells have high requirements on glutamine, because glutamine is an energy source and a carbon source substance which are simultaneously utilized by cells, is an amino acid which is necessary for synthesizing nucleic acid and protein by the cells, and in the absence of glutamine, cells are ill-grown and die. The glutamine that can be used by the cells is an L-isomer, and D-glutamine cannot be used. In some embodiments, the mesenchymal stem cell serum-free medium of the present invention has an L-glutamine content of about 2-4mM, such as 2mM, 2.5mM, 3mM, 3.5mM, 4mM, or any value within a range ending with any of the above values. In a preferred embodiment, the mesenchymal stem cells of the present invention have a content of L-glutamine of about 4mM in serum-free medium.
The mesenchymal stem cell serum-free medium of the present invention may further comprise a growth factor. Growth factors are supplementary factors necessary to maintain cell survival, proliferation and differentiation in vitro. Growth factors are potent mitogens and can shorten cell population doubling times. Polypeptide growth factors and steroid growth factors can be classified according to chemical properties. Growth factors that can be added to the serum-free medium of mesenchymal stem cells of the present invention include, but are not limited to, polypeptide growth factors and steroid growth factors, such as natural growth factors or recombinant growth factors obtained by genetic recombination means. In some embodiments, the mesenchymal stem cell serum-free medium of the present invention comprises a polypeptide selected from the group consisting of Epidermal Growth Factor (EGF); fibroblast Growth Factor (FGF), such as basic fibroblast growth factor (bFGF); transforming Growth Factor (TGF), such as transforming growth factor-beta (TGF-beta); platelet derived growth factor-BB (PDGF-BB) and Nerve Growth Factor (NGF), and the like. In some embodiments, the mesenchymal stem cell serum-free medium of the present invention comprises Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), transforming growth factor- β (TGF- β), and platelet-derived growth factor-BB (PDGF-BB). In some embodiments, the amount of EGF in the serum-free medium of mesenchymal stem cells of the present invention is about 5-20ng/ml, for example about 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 within a range ending with any of the above. In a preferred embodiment, the mesenchymal stem cell serum-free medium of the present invention has an EGF content of about 20ng/ml. In some embodiments, the amount of bFGF in the serum-free medium of mesenchymal stem cells of the present invention is about 5-20ng/ml, e.g., about 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 within a range ending with any of the above. In a preferred embodiment, the mesenchymal stem cells of the present invention have a bFGF content of about 20ng/ml in serum-free medium. In some embodiments, the amount of TGF- β in the serum-free medium of mesenchymal stem cells of the present invention is about 5-15ng/ml, such as about 5ng/ml, 6ng/ml, 7ng/ml, 8ng/ml, 9ng/ml, 10ng/ml, 11ng/ml, 12ng/ml, 13ng/ml, 14ng/ml, 15ng/ml, or any value within a range ending with any of the above. In a preferred embodiment, the mesenchymal stem cells of the present invention have a TGF-beta content of about 10ng/ml in serum-free medium. In some embodiments, the amount of PDGF-BB in the serum-free medium of the mesenchymal stem cells of the present invention is about 5-15ng/ml, such as about 5ng/ml, 6ng/ml, 7ng/ml, 8ng/ml, 9ng/ml, 10ng/ml, 11ng/ml, 12ng/ml, 13ng/ml, 14ng/ml, 15ng/ml, or any value within the range ending with any of the above. In a preferred embodiment, the mesenchymal stem cells of the present invention have a PDGF-BB content of about 10ng/ml in serum-free medium.
The serum-free medium of mesenchymal stem cells of the present invention may further comprise glutathione, such as reduced glutathione. Reduced glutathione can participate in the in vivo redox process. Under the action of glutathione transferase, reduced glutathione can be combined with peroxide and free radical to damage the antioxidant to sulfhydryl and protect sulfhydryl-containing protein and sulfhydryl-containing enzyme in cell membrane from damage and also resist damage of free radical to vital organs. Glutathione participates in tricarboxylic acid circulation and sugar metabolism in the body, so that the human body obtains high energy. It can activate and protect various enzymes, such as cholinesterase in vivo and other sulfhydryl enzymes, and prevent body from poisoning by iodoacetic acid, mustard gas, free radicals, heavy metals, epoxide and other harmful substances, so as to promote metabolism of saccharide, fat and protein, and influence cell metabolism. In some embodiments, the amount of reduced glutathione in the mesenchymal stem cell serum-free medium of the present invention is about 80-120 μg/L, for example about 80 μg/L, 85 μg/L, 90 μg/L, 95 μg/L, 100 μg/L, 105 μg/L, 110 μg/L, 115 μg/L, 120 μg/L, or any value within a range ending with any of the above values. In a preferred embodiment, the content of reduced glutathione in the serum-free medium of the mesenchymal stem cells of the present invention is about 100. Mu.g/L.
