CN107083358A - A kind of cell culture medium and the application in umbilical cord mesenchymal stem cells culture - Google Patents
A kind of cell culture medium and the application in umbilical cord mesenchymal stem cells culture Download PDFInfo
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- CN107083358A CN107083358A CN201710438958.2A CN201710438958A CN107083358A CN 107083358 A CN107083358 A CN 107083358A CN 201710438958 A CN201710438958 A CN 201710438958A CN 107083358 A CN107083358 A CN 107083358A
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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
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- 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
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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Abstract
The present invention relates to technical field of stem cell culture, more particularly to a kind of cell culture medium and the application in umbilical cord mesenchymal stem cells culture.The present invention addition, EGF, NEAA, glutamine and conditioned medium in basal medium, each component serve good facilitation to the growth of dental pulp stem cell jointly.Experiment shows, the medium culture umbilical cord mesenchymal stem cells provided using the present invention, the cell cycle can be shortened, improve ability of cell proliferation, improve the purity and dryness of stem cell, and the culture medium, without animal derived components such as hyclones, the stem cell obtained by the culture medium is suitable for clinical practice.
Description
Technical field
The present invention relates to technical field of stem cell culture, more particularly to a kind of cell culture medium and dry thin in umbilical cord mesenchyma
Application in born of the same parents' culture.
Background technology
Umbilical cord mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) refer to be present in neonatal umbilical cord tissue
In a kind of versatile stem cell, with higher differentiation potential, can be broken up to multiple directions.It is in bone, cartilage, flesh
There is wide potential applicability in clinical practice in terms of the organizational projects such as meat, tendon, ligament, nerve, liver, endothelium and cardiac muscle.Have been reported that from
MSCs is isolated in people's umbilical cord, and cell content, multiplication capacity are better than bone marrow MSCs, immunogenicity is lower than bone marrow MSCs, and
With convenient material drawing, the advantages of no ethics is disputed on, therefore increasingly by the concern of research workers.
At present, to cultivating system mainly culture of the application containing animal blood serum (such as hyclone) of umbilical cord mesenchymal stem cells
Contain the growth factor needed for cell growth, hormone, carrier protein, anchoring factor, trace element and other battalion in base, serum
Material is supported, cell growth can be promoted, but there is carrying pathogen in the culture medium containing serum, utilize the dry of animal blood serum culture
Which kind of cell, its internal structure can occur and change on the knees of the gods, to avoid the pathogen contamination such as virus in culture containing animal blood serum
With the allergic reaction caused by foreign sera, have been reported that develop the culture medium for not containing serum to cultivate stem cell at present, but
Income effect is not satisfactory.
Therefore, further exploitation is very necessary suitable for the culture medium without serum of umbilical cord stem cells.
The content of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of cell culture medium and dry in umbilical cord mesenchyma
Animal blood serum is not included in application in cell culture, the culture medium that the present invention is provided, the culture medium provided using the present invention is trained
Support the umbilical cord mesenchymal stem cells dryness obtained and keep good, the cell cycle is short, and multiplication capacity is strong.
The cell culture medium that the present invention is provided includes basal medium, EGF, NEAA, glutamine and conditioned medium；
The conditioned medium is with gained after DMEM/F12 base culture base umbilical cord mesenchyma mescenchymal stem cells
Supernatant.
In some embodiments, the preparation method of conditioned medium that the present invention is used for：P3 is filled between umbilical cord mesenchyma
It is inoculated with after the recovery of matter stem cell into DMEM/F12 basal mediums in 5%CO2, 37 DEG C, the CO that saturated humidity is 95%2Incubator
Middle culture；Collected per 24h after culture supernatant, be supplemented new DMEM/F12 basal mediums；Collect 3 times altogether.
Specifically, the preparation method for the conditioned medium that the present invention is used includes：
Step 1：P3 is taken for umbilical cord mesenchyma mescenchymal stem cell, it is inoculated in 15cm culture dishes, by mescenchymal stem cell
It is transferred to 5%CO2, 37 DEG C, the CO that saturated humidity is 95%2Recovered in incubator.When mescenchymal stem cell degree of converging up to 80%~
When 90%；Cell is cleaned 2 times with cell cleaning fluid；
Step 2：20ml DMEM/F12 basal mediums are added in each culture dish, mescenchymal stem cell is transferred to 5%
CO2, 37 DEG C, the CO that saturated humidity is 95%2Cultivated in incubator；
Step 3：After 24h, culture supernatant is collected in receiving flask, and the new DMEM/F12 bases of 20ml are added in each culture dish
Basal culture medium；
Step 4：Again after 24h, culture supernatant is collected in receiving flask, and the new DMEM/F12 of 20ml are added in each culture dish
Basal medium；
Step 5：Again after 24h, culture supernatant is collected in receiving flask；Merge the culture supernatant of 3 collections, obtain condition
Culture medium.
In the present invention, the volume fraction of conditioned medium is 25%~55%.
