CN110317779A - The umbilical cord mesenchymal stem cells culture medium of serum-free - Google Patents
The umbilical cord mesenchymal stem cells culture medium of serum-free Download PDFInfo
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Abstract
The invention discloses a kind of umbilical cord mesenchymal stem cells culture mediums of serum-free, for cultivating umbilical cord mesenchymal stem cells, are made of serum-free minimal medium and adding ingredient；Adding ingredient is recombinant human fibroblast growth factor hFGF, recombinant human epidermal growth factor hEGF, insulin hI, L-Glutamine, 2 mercapto ethanol, sodium selenite, transferrins, human serum albumins, fibronectin, astragalus polyose APS, IL-3, IL-6, IL-r, ginseng extract, rose extract, jasmine flower extract, vitamin C.
Description
Technical field
The present invention relates to the research field of stem cell, more particularly to it is a kind of it is novel, be efficiently suitable for umbilical cord mesenchyma
The serum free medium of stem cell large-scale culture.
Background technique
Umbilical cord mesenchymal stem cells, which refer to, is present in one of neonatal umbilical cord tissue multipotential stem cell, and cell surface is strong
Expression specificity marks CD90, CD73, CD105, do not express label CD45, CD14 of hematopoiesis system or interior skin system cell, CD11,
CD34, CD19, low expression or do not express major histocompatibility antigen HLA-DR.Studies have shown that umbilical cord mesenchymal stem cells tool
There is higher differentiation potential, can be broken up to multiple directions such as bone, cartilage, muscle, tendon, ligament, nerve, liver endothelium and the heart
Flesh etc..Compared with mesenchymal stem cell, umbilical cord mesenchymal stem cells content and proliferative capacity are superior to the latter, and immunogene
Property it is low, materials are convenient, without ethics dispute the advantages that, therefore in mescenchymal stem cell have higher clinical value.
In practical clinical, no exogenous pollution and cell quantity be guarantee umbilical cord mesenchymal stem cells treatment must
Want factor.Currently, the cultivating system of umbilical cord mesenchymal stem cells mostly contains animal blood serum (such as fetal calf serum), on the one hand due to
The presence of unknown albumen in serum, signal transduction and cell surface marker to cell form interference, on the other hand using addition
The stem cell of serum free culture system is transplanted in vivo, may cause the cross-infection of pathogen, it is therefore desirable to it is bright to develop chemical component
True serum free medium.
Summary of the invention
The object of the present invention is to provide a kind of for cultivating the culture medium of umbilical cord mesenchymal stem cells.Solve existing culture medium
Cultivate the existing proliferation of umbilical cord mesenchymal stem cells slowly, cellular morphology and biological character etc. easily change, and are unable to satisfy
The problem of clinical application demand of stem cell.
It is an object of the present invention to provide a kind of umbilical cord mesenchymal stem cells culture mediums of serum-free, including DMEM/
F12,1~20ng/mL recombinant human fibroblast growth factor, 1~20 μ g/mL recombinant human epidermal growth factor, 1~20 μ g/
ML insulin, 1~10mM L-Glutamine, 50~200 μM of 2 mercapto ethanols, 20~50nM sodium selenite, 10~30 μ g/ml
Transferrins, 10~100mg/mL human serum albumins, 50~200 μ g/mL fibronectin, 50~200ug/ml astragalus polyose
APS, 10~50mg/L IL-3,10~50mg/L IL-6,45~55 μ g/L IL-r, 10~50mg/L ginseng extract, 10~
50mg/L rose extract, 10~50mg/L jasmine flower extract, 5~8mM vitamin C.
Preferably, it is recombinated in culture medium containing 5~15ng/mL recombinant human fibroblast growth factor, 5~15 μ g/mL
HEGF, 5~18 μ g/mL insulin, 5~8mM L-Glutamine, 80~150 μM of 2 mercapto ethanols, 25~
40nM sodium selenite, 15~25 μ g/ml transferrins, 30~80mg/mL human serum albumins, 80~150 μ g/mL fibre adhesion eggs
White, 80~150ug/ml astragalus polyose APS.