As used herein, the term "hydrocortisone," also known as cortisone, refers to an adrenocortical hormone agent having a glucocorticoid action. In mesenchymal stem cell culture, hydrocortisone is a commonly used additive that can affect cell signaling pathways and thus cell function. Can also inhibit immune and inflammatory reactions of cells, thereby reducing stress on cells in culture and improving survival rate and growth rate of stem cells. In some embodiments, the serum-free medium of mesenchymal stem cells of the present invention comprises hydrocortisone in an amount of about 0.5-1.5mg/L, for example about 0.5mg/L, 0.6mg/L, 0.7mg/L, 0.8mg/L, 0.9mg/L, 1.0mg/L, 1.1mg/L, 1.2mg/L, 1.3mg/L, 1.4mg/L, 1.5mg/L, or any value within the range ending with any value of the foregoing. In a preferred embodiment, the content of hydrocortisone in the serum-free medium of mesenchymal stem cells of the present invention is about 1.0mg/L.
In some embodiments, the mesenchymal stem cell serum-free medium of the present invention further comprises other additives such as vitamins, glucose and salts including inorganic and organic salts, and the like.
Vitamins are a class of bioactive substances that maintain cell growth and have a significant effect on cell metabolism. They form large enzyme prosthetic groups or coenzymes in cells, and without vitamins, the enzymes are inactive and metabolic activity will not be able to proceed. The vitamin which can be added to the serum-free medium of mesenchymal stem cells of the present invention may be vitamin C, for example, commercially available vitamin C products which are commercially available, and vitamin C has an antioxidant effect. In some embodiments, the amount of vitamin C in the serum-free medium of mesenchymal stem cells of the present invention is about 15-25mg/L, for example about 15mg/L, 16mg/L, 17mg/L, 18mg/L, 19mg/L, 20mg/L, 21mg/L, 22mg/L, 23mg/L, 24mg/L, 25mg/L, or any value within the range ending with any of the above values. In a preferred embodiment, the vitamin C is present in an amount of about 20mg/L.
Glucose provides an energy source for the growth of mesenchymal stem cells in culture medium. In some embodiments, the amount of glucose in the serum-free medium of mesenchymal stem cells of the present invention is about 20-30mM, e.g., about 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, or any value within a range ending with any of the above. In a preferred embodiment, the glucose content is about 25mM.
Salts such as inorganic or organic salts are capable of maintaining the osmotic balance of the culture medium and are involved in the metabolic activity of the cells. Mainly comprises Na + 、K + 、Ca 2+ 、Mg 2+ Etc. Na (Na) + Is the most predominant cation in extracellular fluid and has a decisive effect on maintaining the osmotic pressure constant. K (K) + Mainly distributed in intracellular fluid, intracellular K + Are necessary for activating certain enzymes and are in acid-base equilibrium regulating the intracellular environmentAnd has great significance. Ca (Ca) 2+ The role in extracellular fluid is to adhere cells within tissues to each other, and to participate in a number of important cellular physiological activities within the cells, such as conduction, participation in muscle cell contraction, etc. Mg of 2+ Is an important component constituting the cell matrix and has important significance for the mutual stable combination between cells. Phosphorus compounds play an important role in the regulation of cellular material metabolism and 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 serum-free medium of mesenchymal stem cells of the present invention is about 1-2mM, e.g., about 1mM, 2mM, or any value within a range ending with any of the above. In a preferred embodiment, the sodium pyruvate is present in an amount of about 1mM.
In some embodiments, sodium selenite is included in the mesenchymal stem cell serum-free medium of the present invention. In some embodiments, the content of sodium selenite in the serum-free medium of mesenchymal stem cells of the present invention is about 25-35 mg/L, e.g., about 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL, 30mg/mL, 31mg/mL, 32mg/mL, 33mg/mL, 34mg/mL, 35mg/mL, or any value within a range ending with any of the above. In a preferred embodiment, the sodium selenite is present in an amount of about 30mg/mL.
