CN107151647A - A kind of epidermal cell large-scale cultivation method - Google Patents
A kind of epidermal cell large-scale cultivation method Download PDFInfo
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Abstract
The invention discloses a kind of epidermal cell large-scale cultivation method, the epidermis seed cell after amplification and nutrient solution are mixed after being resuspended, are uniformly inoculated in cell factory, are put into CO2Incubator, in 37 DEG C, 5% CO2Cultivated under concentration conditions, according to harvesting after passage demand culture for a period of time；To cell factory add digestive juice, be incubated to most cells it is no longer adherent when, immediately by liquid in factory be transferred in the container equipped with terminate liquid terminate digest；Then continuously add the digestive juice of same volume to cell factory, be incubated to all cells it is no longer adherent when, then add into cell factory the terminate liquid of same volume and terminate digestion；The all liq after terminating twice is collected, centrifugation removal of impurities obtains the good epidermal cell of large-scale culture.The present invention once culture can to obtain a large amount of state consistencies good, heteroproteose cell content is considerably less or without heteroproteose cell, and the epidermal cell of steady quality saves incubation time, reduces pollution risk.
Description
Technical field
The invention belongs to technical field of cell culture, and in particular to a kind of epidermal cell large-scale cultivation method.
Background technology
Epidermal cell is the raw material and research tool that biomedical and field of tissue engineering technology is commonly used, wherein medicine and cosmetic
Screening, the assessment of potency, safety evaluatio of raw material etc. all refer to the application of epidermal cell in product exploitation.For example, in tissue
The structure of the various organizational project vitro detection skin models of engineering field, the organization engineering skin for disease treatments such as burn and scalds
Structure, the culture of cell patch etc..It is good that use above is both needed to a large amount of state consistencies, and cell purity is high, the table of steady quality
Chrotoplast.
It is rarely reported currently for the large-scale cultivation method of epidermal cell, the large-scale culture of cell should prevent dirt first
Dye, obtains sufficient amount cell, it is ensured that cell quality is stable, state consistency is to obtain reliable experimental data and the premise of product.
Current conventional cell cultural method has following several：
（1）Microcarrier training method：Need to be using complete set of equipments such as reactors, cost is high, and the construction period is long, is unfavorable for amplification, by
Laboratory bottle, which is supported, turns large-scale production operating process complexity, and technical requirements are high, and parameter determines that the time is long.
（2）Using trophoderm culture：It is mixed into more heteroproteose cell unavoidably in cell acquisition process, it is impossible to obtain purity high
Epidermal cell, it is impossible to be used in for Journal of Sex Research, various cells mix in large-scale production, and cell proportion is inconsistent, it is impossible to obtain
The clear and definite quantity of certain cell.In trophocyte's breeding is suppressed using mitomycin processing, due to the inhibitory action
Non-selectivity, therefore residual mitomycin can equally have an impact to propagation；According to irradiation, dose fluctuations can not be controlled accurately, such as
Really lethal Fb quantity is not enough or excessive, can directly affect nutritional ingredient needed for epidermal growth, factor concentration, and epidermis is thin
Born of the same parents have contact inhibition characteristic, if trophocyte's excessive proliferation, the acquisition quantity of epidermal cell are directly affected, moreover, irradiated
The sterile of journey is difficult to ensure that；Living cells general unit in irradiation can not meet ageing requirement.For example, in Chinese patent
CN200910155159 adds trophoblastic method containing serum free culture system, this method nutrient solution complicated component, trophoderm using microcarrier
Cell category is more, and handling process is cumbersome or is difficult to quantify, and Determination of Calcium in Serum ion can be obviously promoted epidermal cell differentiation.
（3）Serum-containing media：Calcium ion in serum is a key factor for promoting epidermal cell to break up, while carefully
The following amplification number of times for the cell that born of the same parents' differentiation is also limited.For example, mainly passing through training in Chinese patent CN201610 364492
Support the exploitation of base to reduce cost, so that large-scale culture becomes feasible from cost consideration angle, but be not directed to specific cell
Content in terms of Quality advance or in stable condition or purity.
According to above-mentioned classical culture protocols, inoculum density is too low in large-scale culture, and cell is difficult survival, it is also difficult to obtained
Obtain steady quality, the epidermal cell that state consistency is good, purity is high.
