CN107083355A - A kind of feeder cells and preparation method and application - Google Patents
A kind of feeder cells and preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of feeder cells and preparation method thereof, and this method comprises the following steps：（1）The culture of cell：L cell is seeded in culture dish, nutrient solution is added and is cultivated, when cell density reaches 80 90%, stops culture, the cell cultivated；（2）Using methyl alcohol process：Culture dish is taken out, removes nutrient solution, the cell of culture is cleaned with phosphate buffer, then removes phosphate buffer, methanol is added, places at room temperature after 5min, removes methanol, 3 6min are stood on superclean bench, culture dish are sealed with sealed membrane, that is, obtain feeder cells.The present invention also provides the application that multipotential stem cell culture is carried out using the feeder cells.This method has the advantages that the repeatable utilization of feeder cells simple, time saving, with low cost, to prepare, moreover it is possible to avoid cross pollution.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of feeder cells and preparation method and application.
Background technology
Multipotential stem cell, including embryonic stem cell（ES）With induced multi-potent stem cell (iPS), with self-renewing and multidirectional
The ability of differentiation, so as to be had broad application prospects in organizational project and regenerative medicine field.Multipotential stem cell in vitro culture
Feeder cells are needed as matrix, to provide nutrition.Evans and Kaufman is in the inner cell mass from mouse in 1981 point
During from obtaining mouse embryo stem cell, with reference to internal microenvironment, one layer of mouse handled by mitomycin C is spread in culture dish
Fetal fibroblast is as feeder layer, and treated mouse fetal fibroblast loses division growth ability, can be with
Many supports are provided for the adherent of embryonic stem cell, propagation and signal transduction etc., multipotential stem cell self-renewing energy is maintained
Power.So, all it is that to continue to use treated feeder layer culture thin at present, including human embryo stem cell and induced multi-potent stem cell
Born of the same parents.
At present, the preparation of feeder cells is small by what is be separately cultured based on mitomycin C or radiation exposure processing
Mouse fetal fibroblast（MEF）, handled by mitomycin C or radiation exposure, pancreatin digestion counts, again meets MEF
Plant to being covered with advance in the culture dish of gelatin, cultivated by least 12 hours, after cell attachment expansion, can just obtain feeder layer thin
Born of the same parents, obtained feeder cells can not be preserved at room temperature, it is necessary to immediately using or be stored in liquid nitrogen, condition of storage is tight
Lattice；And multipotential stem cell digests in passage through pancreatin, feeder cells can be also digested, so ready-made feeder layer can only
With once.Traditional raising layer manufacturing method thereof, through closing up mouse, separate 12.5 days pregnant mouse MEF cells, MEF is thin
Born of the same parents' Secondary Culture, mitomycin C processing 2-3h, whole flow process need 2-3 time-of-weeks.Mitomycin C is expensive, whole to make
Process is complicated, and work of giving up takes, the problem of also there is cell cross pollution.Although radiation exposure processing can be thin with mass disposal
Born of the same parents, but common laboratory does not possess experiment condition, with limitation.
Although establishing many feeder free systems in recent years to cultivate multipotential stem cell, in these systems
Costly, and the phenomenons such as cell differentiation and apoptosis occur in some multipotential stem cells to cellular matrix price, it is impossible to dimension well
The self-renewing of stem cell is held, therefore, traditional feeder layer cultural method can not be also substituted completely.
The content of the invention
For the defect of prior art, the present invention provides a kind of feeder cells and preparation method thereof, using methyl alcohol process
The fibroblast of culture, this method has the advantages that simple, time saving, with low cost, and the feeder cells prepared can be weighed
It is multiple to utilize.The present invention is also provided is cultivating the application in multipotential stem cell field by the feeder cells.
The present invention is achieved by the following technical solutions.