In some embodiments, the mesenchymal stem cell serum-free medium of the present invention further optionally comprises a balance of water. It will be appreciated by those skilled in the art that during the formulation of serum-free media, the volume obtained after formulation may be less than the sum of the volumes of the components due to factors such as the miscibility of the mixture between the liquids and the dissolution of the solid components in the liquid components, in which case a serum-free medium of mesenchymal stem cells having the desired total volume may be obtained by supplementing the balance of water.
It will be appreciated by those skilled in the art that the components contained in the serum-free medium of mesenchymal stem cells of the present invention may be commercially available products.
In some embodiments, the mesenchymal stem cell serum-free medium of the present invention comprises the following components and the amounts are as follows:
in some embodiments, the mesenchymal stem cell serum-free medium of the present invention comprises the following components and the amounts are as follows:
in some embodiments, the mesenchymal stem cell serum-free medium of the present invention is a two-pack formulation. The double-group agents are stored separately and are mixed uniformly before use, and the double-group agents are used immediately. In some embodiments, the mesenchymal stem cell serum-free medium of the present invention is a two-pack formulation, wherein the first pack is a basal medium pack comprising basal medium; the second group of agents is a nutritional supplement group comprising other components than the basal medium. The component content of each formulation is defined herein.
In some embodiments, the dual agents are stored separately upon storage, wherein the basal medium agents are stored at a temperature of 2-8 ℃, and the nutritional supplement agents are stored at a temperature of-18 to-22 ℃, such as-20 ℃.
Preparation of serum-free medium for mesenchymal stem cells
In another aspect, the invention provides a method for preparing a serum-free medium of mesenchymal stem cells, comprising the steps of:
The components are added into the basal medium in proportion.
In some alternative embodiments, the invention provides a method of preparing a serum-free medium of mesenchymal stem cells, comprising:
1) Mixing other components except the basic culture medium in proportion to prepare a nutrition additive; and
2) And (3) adding the nutritional additive prepared in the step (1) into the basic culture medium.
In some embodiments, the components may be dissolved in water or an organic solvent, such as DMSO, prior to addition. In some embodiments, EGF, bFGF, TGF-beta, PDGF-BB, glucose, etc. are dissolved with cell culture water prior to addition. In some embodiments, hydrocortisone is dissolved with DMSO prior to addition.
In some embodiments, during each step of the above method, agitation or means well known in the art is optionally performed during component addition to promote uniform mixing of the components.
In some embodiments, in each step of the above method, the components optionally may be filtered after mixing using filtration methods known in the art, such as filtration using a filter membrane. In some embodiments, filtration is performed using a filter membrane having a pore size no greater than 0.22 μm. In some embodiments, filtration is performed using a filter membrane having a pore size of 0.22 μm.
The serum-free medium of mesenchymal stem cells of the present invention may be used immediately after preparation, alternatively, may be stored in a low-temperature environment for subsequent use. In some embodiments, the mesenchymal stem cell serum-free medium dual-group agents of the present invention are stored separately upon storage, wherein the basal medium group agent is stored at a temperature of 2-8 ℃, and the nutritional supplement group agent is stored at a temperature of-18 to-22 ℃, e.g., -20 ℃.
Culture method of mesenchymal stem cells
In still another aspect, the present invention provides a method for culturing mesenchymal stem cells, wherein the culture is performed using the mesenchymal stem cell serum-free medium of the present invention.
The serum-free medium for the mesenchymal stem cells is not only suitable for two-dimensional culture of the mesenchymal stem cells, but also can be applied to three-dimensional culture of the mesenchymal stem cells, in particular to large-scale three-dimensional culture.
As used herein, the term "two-dimensional culture" refers to a culture in which cells are exposed to conditions compatible with cell growth and allowing cells to grow in a monolayer. Devices suitable for such growth are known 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 plates.
As used herein, the term "three-dimensional culture" refers to co-culturing a carrier having a three-dimensional structure of different materials with various different kinds of cells in vitro, so that the cells can migrate and grow in the three-dimensional spatial structure of the carrier to form a three-dimensional cell-carrier complex, and the characteristics of the cells on the wall of the culture process are changed or reduced, so that the cells can obtain more living space in space and the cell contact inhibition is reduced.
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, skeletal 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, and the like.
The two-dimensional culture method comprises the following steps,
1) Uniformly mixing a cell suspension containing cells with a mesenchymal stem cell complete culture medium;
2) Collecting cells, and re-suspending the cells with a mesenchymal stem cell complete medium;
3) Inoculating cells into a cell culture container, adding a mesenchymal stem cell complete culture medium, and placing the cells in an incubator for culture;
4) The cells were harvested.
