CN106434543A - Culture medium and cell cultural method - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention relates to the field of cells, in particular to a culture medium and a cell cultural method. The culture medium comprises a serum-free medium, a basic fibroblast growth factor (bFGF) and an epidermal growth factor (EGF). On the basis of adopting a mesenchymal stem cell serum-free medium, the two growth factors, i.e., the bFGF and the EGF are added, so that the risk brought by animal-derived serum can be avoided, and the in vitro amplification ability of gingiva-derived mesenchymal stem cells (GMSCs) is effectively improved.
Description
Technical field
The present invention relates to cell field, particularly to a kind of culture medium and cell culture processes.
Background technology
Mescenchymal stem cell is the stem cell that a class has self renewal, propagation and multi-lineage potential, first in 1966
First found from bone marrow by Friedenstein etc..Numerous studies find, mescenchymal stem cell not only can be divided into multiple
The cell of stroma, such as bone, fat, cartilage, muscle, tendon and ligament etc..Simultaneously acceptable under certain inductive condition
Transdifferentiationof is ectoderm and endoderm cell.As epithelial cell, neuronal cell, neurogliocyte, vascular endothelial cell,
Epidermal stem cells etc..Current study show that, source for mesenchymal stem cells is quite varied, in addition to bone marrow, fat, umbilical cord Wharton jelly,
Placenta Hominiss, amniotic membrane, muscle, ligament etc. are all separated to turn out.Numerous studies prove the cranium Maxillary region of human body and animal in recent years, special
It is not in Odontogenic cysts tissue, to there is the mescenchymal stem cell with special differentiation and regeneration function.In recent years because it is in organizational project
Receive much concern with the preclinical study of cell replacement therapy.
People's gingiva mescenchymal stem cell (gingiva-derived mesenchymal stem cells, GMSCs) is group
Become the main mesenchymal cell of gingival connective tissue.It is derived from mesoblastic fibroblast, not only have active from
The ability that I updates, and also there is the function of the extracellular matrix such as synthesis and degraded collagen：I type and III Collagen Type VI, fiber glue
Even albumen etc..Therefore GF plays a very important role in many physiology and pathological process, and collagen can be maintained dynamically to put down
Weighing apparatus, adjusts cell interaction, protection and reparation gingiva tissue, maintains gingiva tissue self stability.
The irreversibility that periodontal disease causes supporting tissue around tooth is damaged, and is now widely used in controlling of periodontal regenerative
Treatment method fails to fully achieve the physiological of periodontal tissue and functional regeneration.Periodontal tissue engineering technology develop into periodontal
The reparation of defective tissue and regeneration provide new thinking and space., since being found, it is good dry for periodontal ligament stem cell
Cellularity just has been obtained for being widely recognized as, but its limited source, need to have tooth pulled out and could obtain periodontal membrane tissue so as to clinical practice
Potential be restricted.And in tissue engineering research, seek the always research of wide material sources, convenient seed cell of drawing materials
Emphasis and core.Be both the gingiva tissue in periodontal tissue source, then have draw materials conveniently, organization healing ability strong and can
The feature that no scar heals.Therefore, the gingiva mescenchymal stem cell from gingiva tissue received highest attention in recent years.
It is 10% hyclone+DMEM/F12 that the cultural method that the current amplification in vitro of GMSCs adopts is mostly volume fraction
Or volume fraction is 10% hyclone+low sugar DMEM culture medium, but because hyclone increased virus infection and xenogenesis egg
, thus there are potential risks in clinical practice in the possibility of led to allogene immunne response in vain.
Content of the invention
In view of this, the present invention provides a kind of culture medium and cell culture processes.The present invention is open a kind of more efficiently
GMSCs amplification in vitro method, on the basis of using mesenchymal stem cell serum-free culture medium, add proper proportion bFGF and
Two kinds of somatomedin of EGF, had both avoided the risk that animal sources serum is brought, and more effective raising GMSCs amplification in vitro energy
Power.