A kind of preparation method of feeder cells, this method comprises the following steps：
（1）The culture of cell：L cell is seeded in culture dish, nutrient solution is added, in 37 DEG C, 5%CO2It is thin
Cultivated in born of the same parents' incubator, when cell density reaches 80-90%, stop culture, the cell cultivated；
（2）Using methyl alcohol process：Culture dish is taken out, removes nutrient solution, by step（1）The obtained cell phosphoric acid being separately cultured
Salt buffer is cleaned, and then removes phosphate buffer, adds methanol, removes methanol after placing 5min at room temperature, in ultra-clean work
Make to stand 3-6min on platform, methanol is volatilized, cover culture dish lid, sealed culture dish with sealed membrane, that is, obtain feeder layer thin
Born of the same parents, it is standby.
Preferably, step（2）The methanol of middle addition is 4 DEG C of methanol.
Preferably, step（2）The middle volume and step for adding methanol（1）The volume ratio of middle nutrient solution is 1:2.
Preferably, step（1）Middle cell inoculum concentration is each 35mm culture dishes inoculation 5 × 104Individual l cell,
Correspondence adds 2mL nutrient solution.
Preferably, step（1）In l cell be primary separation mouse fetal fibroblast, the training
Nutrient solution is made up of high glycoform DMEM and hyclone, and the volume ratio of high glycoform DMEM and hyclone is：17:3.
Preferably, step（1）In l cell be to build the l cell for being, the nutrient solution is by height
Sugar-type DMEM and calf serum are constituted, and the volume ratio of high glycoform DMEM and calf serum is：9:1.
A kind of feeder cells, are prepared using above-mentioned preparation method.
A kind of application of feeder cells, is the culture that multipotential stem cell is carried out using above-mentioned feeder cells.
By methyl alcohol process, cell death, form is shunk, and is added corresponding multipotential stem cell nutrient solution, can be returned to again
Normal morphology.
When carrying out the culture of multipotential stem cell using above-mentioned feeder cells, concretely comprise the following steps：By the above-mentioned feeding prepared
The sealed membrane for supporting confluent monolayer cells removes, and adds corresponding multipotential stem cell nutrient solution, is then inoculated with multipotential stem cell, changes one within every two days
Secondary nutrient solution, is cultivated according to prior art.The death after methyl alcohol process due to feeder cells, so
It is thin relative to feeder layer prepared by conventional method for cultivating the nutrient solution for not consuming multipotential stem cell during multipotential stem cell additionally
Born of the same parents will save the nutrient solution of half, and in multipotential stem cell had digestive transfer culture, the digestive ferment such as pancreatin will not destroy the feeder layer
Cellular morphology, it is possible to reuse 2-3 times.
Advantages of the present invention：
（1）The method that the present invention is provided is simple to operate, and cost is low, and the used time is short.The present invention uses methanol with low cost, will grow
Cell processing 5min in culture dish, it is to avoid using bed board during mitomycin C processing, again digestion the step such as count
Suddenly, simple to operate, efficiency is improved.
（2）Feeder cells prepared by the present invention can be reused.Cell is fixed on culture after methyl alcohol process
The bottom of ware, so, in multipotential stem cell culture, i.e., handled using pancreatin after multipotential stem cell is digested, by fixed feeding
The form that foster confluent monolayer cells remain to keep complete is not destroyed, it is possible to reused 2-3 times.
（3）When feeder cells prepared by the present invention are used for multipotential stem cell culture, nutrient solution is saved.Conventional method system
Standby feeder cells, during for multipotential stem cell culture, feeder cells and multipotential stem cell are required for consuming nutrient solution, need
Change nutrient solution daily.Feeder cells prepared by the present invention are when for multipotential stem cell culture, and only multipotential stem cell needs
Nutrient solution, because feeder cells have been killed through methyl alcohol process, it is not necessary to consume nutrient solution, changes a nutrient solution in every two days.
