CN109385397A - A kind of serum free medium and preparation method thereof for cultivating mescenchymal stem cell - Google Patents
A kind of serum free medium and preparation method thereof for cultivating mescenchymal stem cell Download PDFInfo
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Abstract
The application belongs to stem cells technology field, and in particular to a kind of serum free medium and preparation method thereof for cultivating mescenchymal stem cell.It is provided by the present invention it is a kind of cultivate mescenchymal stem cell serum free medium, include: human serum albumin, epithelical cell growth factor and stem cell factor.The medium component determines, can avoid the interference of inhibiting factor the TGF-b concentration difference and position ingredient of serum, reduces anaphylactogen；The present invention may advantageously facilitate the proliferation of mescenchymal stem cell and maintain its cell stemness using the self-characteristic of stem cell factor so that the serum free medium specific aim is very strong.
Description
Technical field
The invention belongs to stem cells technology fields, and in particular to it is a kind of cultivate mescenchymal stem cell serum free medium and
Preparation method.
Background technique
Mescenchymal stem cell is derived from the mesoderm and ectodermic a kind of cell of mesoderm growing early stage, have self-replacation,
The characteristics such as self-renewing, pluripotency, hematopoiesis support and immunoregulation.Currently, mainly from fat, marrow, umbilical cord,
Mescenchymal stem cell is extracted in placenta tissue.Still there is Multidirectional Differentiation after mescenchymal stem cell continuous passage culture and freezen protective
Potential is the various diseases of clinical treatment, such as the treatment of the nervous system disease, nephrosis, autoimmune disease, malignant tumour mentions
New approach is supplied.From the mescenchymal stem cell of people's amnion, materials are convenient, abundance, are easy to acquire and transport, cost
It is low, it is harmless to donor and be not related to the advantages such as ethics problem, it is easily isolated and to separate cell concentration big, become following dry
Cell is in medical applications with the ideal chose of great potential.
Contain human serum or animal blood serum in current mescenchymal stem cell culture medium prescription, in serum non-principal component compared with
It is more, it could even be possible to include some unknown anaphylactogens and pathogen, on the one hand understand interference experiment as a result, be on the other hand can not
Guarantee the safety for the cell that it is cultivated, clinical research can not be applied to.The present invention is intended to find a kind of significant effect, safety
Height is suitble to the serum free medium of the amnion mesenchymal stem cell of culture clinical application.
Summary of the invention
In view of this, the main purpose of the present invention is to provide a kind of serum free medium for cultivating mescenchymal stem cell,
For solving the problems, such as that existing culture medium safety is poor, being not suitable for being applied to clinically cultivate amnion mesenchymal stem cell.
In order to solve the above-mentioned technical problem, one aspect of the present invention provides a kind of serum-free for cultivating mescenchymal stem cell
Culture medium includes: human serum albumin, epithelical cell growth factor, stem cell factor and basal medium.
Preferably, the concentration of volume percent of the human serum albumin is 2%~4%.
Preferably, the concentration of the epithelical cell growth factor is 5~15ng/mL.
Preferably, the concentration of the stem cell factor is 1~2mg/mL.
Preferably, the basal medium is low sugar DMEM.
Preferably, the stem cell factor is the freeze-dried powder of umbilical cord mesenchymal stem cells culture solution；
The umbilical cord mesenchymal stem cells are the first generation to the 5th generation umbilical cord mesenchymal stem cells.
On the other hand, the present invention also provides the preparation methods of above-mentioned serum free medium, and human serum albumin, epidermis is thin
The intracellular growth factor, stem cell factor and basal medium mixing, obtain the serum free medium.
Another aspect, the present invention also provides a kind of methods for cultivating mescenchymal stem cell, and mescenchymal stem cell is inoculated with
It is cultivated in the serum free medium that above-mentioned serum free medium or above-mentioned preparation method obtain.
In the present invention, mescenchymal stem cell is preferably amnion mesenchymal stem cell, and more preferably human amnion mesenchymal is dry thin
Born of the same parents.
Preferably, the CO that the condition of the culture is 37 DEG C and volumetric concentration is 5%2。
Preferably, the cell density of the inoculation is 5 × 104A/mL.
The present invention provides a kind of serum free mediums for cultivating mescenchymal stem cell, and include: human serum albumin, epidermis are thin
The intracellular growth factor and stem cell factor.The medium component determines that the inhibiting factor TGF-b concentration that can avoid serum is different
With the interference of position ingredient, anaphylactogen is reduced；The present invention using stem cell factor self-characteristic, so that the serum-free is trained
It is very strong to support base specific aim, may advantageously facilitate the proliferation of amnion mesenchymal stem cell and maintain its cell stemness.
Specific embodiment
Technical solution of the present invention is clearly and completely described below in conjunction with the specific embodiment of the invention, it is clear that
Described embodiment is a part of the embodiment of the present invention, instead of all the embodiments.Those skilled in the art should manage
Solution, modifies to specific embodiments of the present invention or is replaced on an equal basis to some technical characteristics, without departing from the present invention
The spirit of technical solution should all cover in the scope of protection of the invention.
