CN109182262A - A kind of mesenchymal stem cell serum-free culture medium - Google Patents
A kind of mesenchymal stem cell serum-free culture medium Download PDFInfo
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- CN109182262A CN109182262A CN201811115805.5A CN201811115805A CN109182262A CN 109182262 A CN109182262 A CN 109182262A CN 201811115805 A CN201811115805 A CN 201811115805A CN 109182262 A CN109182262 A CN 109182262A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The present invention relates to the technical fields of cell culture, disclose a kind of mesenchymal stem cell serum-free culture medium, including basal medium and additive, and the additive includes serum substitute, other albumen and trophic factors；A kind of mesenchymal stem cell serum-free culture medium provided by the invention has the advantage that (1), serum free medium without animal blood serum or other heterologous proteins, reduce the contaminated danger of cell preparation.(2), the serum free medium that the present invention configures can reach import level in cell culture effect, and basal medium source abundance is not by market clout.(3), simplify cell separating step, the cell after tissue digestion can be directly used for originally culture, and guarantee to cultivate the specificity of cell.(4), using culture medium of the invention, cell keeps good adherence quality, shows the fibroblast form of shuttle shape, at the same be commissioned to train more it is feeding after, cell can still keep normal condition.
Description
Technical field
The present invention relates to the technical field of cell culture, especially a kind of mesenchymal stem cell serum-free culture medium.
Background technique
Mescenchymal stem cell (MSC) is mesoderma origin with self-replacation, Multidirectional Differentiation and immunoloregulation function
Multipotential stem cell.MSC has the characteristic of low immunogenicity, it is possible to escape the identification of T cell.Furthermore MSC can inhibit IL-2
The NK cell activation of mediation avoids Delayed onset graft host response to reach immune tolerance.MSC can pass through secretory cell
The factor changes tissue microenvironment, promotes injury repair.In recent years, MSC is clinically widely used, white treating
The malignant hematologic diseases such as blood disease, alpastic anemia, difficult therapeutic immunologic deficiency disease, certain genetic diseases and histoorgan
A variety of diseases such as injury repair have significant curative effect.The mechanism of action of stem cell, which mainly passes through, to be inhibited cell death, promotes
Cell differentiation, proliferation are to realize the reparation of damaged tissues.
For peripheral blood of the clinical mescenchymal stem cell after marrow, umbilical cord, fat and stem cell mobilization.By
It is Biohazard Waste in umbilical cord, therefore abundance materials are convenient.And have no adverse effect to donor, the limitation of no ethics problem.
Mescenchymal stem cell rich content in umbilical cord, undifferentiated degree is high, therefore differentiation capability is strong and is more conducive to culture expansion in vitro
Increase, sufficient cell origin can be provided with clinical to test.
The content of mescenchymal stem cell in the tissue is extremely low, brings limitation to stem-cell research and application.How to obtain
The mescenchymal stem cell of a large amount of high-purities and high activity, is considerable project.International stem-cell therapy association proposes mirror
Determine 3 standards of source of people mescenchymal stem cell: 1) mescenchymal stem cell has attaching to plastic substrates under Standard culture conditions
Property；2) CD105, CD73 and CD90 express in mescenchymal stem cell group, without expressing hematopoietic markers CD45 and CD34；3) through body
Outer inducing mesenchymal stem cell can be to osteoblast, fat cell and Chondrocyte Differentiation.According to these characteristics of stem cell, divide
From purifying and culture amplification of mesenchymal stem cells.
There is no the cultural methods of a mescenchymal stem cell gaining public acceptance, convenient and practical both at home and abroad at present.Mostly
Number still carries out mescenchymal stem cell culture using the culture medium of addition fetal calf serum (FBS).But the foreign sera of this addition
May Carried bacteria, virus etc., there is many security risks.Foreign sera bring heterologous protein, easily causes people
The allergic reaction of body.Some researches show that the albumen that can be swallowed in culture medium in, MSC in vitro incubation, therefore contain tire ox
The culture medium of serum can make to contain bovine serum albumin in MSC cell, receptor can be made to cause to be immunoreacted.The presence of these problems,
People are made to limit using additives such as serum the application for cultivating mescenchymal stem cell in clinic.