The content of the invention
In order to solve drawbacks described above, the present invention can provide a kind of epidermal cell large-scale cultivation method, can be in the short time
The epidermal cell that a large amount of steady qualities, state consistency are good, purity is high is obtained in vitro, is solved prior art and is difficult to advising greatly
The problem of obtaining the epidermal cell that state consistency is good, purity is high of mould.
The technical solution adopted in the present invention is, intensive cell injuring model mode, by controlling inoculum density, training
Nutrient solution addition, increase gas are forced to exchange, change cell dissociation mode etc., for the pH needed for cell provides cultured in vitro, ooze
Saturating pressure, nutriment, Auto-regulator etc., so as to obtain the cell of a large amount of uniform qualities.The epidermal cell large-scale cultivation method
Specifically include following steps：
Step 1, it is inoculated with
Epidermis seed cell and nutrient solution are mixed after being resuspended, are uniformly inoculated in cell factory, it is ensured that every layer of inoculating cell number
Amount is consistent；
Step 2, cultivate
The epidermal cell being inoculated with is put into CO2Incubator, in 37 DEG C, 5% CO2Cultivated under concentration conditions, according to passage
Harvesting after demand culture for a period of time；
Step 3, digest
Digestive juice, CO are added to cell factory237 DEG C of incubations of incubator, when most cells are no longer adherent, immediately by factory
Interior liquid, which is transferred in the container equipped with terminate liquid, terminates digestion；Then the digestion of same volume is continuously added to cell factory
Liquid, CO237 DEG C of incubator be incubated to all cells it is no longer adherent when, then add into cell factory the terminate liquid of same volume
Terminate digestion；
Step 4, centrifuge
The all liq after terminating twice is collected, supernatant is abandoned in centrifugation, is resuspended with PBS balanced salt solutions, supernatant is abandoned in centrifugation again, is obtained
To the good epidermal cell of large-scale culture.
The features of the present invention is also resided in：
Further, the inoculum concentration being inoculated with described in step 1 is：Cell is pressed by every cell factory 0.25L~1.25L liquid volume added
1.6×104/ ml~4 × 104/ ml is inoculated with after being resuspended.In vitro in culture cell, growth of the cell inoculation number to cell has shadow
Ring, suitable inoculum density can promote cell to breed, inoculum density is too low or the too high growing multiplication for being all unfavorable for cell.
If the volume fraction of culture medium and cell is more than 2000：1, growth substance permeates into extracellular excessive, will make IC
Less than minimal concentration, now cell can not breed again, and the pH value of culture medium becomes alkali, suppresses cell growth, cell rounding
Can not be adherent, or even cause cell death etc..If inoculum density is too high, the volume of each cell peripheral culture medium is down to
0.007mm3Hereinafter, due to diffusivity reduction, the concentration increase of intracellular biological matter easily makes metabolite reach saturation, energy
Amount source also exhausts quickly, hinders the propagation of cell.Therefore select suitable inoculum density will be according to the metabolism and growth of cell
Reproduction speed and requirements of one's work and determine.Typically it is metabolized vigorous, fast growth cell（Such as tumour cell）, it is inoculated with dense
Degree is preferably relatively low；And normal tissue cell growth is slower, metabolism is not vigorous enough, and inoculum density can be higher.
Further, the epidermal cell being inoculated with described in step 2 is in CO2When being cultivated in incubator, lead into cell factory
Enter air and CO2, make to keep 5% CO in cell factory2.Cell increasing and metabolic activity with cell quantity in growth course
Reinforcement, constantly discharge CO2, make culture medium souring, pH changes.Cell factory environment is relatively closed, and carbon dioxide hardly possible is overflow
Go out, so, in order to maintain the pH that culture environment is constant, while the need for meeting cell metabolism to oxygen, increasing thin using air pump
The convective exchange of gas in born of the same parents factory, it is ensured that cell growth ambient stable.