In order to realize foregoing invention purpose, the present invention provides technical scheme below：
The invention provides a kind of culture medium, including serum-free medium, bFGF and EGF.
Basic fibroblast growth factor (Basic Fibroblast Growth Factor, bFGF) is containing 155
The mitogenetic cationic polypeptide of aminoacid, molecular weight is 16～18.5KD.BFGF has extensive biological action, right
The growth of various kinds of cell, differentiation and function have an impact, and play a role in normal physiological and pathological process, and its principal biological is made
With having：(1) as angiogenesis factor；(2) wound healing and tissue repair are promoted；(3) promotion organization regeneration；(4) participate in god
Through regeneration etc..Epidermal growth factor (Epidermal Growth Factor, EGF) is a kind of small peptide, by 53 amino acid residues
Composition, EGF is a kind of multi-functional somatomedin, all has strong rush splitting action to Various Tissues cell in vitro in vivo.
In some specific embodiments of the present invention, the concentration of bFGF described in described culture medium is 5～25ng/ml.
In some specific embodiments of the present invention, described in described culture medium, described in culture medium, the concentration of bFGF is
10ng/ml.
In some specific embodiments of the present invention, the concentration of EGF described in described culture medium is 5～25ng/ml.
In some specific embodiments of the present invention, the concentration of EGF described in described culture medium is 10ng/ml.
Present invention also offers application in cultured cells for the described culture medium.
In some specific embodiments of the present invention, described in described application, cell is mescenchymal stem cell.
In some specific embodiments of the present invention, described in described application, cell is gingiva mescenchymal stem cell.
Additionally, present invention also offers a kind of cell culture processes, comprising the steps：
Step 1：Obtain frozen cell；
Step 2：Washing after taking the cell that step 1 obtains to mix with serum-free medium, abandons supernatant, then provides with the present invention
Culture medium mixing recovery, continue culture.
In some specific embodiments of the present invention, in described cell culture processes, the culture medium of present invention offer adds
Entering amount is every 5 × 104Individual cell adds culture medium described in 1mL.
In some specific embodiments of the present invention, recover described in described cell culture processes described frozen for taking
Cell dissolves in 37 DEG C of water-baths, after 1～2min liquid melts, with 10mL serum-free medium diluting cells suspension, 1000r/
Min is centrifuged 5min, abandons supernatant；The culture medium re-suspended cell that the 10mL present invention provides, by 5 × 104Individual/mL density cells inoculation,
In 5%CO2Under conditions of 37 DEG C culture.
In some specific embodiments of the present invention, continue culture described in described cell culture processes for treating that cell is long
Full 80～90%, discard culture fluid, add 0.25% trypsin, digest 1～3min, Microscopic observation cellular contraction becomes bowlder,
Add the culture medium that the present invention provides to terminate digestion, collect cell.
The present invention discloses a kind of more efficiently GMSCs amplification in vitro culture medium and cultural method, is being done using mesenchyme
On the basis of cell non-serum culture medium, add two kinds of somatomedin of bFGF and EGF, especially 10ng/mLbFGF and 10ng/mL
Two kinds of somatomedin of EGF, had both avoided the risk that animal sources serum is brought, and more effective raising GMSCs amplification in vitro energy
Power.
Brief description
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
The accompanying drawing having required use in technology description is briefly described.
Fig. 1 shows comparative example and embodiment 3GMSCs aspect graph；
Fig. 2 shows each group total viable cell average ratio relatively；
Fig. 3 shows each group Cell viability average ratio relatively；
Fig. 4 shows comparative example GMSCs growth curve chart；
Fig. 5 shows embodiment 3GMSCs growth curve chart.
Specific embodiment
The invention discloses a kind of culture medium and cell culture processes, those skilled in the art can use for reference present disclosure,
It is suitably modified technological parameter to realize.Specifically, all similar replacements and change come to those skilled in the art
Say it is it will be apparent that they are considered as including in the present invention.Preferred embodiment has been passed through in the method for the present invention and application
Be described, related personnel substantially can in without departing from present invention, spirit and scope to method described herein and should
Be modified or suitably change and combine, to realize and to apply the technology of the present invention.