（4）Storage is convenient, can store at room temperature, cost-effective.Feeder cells prepared by the present invention can be direct
Utilize, can also preserve at room temperature, without being stored in liquid nitrogen environment.
（5）Cross pollution can be prevented.Feeder cells are after methyl alcohol process, pollution-free source, for multipotential stem cell
During culture, cross pollution can be avoided.
Brief description of the drawings
The form that Fig. 1 mouse multipotential stem cells are cultivated on feeder cells.Wherein, A, B, C are respectively mouse embryonic stem
The form that cell is cultivated on feeder cells prepared by embodiment 1, embodiment 2 and comparative example 1, D, E, F are respectively that mouse lures
Lead the form that multipotential stem cell is cultivated on feeder cells prepared by embodiment 1, embodiment 2 and comparative example 1.
The form that Fig. 2 people's induced multi-potent stem cell and pig induced multi-potent stem cell are cultivated on feeder cells.Wherein, A,
B, C are the shape that people's induced multi-potent stem cell is cultivated on feeder cells prepared by embodiment 1, embodiment 2 and comparative example 1 respectively
State, D, E, F are respectively that pig induced multi-potent stem cell is trained on feeder cells prepared by embodiment 1, embodiment 2 and comparative example 1
Foster form.
The form of Fig. 3 feeder cells and mouse embryo stem cell, wherein, wherein, A, B, C represent that embodiment 1 is made respectively
Feeder cells prepared by standby feeder cells, embodiment 1 are utilized feeder layer prepared by form, embodiment 1 after 1 time
Cell is utilized the form after 2 times；D, E, F represent that mouse embryo stem cell is cultivated on A, B, C feeder cells respectively
Form.
The form of Fig. 4 feeder cells and mouse embryo stem cell, wherein, A, B represent raising prepared by comparative example 1 respectively
Feeder cells prepared by confluent monolayer cells, comparative example 1 are being utilized 1 form after pancreatin digestion；C, D represent small respectively
The form that mouse embryonic stem cell is cultivated on A, B feeder cells respectively.
Fig. 5 mouse embryo stem cells cultivated on feeder layer after alkaline phosphatase（AP）Colored graph, wherein, A, B, C points
Not Biao Shi mouse embryo stem cell cultivated on feeder layer prepared by embodiment 1, embodiment 2 and comparative example 1 after alkaline phosphatase
Enzyme（AP）Colored graph.
Fig. 6 detects the result figure of multipotential stem cell marker gene with q-PCR methods.
Fig. 7 mouse embryo stem cell particular surface marks SSEA-1 expression of results, wherein, A, B, C represent mouse respectively
Embryonic stem cell is more competent on feeder cells prepared by embodiment 1, embodiment 2, comparative example 1 during 30 generation of continuous passage
Cell specific surface mark SSEA-1 expression of results.
Embodiment
Embodiment 1
A kind of feeder cells, its preparation method is as follows：
（1）The culture of cell：By 5 × 104The mouse fetal fibroblast of individual primary separation（Abbreviation MEF）It is seeded in 35mm's
In culture dish, 2mL nutrient solutions are added, in 37 DEG C, 5%CO2Cell culture incubator in cultivate, by 36 hours, treat that cell density reaches
To 80-90%, stop culture；Wherein, the formula of nutrient solution is：By high glycoform DMEM（Brand is：HyClone, lot number is
AB10134659）And hyclone（Brand is Gibco, lot number 548979）The volume of composition, high glycoform DMEM and hyclone
Than for：17:3, wherein hyclone abbreviation FBS；
（2）Using methyl alcohol process：Culture dish is taken out from cell culture incubator, removes nutrient solution, by step（1）It is thin that culture is obtained
Born of the same parents' phosphate buffer（Abbreviation PBS）Cleaning one time, then removes PBS, and 4 DEG C of 1mL methanol is drawn with liquid-transfering gun（Methanol
The precooling in 4 DEG C of refrigerator in advance）, it is adherent to add in culture dish, 5min is placed at room temperature, removes methanol, then in ultra-clean work
Make to stand 3-6min on platform, make methanol volatilization clean, cover culture dish lid, sealed culture dish with sealed membrane, it is standby.