Preferably, 10ng/mL recombinant human fibroblast growth factor, 10 μ g/mL recombinant human epidermals are contained in culture medium
Growth factor, 10 μ g/mL insulin, 5mM L-Glutamine, 100 μM of 2 mercapto ethanols, 30nM sodium selenite, 25 μ g/ml turn
Ferritin, 50mg/mL human serum albumins, 100 μ g/mL fibronectin, 100ug/ml astragalus polyose APS, 30mg/L IL-
3,30mg/L IL-6,50 μ g/L IL-r, 30mg/L ginseng extract, 30mg/L rose extract, 30mg/L Jasmine are extracted
Object, 5mM vitamin C.
There is provided a kind of secondary culture of umbilical chord mesenchymal cells and the methods of amplification for another object of the present invention, specific to walk
It is rapid as follows:
A. the umbilical cord mesenchymal stem cells being stored in cryopreservation tube are removed from liquid nitrogen, put into 37 DEG C of water-baths immediately
In, gentle agitation 2min dissolution, while preheating the umbilical cord mesenchymal stem cells culture medium of above-mentioned serum-free；
B. the umbilical cord mesenchymal stem cells after 37 DEG C thaw the umbilical cord mesenchyma equipped with the above-mentioned serum-free of 8mL is drawn onto do
In the 15ml centrifuge tube of cell culture medium, then is rinsed 1 time and frozen with the umbilical cord mesenchymal stem cells culture medium of the above-mentioned serum-free of 1ml
Pipe reduces cell quantity loss to the greatest extent, and rinsing liquid is added in centrifuge tube together, gently blows even, avoids generating bubble, then 800g
Centrifugation 5 minutes；
C. abandon supernatant, respectively plus 2mL preheating above-mentioned serum-free umbilical cord mesenchymal stem cells culture medium, gently blow
It is even, guarantee that cell suspends completely；In the culture bottle of the cell inoculation of resuspension to T25,3-4ml above-mentioned serum-free will be added
Umbilical cord mesenchymal stem cells culture medium, then cell is shaken and uniformly guarantees that all cells can be uniformly distributed；
D. the cell of recovery is put into 37 DEG C, cultivated in 5%C02 incubator；
E. the umbilical cord mesenchymal stem cells culture medium for stating serum-free is changed after cell is adherent, removes dead cell；In culture bottle
Cell coverage rate be passaged in tissue culture plate by the cell concentration of 8000/square centimeter when reaching 80%~90%, carefully
The growth medium that born of the same parents' culture plate uses is the umbilical cord mesenchymal stem cells culture medium of above-mentioned serum-free.
Technical solution of the present invention realize the utility model has the advantages that the present invention provides a kind of umbilical cord mesenchymal stem cells of serum-free
Culture medium is made of for cultivating umbilical cord mesenchymal stem cells serum-free minimal medium and adding ingredient；Adding ingredient is attached most importance to
Group human fibroblastic growth factor hFGF, recombinant human epidermal growth factor hEGF, insulin hI, L-Glutamine, 2- sulfydryl
Ethyl alcohol, sodium selenite, transferrins, human serum albumins, fibronectin, astragalus polyose APS, IL-3, IL-6, IL-r, people
Conopsea extraction, rose extract, jasmine flower extract, vitamin C.In this free serum culture environment, growth and proliferation of cell speed
Rate is higher, and keeps its stem cell properties, is more advantageous to stem cell and is safely applied to clinical transplantation.
Detailed description of the invention
The mescenchymal stem cell of Fig. 1 culture medium culture of the present invention；
Fig. 2 routinely has blood serum medium；
The commercially available serum free medium of Fig. 3 (Lonza).
Specific embodiment
Explanation that the present invention will be further explained combined with specific embodiments below, but specific embodiment is not to the present invention
It is limited in any way.Unless stated otherwise, reagent, method involved in embodiment are reagent and method commonly used in the art.