To solve the animal derived materials in culture medium, there is research using human serum, umbilical sera, platelet rich plasma, blood
Platelet lysate etc. substitutes fetal calf serum.The main function of these animal blood serum substitutes is to provide growing multiplication institute to cell
Hormone, growth factor, transfer protein and other nutriments needed are done although avoiding animality heterologous protein and introducing mesenchyma
Cell.But these substitutes equally exist some disadvantages: being that these substitute ingredients are indefinite, blood product has batch first
Between difference, it is unstable, and influenced by donor age etc.；Followed by blood product there is also infectious disease and unidentified illness examine it is potential
Risk；In addition human blood limited source limits extensive mescenchymal stem cell culture preparation, is unfavorable for practical clinical.
Summary of the invention
The purpose of the present invention is to provide a kind of mesenchymal stem cell serum-free culture mediums, it is intended to which solution lacks in the prior art
A kind of the problem of weary culture medium that mescenchymal stem cell is prepared convenient for large-scale culture.
The invention is realized in this way a kind of mesenchymal stem cell serum-free culture medium, including basal medium and addition
Object, the additive include amino acid, buffer, albumin, other albumen and trophic factors.
Further, the amino acid includes l-tyrosine (40mg/L), l-cysteine (20mg/L), glycine (10mg/
L), Valine (70mg/L), L-Trp (20mg/L), L-phenylalanine (50mg/L), Serine (50mg/L), L- Soviet Union
Propylhomoserin (75mg/L), Ala-Gln (500mg/L) and L- glycyl-L-glutamine (500mg/L).
Further, the buffer includes L- phosphoglycerol disodium salt hydrate (1.5g/L) and 4- hydroxyethyl piperazine
Ethanesulfonic acid (1.2g/L).
Further, the concentration of the albumin are as follows: 2g/L.
Further, other described albumen are Holo Transferrin (people's Holo-transferrin).
Further, the trophic factors includes Cholesterol (cholesterol), EGF and bFGF.
Further, the concentration of the Holo Transferrin (people's Holo-transferrin) is 2-8mg/L.
Further, the concentration of the Cholesterol (cholesterol) is 0.05-0.2mg/L, and the concentration of the EGF is
The concentration of 0.001mg/L, the bFGF are 0.001mg/L.
Further, the basal medium is DMEM.
Compared with prior art, a kind of mesenchymal stem cell serum-free culture medium provided by the invention, has the advantage that
(1), serum free medium is free of animal blood serum or other heterologous proteins, reduces the contaminated danger of cell preparation
Danger.It is added to specific trophic factors in serum free medium, improve cell amplification efficiency and maintains the life of stem cell
Object characteristic increases the motility rate of mescenchymal stem cell, it is ensured that the quality of cell reaches clinical application standard.
(2), existing using LONZA as the serum free medium of representative in the market, it is mainly monopolized by offshore company, price
Height, and often will appear the situation of the supply of material not in time.And the serum free medium that the present invention configures, the energy in cell culture effect
Reach import level, basal medium source abundance solves the problems, such as this not by market clout.
(3), simplify cell separating step, the cell after tissue digestion can be directly used for originally culture, and guarantee to cultivate
The specificity of cell.
(4), using culture medium of the invention, cell keeps good adherence quality, shows the fibroblast shape of shuttle shape
State, at the same through more be commissioned to train support after, cell can still keep normal condition；Cell growth curve explanation, cell maintain high amplification efficiency；
Immunophenotyping analytic explanation, CD29, CD73, CD90 and CD105 positive expression object are up to 95% or more；And CD14, CD34,
The negative expression analyte detection such as CD45, CD79a and HLA-DR is shown in 2% hereinafter, reaching clinical application level.