The method provided using the embodiment, prepare feeder cells needed about 36h altogether, less than 2 days.
Embodiment 2
A kind of feeder cells, its preparation method is as follows：
（1）The culture of cell：By 5 × 104It is individual to build the l cell for being（Abbreviation NIH3T3）It is seeded in 35mm culture
In ware, 2mL nutrient solutions are added, in 37 DEG C, 5%CO2Cell culture incubator in cultivate, by 24 hours, treat that cell density reaches
During 80-90%, stop culture；Wherein, the formula of nutrient solution is：Be made up of high glycoform DMEM and calf serum, high glycoform DMEM and
The volume ratio of calf serum is： 9:1；
（2）Using methyl alcohol process：Culture dish is taken out from cell culture incubator, removes nutrient solution, by step（1）It is thin that culture is obtained
Born of the same parents' phosphate buffer（Abbreviation PBS）Cleaning one time, then removes PBS, and 4 DEG C of 1mL methanol is drawn with liquid-transfering gun（Methanol
The precooling in 4 DEG C of refrigerator in advance）, it is adherent to add in culture dish, 5min is placed at room temperature, removes methanol, then in ultra-clean work
Make to stand 3-6min on platform, after making methanol volatilization clean, cover culture dish lid, sealed culture dish with sealed membrane, it is standby.
The method provided using the embodiment, prepare feeder cells needs 24h altogether.
Comparative example 1（Traditional preparation methods）
A kind of feeder cells, its preparation method is as follows：
（1）Cell is separately cultured：By 5 × 104The mouse fetal fibroblast of individual primary separation is seeded in 35mm culture
In ware, 2mL nutrient solutions are added, nutrient solution is identical with the nutrient solution in embodiment 1, in 37 DEG C, 5%CO2Cell culture incubator in train
Support, after 36 hours, when cell density reaches 80%, stop culture.
（2）Mitomycin C processing：Cell is taken out from cell culture incubator, removes nutrient solution, fresh nutrient solution 1mL is added,
Mitomycin C 10ug is added, 37 DEG C, 5%CO are put into2Handled 2-4 hours in incubator, then remove nutrient solution, PBS is washed 3 times,
Immediately using or digest, count, dispense, freeze in liquid nitrogen, with when recover again.
Handled using mitomycin C, process is complicated, it is cumbersome by steps such as digestion again, countings.
The l cell for being using building, can also avoid the primary separation of l cell, and mouse into
The primary separation of fibrocyte needs 2-3 weeks, using the l cell that is is built, more can streamline operation, during saving
Between.Take shorter it can be seen that the embodiment of the present invention prepares feeder cells, step is simpler.
The application effect of feeder cells prepared by the one, detection embodiment of the present invention.
1. feeder cells prepared by embodiment 1, embodiment 2 and comparative example 1, are respectively used to mouse multipotential stem cell
Culture, mouse multipotential stem cell is respectively mouse embryo stem cell and mouse induced multi-potent stem cell, and specific cultural method is such as
Under：
Using 35mm culture dishes, 1 × 10 is inoculated with respectively5Individual mouse multipotential stem cell is made to embodiment 1, embodiment 2 and comparative example 1
2mL mouse multipotential stem cell nutrient solutions are separately added on standby feeder cells, in culture dish, are cultivated 3 days respectively, using real
, it is necessary to which every 2 days change a nutrient solution when applying feeder cells prepared by example 1 and embodiment 2；In the feeding prepared using comparative example 1
When supporting confluent monolayer cells, nutrient solution needs to change nutrient solution daily, and cellular morphology is as shown in Figure 1.Wherein, mouse multipotential stem cell culture
Liquid is consisted of the following composition（By taking 100ml nutrient solutions as an example）：High glycoform DMEM 83mL, hyclone 15mL, non-essential amino
1 mL of acid (100x), Glu 1mL（100x）, beta -mercaptoethanol 0.1mM, mouse leukemia inhibiting factor 10ul
（10000x）.