The preparation of 1. culture medium of embodiment
Serum-free minimal medium is DMEM/F12 culture medium；
Adding ingredient formula be 1ng/mL recombinant human fibroblast growth factor, 1 μ g/mL recombinant human epidermal growth because
Son, 1 μ g/mL insulin, 1mM L-Glutamine, 50 μM of 2 mercapto ethanols, 20nM sodium selenite, 10 μ g/ml transferrins,
10mg/mL human serum albumins, 50 μ g/mL fibronectin, 50ug/ml astragalus polyose APS, 10mg/L IL-3,10mg/L
IL-6,45 μ g/L IL-r, 10mg/L ginseng extract, 10mg/L rose extract, 10mg/L jasmine flower extract, 5mM dimension life
Plain C；
Adding ingredient is made into mother liquor the preparation method comprises the following steps: measuring according to the rules by culture medium, and 0.45um membrane filtration is added without blood
Clear minimal medium is uniformly mixed and is tuned into final concentration.
The preparation of 2. culture medium of embodiment
Serum-free minimal medium is DMEM/F12 culture medium；
Adding ingredient formula be 20ng/mL recombinant human fibroblast growth factor, 20 μ g/mL recombinant human epidermals growth because
Son, 20 μ g/mL insulin, 10mM L-Glutamine, 200 μM of 2 mercapto ethanols, 50nM sodium selenite, 30 μ g/ml turn iron egg
White, 100mg/mL human serum albumins, 200 μ g/mL fibronectin, 200ug/ml astragalus polyose APS, 50mg/L IL-3,
50mg/L IL-6,55 μ g/L IL-r, 50mg/L ginseng extract, 50mg/L rose extract, 50mg/L jasmine flower extract,
8mM vitamin C；
Adding ingredient is made into mother liquor the preparation method comprises the following steps: measuring according to the rules by culture medium, and 0.45um membrane filtration is added without blood
Clear minimal medium is uniformly mixed and is tuned into final concentration.
The preparation of 3. culture medium of embodiment
Serum-free minimal medium is DMEM/F12 culture medium；
Adding ingredient formula be 5ng/mL recombinant human fibroblast growth factor, 5 μ g/mL recombinant human epidermals growth because
Son, 5 μ g/mL insulin, 5mM L-Glutamine, 80 μM of 2 mercapto ethanols, 25nM sodium selenite, 15 μ g/ml transferrins,
30mg/mL human serum albumins, 80 μ g/mL fibronectin, 80ug/ml astragalus polyose APS, 50mg/L IL-3,50mg/L
IL-6,55 μ g/L IL-r, 50mg/L ginseng extract, 50mg/L rose extract, 50mg/L jasmine flower extract, 8mM dimension life
Plain C；
Adding ingredient is made into mother liquor the preparation method comprises the following steps: measuring according to the rules by culture medium, and 0.45um membrane filtration is added without blood
Clear minimal medium is uniformly mixed and is tuned into final concentration.
The preparation of 4. culture medium of embodiment
Serum-free minimal medium is DMEM/F12 culture medium；
Adding ingredient formula be 15ng/mL recombinant human fibroblast growth factor, 15 μ g/mL recombinant human epidermals growth because
Son, 18 μ g/mL insulin, 8mM L-Glutamine, 150 μM of 2 mercapto ethanols, 40nM sodium selenite, 25 μ g/ml turn iron egg
White, 80mg/mL human serum albumins, 150 μ g/mL fibronectin, 150ug/ml astragalus polyose APS, 50mg/L IL-3,
50mg/L IL-6,55 μ g/L IL-r, 50mg/L ginseng extract, 50mg/L rose extract, 50mg/L jasmine flower extract,
8mM vitamin C；
Adding ingredient is made into mother liquor the preparation method comprises the following steps: measuring according to the rules by culture medium, and 0.45um membrane filtration is added without blood
Clear minimal medium is uniformly mixed and is tuned into final concentration.