Further, digestive juice described in step 3 is tryplE digestive juices, and the digestive juice volume added twice is equal, twice
The digestive juice total amount of addition is 50 ~ 250ml.Accordingly, the terminate liquid is serum-free medium, the termination liquid added every time
Product is equal with the digestive juice volume added every time.Cell contacts digestive juice time difference, cell state difference in digestion process
The time for being digested can be caused to have differences, the cell digested continues to contact with digestive juice that cell can be caused for a long time
Membrane surface proteins are largely decomposed, and cause cell death or form floccule, and suction-operated of the floccule to cell is extremely strong, greatly
Amount cell is adsorbed to cause cell harvesting amount to reduce, and the cell adsorbed after post incoulation due to that adherent can not stretch, also can
It is dead.Therefore, the present invention is using digestion method twice, it is to avoid is first digested the cell got off and is contacted and shadow with digestive juice again for a long time
Ring cell quality.
Further, centrifugation leaves the heart 6 minutes for per minute 800 described in step 3.
The inventive method is applied to the large-scale culture of all epidermal cells, such as human epidermal cell, mouse epidermal cells, big
Mouse epidermal cell, pig epidermal cell and ox epidermal cell etc..Meanwhile, the present invention can select the table of initial inoculation according to passage demand
Chrotoplast algebraical sum inoculum density.
Beneficial effects of the present invention include it is following some：
（1）Once culture can obtain that a large amount of state consistencies are good, and heteroproteose cell content is considerably less or without heteroproteose cell, steady quality
Epidermal cell.
（2）Incubation time is saved, pollution risk is reduced.Cultivated with conventional flask, because most cells are in body
The Density dependence and contact inhibition phenomenon of outer culture, cell-seeding-density can not be too low, and cell access amount is big, reaches passage density
Time it is short, reaching passage density be accomplished by being passed on, otherwise cell begin to differentiation or apoptosis, this is not reaching in cell
It is undesirable before to usage quantity.And it is about the 1/3~1/6 of conventional method to use cell inoculum concentration of the present invention, in training
Support in container and grow 2-4 days, bred for about 3 generations from the comparison cell of harvesting quantity and inoculum concentration.Conventional method then needs
At least 6 days, and need within least every 2 days to pass on once, it is cumbersome, increase the probability of cell contamination.And reduce and passing
The mechanical damage caused for human users in digestion process to cell and the damage of the cell of digestive juice.
（3）Early investment is few, has saved equipment construction and condition of culture gropes the time.
Brief description of the drawings
Fig. 1 is the preoperative cell state figure of passage in the embodiment of the present invention 1；
Fig. 2 is the cellular morphology comparison diagram of conventional method and the method for the invention；Wherein, A is the P7 cell shapes of conventional method
State figure, B is the P14 cellular morphology figures of conventional method, and C is the P7 cellular morphology figures of the method for the invention, and D is of the present invention
The P14 cellular morphology figures of method；
Fig. 3 is the cell growth curve of conventional method and the method for the invention.
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description, but the present invention is not limited to
These embodiments.
The inventive method is applied to the large-scale culture of all epidermal cells, such as human epidermal cell, mouse epidermal cells, big
Mouse epidermal cell, pig epidermal cell and ox epidermal cell etc..
Embodiment 1
The present embodiment is illustrated exemplified by cultivating human epidermal cell.
1st, the acquisition of seed coat cell
Take less than 8 years old boy's Post operation health tissues about 2cm2, desinfection chamber is sent in 2 hours, is carried out with 75% alcohol-pickled 2-3,
Embathed in PBS solution, subcutaneous connective tissue is removed with scissors, cutting is organized as to the leather strap of the mm sizes of 3 mm × 9, by every
Bar 1ml digestive juices tryplE adds 1.2U/ml Dispase, and 4 DEG C of lucifuge digests 16-17 hours.
Separate epidermis and corium：Epidermis is torn in sterile working.
Collect epidermal cell：The epidermis torn is collected in container, 10 are digested at 36-37 DEG C with 25 ~ 65ml tryplE
Min, per 2min under concussion 3, digestion terminates to add and the isometric terminate liquid of digestive juice（Serum-free medium）, soft piping and druming, mistake
Liquid is transferred to centrifuge tube after filter, takes 10ul suspensions to count respectively, centrifugation, is centrifuged 6 minutes by 800 turns per minute.Centrifugation end is pressed
Every bottle 4 × 104~7 × 104Individual/ml inoculations.
Cell inoculated and cultured：Use cultured epidermal cell liquid（KcGrowth2500）By in each bottle 4 × 104~7 × 104Individual/
Cell is resuspended in ml, and the mL of suspension 10 is added altogether, makes cell dispersed.Blake bottle is placed in 37 DEG C, 5% CO2Carried out in incubator
Culture, 24h changes nutrient solution, and now cell marking is P0 generations.