Detailed description of the invention
Fig. 1 is the mescenchymal stem cell provided in an embodiment of the present invention using mesenchymal stem cell serum-free culture medium culture
Form observes result figure；
Fig. 2 is the mescenchymal stem cell provided in an embodiment of the present invention using mesenchymal stem cell serum-free culture medium culture
Growth curve chart；
Fig. 3 is the mescenchymal stem cell provided in an embodiment of the present invention using mesenchymal stem cell serum-free culture medium culture
Cellular immunity testing result figure.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.
The same or similar label correspond to the same or similar components in the attached drawing of the present embodiment；In description of the invention
In, it is to be understood that if there is the orientation or positional relationship of the instructions such as term " on ", "lower", "left", "right" for based on attached drawing institute
The orientation or positional relationship shown, is merely for convenience of description of the present invention and simplification of the description, rather than the dress of indication or suggestion meaning
It sets or element must have a particular orientation, be constructed and operated in a specific orientation, therefore describe the use of positional relationship in attached drawing
Language only for illustration, should not be understood as the limitation to this patent, for the ordinary skill in the art, can be with
The concrete meaning of above-mentioned term is understood as the case may be.
Realization of the invention is described in detail below in conjunction with specific embodiment.
Referring to Fig.1 shown in -3, preferred embodiment is provided for the present invention.
The present invention provides a kind of mesenchymal stem cell serum-free culture medium, which includes basal medium and addition
Object.Basal medium is DMEM, and additive includes amino acid, buffer, albumin, other albumen and trophic factors.
Amino acid includes l-tyrosine, l-cysteine, glycine, Valine, L-Trp, L-phenylalanine, L-
Propylhomoserin, L-threonine, Ala-Gln and L- glycyl-L-glutamine.
Buffer includes L- phosphoglycerol disodium salt hydrate and 4- hydroxyethyl piperazineethanesulfonic acid.
Other albumen include Holo Transferrin (people's Holo-transferrin), and trophic factors includes Cholesterol
(cholesterol), EGF and bFGF.
The preferred concentration of these additives are as follows: Holo Transferrin 2-8mg/L, Cholesterol 0.05-
0.2mg/L, EGF 0.001mg/L and bFGF 0.001mg/L.
A kind of mesenchymal stem cell serum-free culture medium provided by the invention, has the advantage that
(1), serum free medium is free of animal blood serum or other heterologous proteins, reduces the contaminated danger of cell preparation
Danger.It is added to specific trophic factors in serum free medium, improve cell amplification efficiency and maintains the life of stem cell
Object characteristic increases the motility rate of mescenchymal stem cell, it is ensured that the quality of cell reaches clinical application standard.
(2), existing using LONZA as the serum free medium of representative in the market, it is mainly monopolized by offshore company, price
Height, and often will appear the situation of the supply of material not in time.And the serum free medium that the present invention configures, the energy in cell culture effect
Reach import level, basal medium source abundance solves the problems, such as this not by market clout.
(3), simplify cell separating step, the cell after tissue digestion can be directly used for originally culture, and guarantee to cultivate
The specificity of cell.
(4), using culture medium of the invention, cell keeps good adherence quality, shows the fibroblast shape of shuttle shape
State, at the same through more be commissioned to train support after, cell can still keep normal condition；Cell growth curve explanation, cell maintain high amplification efficiency；
Immunophenotyping analytic explanation, CD29, CD73, CD90 and CD105 positive expression object are up to 95% or more；And CD14, CD34,
The negative expression analyte detection such as CD45, CD79a and HLA-DR is shown in 2% hereinafter, reaching clinical application level.
The following are specific embodiments provided by the invention:
Mesenchymal stem cell serum-free culture medium includes basal medium and additive.Basal medium is DMEM, addition
Object includes amino acid, buffer, albumin, other albumen and trophic factors.
Amino acid includes l-tyrosine (40mg/L), l-cysteine (20mg/L), glycine (10mg/L), Valine
(70mg/L), L-Trp (20mg/L), L-phenylalanine (50mg/L), Serine (50mg/L), L-threonine (75mg/
L), Ala-Gln (500mg/L) and L- glycyl-L-glutamine (500mg/L).