Embodiment 1
A kind of serum free medium for cultivating mescenchymal stem cell is present embodiments provided, formula and preparation process are such as
Under:
1, it is formulated
2, preparation process
1) preparation of stem cell factor
The human amnion mesenchymal stem cell for taking P2 is diluted with the low sugar DMEM containing 10%FBS, then presses 1 × 105~
10×105The cell density of cell/mL is inoculated in culture bottle, is cultivated on a large scale.
When cell growth fusion degree is up to 80% or more, to use the training of 1640 Selective agar mediums instead after twice of cell of PBS cleaning
Cell is supported, liquid is changed within 3 days one, collects the culture solution of culture cell.
Culture solution is removed into large particulate matter using 0.45 μm of membrane filtration, obtains first-time filtrate.Using 0.22 μm of filter membrane
First-time filtrate is filtered to remove 3KDa or less finely ground particle substance, obtains secondary filtrate.It is carried out using Slice200 slipstream device dense
It contracts secondary filtrate, the time of circulating filtration is 3h, and freeze-dried powder is made with freeze dryer in concentrate, and the stem cell for obtaining the present embodiment is raw
The long factor, for use.
2) select DMEM low sugar as basic culture medium.
3) human serum albumin is taken, the DMEM low sugar of 500mL is added, it is raw to sequentially add the epidermis that concentration is 1000ng/mL
The long factor (EGF) and stem cell factor mix, obtain serum free medium, be placed in 4 DEG C of preservations.
In use, human amnion mesenchymal stem cell is pressed 5 × 104The cell density of a/mL is inoculated in above-mentioned serum-free training
It supports in base, and in 37 DEG C, 5%CO2Under cultivated.
Embodiment 2
A kind of serum free medium for cultivating mescenchymal stem cell is present embodiments provided, formula and preparation process are such as
Under:
1, it is formulated
2, preparation process
1) preparation of stem cell factor
The human amnion mesenchymal stem cell for taking P2 is diluted with the low sugar DMEM containing 10%FBS, then presses 1 × 105~
10×105The cell density of cell/mL is inoculated in culture bottle, is cultivated on a large scale.
When cell growth fusion degree is up to 80% or more, to use the training of 1640 Selective agar mediums instead after twice of cell of PBS cleaning
Cell is supported, liquid is changed within 3 days one, collects the culture solution of culture cell.
Culture solution is removed into large particulate matter using 0.45 μm of membrane filtration, obtains first-time filtrate.Using 0.22 μm of filter membrane
First-time filtrate is filtered to remove 3KDa or less finely ground particle substance, obtains secondary filtrate.It is carried out using Slice200 slipstream device dense
It contracts secondary filtrate, the time of circulating filtration is 3h, and freeze-dried powder is made with freeze dryer in concentrate, and the stem cell for obtaining the present embodiment is raw
The long factor, for use.
2) select DMEM low sugar as basic culture medium.
3) human serum albumin is taken, the DMEM low sugar of 500mL is added, it is raw to sequentially add the epidermis that concentration is 1000ng/mL
The long factor (EGF) and stem cell factor mix, obtain serum free medium, be placed in 4 DEG C of preservations.
In use, human amnion mesenchymal stem cell is pressed 5 × 104The cell density of a/mL is inoculated in above-mentioned serum-free training
It supports in base, and in 37 DEG C, 5%CO2Under cultivated.
Embodiment 3
A kind of serum free medium for cultivating mescenchymal stem cell is present embodiments provided, formula and preparation process are such as
Under:
1, it is formulated
2, preparation process
1) preparation of stem cell factor
The human amnion mesenchymal stem cell for taking P2 is diluted with the low sugar DMEM containing 10%FBS, then presses 1 × 105~
10×105The cell density of cell/mL is inoculated in culture bottle, is cultivated on a large scale.
When cell growth fusion degree is up to 80% or more, to use the training of 1640 Selective agar mediums instead after twice of cell of PBS cleaning
Cell is supported, liquid is changed within 3 days one, collects the culture solution of culture cell.
Culture solution is removed into large particulate matter using 0.45 μm of membrane filtration, obtains first-time filtrate.Using 0.22 μm of filter membrane
First-time filtrate is filtered to remove 3KDa or less finely ground particle substance, obtains secondary filtrate.It is carried out using Slice200 slipstream device dense
It contracts secondary filtrate, the time of circulating filtration is 3h, and freeze-dried powder is made with freeze dryer in concentrate, and the stem cell for obtaining the present embodiment is raw
The long factor, for use.
2) select DMEM low sugar as basic culture medium.
3) human serum albumin is taken, the DMEM low sugar of 500mL is added, it is raw to sequentially add the epidermis that concentration is 1000ng/mL
The long factor (EGF) and stem cell factor mix, obtain serum free medium, be placed in 4 DEG C of preservations.