The mesenchymal stem cell complete medium is the mesenchymal stem cell serum-free medium of the present invention, the composition of which is as described in the above aspect.
In some embodiments, the cells to be cultured may be cryopreserved cells. Prior to culturing, the cells are thawed according to procedures well known to those skilled in the art, e.g., the frozen cells are placed in a 37 ℃ water bath and thawed by shaking, e.g., the ice crystals in the cell suspension are removed by visual inspection as soon as they have completely disappeared, thereby obtaining a cell suspension comprising cells that can be continued for cell culture.
In some embodiments, the mesenchymal stem cell complete medium is used immediately after preparation at room temperature. In some embodiments, the mesenchymal stem cell complete medium has been restored to room temperature for use after storage at low temperature.
In some embodiments, the mesenchymal stem cell complete medium is added by dropwise addition and mixed homogeneously with the cell suspension.
In some embodiments, the cells are collected by centrifugation, for example, at 200 Xg for 5 min.
In some embodiments, the cells are precisely counted after being resuspended using a complete medium of mesenchymal stem cells.
In some embodiments, the inoculation density may be 6000-10000/cm 2 For example 6000/cm 2 、7000/cm 2 、8000/cm 2 、9000/cm 2 、10000/cm 2 Or any value within a range from any value recited above as an endpoint. In a preferred embodiment, the inoculation density is 8000/cm 2 。
In some embodiments, the culturing is performed using conventional incubation conditions in the art, e.g., at 37 ℃,5% CO 2 Concentration, saturated humidity in incubator.
In some embodiments, the continuous culture time varies with the time required for the cell confluence to reach a suitable standard, e.g., 75-90% or 80-85%, e.g., 2 days, 3 days, 4 days, etc., after which the cells are harvested following routine methods in the art, e.g., 75-90% or 80-85%, or alternatively, subsequent steps such as selection passaging may be performed.
In a specific embodiment, the two-dimensional culture method of mesenchymal stem cells of the present invention comprises the following steps.
And (3) sucking the cell suspension into a centrifuge tube, adding the mesenchymal stem cell complete culture medium which is restored to room temperature, and gently and uniformly mixing. Cells were collected by centrifugation, and then the supernatant was aspirated, and the cells were resuspended in mesenchymal stem cells complete medium. Then inoculating the cells into a cell culture container, adding a proper amount of fresh mesenchymal stem cell complete culture medium which is restored to room temperature, and placing the cells into an incubator for culture. Serial culture to appropriate cell confluence may be selected for passage.
The three-dimensional culture method comprises the following steps,
1) Preparing a microcarrier: placing the microcarrier in a culture container, and adding a mesenchymal stem cell complete culture medium to uniformly disperse the microcarrier;
2) Cell inoculation and culture: adding the cell suspension into a culture container, and placing the culture container into an incubator for culture;
3) The cells were harvested.
The mesenchymal stem cell complete medium is the mesenchymal stem cell serum-free medium of the present invention, and the composition thereof is as described above.
In some embodiments, the cells to be cultured may be cryopreserved cells. Prior to culturing, the cells are thawed according to procedures well known to those skilled in the art, e.g., the frozen cells are placed in a 37 ℃ water bath and thawed by shaking, e.g., the ice crystals in the cell suspension are removed by visual inspection as soon as they have completely disappeared, thereby obtaining a cell suspension comprising cells that can be continued for cell culture.
In some embodiments, the mesenchymal stem cell complete medium is used immediately after preparation at room temperature. In some embodiments, the mesenchymal stem cell complete medium has been restored to room temperature for use after storage at low temperature.
In some embodiments, the culturing is performed according to three-dimensional culturing conventional conditions using a reactor conventional in the art. In some embodiments, the parameters of the reactor are set to combine variable speed culture and constant speed culture, e.g., 40rpm,5min,1rpm,2h, for 1 day. After the complete medium of mesenchymal stem cells is optionally replenished, the reactor is set at a constant speed of 40rpm, and the culture is continued for several days, for example, 1 day, 2 days, 3 days, 4 days, and the time to stop the culture is determined based on the result of the cell sampling test.
In some embodiments, cell sampling assays may be performed using procedures conventional in the art. For example, in particular, a small amount of microcarrier suspension is pipetted into a microwell plate, such as a 96-well plate, and cell observation is performed by fluorescent label staining.