2nd, epidermis is expanded
Treat that seed cell degrees of fusion reaches 50-65%, discard nutrient solution；Digestive juice tryplE, digestive juice are added in blake bottle
Cell is transferred to CO by minimum amount to cover whole cell culture face237 DEG C of incubation 5min of incubator, treat that 80-90%'s is thin
Born of the same parents are no longer adherent, add terminate liquid（Serum-free medium）, digestion is terminated, culture can not be rocked energetically in digestion operating process
Bottle, in order to avoid mechanical damage is caused to cell, or cell flocculation influence cell harvesting amount.Blown and beaten 6-8 times repeatedly with elbow straw,
Collect, should successively be carried out by from top to bottom during piping and druming, it is to avoid bubble is produced, 800rpm/min centrifugations 6min；After centrifugation terminates, abandon
Supernatant, cell precipitation is resuspended with serum free medium, is mixed, is taken 2 10 μ l cell suspensions to be counted respectively with cell counter, is counted
Number when can be dispelled with 5ml serum free mediums precipitation after add remaining liq again, during sampling respectively from the cell suspension of mixing,
Hypomere respectively takes two samples to carry out countings and averaged, and reduces the inaccurate of caused by extract operation count results.
3rd, cell culture
A cell is often passed on according to step 2 and breeds about 1 times, cell is reached into P4 generations, when P4 is resuspended for cell and is inoculated with, by every
Cell is pressed 2 × 10 by cell factory 0.25L liquid volume added4After/ml is resuspended, it is inoculated in cell factory.PBS or training are used before inoculation
Nutrient solution soaks cell attachment face, is conducive to cell inoculation uniform.Suspension should be rocked before inoculation uniformly, liquid is uniformly divided to
Each layer, it is ensured that be seeded to every confluent monolayer cells quantity consistent.
The cell being inoculated with is put into CO2Incubator, 37 DEG C, 5% gas concentration lwevel carry out culture 24 hours；Culture 24
Unscrew the lid of culture vessel one end after hour, air pump is put into CO2Other one layer in incubator, in another termination of culture vessel
Air filtration filter, air pump is connected with filter inlet end, adjusts gas transmission parameter, it is to avoid the container inner pressure caused by gas transmission is too fast
Rise and damage container, pollute.Cell fusion degree reaches 50~60% harvestings after cultivating 4-5 days.
Sterile working adds tryplE digestive juices 25ml into cell factory（Also volume available compares 4:1 mass fraction is
The digestive juice that 0.25% trypsase and 0.1%EDTA are voluntarily prepared）, digestive juice is divided equally to interlayer rapidly, rocking makes digestive juice
Cover whole cell culture face；It is transferred to CO237 DEG C of incubation 4min of incubator, treat that 60-70% cell is no longer adherent, move into pre-
First added with 25ml terminate liquids（It is preferred that serum-free medium, it is possible to use the DMEM nutrient solutions containing serum）Centrifuge tube.Again to thin
TryplE digestive juice 25ml are added in born of the same parents factory, 1~2min is incubated, the identical terminate liquids of 25ml are added, and it is rapid by terminate liquid
Respectively to interlayer, rock and mixed with digestive juice.The all liq after terminating twice is collected, per minute 800 leave the heart 6 minutes；From
Hearty cord beam abandons supernatant, is resuspended and counted with PBS balanced salt solutions, and step 3 is repeated after centrifugation and is carried out continuously culture, is obtained successively
P7, P14 are for cell human epidermal cell.
Large-scale culture also conventionally is carried out to the human epidermal cell got, step is as follows：In seed cell
A cell is often passed on according to step 2 after acquisition and breed about 1 times, cell is reached into P7, P14 generation, equally obtain P7, P14 generation thin
Born of the same parents' human epidermal cell.Two methods incubation time and cell harvesting amount are counted such as table 1.
The present invention of table 1 and conventional method cell harvesting amount compare
Count results are shown, harvest the cell of identical generation, this method is saved about 2-3 days time than classical culture protocols, identical
Incubation time this method cell amount to obtain is substantially reduced 2-4 times more than conventional method while reducing passage, digestion operation
Pollution risk.The cell of harvest can be directly used for follow-up skin and build in production or cell detection experiment.