Buffer includes L- phosphoglycerol disodium salt hydrate (1.5g/L) and 4- hydroxyethyl piperazineethanesulfonic acid (1.2g/
L)。
Furthermore the concentration of albumin are as follows: 2g/L.
Other albumen include Holo Transferrin (people's Holo-transferrin), and trophic factors includes Cholesterol
(cholesterol), EGF and bFGF.
The concentration of these additives are as follows: Holo Transferrin 5mg/L, Cholesterol 0.125mg/L, EGF
0.001mg/L and bFGF 0.001mg/L.
According to the above-mentioned serum free medium for preparing mescenchymal stem cell, and primary, passage for mescenchymal stem cell
And amplification cultivation.The present invention uses umbilical cord derived mesenchymal stem cell as technology objective for implementation material, square according to the following steps
Separation, originally culture, passage and the amplification cultivation of case progress umbilical cord mesenchymal stem cells.Pass through the thin of record cultured cell line
Born of the same parents' form, growth curve and flow cytometry cell surface antigen, analysis are expanded using serum free medium provided by the invention
Increase the effect of mescenchymal stem cell (result is as shown in Figure 1, Figure 2 and Figure 3).
Umbilical cord mesenchymal stem cells separation:
1) it uses PBS sufficiently to rinse repeatedly to remove remaining blood after umbilical cord tissue is removed, and is cut into tiny tissue
Fragment (1mm3)；
2) after pancreatin digestive juice being added, 37 DEG C persistently digest 30min, 800rpm, are centrifuged 5min, abandon supernatant；
3) cell is resuspended, and moves into the centrifuge tube containing separating liquid, 900rpm is centrifuged 5min, draws tunica albuginea layer；
4) PBS is washed twice, 900rpm, is centrifuged 5min；
5) cell is resuspended in culture solution, by 1-2 × 10 every square centimeter6A cell inoculation is in 175cm2Culture bottle, 37 DEG C, 5%
CO2Culture；
6) cell changes liquid for the first time after 3 days, rinses 2 cells to remove non-adherent growth with PBS；
7) it is changed the liquid once within every 3-4 days after, when cell grows into culture bottle 80%-90% area, digestion harvest cell；
8) culture solution is abandoned, PBS is washed 2 times, and digestive juice digestion, microscopically observation cell dissociation situation (microscope is added
Under gap between visible mescenchymal stem cell be gradually increased, there is the final rounded levitating that bounces back in the cytoplasm of cell, and slightly shakes
Culture bottle is shaken, cell falls off from culture bottle wall), cast-off cells are moved into centrifuge tube, 900rpm is centrifuged 5min, abandons supernatant；
9) it is precipitated as the mescenchymal stem cell of harvest, is primary cultured cell.
The passage and amplification cultivation of umbilical cord mesenchymal stem cells
1) originally culture mescenchymal stem cell is resuspended in culture solution, by 1-2 × 10 every square centimeter6A cell, is inoculated in
175cm2Culture bottle, 37 DEG C, 5%CO2Culture；
2) when cell grows full culture bottle 80%-90% area, digestion harvest cell；
3) harvest mescenchymal stem cell, by 1:3 ratio progress cell passage, 37 DEG C, 5%CO2Culture.
4) it when growth of mesenchymal stem cells expires culture bottle 80%-90% area, repeats the above steps again, it is thin to carry out
The amplification cultivation of born of the same parents.
Flow cytomery umbilical cord mesenchymal stem cells
1) harvest cultivates cell when growing to 80% degrees of fusion, digestion harvest cell, and PBS is washed 2 times；
2) plus cell is resuspended in PBS, and adjustment cell concentration is 1 × 106A/ml；
3) antibody is added, is incubated at room temperature 30min；PBS is washed 1 time, and centrifugation removes extra antibody；
4) cell is resuspended with 500ulPBS, and is transferred in streaming pipe, flow cytometer detects and records result.Streaming
Cell instrument detects following Immunophenotyping: the expression positive CD29, CD73, CD90 and CD105；Expression feminine gender CD14, CD34,
CD45、CD79a、HLA-DR。
Analyze result
1) cellular morphology is observed
As shown in Figure 1, cell keeps good adherence quality, shows the fibroblast form of shuttle shape, while through mostly generation
After culture, cell can still keep normal condition.