In some embodiments, the volume fraction of conditioned medium is 25%~35%；
In other embodiments, the volume fraction of conditioned medium is 35%~45%；
In other embodiments, the volume fraction of conditioned medium is 45%~50%；
In other embodiments, the volume fraction of conditioned medium is 50%~55%.
Epithelical cell growth factor (Epidermalgrowthfactor, EGF) can promote epithelial cell, fibroblast
Increment；Strengthen the vigor of epidermal cell；Delay the aging of epidermal cell.The EGF of high activity could promote point of Skin Cell
Split and grow, it can also stimulate the synthesis and secretion of some extracellular macromoleculars (such as hyaluronic acid and collagen are white) in addition.
In the present invention, EGF concentration is 4.5ng/mL~5.5ng/mL.
Nonessential amino acid (non-essentialaminoacid, NEAA), includes nonessential ammonia needed for 7 kinds of cell culture
Base acid, i.e. aspartic acid, asparagine, glycine, proline, glutamic acid, serine, hydroxyproline.Nonessential amino acid
Addition can increase the reproductive capacity of cell, extend the life span of cell.NEAA can buy for voluntarily configuration or market, this
Invention is not limited this, and it is implemented all within protection scope of the present invention.The NEAA that the present invention is used is purchased from sigma's
100×NEAA.In the present invention, NEAA volume fraction is 1%.
Glutamine, scientific name 2- amino -4- carbamyl butyric acid, English Glutamine (Gln).Glutamine is in cell
Played an important role in metabolic process, Glu can be used as the energy source of culture cell, the synthesis of participation protein and core
Acid metabolic.Glutamine can be bought for voluntarily configuration or market, and the present invention is not limited this, and it is implemented all in the present invention
Protection domain within.The glutamine that the present invention is used is purchased from sigma 100 × glutamine.In the present invention, paddy ammonia
The volume fraction of acid amides is 1%.
In the present invention, basal medium is DMEM/F12 culture mediums.
In some embodiments, the cell culture medium that the present invention is provided includes：
In some embodiments, the cell culture medium that the present invention is provided includes：
In some embodiments, the cell culture medium that the present invention is provided includes：
In some embodiments, the cell culture medium that the present invention is provided includes：
The cell culture medium that the present invention is provided can promote the multiplication capacity of umbilical cord mesenchymal stem cells, and can effectively protect
Hold the dryness of stem cell.Experiment shows that the cell culture medium provided using the present invention is cultivated umbilical cord mesenchymal stem cells
Application of the cell culture medium that the present invention is provided in culture umbilical cord mesenchymal stem cells.
The umbilical cord mesenchymal stem cells are human umbilical cord mesenchymal stem cells.
Present invention also offers a kind of method for cultivating umbilical cord mesenchymal stem cells, the cell culture medium provided with the present invention
Umbilical cord mesenchymal stem cells are cultivated, culture to attached cell degree of converging is passed on up to 80%~90%.
In the present invention, the condition of culture is 37 DEG C, CO2Volume fraction 5%, incubation time is 48h~72h.
In the present invention, after passage is using mass fraction as 0.25% trypsin digestion and cell, according to 1 × 105Individual/ml
Density the culture medium that provides of the present invention be provided cultivated.
The present invention addition, EGF, NEAA, glutamine and conditioned medium in basal medium, each component is jointly to tooth
The growth of marrow stem cell serves good facilitation.Experiment shows, between the medium culture umbilical cord provided using the present invention
Mesenchymal stem cells, can shorten the cell cycle, improve ability of cell proliferation, improve the purity and dryness of stem cell, and the culture
Base is without animal derived components such as hyclones, and the stem cell obtained by the culture medium is suitable for clinical practice.
Embodiment
The invention provides a kind of cell culture medium and the application in umbilical cord mesenchymal stem cells culture, art technology
Personnel can use for reference present disclosure, be suitably modified technological parameter realization.In particular, all similar replacements and change
Dynamic apparent to those skilled in the art, they are considered as being included in the present invention.The present invention method and should
With being described by preferred embodiment, related personnel substantially can be not departing from present invention, in spirit and scope
Methods herein and application are modified or suitably change is with combining, to realize and apply the technology of the present invention.
The examination material that the present invention is used is all common commercially available product, can all be bought in market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1~4
1), preparation condition culture medium：
Step 1：P3 is taken for umbilical cord mesenchyma mescenchymal stem cell, it is inoculated in 15cm culture dishes, by mescenchymal stem cell
It is transferred to 5%CO2, 37 DEG C, the CO that saturated humidity is 95%2Recovered in incubator.When mescenchymal stem cell degree of converging up to 80%~
When 90%；Cell is cleaned 2 times with cell cleaning fluid；
Step 2：20ml DMEM/F12 basal mediums are added in each culture dish, mescenchymal stem cell is transferred to 5%
CO2, 37 DEG C, the CO that saturated humidity is 95%2Cultivated in incubator；
Step 3：After 24h, culture supernatant is collected in receiving flask, and the new DMEM/F12 bases of 20ml are added in each culture dish
Basal culture medium；
Step 4：Again after 24h, culture supernatant is collected in receiving flask, and the new DMEM/F12 of 20ml are added in each culture dish
Basal medium；
Step 5：Again after 24h, culture supernatant is collected in receiving flask；Merge the culture supernatant of 3 collections, obtain condition
Culture medium.
