CN106947737A - A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium - Google Patents
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium Download PDFInfo
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Abstract
The invention discloses a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, including UltraCULTURE serum free mediums, L glutamine, bFGF, Ultroser G.A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium proposed by the present invention, it is to avoid toxic action and serum source contact scar of the serum to cell, is conducive in vitro culture human adipose's mescenchymal stem cell, keeps the characteristic of stem cell.
Description
Technical field
The present invention relates to technical field of cell culture, more particularly to a kind of human umbilical cord mesenchymal stem cells serum-free is complete
Culture medium.
Background technology
Umbilical cord mesenchymal stem cells (Mesnchymal Stem Cells, MSCs) refer to be present in neonatal umbilical cord tissue
In a kind of versatile stem cell, it can be divided into many kinds of histocytes, with wide potential applicability in clinical practice.How to ensure
The culture and passage of umbilical cord mesenchymal stem cells, are the premises for researching and developing umbilical cord mesenchymal stem cells various functions.
Cell culture medium is to cultivate supply cytotrophy and the basic substance for promoting germiparity in cell, is also that culture is thin
Intracellular growth and the living environment of breeding.Cell culture medium is divided into natural medium and synthetic media, most artificial synthesis
Culture medium need to add serum when using.
Serum free medium, be exactly from cellular environment without serum, but in some applications need addition grow
The main component of the factor or serum replacement, i.e. serum：Adhesion factor, growth factor, required nutriment and hormone etc..Nothing
Blood serum medium can reduce the unfavorable factor that serum is brought, and make the condition of cell culture more stable.Therefore, serum free medium and
Free serum culture turns into a main trend of current field of cell culture.
The market of serum free medium is huge, potentiality are unlimited, but is due to home made article quality and prestige problem so that for many years
Carry out the domestic serum free medium for buying import always, how to break this situation, occupy market, be badly in need of solving one
Big problem.
Chinese patent, the A of application notification number CN 105200009, discloses a kind of training of people's umbilical cord stem cells serum-free basis
Support base, comprising serum-free animal cell culture medium, growth factor, cell culture additive, trace element, with population doublings when
Between it is short, the characteristics of cell growth form is good, mitochondrial function is strong.
The content of the invention
The technical problem existed based on background technology, it is complete that the present invention proposes a kind of human umbilical cord mesenchymal stem cells serum-free
Full culture medium, it is to avoid toxic action and serum source contact scar of the serum to cell, is conducive to filling between in vitro culture human adipose
Matter stem cell, keeps the characteristic of stem cell.
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, including UltraCULTURE serum free mediums,
Glu, bFGF, serum replacement.
It is preferred that, the concentration of the Glu is 0.01-0.03mol/L.
It is preferred that, the concentration of the bFGF is 0.02-0.05ng/mL.
It is preferred that, the serum replacement is Ultroser G.
It is preferred that, the Ultroser G are (1-5) with the volume ratio of UltraCULTURE serum free mediums: 50.
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium can be applied to cultivate human umbilical cord mesenchymal stem cells.
In the universal serum free mediums of UltraCULTURE, the content of albumen is 3mg/ml, without Glu.
Ultroser G can as serum replacement, comprising material necessary to Growth of Biologic Cell, including growth factor,
Adhesion factor, hormone, associated proteins, vitamin, trace quantity mineral substance element etc., its bioactivity are 5 times of serum.
The uniformity of Ultroser G components, the stability of batch makes the repeatability raising of experimental result.Ultroser G are typically done
Into the form of freeze-dried powder, with the longer shelf-life, cost can be reduced with volume procurement.Serum replacement compatibility market master
Cell culture medium is flowed, while improving cell culture quality.
Glu is the necessary amino acid of cell growth in serum free medium, is provided for the fat stem cell of culture
Important energy source, takes off after amino, and Glu can originate as the energy of culture cell, the synthesis of participation protein
And nucleic acid metabolism.Glu in the solution through that can degrade after a period of time, and degradation rate becomes with storage temperature, L- paddy ammonia
The degraded of acid amides causes the formation of ammonia, and ammonia has toxicity for some cells, so the amount of Glu will be controlled most
Suitable concentration.
BFGF is basic fibroblast growth factor, is a kind of fissional heparin-binding protein of rush, can induce many
Plant the survival of a variety of maincenters and peripheral neurons in propagation and the differentiation of cell, energy Extending culture liquid.
Compared with prior art, the device have the advantages that being：
Added in a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium proposed by the present invention, serum free medium
The composition and content of cell growth, which can be promoted, can determine and control, it is ensured that the accuracy of experimental result, repeatability
And stability.Serum free medium proposed by the present invention, it is to avoid toxic action and serum source contact scar of the serum to cell, has
Beneficial in vitro culture human adipose's mescenchymal stem cell, the characteristic of stem cell is kept.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph of commercially available serum free medium culture human umbilical cord mesenchymal stem cells；
Fig. 2 is the scanning electron microscope (SEM) photograph of the serum-free base culture human umbilical cord mesenchymal stem cells of the gained of embodiment 4.
Embodiment
The present invention is made with reference to specific embodiment further to explain.