The form that mouse multipotential stem cell is cultivated on feeder cells prepared by embodiment 1, embodiment 2 and comparative example 1
Compare, see Fig. 1.Wherein, A, B, C are respectively the raising that mouse embryo stem cell is prepared in embodiment 1, embodiment 2 and comparative example 1
The form cultivated on confluent monolayer cells, D, E, F are respectively that mouse induced multi-potent stem cell is made in embodiment 1, embodiment 2 and comparative example 1
The form cultivated on standby feeder cells.A, B, C and D, E, F in Fig. 1, mouse multipotential stem cell are prepared in the present invention
Feeder layer on cultivate after, cell edges are more neat, more there is third dimension, more standard compliant multipotential stem cell.The He of embodiment 1
Feeder cells prepared by embodiment 2 are in the culture applied to multipotential stem cell, and its cellular morphology, culture effect are better than biography
Feeder layer prepared by system method.
2. feeder cells prepared by embodiment 1, embodiment 2 and comparative example 1, are respectively used to people's induced multi-potent stem cell
With the culture of pig induced multi-potent stem cell, specific cultural method is as follows：
Using 35mm culture dishes, 1 × 10 is inoculated with respectively5Personal induced multi-potent stem cell and pig induced multi-potent stem cell are to embodiment
1 and comparative example 1 prepare feeder cells on, in culture dish add 2mL multipotential stem cell nutrient solutions, respectively cultivate 4 days.Adopting
, it is necessary to which every 2 days change a nutrient solution during feeder cells prepared with embodiment 1；It is thin in the feeder layer prepared using comparative example 1
During born of the same parents, nutrient solution needs to change nutrient solution daily, wherein,
Human pluripotent stem cells culture formula of liquid（By taking the composition of 100ml nutrient solutions as an example）：DMEM/F12 78mL, serum substitute
KSR 20mL, nonessential amino acid 1mL, Glu 1mL（100x）, beta -mercaptoethanol 0.1mM, 1ug human alkaline fibroblast
Porcine HGF, wherein DMEM/F12 are a kind of basic culture solutions.
Pig induced multi-potent stem cell culture formula of liquid（By taking the composition of 100ml nutrient solutions as an example）：High glycoform DMEM 83mL,
Hyclone 15mL, L- glutamine 1mL, nonessential amino acid 1mL, beta -mercaptoethanol 0.1mM, mouse leukemia inhibiting factor
10uL（10000x）, 2uM SB031542,3uM CHIR99021,1ug human alkaline fibroblast growth factors.Wherein,
SB031542 and CHIR99021 are micromolecular inhibitors.
The feeding that people's induced multi-potent stem cell and pig induced multi-potent stem cell are prepared in embodiment 1, embodiment 2 and comparative example 1
Support the form cultivated on confluent monolayer cells to compare, see Fig. 2, A, B, C are people's induced multi-potent stem cell respectively in embodiment 1, the and of embodiment 2
The form cultivated on feeder cells prepared by comparative example 1, D, E, F are respectively pig induced multi-potent stem cell in embodiment 1, implementation
The form cultivated on feeder cells prepared by example 2 and comparative example 1.As shown in Figure 2, people's induced multi-potent stem cell and pig induction are more
After energy stem cell is cultivated on feeder layer prepared by the present invention, cell edges are more neat, more there is third dimension.Embodiment 1 and reality
The feeder cells of the preparation of example 2 are applied in the culture applied to multipotential stem cell, its cellular morphology, culture effect are better than tradition
Feeder layer prepared by method.