In some embodiments, cell harvesting may be performed using procedures conventional in the art. In some embodiments, the microcarriers used are microcarriers that can be lysed with a lysate, after all of the microcarriers have been degraded, the cell suspension is collected, centrifuged, and the supernatant discarded to harvest the cells. Cells may also be resuspended in PBS, centrifuged again, and the supernatant discarded, optionally after repeated several times to harvest the cells. After harvesting the cells, they can be resuspended to a suitable density for later use.
In some embodiments, cell counts are optionally included, and relevant parameters such as number of living cells, cell viability, cell diameter, etc. are detected. In a specific embodiment, a small amount of cell suspension which is resuspended once after being lysed is taken and is placed in a cell counter for counting, and finally relevant parameters such as the number of living cells, the cell viability, the cell diameter and the like are detected.
In a specific embodiment, the three-dimensional culture method of mesenchymal stem cells of the present invention comprises the steps of:
1. Micro slide and cell preparation: putting the micro-slide into a culture bottle, adding a mesenchymal stem cell complete culture medium, and shaking the culture bottle to uniformly disperse the micro-slide;
2. cell inoculation and culture: adding the cell suspension into a culture bottle, supplementing a complete medium of the mesenchymal stem cells, and placing the culture bottle into an incubator for culture, wherein the complete medium of the mesenchymal stem cells is optionally supplemented during the culture;
3. optionally cell sampling observations: sucking a small amount of microcarrier suspension by using a sterile pipette, placing the microcarrier suspension in a microplate, and observing cells by a fluorescent labeling staining method;
4. cell harvesting: stopping stirring the bioreactor, adding a lysate to crack and digest after the microcarrier is settled, collecting a cell suspension after the microcarrier is completely degraded, optionally performing the steps of centrifugation, discarding the supernatant, re-suspending cells by PBS, re-centrifugation, discarding the supernatant and the like, and optionally re-suspending the cells to a proper density according to the requirement for later use;
5. cell count optionally: taking a small amount of cell suspension which is resuspended once after being lysed, placing the cell suspension in a cell counter for counting, and finally detecting relevant parameters such as the number of living cells, the cell activity rate, the cell diameter and the like.
Compared with the two-dimensional cell culture, the three-dimensional cell culture has a plurality of advantages: 1. the three-dimensional culture cell is closer to the in-vivo environment, the cells can form more complex tissue structures in the three-dimensional structure, and the three-dimensional culture cell is closer to the internal environment of a human body, so that the three-dimensional culture cell has more biological significance and clinical value. 2. Better cell-cell and cell-matrix interactions: the three-dimensional culture can make the cells perform cell-cell and cell-matrix interactions better, thereby better simulating in vivo biological environment, increasing communication between cells and being beneficial to the plasticity of growth and differentiation. 3. Better drug screening, three-dimensional culture can better simulate in vivo biological environment, so that the method is more suitable for drug screening. The method can help to better predict the curative effect and safety of the new medicine, thereby better improving the research and development efficiency of the medicine. 4. Better tissue engineering and regenerative medicine application, and three-dimensional culture can provide better models and methods for tissue engineering and regenerative medicine. By three-dimensional culture, multiple cell types can be combined into complex tissue structures, which is a great aid in studying intercellular interactions and cell differentiation. In addition, three-dimensional culture can provide a better method for tissue repair and regeneration, for example, tissue engineering using stem cells and pluripotent cells.
For the three-dimensional cell culture, a three-dimensional culture system comprising microcarriers may be used. As used herein, the term "microcarrier" refers to a supporting matrix particle that allows for the growth of adherent cells, such as mesenchymal stem cells, in a bioreactor. In the present invention, the microcarrier may be a sphere, cylinder or flat carrier. Microcarriers may be solid or porous. The microcarrier can be 10-1000 microns porous microsphere, wherein the porous permeable structure formed by a plurality of pores with diameters larger than 10 microns are mutually communicated to wrap cells, and the porous structure of the microcarrier provides nest protection for the cells from external adverse factors like the relationship between a honeycomb and bees.
Microcarriers may be made of a variety of different materials. In some embodiments, the microcarriers are made of a non-biodegradable material, such as, but not limited to, cellulose, DEAE-dextran, hydroxylated methacrylate, polyacrylamide, polystyrene, plastic, glass, ceramic, and silicone. In some embodiments, the microcarriers are made of biodegradable materials, such as, but not limited to, collagen, alginate, dextran, gellan gum, and gelatin. These microcarrier materials, as well as different surface chemistries, can affect cell behavior, including morphology and proliferation.