The preparation of 5. culture medium of embodiment
Serum-free minimal medium is DMEM/F12 culture medium；
Adding ingredient formula be 10ng/mL recombinant human fibroblast growth factor, 10 μ g/mL recombinant human epidermals growth because
Son, 10 μ g/mL insulin, 5mM L-Glutamine, 100 μM of 2 mercapto ethanols, 30nM sodium selenite, 25 μ g/ml turn iron egg
White, 50mg/mL human serum albumins, 100 μ g/mL fibronectin, 100ug/ml astragalus polyose APS, 30mg/L IL-3,
30mg/L IL-6,50 μ g/L IL-r, 30mg/L ginseng extract, 30mg/L rose extract, 30mg/L jasmine flower extract,
5mM vitamin C；
Adding ingredient is made into mother liquor the preparation method comprises the following steps: measuring according to the rules by culture medium, and 0.45um membrane filtration is added without blood
Clear minimal medium is uniformly mixed and is tuned into final concentration.
As needed, the culture medium provided by the present invention for cultivating umbilical cord mesenchymal stem cells can be by with liquid shape
The serum-free minimal medium existing for formula and the in solid form existing adding ingredient composition, are also possible to liquid
The fluid nutrient medium formed after the adding ingredient is added in the serum-free minimal medium existing for body form.
6. secondary culture of embodiment and the method for amplification, the specific steps are as follows:
A. the umbilical cord mesenchymal stem cells being stored in cryopreservation tube are removed from liquid nitrogen, put into 37 DEG C of water-baths immediately
In, gentle agitation 2min dissolution, while preheating the umbilical cord mesenchymal stem cells culture medium of above-mentioned serum-free；
B. the umbilical cord mesenchymal stem cells after 37 DEG C thaw the umbilical cord mesenchyma equipped with the above-mentioned serum-free of 8mL is drawn onto do
In the 15ml centrifuge tube of cell culture medium, then is rinsed 1 time and frozen with the umbilical cord mesenchymal stem cells culture medium of the above-mentioned serum-free of 1ml
Pipe reduces cell quantity loss to the greatest extent, and rinsing liquid is added in centrifuge tube together, gently blows even, avoids generating bubble, then 800g
Centrifugation 5 minutes；
C. abandon supernatant, respectively plus 2mL preheating above-mentioned serum-free umbilical cord mesenchymal stem cells culture medium, gently blow
It is even, guarantee that cell suspends completely；In the culture bottle of the cell inoculation of resuspension to T25,3-4ml above-mentioned serum-free will be added
Umbilical cord mesenchymal stem cells culture medium, then cell is shaken and uniformly guarantees that all cells can be uniformly distributed；
D. the cell of recovery is put into 37 DEG C, cultivated in 5%CO2 incubator；
E. the umbilical cord mesenchymal stem cells culture medium for stating serum-free is changed after cell is adherent, removes dead cell；In culture bottle
Cell coverage rate be passaged in tissue culture plate by the cell concentration of 8000/square centimeter when reaching 80%~90%, carefully
The growth medium that born of the same parents' culture plate uses is the umbilical cord mesenchymal stem cells culture medium of above-mentioned serum-free.
The test of 7. ability of cell proliferation of embodiment
Experimental method: with Ce11Counting Kit (CCK-8) kit above-mentioned culture medium more of the invention and commercially available nothing
Serum and routine have the ability of cell proliferation after blood serum medium culture；
1) different formulations culture medium is added into culture plate, is placed on for inoculating cell suspension (hole 100uL/) in 96 orifice plates
Preculture is for 24 hours (37 DEG C, 5%C02) in incubator；
2) 10ul CCK-8 solution is added in every hole；
3) culture plate is placed in incubator and is incubated for 3 hours；
It is measured with microplate reader for the absorbance at 450nm.