In use, human amnion mesenchymal stem cell is pressed 5 × 104The cell density of a/mL is inoculated in above-mentioned serum-free training
It supports in base, and in 37 DEG C, 5%CO2Under cultivated.
Test case
According to the following table 1, three kinds of tested culture mediums are prepared respectively, wherein A group culture medium is the free serum culture of embodiment 1
Base, B group culture medium are common culture medium in the prior art, and C group culture medium is blank control group, and formula is as shown in table 1:
Table 1
| A group | B group | C group |
| Low sugar DMEM | Low sugar DMEM | Low sugar DMEM |
| 3vol% human serum albumin | 10%FBS | |
| 10mg/mL EGF, 1.5mg/mL stem cell factor |
Taking convergence degree is that the human amnion mesenchymal stem cell in 80% P1 generation is added after washing 2 times with PBS into cell
0.015mL/cm20.25% pancreatin+0.04%EDTA digest 2min, terminate enzyme with the complete medium of 10 times of digestive juices
Solution, sampling count, and are divided into three pipes, and 500g is centrifuged 5min, discards supernatant.According to count results, in corresponding pipe respectively
Tri- kinds of culture mediums of A, B, C are added, and adjusting cell density is 5 × 104Cell/mL gently takes 20mL to connect respectively after piping and druming uniformly
Kind is transferred to 37 DEG C, 5%CO in the culture dish of 15cm2Incubator in cultivate.
After cultivating 72h, after washing 2 times with PBS, 0.015mL/cm is added into cell20.25% pancreatin+0.04%
EDTA digests 2min, terminates enzymatic hydrolysis with the DMEM-F12 of 10 times of digestive juices, sampling is set after 0.4% trypan blue 1:1 dyeing
Under microscope, viable count and dead cell number are calculated, wherein Cell viability=viable count/total cell number × 100%.
Table 2 is that the cell quantity for the human amnion mesenchymal stem cell being respectively adopted under three kinds of culture medium cultures and its cell are lived
The result of rate.
Table 2
| Group | A group | B group | C group |
| Cell quantity | 5.6×106It is a | 4.4×106It is a | 1.2×106It is a |
| Cell viability | 96.65% | 92.62% | 69.74% |
If table 2 shows, the cell quantity and Cell viability of A group and B group are obviously higher than C group；The Cell viability of A group and B group
There is no notable difference, however the cell quantity of A group is higher than B group, illustrates that culture medium of the present invention can effectively facilitate human amnion mesenchymal
The proliferation of stem cell.The experimental result of embodiment 2,3 is similar to Example 1.
Claims (10)
1. a kind of serum free medium for cultivating mescenchymal stem cell is, characterized by comprising: human serum albumin, epidermal cell
Growth factor, stem cell factor and basal medium.
2. serum free medium according to claim 1, which is characterized in that the percent by volume of the human serum albumin is dense
Degree is 2%~4%.
3. serum free medium according to claim 1, which is characterized in that the concentration of the epithelical cell growth factor is
2~15mg/mL.
4. serum free medium according to claim 1, which is characterized in that the concentration of the stem cell factor is 1
~2mg/mL.
5. serum free medium according to claim 1, which is characterized in that the basal medium is low sugar DMEM.
6. serum free medium according to claim 1, which is characterized in that the stem cell factor fills between umbilical cord
The freeze-dried powder of matter stem cell medium；
The umbilical cord mesenchymal stem cells are the first generation to the 5th generation umbilical cord mesenchymal stem cells.
7. the preparation method of serum free medium described in claim 1 to 6 any one, which is characterized in that by human serum albumin,
Epithelical cell growth factor, stem cell factor and basal medium mixing, obtain the serum free medium.
8. a kind of method for cultivating mescenchymal stem cell, which is characterized in that mescenchymal stem cell is inoculated in claim 1 to 6
It is trained in the serum free medium that preparation method described in serum free medium described in any one or claim 7 obtains
It supports.
9. according to the method described in claim 8, it is characterized in that, the condition of the culture is 37 DEG C and volumetric concentration is 5%
CO2。
10. according to the method described in claim 8, it is characterized in that, the cell density of the inoculation is 5 × 104A/mL.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109943526A (en) * | 2019-05-23 | 2019-06-28 | 广州赛莱拉干细胞科技股份有限公司 | A kind of serum-free peptide composition promoting mescenchymal stem cell proliferation |
| CN110804587A (en) * | 2019-11-27 | 2020-02-18 | 浙江卫未生物医药科技有限公司 | Method for modeling production of mesenchymal stem cells by adopting improved microcarrier cell culture method |
| CN116836920A (en) * | 2023-08-21 | 2023-10-03 | 佛山市生物医学工程学会 | Serum-free culture medium and method for preparing mesenchymal stem cells by using same |
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Cited By (4)
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| CN116836920B (en) * | 2023-08-21 | 2024-05-24 | 广东横琴粤澳深度合作区齐美国际干细胞医院有限公司 | Serum-free culture medium and method for preparing mesenchymal stem cells by using same |
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Application publication date: 20190226 |