Microcarriers useful for three-dimensional cell culture are commercially available from manufacturers such as Global Cell Solutions, GE Healthcare, cultispher Percell, soloHill Engineering, situo Va (Cytiva), beijing Hua niche, and the like. In some embodiments, the microcarrier used in the three-dimensional culture is a three-dimensional porous microcarrier, preferably having a particle size of 80-400 μm and a pore size of 30-50 μm. The serum-free medium for mesenchymal stem cells of the present invention can be suitably used for three-dimensional culture of mesenchymal stem cells using various commercially available microcarriers for three-dimensional culture.
It should be understood that the method of culturing mesenchymal stem cells using the serum-free medium of the present invention should not be limited to the above-described embodiments. The mesenchymal stem cell culture method in which the serum-free medium of the mesenchymal stem cell of the present invention is used should fall within the scope of the present invention.
Application of serum-free medium of mesenchymal stem cells in culturing mesenchymal stem cells
In yet another aspect, the invention provides the use of a serum-free medium of mesenchymal stem cells for culturing mesenchymal stem cells. In some embodiments, the culturing is two-dimensional. In some embodiments, the culturing is three-dimensional.
Examples
The invention will now be described in further detail by way of examples with reference to the accompanying drawings. Unless otherwise indicated, the methods and materials of the examples described below are all conventional products available commercially. Those skilled in the art will appreciate that the methods and materials described below are exemplary only and should not be construed as limiting the scope of the invention.
The platelet lysate used in the following examples was EliteGro-adv from EliteCell Biomedical Corp.
EXAMPLE 1 Effect of different concentrations of plant protein hydrolysates on proliferation of MSCs
The culture medium is prepared by adopting the formula, and umbilical cord mesenchymal stem cells are cultured according to a conventional culture method. Human umbilical cord mesenchymal stem cells P5 are recovered, inoculated (inoculum size 2×10) 5 And then cultured in a T25 flask for 3 days, and proliferation factors are detected.
TABLE 1
The results in Table 1 show that the proliferation factor is higher and the culture effect is better when the concentration of the plant protein hydrolysate is 5 g/L.
Example 2
The present example provides a serum-free medium (HK-SFM) of MSC, the basal medium is alpha-MEM, the content is 95% (v/v), and the other components and concentrations are respectively:
the culture medium is prepared by adopting the formula, and umbilical cord mesenchymal stem cells are cultured according to a conventional culture method.
EXAMPLE 3 Primary isolation culture of umbilical cord mesenchymal Stem cells
A mesenchymal stem cell complete medium, a plurality of sterile petri dishes (6-10), a medical sterile alcohol 1 bottle, a physiological saline 1 bottle, a tool kit (2 scissors, 2 tweezers), and a retrieved umbilical cord (placed on the umbilicus) were prepared as in example 2With preservation solution) are transferred into a biosafety cabinet together. Absorbing and discarding umbilical cord preservation solution in the umbilical cord preservation bottle, adding medical disinfection alcohol (75%) to completely immerse umbilical cord, and soaking and disinfecting for 2 minutes. The umbilical cord is taken out and placed in a sterile culture dish, and is washed with normal saline for 2-3 times, and residual umbilical cord serum is washed clean. Then cutting the umbilical cord into about 2-3 cm segments, washing with physiological saline for 2-3 times again, and washing the residual umbilical serum. The umbilical cord was cut along the vein and the vein wall removed, the umbilical cord was fully deployed after complete vein wall removal, then 2 arteries were removed, and after complete vein and artery removal, the Wharton's jelly was carefully isolated, taking care to avoid the epidermis. Transferring the separated Whatman gum into 50 mL centrifuge tube, adding 3-5 drops of physiological saline to keep moist, and cutting Whatman gum into 2-3 mm pieces by using elbow surgical scissors 3 Size, and then weighed. Adding sheared Whatman gel into the complete medium of mesenchymal stem cells of example 2, re-suspending, inoculating into a culture flask, and placing into an incubator (37 ℃ C., 5% CO) 2 Saturated humidity). On day 5 after inoculation, cells start to climb out from tissue blocks, the culture flask is vertically inclined for 30 degrees, the tissue blocks naturally settle to one corner of the culture flask, the supernatant is sucked and removed, fresh and rewarmed complete culture medium of the mesenchymal stem cells of the example 2 is slowly added, and the mixture is gently mixed and placed back into the culture box for continuous culture. The 9 th to 10 th days after inoculation, the state of the climbing out cells is good, stacking growth is started, a culture flask is vertically inclined for 30 degrees, a tissue block naturally settles to one corner of the culture flask, the supernatant is sucked and removed, fresh and re-warmed mesenchymal stem cell complete culture medium of example 2 is slowly added, the mixture is gently mixed, and the mixture is placed back into an incubator for continuous culture, and the culture can be carried out about day 14 (see figure 1). The culture supernatant and the tissue mass were aspirated, washed 1 time with physiological saline, and aspirated. Adding rewarmed digestive juice (scientific grade 0.125% trypsin digestive juice), digesting at 37deg.C for 4-5 min, adding isovolumetric enzyme inhibitor/complete culture medium to stop digestion, collecting cell, centrifuging (200 Xg, 5 min), counting, and harvesting 4.46×10 5 Cells (see Table 2), cells were frozen.