Experimental result:
(1) spectrophotometric analysis conclusion: absorbance value is higher than control group, and absorbance value is higher, and cell concentration is bigger, explanation
Using the Serum-free complete medium culture cell in the present invention, cell can be made to have stronger proliferative capacity.(being shown in Table 1)
1 light absorption value result of table
(2) phase contrast microscope interpretation of result: as Fig. 1-3 comparison as it can be seen that culture medium culture of the present invention cellular morphology compared with
Good, the adherent better performances of cell, adherent suppression is unobvious, good to Tissue Culture Flask adaptability.
Those skilled in the art should understand that: the above embodiments are only used to illustrate the technical solution of the present invention., and
It is non-that it is limited；Although present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can
To modify the technical solutions described in the foregoing embodiments, or some or all of the technical features are carried out etc.
With replacement；And these are modified or replaceed, the range of it does not separate the essence of the corresponding technical solution claims of the present invention.
Claims (4)
1. a kind of umbilical cord mesenchymal stem cells culture medium of serum-free, which is characterized in that including DMEM/F12,1~20ng/mL weight
Group human fibroblastic growth factor, 1~20 μ g/mL recombinant human epidermal growth factor, 1~20 μ g/mL insulin, 1~10mM
L-Glutamine, 50~200 μM of 2 mercapto ethanols, 20~50nM sodium selenite, 10~30 μ g/ml transferrins, 10~
100mg/mL human serum albumins, 50~200 μ g/mL fibronectin, 50~200ug/ml astragalus polyose APS, 10~50mg/
L IL-3,10~50mg/L IL-6,45~55 μ g/L IL-r, 10~50mg/L ginseng extract, 10~50mg/L rose mention
Take object, 10~50mg/L jasmine flower extract, 5~8mM vitamin C.
2. the umbilical cord mesenchymal stem cells culture medium of serum-free according to claim 1, which is characterized in that contain in culture medium
There are 5~15ng/mL recombinant human fibroblast growth factor, 5~15 μ g/mL recombinant human epidermal growth factors, 5~18 μ g/mL
Insulin, 5~8mM L-Glutamine, 80~150 μM of 2 mercapto ethanols, 25~40nM sodium selenite, 15~25 μ g/ml turn
Ferritin, 30~80mg/mL human serum albumins, 80~150 μ g/mL fibronectin, 80~150ug/ml astragalus polyose
APS。
3. the umbilical cord mesenchymal stem cells culture medium of serum-free according to claim 2, which is characterized in that contain in culture medium
There are 10ng/mL recombinant human fibroblast growth factor, 10 μ g/mL recombinant human epidermal growth factors, 10 μ g/mL insulin, 5mM
L-Glutamine, 100 μM of 2 mercapto ethanols, 30nM sodium selenite, 25 μ g/ml transferrins, 50mg/mL human serum albumins,
100 μ g/mL fibronectin, 100ug/ml astragalus polyose APS, 30mg/L IL-3,30mg/L IL-6,50 μ g/L IL-r,
30mg/L ginseng extract, 30mg/L rose extract, 30mg/L jasmine flower extract, 5mM vitamin C.