The recycling effect of feeder cells prepared by the two, present invention
1. feeder cells prepared by embodiment 1 are preserved 60 days at room temperature, reused, cultivate mouse embryonic stem
Cell.Its cultural method is with above-mentioned（The 1st in the application effect of feeder cells prepared by the one, detection embodiment of the present invention
Point）.The form of feeder cells and mouse embryo stem cell as shown in figure 3, wherein, wherein, A, B, C are represented respectively, embodiment 1
Feeder cells prepared by the feeder cells of preparation, embodiment 1 are utilized raising prepared by form, embodiment 1 after 1 time
Confluent monolayer cells are utilized the form after 2 times；D, E, F represent that mouse embryo stem cell is trained on A, B, C feeder cells respectively
Foster form.
From the figure 3, it may be seen that B shows that feeder cells are utilized after 1 time, complete form can be still kept；E is shown will be small
The renewed vaccination of mouse embryonic stem cell is on the feeder cells of used 1 time, and mouse embryo stem cell still can normally give birth to
It is long；C shows the feeder cells utilized by 2 times, and its form is still complete；F displays are using the feeder cells for being utilized 2 times
When cultivating mouse embryo stem cell, mouse embryo stem cell still can normal growth, illustrate the feeder cells prepared of the invention
It can reuse.
2. feeder cells prepared by comparative example 1, are repeated 1 times utilization, for cultivating mouse embryo stem cell, raise
The form of confluent monolayer cells and mouse embryo stem cell is shown in Fig. 4, wherein, A, B represent feeder cells, right prepared by comparative example 1 respectively
Feeder cells prepared by ratio 1 are being utilized 1 form after pancreatin digestion；C, D represent mouse embryonic stem respectively
The form that cell is cultivated on A, B feeder cells respectively.
As shown in Figure 4, A displays use comparative example 1（Conventional method）The feeder cells form of preparation, its cellular morphology base
This is complete, and C shows that mouse embryo stem cell is inoculated into the form cultivated on the feeder cells of conventional method preparation；B displays are passed
Feeder cells prepared by system method are using the form after once being digested by pancreatin, and feeder layer prepared by conventional method is thin
Born of the same parents are after pancreatin digests, and cell whole levitating comes, and the basic feeder cells without normal morphology understand mice embryonic by D
The form that stem cell is cultivated on the conventional husbandry confluent monolayer cells utilized once, it can be seen that because feeder cells are floated
Come, exist without feeder cells, so mouse embryo stem cell edge is irregular, differentiation is serious, it is impossible to remain normal many
Energy stem cell morphology, it is seen then that feeder cells prepared by conventional method can not be reused.
Three, mouse embryo stem cells are more after multiple passage on feeder cells prepared by embodiment 1 and embodiment 2
Energy property detection, testing result is shown in Fig. 5, Fig. 6, Fig. 7 respectively.
Wherein, A, B, C represent what mouse embryo stem cell was prepared in embodiment 1, embodiment 2 and comparative example 1 respectively in Fig. 5
Alkaline phosphatase after being cultivated on feeder layer（AP）Colored graph；
Fig. 6 represents to detect the feeder layer that mouse embryo stem cell is prepared in embodiment 1, embodiment 2, comparative example 1 with q-PCR methods
On cell during 20 generation of continuous passage, the testing result of multipotential stem cell marker gene, with the feeder layer prepared using comparative example 1
Cell culture mouse embryo stem cell is used as control；
Expression streaming method detection mouse embryo stem cell is made in embodiment 1, embodiment 2, comparative example 1 respectively by A, B, C in Fig. 7
On standby feeder cells during 30 generation of continuous passage, multipotential stem cell particular surface mark SSEA-1 expression of results, to adopt
The feeder cells culture mouse embryo stem cell prepared with comparative example 1 is used as control.