Before passage operation（P4 generations）Cell take pictures under the microscope（Fig. 1）, traditionally with side of the present invention
Method carries out normally being passaged to P7, P14 for cell state such as（Fig. 2）, as a result show, identical cell uses conventional method and Ben Fa
Bright methods described is cultivated, and conventional method cellular morphology is poorer than the inventive method, and cell edges are spread out, nucleus disperse, core
Benevolence is not obvious, and senile cell accounting is larger, and the cell edges in the method for the invention P14 generations are clear, and senile cell accounting is bright
It is aobvious small compared with conventional method.
The P7 cells of the degrees of fusion about 60% of the 2nd step amplification method amplification of the invention are taken, respectively with conventional method and the present invention
Method presses 1.6E5/cm2Inoculation is cultivated, and traditional group collected a cell according to digestion in 48 hours, and the inventive method is according to phase
With condition of culture be inoculated with 2 containers, every 24 hours digestion collect one, count, 48 collect all according to same culture conditions
2 containers are inoculated with, digestion in every 24 hours collects one, counts, according to container inner cell use ratio, calculate a factory thin
Born of the same parents' amplification amount, is so passaged to P14.Calculate each passage cell and count total amount, draw cell vegetative map.From Fig. 3 cells propagation
Curve sees, same inoculum density, in same time, and the inventive method cell amount to obtain is substantially more than conventional method.With
Cell ageing or differentiation, ability of cell proliferation gradually weaken.Cultural method epidermal cell of the present invention is used with reference to Fig. 2 results explanation
Its passage number can be obviously prolonged, is conducive to obtaining more many cells.
Embodiment 2
The present embodiment is illustrated exemplified by cultivating mouse epidermal cells.
1st, the acquisition of mouse seed coat cell
Newborn mice is taken, cervical dislocation is put to death, immersion 70% ethanol solution sterilization；Remove the manadesma and fat of basal；In
Property protease, 4 DEG C of incubation 12h or so；Corium and epidermis are separated, epidermis 0.25% Trypsin Induced 8min, digestion is collected
Terminate to add isometric terminate liquid（DMEM nutrient solutions containing serum）, the counting of PBS solution suspension, 1000 revs/min, 10 points of centrifugation
Clock.Centrifugation terminates to press every bottle 4 × 10 with K-SFM culture mediums4~5 × 104Individual/cm2Cultivated under the conditions of 37 DEG C of inoculation, 5%CO2, often
2~3d changes liquid；Passed on before cell fusion, 1 week or so about after inoculating cell.24h changes nutrient solution, now carefully
Born of the same parents are labeled as P0 generations.
2nd, cell is expanded
Treat that cell fusion degree reaches 60-70%, abandon nutrient solution；Add digestive juice（0.25% trypsase and 0.01% ethylenediamine tetraacetic
Acetic acid mixture）, digestive juice minimum amount is covers whole cell culture face, and 37 DEG C of incubation 4min treat 80-90% cell not
It is adherent again, add terminate liquid（DMEM nutrient solutions containing serum）, digestion is terminated, suspension, 1000 revs/min, centrifugation is collected
10min；Supernatant is abandoned, cell precipitation is resuspended with K-SFM culture mediums, sampling is counted,
3rd, cell culture
A cell is often passed on according to step 2 and breeds about 1 times, cell is reached into P2 generations, when P2 is resuspended for cell and is inoculated with, by every
Cell is pressed 1.5 × 10 by cell factory 1.25L liquid volume added4After/ml is resuspended, it is inoculated in cell factory.Before inoculation with PBS or
Nutrient solution soaks cell attachment face, is conducive to cell inoculation uniform.Suspension should be rocked uniformly before inoculation, liquid is uniformly divided
To each layer, it is ensured that be seeded to every confluent monolayer cells quantity consistent.
The cell being inoculated with is put into CO2Incubator, 37 DEG C, 5% gas concentration lwevel carry out culture 24 hours, unscrew training
The lid of one end is supported, air pump is put into CO2Other one layer in incubator, in another termination air filtration filter of culture vessel
Device, air pump is connected with filter inlet end, adjusts gas transmission parameter, it is to avoid container inner pressure rises and damaged caused by gas transmission is too fast
Container, is polluted.Cell fusion degree reaches 50~70% harvestings after cultivating 3-6 days.