2) cell growth efficiency
Respectively in cell culture 0h, for 24 hours, 48h, 72h, 96h cell is counted, draw growth curve, such as Fig. 2 institute
Show.
3) Immunophenotyping detects
As shown in figure 3, CD29, CD73, CD90 and CD105 positive expression object are up to 95% or more；And CD14, CD34, CD45,
The negative expression analyte detection such as CD79a and HLA-DR is shown in 2% hereinafter, reaching clinical application level.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (9)
1. a kind of mesenchymal stem cell serum-free culture medium, which is characterized in that including basal medium and additive, the addition
Object includes amino acid, buffer, albumin, other albumen and trophic factors.
2. a kind of mesenchymal stem cell serum-free culture medium as described in claim 1, which is characterized in that the amino acid includes
L-tyrosine (40mg/L), l-cysteine (20mg/L), glycine (10mg/L), Valine (70mg/L), L-Trp
(20mg/L), L-phenylalanine (50mg/L), Serine (50mg/L), L-threonine (75mg/L), L- alanyl-L- paddy ammonia
Amide (500mg/L) and L- glycyl-L-glutamine (500mg/L).
3. a kind of mesenchymal stem cell serum-free culture medium as claimed in claim 2, which is characterized in that the buffer includes
L- phosphoglycerol disodium salt hydrate (1.5g/L) and 4- hydroxyethyl piperazineethanesulfonic acid (1.2g/L).
4. a kind of mesenchymal stem cell serum-free culture medium as claimed in claim 3, which is characterized in that the albumin it is dense
Degree are as follows: 2g/L.
5. a kind of mesenchymal stem cell serum-free culture medium as claimed in claim 4, which is characterized in that other described albumen are
Holo Transferrin (people's Holo-transferrin).
6. a kind of mesenchymal stem cell serum-free culture medium as claimed in claim 5, which is characterized in that the trophic factors packet
Include Cholesterol (cholesterol), EGF and bFGF.
7. a kind of mesenchymal stem cell serum-free culture medium as claimed in claim 6, which is characterized in that the Holo
The concentration of Transferrin (people's Holo-transferrin) is 2-8mg/L.
8. a kind of mesenchymal stem cell serum-free culture medium as claimed in claim 7, which is characterized in that described
The concentration of Cholesterol (cholesterol) is 0.05-0.2mg/L, and the concentration of the EGF is 0.001mg/L, and the bFGF's is dense
Degree is 0.001mg/L.
9. a kind of mesenchymal stem cell serum-free culture medium as described in claim 1-8 any one, which is characterized in that described
Basal medium is DMEM.
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Cited By (5)
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| CN109749992A (en) * | 2019-01-31 | 2019-05-14 | 和携科技(北京)有限公司 | A kind of mesenchymal stem cell serum-free cultural method |
| CN110172440A (en) * | 2019-04-18 | 2019-08-27 | 青岩生物科技(湖州)有限公司 | It is a kind of for cultivating the serum free medium of mesodermal stroma cell |
| CN112961825A (en) * | 2021-02-26 | 2021-06-15 | 江苏普瑞康生物医药科技有限公司 | Serum-free medium and preparation method thereof |
| CN114107187A (en) * | 2021-11-23 | 2022-03-01 | 广东普罗凯融生物医药科技有限公司 | Dental pulp mesenchymal stem cell culture medium and method for applying same |
| CN116410921A (en) * | 2023-02-09 | 2023-07-11 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
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| CN116410921B (en) * | 2023-02-09 | 2024-01-23 | 北京益华生物科技有限公司 | Human umbilical cord mesenchymal stem cell induction culture medium, induction method and application |
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