Alkaline phosphatase staining, can be for tentatively judging multipotency as one of the index for differentiating multipotential stem cell versatility
The versatility of stem cell.As shown in Figure 5, mouse embryo stem cell gives birth to culture in feeder layer prepared by conventional husbandry layer and the present invention
Afterwards, AP dyeing is all red, illustrates feeder cells prepared by embodiment 1 and embodiment 2 and comparative example 1, can maintain many
The alkaline phosphatase activities of energy stem cell.
It will be appreciated from fig. 6 that mouse embryo stem cell is continuously cultivated on feeder cells prepared by embodiment 1 and embodiment 2
During to 20 generation, compared with classical culture protocols, multipotency related gene（OCT4、NANOG、SOX2）There is elevated trend, illustrate real
Apply example 1 and compared with feeder layer prepared by embodiment 2 with feeder layer prepared by comparative example 1, can preferably maintain multipotential stem cell
Versatility.
As shown in Figure 7, mouse embryo stem cell embodiment 1, embodiment 2 prepare feeder cells on continuous passage extremely
During 30 generation, flow cytometer detection mouse multipotential stem cell particular surface mark SSEA-1, with comparative example 1（Conventional method）Compare,
SSEA-1 percentage is higher, illustrates that embodiment 1 is compared with feeder layer prepared by embodiment 2 with feeder layer prepared by comparative example 1,
Be conducive to preferably maintaining the versatility of multipotential stem cell.
It follows that feeder cells prepared by embodiment 1 and embodiment 2, can be well when cultivating multipotential stem cell
The versatility of multipotential stem cell is maintained, its versatility turns out the cell come better than feeder cells prepared by conventional method, real
Feeder cells prepared by example 1 and embodiment 2 are applied, when cultivating multipotential stem cell, cellular morphology is more complete, it is seen then that this hair
The feeder cells of bright offer are better than feeder cells prepared by conventional method.
Claims (8)
1. a kind of preparation method of feeder cells, it is characterised in that：This method comprises the following steps：
（1）The culture of cell：L cell is seeded in culture dish, nutrient solution is added, in 37 DEG C, 5%CO2Cell
Cultivated in incubator, when cell density reaches 80-90%, stop culture, the cell cultivated；
（2）Using methyl alcohol process：Culture dish is taken out, removes nutrient solution, the cell of culture is cleaned with phosphate buffer, then
Remove phosphate buffer, add methanol, place at room temperature after 5min, remove methanol, 3-6min is stood on superclean bench,
Culture dish lid is covered, culture dish is sealed with sealed membrane, that is, obtains feeder cells, it is standby.
2. the preparation method of feeder cells according to claim 1, it is characterised in that：Step（2）The methanol of middle addition
For 4 DEG C of methanol.
3. the preparation method of feeder cells according to claim 1, it is characterised in that：Step（2）Middle addition methanol
Volume and step（1）The volume ratio of middle nutrient solution is 1:2.
4. the preparation method of feeder cells according to claim 1, it is characterised in that：Step（1）Middle cell inoculum concentration
For each 35mm culture dishes inoculation 5 × 104Individual l cell, adds 2mL nutrient solution.
5. the preparation method of the feeder cells according to profit requires 1, it is characterised in that：Step（1）In mouse into fiber
Cell is the mouse fetal fibroblast of primary separation, and the nutrient solution is made up of high glycoform DMEM and hyclone, high sugar
The volume ratio of type DMEM and hyclone is：17:3.
6. the preparation method of feeder cells according to claim 1, it is characterised in that：Step（1）In mouse into fibre
It is to build the l cell for being to tie up cell, and the nutrient solution is made up of high glycoform DMEM and calf serum, high glycoform DMEM
Volume ratio with calf serum is：9:1.
7. a kind of feeder cells, are prepared using any one preparation method described in claim 1-6.
8. a kind of application of feeder cells, is the training that multipotential stem cell is carried out using the feeder cells described in claim 7
Support.
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