Sterile working adds digestive juice 125ml into cell factory（Volume ratio 9:1 trypsase and EDTA, tryptose
Enzyme mass concentration is that 0.25%, EDTA mass concentrations are 0.1%）, digestive juice is divided equally to interlayer rapidly, rocking covers digestive juice
Whole cell culture face；It is transferred to CO237 DEG C of incubation 4min of incubator, treat that 60-70% cell is no longer adherent, move into and add in advance
There are 125ml terminate liquids（It is preferred that serum-free medium, it is possible to use the DMEM nutrient solutions containing serum）Centrifuge tube.Again to cell work
Add same digestive juice 125ml in factory, be incubated 2min, add the identical terminate liquids of 125ml, and it is rapid by terminate liquid divide equally to
Interlayer, rocks and is mixed with digestive juice.The all liq after terminating twice is collected, 1000 revs/min, 10min is centrifuged；Centrifugation terminates
Supernatant is abandoned, is resuspended and counted with PBS balanced salt solutions, step 3 is repeated after centrifugation and is carried out continuously culture, large-scale culture is obtained
Mouse epidermal cells.
Embodiment 3
The present embodiment is illustrated exemplified by cultivating ox epidermal cell.
1st, the acquisition of ox kind sublist chrotoplast
Take the ears of an ox or cow inner skin 1cm2Left and right, with 75% alcohol-pickled 1min, is rinsed 3 times with PBS, tissue is entered into 0.25% pancreas egg
In white enzyme, 4 DEG C overnight, are taken off epidermis, with 0.25% trypsase and 0.01% ethylenediamine tetra-acetic acid mixed liquor by the epidermis of collection
Individual cells suspension is digested to, the filtering of 200 mesh is centrifuged, counted, by 3 × 10 6/ bottle is inoculated with, and adds be added with EGF, cow's serum altogether
The mL of MCDB153 nutrient solutions 10 of the additives such as albumin, hydrocortisone, 37 DEG C, 5% CO are placed in by blake bottle2In incubator
Cultivated, 24h changes nutrient solution, now cell marking is P0 generations.
2nd, cell is expanded
Treat that seed cell degrees of fusion reaches 50-65%, discard nutrient solution；Digestive juice is added in blake bottle（0.25% trypsase
With 0.01% ethylenediamine tetra-acetic acid mixed liquor）, 37 DEG C of incubator incubation 6min treat that most cells are no longer adherent, terminate digestion,
Collect suspension after terminating, 800rpm/min centrifugations 6min；Supernatant is abandoned, is resuspended and counted with MCDB153 nutrient solutions.
3rd, cell culture
A cell is often passed on according to step 2 and breeds about 1 times, cell is reached into P6 generations, when P6 is resuspended for cell and is inoculated with, by every
Cell is pressed 2.8 × 10 by cell factory 0.5L liquid volume added4/ ml reclosing kind cell factories.Preceding nutrient solution is inoculated with by cell attachment
Face is soaked, and is conducive to cell inoculation uniform.Suspension should be rocked uniformly, liquid uniformly be divided to each layer before inoculation, it is ensured that inoculation
It is consistent to every confluent monolayer cells quantity.
The cell being inoculated with is put into CO2Incubator, 37 DEG C, 5% gas concentration lwevel carry out culture unscrew culture within 24 hours
The lid of one end, CO is put into by air pump2Other one layer in incubator, in another termination air filtration filter of culture vessel,
Air pump is connected with filter inlet end, gas transmission parameter is adjusted, it is to avoid container inner pressure rises and damages appearance caused by gas transmission is too fast
Device, is polluted.Cell fusion degree reaches 50~60% harvestings after cultivating 4-7 days.