Embodiment 1
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, from UltraCULTURE serum free mediums
500ml, Ultroser G are 20mL, then require to weigh each component according to following final concentration：
The concentration of Glu is 0.01mol/L；
BFGF concentration is 0.02ng/mL.
A kind of preparation method of human umbilical cord mesenchymal stem cells Serum-free complete medium：, will in aseptic safe cabinet
Ultroser G are dissolved in UltraCULTURE serum free mediums；Then Glu is sequentially added；Treat that solution mixing is equal
BFGF is added after even；It is finally standby with the filtering of 0.22um filters.
Embodiment 2
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, from UltraCULTURE serum free mediums
500ml, Ultroser G are 50mL, then require to weigh each component according to following final concentration：
The concentration of Glu is 0.03mol/L；
BFGF concentration is 0.05ng/mL.
Preparation method be the same as Example 1.
Embodiment 3
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, from UltraCULTURE serum free mediums
500ml, Ultroser G are 30mL, then require to weigh each component according to following final concentration：
The concentration of Glu is 0.02mol/L；
BFGF concentration is 0.04ng/mL.
Preparation method be the same as Example 1.
Embodiment 4
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, from UltraCULTURE serum free mediums
500ml, Ultroser G are 10mL, then require to weigh each component according to following final concentration：
The concentration of Glu is 0.02mol/L；
BFGF concentration is 0.03ng/mL.
Preparation method be the same as Example 1.
Embodiment 5
A kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, from UltraCULTURE serum free mediums
500ml, Ultroser G are 40mL, then require to weigh each component according to following final concentration：
The concentration of Glu is 0.01mol/L；
BFGF concentration is 0.03ng/mL.
Preparation method be the same as Example 1.
First, ability of cell proliferation is tested
Experimental method：After Cell Counting Kit (CCK-8) kit comparing embodiment 1-5 medium cultures
Ability of cell proliferation；
1) inoculating cell suspension (the 100uL/ holes) in 96 orifice plates, different formulations culture medium is added into culture plate, is placed on
Preculture 24h (37 DEG C, 5%CO in incubator2)；
2) 10uL CCK-8 solution is added to every hole；
3) culture plate is placed in incubator and be incubated 3 hours；
4) absorbance at 450nm is determined with ELIASA.
Experimental result：
The light absorption value result of table one
| Group | Control group | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Light absorption value | 0.531 | 0.693 | 0.732 | 0.716 | 0.746 | 0.722 |
Note：Control group is commercially available serum free medium.
Experiment conclusion：Embodiment 1-5 absorbance is above control group, and absorbance is higher, and cell concentration is bigger, says
The bright Serum-free complete medium culture cell using in the present invention, can make cell possess stronger multiplication capacity.
2nd, ESEM interpretation of result
It can obtain to draw a conclusion according to Fig. 1 and 2：
Serum-free complete medium obtained by the present invention is compared with commercially available serum free medium, more conducively vitro culture of human
Umbilical cord mesenchymal stem cells, its cell quantity is more and growth conditions are excellent.
The foregoing is intended to be a preferred embodiment of the present invention, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent or change, should all be included within the scope of the present invention.
Claims (6)
1. a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium, it is characterised in that including UltraCULTURE without blood
Clear culture medium, Glu, bFGF, serum replacement.
2. a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium according to claim 1, it is characterised in that institute
The concentration for stating Glu is 0.01-0.03mol/L.
3. a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium according to claim 1, it is characterised in that institute
The concentration for stating bFGF is 0.02-0.05ng/mL.
4. a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium according to claim 1, it is characterised in that institute
Serum replacement is stated for Ultroser G.
5. a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium according to claim 1 or 4, its feature exists
In the Ultroser G are (1-5) with the volume ratio of UltraCULTURE serum free mediums: 50.
6. a kind of human umbilical cord mesenchymal stem cells Serum-free complete medium according to claim any one of 1-4 can be applied
In culture human umbilical cord mesenchymal stem cells.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161520A (en) * | 2018-08-17 | 2019-01-08 | 北昊干细胞与再生医学研究院有限公司 | The isolated culture method of mescenchymal stem cell and corresponding store method and serum free medium |
| CN111424011A (en) * | 2020-04-17 | 2020-07-17 | 深圳市旷逸生物科技有限公司 | Three-dimensional culture method capable of maintaining cell morphology of umbilical cord mesenchymal stem cells |
| CN111662866A (en) * | 2020-06-04 | 2020-09-15 | 广州同康生物科技有限公司 | Culture medium for human amniotic mesenchymal stem cells |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109161520A (en) * | 2018-08-17 | 2019-01-08 | 北昊干细胞与再生医学研究院有限公司 | The isolated culture method of mescenchymal stem cell and corresponding store method and serum free medium |
| CN111424011A (en) * | 2020-04-17 | 2020-07-17 | 深圳市旷逸生物科技有限公司 | Three-dimensional culture method capable of maintaining cell morphology of umbilical cord mesenchymal stem cells |
| CN111662866A (en) * | 2020-06-04 | 2020-09-15 | 广州同康生物科技有限公司 | Culture medium for human amniotic mesenchymal stem cells |
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