The GMSCs of the present invention culture medium that effectively amplification method is used in vitro do not contain animal sources serum, it is to avoid draw
Enter pollution and the risk of anaphylactogen, there is compared with regular growth cultural method higher clinical safety.
The effective amplification method of GMSCs of the present invention more effectively improves GMSCs amplification in vitro ability.
In culture medium that the present invention provides and cell culture processes, raw materials used and reagent all can be buied by market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
Take the GMSCs that the 3rd generation is frozen, cryopreservation tube takes out and is immediately placed in dissolving in 37 DEG C of water-baths, needs not in course of dissolution
Break and rock cryopreservation tube.After 1-2min liquid melts, with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension (with training
Foster base washes one time cryopreservation tube), 1000r/min is centrifuged 5min, abandons supernatant.10mL complete medium re-suspended cell, by 5 × 104
Individual/mL density cells are inoculated in 10cm culture dish, put into 5%CO237 DEG C of cultures of incubator.Treat that cell covers with 80-90%, use
Suction pipe is inhaled and is abandoned old culture fluid, adds 2～3mL 0.25% trypsin, digests 1～3 minute, Microscopic observation cellular contraction becomes round
When, add 5mL complete medium to terminate digestion immediately, collect cell, carry out cell counting.
Complete medium formula is：5ng/mLbFGF+5ng/mL EGF+ mesenchymal stem cell serum-free culture medium.(its
In, the meaning of 5n g/mL is：5ng somatomedin is contained, similarly hereinafter in every milliliter of mesenchymal stem cell serum-free culture medium.)
Embodiment 2
Take the GMSCs that the 3rd generation is frozen, cryopreservation tube takes out and is immediately placed in dissolving in 37 DEG C of water-baths, needs not in course of dissolution
Break and rock cryopreservation tube.After 1-2min liquid melts, with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension (with training
Foster base washes one time cryopreservation tube), 1000r/min is centrifuged 5min, abandons supernatant.10mL complete medium re-suspended cell, by 5 × 104
Individual/mL density cells are inoculated in 10cm culture dish, put into 5%CO237 DEG C of cultures of incubator.Treat that cell covers with 80-90%, use
Suction pipe is inhaled and is abandoned old culture fluid, adds 2～3mL 0.25% trypsin, digests 1-3 minute, and Microscopic observation cellular contraction becomes round
When, add 5mL complete medium to terminate digestion immediately, collect cell, carry out cell counting.
Complete medium formula is：10ng/mLbFGF+5ng/mLEGF+ mesenchymal stem cell serum-free culture medium.
Embodiment 3
Take the GMSCs that the 3rd generation is frozen, cryopreservation tube takes out and is immediately placed in dissolving in 37 DEG C of water-baths, needs not in course of dissolution
Break and rock cryopreservation tube.After 1-2min liquid melts, with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension (with training
Foster base washes one time cryopreservation tube), 1000r/min is centrifuged 5min, abandons supernatant.10mL complete medium re-suspended cell, by 5 × 104
Individual/mL density cells are inoculated in 10cm culture dish, put into 5%CO237 DEG C of cultures of incubator.Treat that cell covers with 80-90%, use
Suction pipe is inhaled and is abandoned old culture fluid, adds 2～3mL0.25% trypsin, digests 1-3 minute, and Microscopic observation cellular contraction becomes bowlder,
Add 5mL complete medium to terminate digestion immediately, collect cell, carry out cell counting.
Complete medium formula is：10ng/mLbFGF+10ng/mLEGF+ mesenchymal stem cell serum-free culture medium.