4. a kind of method of the secondary culture and amplification of umbilical chord mesenchymal cells, the specific steps are as follows:
A. the umbilical cord mesenchymal stem cells being stored in cryopreservation tube are removed from liquid nitrogen, are put into 37 DEG C of water-baths immediately, gently
Micro- shake 2min dissolution, while preheating the umbilical cord mesenchymal stem cells culture medium of serum-free described in claim 1-3；
B. the umbilical cord mesenchymal stem cells after 37 DEG C thaw are drawn onto the umbilical cord equipped with serum-free described in 8mL claim 1-3
In the 15ml centrifuge tube of mescenchymal stem cell culture medium, then the umbilical cord mesenchyma of the serum-free described in 1ml claim 1-3 is dry thin
Born of the same parents' culture medium rinses 1 cryopreservation tube, reduces cell quantity loss to the greatest extent, and rinsing liquid is added in centrifuge tube together, gently blows even, keeps away
Exempt to generate bubble, then 800g is centrifuged 5 minutes；
C. abandon supernatant, respectively plus 2mL preheating claim 1-3 described in serum-free umbilical cord mesenchymal stem cells culture medium,
Gently blow it is even, guarantee cell suspend completely；By in the culture bottle of the cell inoculation of resuspension to T25,3-4ml right is added
It is required that the umbilical cord mesenchymal stem cells culture medium of serum-free described in 1-3, then cell is shaken and uniformly guarantees that all cells can be equal
Even distribution；
D. the cell of recovery is put into 37 DEG C, cultivated in 5%C02 incubator；
E. the umbilical cord mesenchymal stem cells culture medium of serum-free described in claim 1-3 is changed after cell is adherent, removes dead cell；Training
It supports and is passaged to tissue culture plate by the cell concentration of 8000/square centimeter when the cell coverage rate in bottle reaches 80%90%
In, the growth medium that tissue culture plate uses is the umbilical cord mesenchymal stem cells culture medium of serum-free described in claim 1-3.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910500463.7A CN110317779A (en) | 2019-06-06 | 2019-06-06 | The umbilical cord mesenchymal stem cells culture medium of serum-free |
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Cited By (6)
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| CN111996164A (en) * | 2020-09-10 | 2020-11-27 | 聊城市人民医院 | Serum-free anti-aging culture medium for mesenchymal stem cells |
| CN113133447A (en) * | 2021-05-20 | 2021-07-20 | 郑州优倍得生物科技有限公司 | Cryopreservation liquid and cryopreservation method for umbilical cord mesenchymal stem cells |
| CN113151165A (en) * | 2021-05-14 | 2021-07-23 | 河北驰熙科技发展有限公司 | Culture medium and culture method for human umbilical cord mesenchymal stem cell amplification |
| CN113648276A (en) * | 2021-09-10 | 2021-11-16 | 广州市万千粉丝化妆品有限公司 | Preparation method of serum protein complete culture solution of mesenchymal cells and application of serum protein complete culture solution in cosmetics |
| WO2022099474A1 (en) * | 2020-11-10 | 2022-05-19 | 南通优元美术图案设计有限公司 | Placenta mesenchymal stem cell cryogenic vial containing ginseng polysaccharide coating |
| CN114807028A (en) * | 2022-06-02 | 2022-07-29 | 广州美健生物技术有限公司 | Serum-free mesenchymal stem cell culture medium and stem cell culture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111996164A (en) * | 2020-09-10 | 2020-11-27 | 聊城市人民医院 | Serum-free anti-aging culture medium for mesenchymal stem cells |
| WO2022099474A1 (en) * | 2020-11-10 | 2022-05-19 | 南通优元美术图案设计有限公司 | Placenta mesenchymal stem cell cryogenic vial containing ginseng polysaccharide coating |
| CN113151165A (en) * | 2021-05-14 | 2021-07-23 | 河北驰熙科技发展有限公司 | Culture medium and culture method for human umbilical cord mesenchymal stem cell amplification |
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| CN113133447A (en) * | 2021-05-20 | 2021-07-20 | 郑州优倍得生物科技有限公司 | Cryopreservation liquid and cryopreservation method for umbilical cord mesenchymal stem cells |
| CN113648276A (en) * | 2021-09-10 | 2021-11-16 | 广州市万千粉丝化妆品有限公司 | Preparation method of serum protein complete culture solution of mesenchymal cells and application of serum protein complete culture solution in cosmetics |
| CN114807028A (en) * | 2022-06-02 | 2022-07-29 | 广州美健生物技术有限公司 | Serum-free mesenchymal stem cell culture medium and stem cell culture method |
| CN114807028B (en) * | 2022-06-02 | 2022-11-01 | 深圳格泰赛尔生物科技有限公司 | Serum-free mesenchymal stem cell culture medium and stem cell culture method |
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