Sterile working adds 50ml digestive juices into cell factory（Volume ratio 2:1 mass fraction is 0.25% trypsase
And 0.1%EDTA）（Volume ratio 2:1 trypsase and EDTA, trypsase mass concentration is that 0.25%, EDTA mass concentrations are
0.1%）, digestive juice is divided equally to interlayer rapidly, rocking makes digestive juice cover whole cell culture face；It is transferred to CO2Incubator 37
DEG C be incubated 6min, treat that 60-70% cell is no longer adherent, move into advance added with 50ml terminate liquids（It is preferred that serum-free medium,
The DMEM nutrient solutions containing serum can be used）Centrifuge tube.Same digestive juice 50ml is added into cell factory again, is incubated
3min, adds the identical terminate liquids of 50ml, and rapidly divides equally terminate liquid to interlayer, rocks and digestive juice is mixed.Collect twice
All liq after termination, per minute 800 leave the heart 6 minutes；Supernatant is abandoned in centrifugation end, is resuspended and counted with PBS balanced salt solutions,
Step 3 is repeated after centrifugation and is carried out continuously culture, the ox epidermal cell of large-scale culture is obtained.
Meanwhile, the present invention can be it is determined that by changing cell inoculum concentration within the specific limits in the case of target cell generation
Cell generation, for example, need P7 for cell needed for being reached with cell growth time, can be by 9 × 106Individual/factory's inoculation P5 generations
Cell culture 3 days, can also be by 5 × 106Individual/factory is inoculated with P4 for cell culture 4 days.
Although embodiment of the present invention is described above in association with accompanying drawing, the invention is not limited in above-mentioned
Specific embodiment, above-mentioned specific embodiment is only schematical, guiding, rather than restricted.This area
Those of ordinary skill under the enlightenment of this specification, in the case where not departing from the scope that the claims in the present invention are protected,
The form of many kinds can also be made, these belong to the row of protection of the invention.
Claims (7)
1. a kind of epidermal cell large-scale cultivation method, it is characterised in that comprise the following steps：
Step 1, it is inoculated with
Epidermis seed cell and nutrient solution are mixed after being resuspended, are uniformly inoculated in cell factory, it is ensured that every layer of inoculating cell number
Amount is consistent；
Step 2, cultivate
The epidermal cell being inoculated with is put into CO2Incubator, in 37 DEG C, 5% CO2Cultivated under concentration conditions, according to passage need
Seek harvesting after culture a period of time；
Step 3, digest
Digestive juice, CO are added to cell factory237 DEG C of incubations of incubator, when most cells are no longer adherent, immediately by factory
Liquid, which is transferred in the container equipped with terminate liquid, terminates digestion；Then the digestive juice of same volume is continuously added to cell factory,
CO237 DEG C of incubator be incubated to all cells it is no longer adherent when, then add into cell factory same volume terminate liquid it is whole
Only digest；
Step 4, centrifuge
The all liq after terminating twice is collected, supernatant is abandoned in centrifugation, is resuspended with PBS balanced salt solutions, supernatant is abandoned in centrifugation again, is obtained
To the good epidermal cell of large-scale culture.
2. epidermal cell large-scale cultivation method according to claim 1, it is characterised in that what is be inoculated with described in step 1 connects
Kind of amount is：Cell is pressed 1.6 × 10 by every cell factory 0.25L~1.25L liquid volume added4/ ml~4 × 104/ ml is resuspended and is followed by
Kind.
3. epidermal cell large-scale cultivation method according to claim 1, it is characterised in that be inoculated with described in step 2
Epidermal cell is in CO2When being cultivated in incubator, air and CO are passed through into cell factory2, make to keep 5% CO in cell factory2。
4. epidermal cell large-scale cultivation method according to claim 1, it is characterised in that digestive juice is described in step 3
TryplE digestive juices, the digestive juice volume added twice is equal, and the digestive juice total amount added twice is 50 ~ 250ml.
5. the epidermal cell large-scale cultivation method according to claim 1 or 4, it is characterised in that terminate liquid described in step 3
For serum-free medium, the terminate liquid volume added every time is equal with the digestive juice volume added every time.
6. the epidermal cell large-scale cultivation method according to claim 1 or 4, it is characterised in that centrifugation described in step 3 is
Per minute 800 ~ 1000 leave the heart 6 ~ 10 minutes.
7. the epidermal cell large-scale cultivation method according to any one in claim 1-4, it is characterised in that the table
Chrotoplast is human epidermal cell, mouse epidermal cells, rat Epithelial Cell, pig epidermal cell or ox epidermal cell.
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| CN115354012A (en) * | 2022-08-08 | 2022-11-18 | 青岛海尔生物医疗科技有限公司 | Cell passage process |
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