Embodiment 4
Take the GMSCs that the 3rd generation is frozen, cryopreservation tube takes out and is immediately placed in dissolving in 37 DEG C of water-baths, needs not in course of dissolution
Break and rock cryopreservation tube.After 1-2min liquid melts, with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension (with training
Foster base washes one time cryopreservation tube), 1000r/min is centrifuged 5min, abandons supernatant.10mL complete medium re-suspended cell, by 5 × 104
Individual/mL density cells are inoculated in 10cm culture dish, put into 5%CO237 DEG C of cultures of incubator.Treat that cell covers with 80-90%, use
Suction pipe is inhaled and is abandoned old culture fluid, adds 2～3mL0.25% trypsin, digests 1-3 minute, and Microscopic observation cellular contraction becomes bowlder,
Add 5mL complete medium to terminate digestion immediately, collect cell, carry out cell counting.
Complete medium formula is：15ng/mLbFGF+10ng/mL EGF+ mesenchymal stem cell serum-free culture medium.
Embodiment 5
Take the GMSCs that the 3rd generation is frozen, cryopreservation tube takes out and is immediately placed in dissolving in 37 DEG C of water-baths, needs not in course of dissolution
Break and rock cryopreservation tube.After 1-2min liquid melts, with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension (with training
Foster base washes one time cryopreservation tube), 1000r/min is centrifuged 5min, abandons supernatant.10mL complete medium re-suspended cell, by 5 × 104
Individual/mL density cells are inoculated in 10cm culture dish, put into 5%CO237 DEG C of cultures of incubator.Treat that cell covers with 80-90%, use
Suction pipe is inhaled and is abandoned old culture fluid, adds 2～3mL 0.25% trypsin, digests 1-3 minute, and Microscopic observation cellular contraction becomes round
When, add 5mL complete medium to terminate digestion immediately, collect cell, carry out cell counting.
Complete medium formula is：10ng/mLbFGF+15ng/mL EGF+ mesenchymal stem cell serum-free culture medium.
Embodiment 6
Take the GMSCs that the 3rd generation is frozen, cryopreservation tube takes out and is immediately placed in dissolving in 37 DEG C of water-baths, needs not in course of dissolution
Break and rock cryopreservation tube.After 1-2min liquid melts, with 10mL mesenchymal stem cell serum-free culture medium diluting cells suspension (with training
Foster base washes one time cryopreservation tube), 1000r/min is centrifuged 5min, abandons supernatant.10mL complete medium re-suspended cell, by 5 × 104
Individual/mL density cells are inoculated in 10cm culture dish, put into 5%CO237 DEG C of cultures of incubator.Treat that cell covers with 80-90%, use
Suction pipe is inhaled and is abandoned old culture fluid, adds 2～3mL 0.25% trypsin, digests 1-3 minute, and Microscopic observation cellular contraction becomes round
When, add 5mL complete medium to terminate digestion immediately, collect cell, carry out cell counting.
Complete medium formula is：25ng/mLbFGF+25ng/mL EGF+ mesenchymal stem cell serum-free culture medium.
Comparative example
Take the GMSCs that the 3rd generation is frozen, cryopreservation tube takes out and is immediately placed in dissolving in 37 DEG C of water-baths, needs not in course of dissolution
Break and rock cryopreservation tube.After 1-2min liquid melts, (use culture medium with 10mL DMEM/F12 basal medium diluting cells suspension
Cryopreservation tube is washed one time), 1000r/min is centrifuged 5min, abandons supernatant.10mL complete medium re-suspended cell, by 5 × 104Individual/ml
Density cells are inoculated in 10cm culture dish, put into 5%CO237 DEG C of cultures of incubator.Treat that cell covers with 80-90%, use suction pipe
Old culture fluid is abandoned in suction, adds 2～3mL0.25% trypsin, digests 1-3 minute, and Microscopic observation cellular contraction becomes bowlder, immediately
Add 5mL complete medium to terminate digestion, collect cell, carry out cell counting.
Complete medium formula is：10% hyclone+DMEM/F12 basal medium.