TABLE 2
Example 4 two-dimensional serial passage of umbilical cord mesenchymal Stem cells
This example demonstrates the two-dimensional serial passage capacity of cells of the serum-free medium provided in example 2 versus the control medium. The control group was a commercially available medium containing no vegetable protein hydrolysate but containing platelet lysate.
The experimental method comprises the following steps: human umbilical cord mesenchymal stem cells P2 are recovered, inoculated (inoculum size 2×10) 5 And then cultured in a T25 culture flask for 3 days, and the cell viability, proliferation factor and cell diameter are detected by a cell counter. Continuously transmitting to the 8 th generation. Experimental results figures 2-5 show: the cell viability of each generation of HK-SFM and control MSCs is higher, but the MSCs are long fusiform with uniform morphology by using the HK-SFM culture medium, maintain higher multiplication times, proliferate more than 10 times and maintain lower cell diameter.
Example 5 flow cytometer detection of two-dimensional cultured cell phenotypes
This example provides a test for the phenotype of P8-generation cells in serum-free medium as provided in example 2 versus control medium (medium commercially available without plant protein hydrolysate but with platelet lysate). The cells detected in this example were mesenchymal stem cells cultured in example 4.
The experimental method comprises the following steps: the P8 generation cells in the serum-free culture medium of the experimental group and the culture medium of the control group are digested and collected by TrypLE, and are respectively incubated with surface antibodies of fluorescent markers such as CD73, CD90, CD105, CD14, CD19, CD34, CD45, HLA-DR and the like, and FITC and PE marked mouse IgG isotype antibodies are used as controls; incubating at 4 ℃ for 45min, and centrifugally collecting cells; after 3 washes with PBS buffer, the cells were resuspended in 400. Mu.L PBS buffer and analyzed by flow cytometry on the machine. Experimental results table 3 shows: both HK-SFM and control MSCs highly express mesenchymal stem cell specific antigens CD73, CD90 and CD105, and low express surface markers HLA-DR related to hematopoietic and endothelial cell surface markers CD14, CD19, CD34 and CD45 and graft immune rejection, which accords with the description of MSC identification in 2006.
TABLE 3 Table 3
EXAMPLE 6 three-dimensional serial passage of umbilical cord mesenchymal Stem cells
This example provides a test for the three-dimensional serial passage capacity of cells in serum-free medium as provided in example 2 versus control medium (medium commercially available without plant protein hydrolysate but with platelet lysate).
The experimental method comprises the following steps: human umbilical cord mesenchymal stem cells P2 are recovered, inoculated (inoculum size 6×10) 5 And then, culturing in a T75 culture flask for 3 days, inoculating in a 125ml rotary flask, and the cell inoculum size is 2.5X10% 6 100mg of microcarriers, 50 volumes ml of medium, are grown at medium speed (40 rpm,5min;1rpm,2 h). 25ml of culture medium was supplemented after 1 day (day 1), the rotation speed was changed to 40rpm at a constant speed, and the culture was continued for 3 days (day 4) and then stained and counted, and the culture was continuously transferred to the 6 th generation. Experimental results figures 6-9 show: the cell viability was higher for both HK-SFM and control generations of MSCs (FIGS. 6-7), but MSCs maintained higher fold-increase (FIG. 8) and lower cell diameters (FIG. 9) using HK-SFM medium.
Example 7 flow cytometer detection of phenotypes of three-dimensional cultured cells
This example provides a three-dimensional P6 generation cell phenotype test of serum-free medium provided in example 2 versus control medium (commercially available medium without plant protein hydrolysate but with platelet lysate). The cells detected in this example were mesenchymal stem cells cultured in example 6.