2), according to the recipe configuration culture medium of table 1.
The embodiment 1~4 of table 1
3), culture medium quality testing
Microorganism detection and endotoxin detection detection are carried out to culture medium made from embodiment 1~4.Wherein, microorganism is examined
(bacterium, fungi) is surveyed to be detected according to the 4th general rule 1100 of version Pharmacopoeia of People's Republic of China in 2015；Endotoxin detects root
Detected according to the 4th general rule 1143 of version Pharmacopoeia of People's Republic of China in 2015.Testing result：Embodiment 1~4 is obtained to be trained
Base is supported in bacterium, fungi and endotoxic detection, is negative.Illustrate the up-to-standard of the culture medium.
4), cell culture effect detection
P2 is inoculated in culture medium made from embodiment 1~4 for umbilical cord mesenchymal stem cells respectively, inoculum density is 1 ×
105Individual/ml, the condition of culture is 37 DEG C, CO2Then volume fraction 5%, saturated humidity culture to degree of converging 100% is observed thin
Born of the same parents' dryness, Cell viability.As a result such as table 2：
The cell culture result of table 2
| Group | Flow cytometer detection result | Form | The time required to degree of converging 100% | Cell viability |
| Embodiment 1 | It is qualified | Typically | 3~4 days | 96.91% |
| Embodiment 2 | It is qualified | Preferably | 3 days | 96.96% |
| Embodiment 3 | It is qualified | It is best | 2~2.5 days | 97.21% |
| Embodiment 4 | It is qualified | It is best | 2~2.5 days | 97.15% |
The streaming is qualified refer to CD73+ >=95%, CD90+ >=95%, CD105+ >=95%, HLA-DR+≤2%,
CD45+≤2%, CD34+≤2%, CD11b+≤2%, CD19+≤2%；
Form refers to whether cellular morphology is fusiformis, and whether shape is homogeneous.
As a result show, the medium culture umbilical cord mesenchymal stem cells provided with the present invention, gained cell dryness keeps good
Good, form meets umbilical cord mesenchymal stem cells form, and degree of converging reached 100% required time less than 4 days, and Cell viability is higher than
96%.
It the above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art
Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of cell culture medium, it is characterised in that including basal medium, EGF, NEAA, glutamine and conditioned medium；
The conditioned medium is with gained supernatant after DMEM/F12 base culture base umbilical cord mesenchyma mescenchymal stem cells
Liquid.
2. cell culture medium according to claim 1, it is characterised in that the volume fraction of the conditioned medium is 25%
~55%.
3. cell culture medium according to claim 1, it is characterised in that the concentration of the EGF be 4.5ng/mL~
5.5ng/mL。
4. cell culture medium according to claim 1, it is characterised in that the volume fraction of the NEAA is 1%.
5. cell culture medium according to claim 1, it is characterised in that the volume fraction of the glutamine is 1%.
6. cell culture medium according to claim 1, it is characterised in that the basal medium is cultivated for DMEM/F12
Base.
7. application of any one of claim 1~6 cell culture medium in culture umbilical cord mesenchymal stem cells.
8. a kind of method for cultivating umbilical cord mesenchymal stem cells, it is characterised in that with any one of claim 1~6 cell
Medium culture umbilical cord mesenchymal stem cells, culture to attached cell degree of converging is passed on up to 80%~90%.
9. method according to claim 8, it is characterised in that the condition of the culture is 37 DEG C, CO2Volume fraction 5%,
Cultivate 48h~72h passages.
10. method according to claim 8, it is characterised in that tryptose of the passage using mass fraction as 0.25%
After enzymic digestion cell, according to 1 × 105The culture medium that individual/ml density is seeded to described in any one of claim 1~6 is trained
Support.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343660A (en) * | 2018-04-08 | 2019-10-18 | 生物角（厦门）科技有限公司 | A kind of mesenchymal stem cell serum-free culture medium composition |
| CN111004777A (en) * | 2019-12-10 | 2020-04-14 | 广东国科细胞科技有限公司 | Material supplementing and culturing method for mesenchymal stem cell culture |
| CN113621566A (en) * | 2021-06-16 | 2021-11-09 | 广州赛莱拉干细胞科技股份有限公司 | Umbilical cord mesenchymal stem cell separation culture method |
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| CN110343660A (en) * | 2018-04-08 | 2019-10-18 | 生物角（厦门）科技有限公司 | A kind of mesenchymal stem cell serum-free culture medium composition |
| CN111004777A (en) * | 2019-12-10 | 2020-04-14 | 广东国科细胞科技有限公司 | Material supplementing and culturing method for mesenchymal stem cell culture |
| CN113621566A (en) * | 2021-06-16 | 2021-11-09 | 广州赛莱拉干细胞科技股份有限公司 | Umbilical cord mesenchymal stem cell separation culture method |
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