Embodiment 8
The statistical data of comparative example and each embodiment is compared.Result shows, embodiment 1, embodiment 6 and contrast
Example has significant difference (*, p<0.05)；Remaining embodiment and comparative example have pole significant difference (* *, p<0.01)；Embodiment 3
There are pole significant difference (※, p with other embodiment<0.01).
Result is as shown in table 1 and Fig. 2：
The each experimental group cell counts of table 1
Note：* represent p<0.05；*, ※ represent p<0.01.
The each experimental group Cell viability meansigma methodss of table 2
Note：*, ※ represents p<0.01.
Table 2 and Fig. 3 result show, there was no significant difference for embodiment 1, embodiment 6 Cell viability and comparative example；Embodiment 2,
4th, 5 have pole significant difference (*, p with comparative example, embodiment 1,6<0.01)；Embodiment 3 and remaining each group all have pole significance poor
Different (※, p<0.01).
Embodiment 9
Take P3 for GMSCs, be respectively adopted the culture medium prescription in comparative example and embodiment 3, cell presses 1 × 104Individual/mL is close
Degree is inoculated in 24 orifice plates, changes liquid once within the 4th day.Daily cell of collecting carries out cell counting, collects every time and calculates 3 holes, continuously
7 days.Draw cell growth curve.
Experimental result is as shown in Fig. 1～Fig. 3, table 3～table 4.
The daily count results of table 3 comparative example GMSCs
The daily count results of table 4 embodiment 3 GMSCs
Note：* represent p<0.01.
The data creating growth curve that comparative example is obtained with embodiment 3 cell counting, result show (table 3, table 4, Fig. 4,
Fig. 5), mescenchymal stem cell is met using the GMSCs growth curve of the mesenchymal stem cell serum-free culture medium culture of the present invention
The rule of proliferation of " S " type；Cell culture the 5th, 6,7 days, experimental group 3 GMSCs proliferation activity (same Initial seeding density,
Cultivated 5,6,7 days, cell counting compare understand.) (*, p is significantly increased than the GMSCs of comparative example<0.01), illustrate this
Used in invention, culture medium prescription is more suitable for GMSCs cellar culture.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
For member, 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
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of culture medium is it is characterised in that include serum-free medium, bFGF and EGF.
2. culture medium according to claim 1 is it is characterised in that the concentration of described bFGF is 5～25ng/ml.
3. culture medium according to claim 1 and 2 is it is characterised in that the concentration of described EGF is 5～25ng/ml.
4. application in cultured cells for the culture medium according to any one of claims 1 to 3.
5. application according to claim 4 is it is characterised in that described cell is mescenchymal stem cell.
6. application according to claim 5 is it is characterised in that described cell is gingiva mescenchymal stem cell.
7. a kind of cell culture processes are it is characterised in that comprise the steps：
Step 1：Obtain frozen cell；
Step 2：Washing after taking the cell that step 1 obtains to be mixed with serum-free medium, is abandoned supernatant, then is appointed with claims 1 to 3
Culture medium mixing recovery described in one, continues culture.
8. cell culture processes according to claim 7 are it is characterised in that culture described in any one of claims 1 to 3
The addition of base is every 5 × 104Individual cell adds culture medium described in 1mL.
9. cell culture processes according to claim 7 it is characterised in that described recovery be take described frozen cell in
Dissolve in 37 DEG C of water-baths, after 1～2min liquid melts, with 10mL serum-free medium diluting cells suspension, 1000r/min centrifugation
5min, abandons supernatant；Culture medium re-suspended cell as described in any one of claims 1 to 3 for the 10ml, by 5 × 104Individual/mL density is thin
Born of the same parents inoculate, in 5%CO2Under conditions of 37 DEG C culture.
10. cell culture processes according to claim 7 are it is characterised in that described continuation is cultivated as treating that cell covers with 80
～90%, discard culture fluid, add 0.25% trypsin, digest 1～3min, Microscopic observation cellular contraction becomes bowlder, add
Culture medium as described in any one of claims 1 to 3 terminates digestion, collects cell.
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