The experimental method comprises the following steps: collecting P8 generation cells in serum-free culture medium of experimental group and culture medium of control group by pancreatin digestion, respectively incubating with surface antibodies of fluorescent markers such as CD73, CD90, CD105, CD14, CD19, CD34, CD45 and HLA-DR, and taking FITC and PE labeled mouse IgG isotype antibodies as controls; incubating at 4 ℃ for 45 min, and centrifugally collecting cells; after 3 washes with PBS buffer, the cells were resuspended in 400. Mu.L PBS buffer and analyzed by flow cytometry on the machine. The experimental results table 4 shows: both HK-SFM and control MSCs highly express mesenchymal stem cell specific antigens CD73, CD90 and CD105, and low express surface markers HLA-DR related to hematopoietic and endothelial cell surface markers CD14, CD19, CD34 and CD45 and graft immune rejection, which accords with the description of MSC identification in 2006.
TABLE 4 Table 4
The invention can achieve the effects of
The culture medium can maintain higher cell activity and proliferation times during continuous passage of two-dimensional culture and three-dimensional culture of human umbilical mesenchymal stem cells.
The embodiments of the present invention are not limited to the examples described above, and those skilled in the art can make various changes and modifications in form and detail, which are considered to fall within the scope of the present invention, without departing from the spirit and scope of the present invention.
Claims (14)
1. A serum-free medium for mesenchymal stem cells, comprising the following components in the following amounts: based on the total volume of the serum-free culture medium of the mesenchymal stem cells,
2. the mesenchymal stem cell serum-free medium of claim 1, wherein the basal medium is an alpha-MEM medium.
3. The serum-free medium of mesenchymal stem cells according to claim 1 or 2, wherein the platelet lysate is present in an amount of 1-5% (v/v); and the content of the plant protein hydrolysate is 5-10g/L.
4. The mesenchymal stem cell serum-free medium of claim 1 or 2, wherein the platelet lysate is present in an amount of 2% (v/v); and the content of the plant protein hydrolysate is 5g/L.
5. The mesenchymal stem cell serum-free medium of claim 1 or 2, wherein the L-glutamine content is 4mM; the content of the reduced glutathione is 100 mug/L; the hydrocortisone content was 1.0mg/L.
6. The mesenchymal stem cell serum-free medium according to claim 1 or 2, wherein the EGF content is 20ng/ml; bFGF content is 20ng/ml; TGF-beta content is 10ng/ml; PDGF-BB content was 10ng/ml.
7. The mesenchymal stem cell serum-free medium according to claim 1 or 2, wherein the content of recombinant human insulin is 10mg/L; the content of progesterone was 20ng/L.
8. The serum-free medium for mesenchymal stem cells according to claim 1 or 2, wherein the recombinant human transferrin content is 20mg/L.
9. The serum-free medium for mesenchymal stem cells according to claim 1 or 2, wherein the content of vitamin C is 20mg/L; glucose content was 25mM; sodium pyruvate content was 1mM; the content of sodium selenite is 30mg/L.
10. The serum-free medium for mesenchymal stem cells according to claim 1 or 2, comprising the following components and the contents are as follows: based on the total volume of the serum-free culture medium of the mesenchymal stem cells,
11. a method of preparing a serum-free medium of mesenchymal stem cells according to any one of claims 1-10, comprising:
the components are added into the basal medium in proportion.
12. A two-dimensional culture method of mesenchymal stem cells comprises the following steps,
1) Uniformly mixing a cell suspension containing cells with a mesenchymal stem cell complete culture medium;
2) Collecting cells, and re-suspending the cells with a mesenchymal stem cell complete medium;
3) Inoculating cells into a cell culture container, adding a mesenchymal stem cell complete culture medium, and placing the cells in an incubator for culture; and
4) Harvesting the cells;
Wherein the mesenchymal stem cell complete medium is a mesenchymal stem cell serum-free medium according to any one of claims 1-10.
13. A three-dimensional culture method of mesenchymal stem cells, comprising the steps of:
1) Preparing a microcarrier: placing the microcarrier in a culture container, and adding a mesenchymal stem cell complete culture medium to uniformly disperse the microcarrier;
2) Cell inoculation and culture: adding the cell suspension into a culture container, and placing the culture container into an incubator for culture; and
3) Harvesting the cells;
wherein the mesenchymal stem cell complete medium is a mesenchymal stem cell serum-free medium according to any one of claims 1-10.
14. Use of a mesenchymal stem cell serum-free medium according to any one of claims 1-10 for culturing mesenchymal stem cells.
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