CN109439628A - Limbal stem cell primary culture method - Google Patents
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Abstract
The present invention relates to a kind of limbal stem cell primary culture methods.It is characterized in that through gentamycin solution, treated that corneal limbal tissue shreds to be placed in tissue digestion liquid digests, filtering, and the corneal limbal tissue cell precipitation obtained after being centrifuged filtrate carries out of short duration freeze, remaining tissue block (i.e. filter residue) will be filtered again to be resuspended with limbal stem cell trophoderm culture solution, it is inoculated in and is cultivated with the pretreated tissue culture plate of coating buffer, obtain corneal limbus fibroblast, then the corneal limbus fibroblast for growing to logarithmic phase is handled with mitomycin c solution, limbal stem cell trophoderm is made, it finally recovers the of short duration histocyte frozen, after being resuspended with limbal stem cell originally culture liquid, it is seeded in be covered in the trophoblastic tissue culture plate of limbal stem cell and carries out originally culture to obtain the final product.The limbal stem cell is highly-safe, stability is good, fully meets the high request of clinical treatment.
Description
Technical field
The invention belongs to technical field of stem cell culture, and in particular to a kind of limbal stem cell primary culture method.
Background technique
The study found that limbal stem cell is located in the unique wavy shaped configuration of corneal limbus basal layer " Vogt fence ", it is
The source that corneal epithelial cell updates.Limbal stem cell, which constantly updates, is able to maintain the successional water of corneal limbal epithelial cell
Centripetal and upward vertical movement is put down, to guarantee the normal of the complete and function of corneal epithelium structure.And corneal limbus is dry thin
The proliferation pressure of born of the same parents is able to suppress growing into for conjunctival epithelial cell, and prevents the conjunctiva vascular invasion of cornea edge.When various damages
Hurt factor, such as chemical burn, mechanical damage, cause pathogeny imcrobe infection, will lead to the missing of limbal stem cell or influences it
Existence microenvironment and when its physiological function being made to be badly damaged, will cause corneal epithelium structure that cannot rebuild, conjunctival epithelium and blood vessel
Screen migrates repairing corneal surface, leads to the opacity of the cornea, visual function decline.Therefore, it is necessary to obtain energy in vitro by originally culture
Enough fast breedings and the limbal stem cell for keeping stem cell attribute, with for the treatment of limbal deficiency/lesion, this is also existing
Modern clinical transplantation treats the key link of such disease.
Currently, the primary culture method of limbal stem cell is more: the in-vitro separation of limbal stem cell generally uses group
It knits block and moves out that (enzyme digestion includes IV Collagenase Type joint trypsase, neutral proteinase joint tryptose for method and enzyme digestion
Enzyme etc.)；The trophoderm that culture uses mainly includes mouse NIH 3T3 cytotrophoblast, mouse embryonic fibroblasts trophoderm
Deng；Culture solution ingredient generally comprise serum, growth factor, cholera toxin, insulin, 3- iodine thyronine, hydrogenation can
Pine etc..
Though the cultural method of existing above-mentioned limbal stem cell can obtain epithelial cell, all there is certain limitation:
In the in-vitro separation link that originally culture is related to, it is easy to be mixed with other types using the cell that the tissue block method of moving out obtains
Cell, such as fibroblast, and the fibroblastic growth speed is exceedingly fast and cell category is caused to mix；Above-mentioned trypsase
For endopeptidase, there is selective hydrolysis effect to arginine and lysine peptide chain.Due to containing non-specific suppression in serum
Peptase, therefore trypsase can not digest normal tissue in the presence of having serum, if but tissue is placed in no blood for a long time
It is digested in clear digestive juice, the decline of cell viability certainly will be will cause；In terms of trophoblastic selection, NIH 3T3 cell
It is external non-human archeocyte with embryo fibroblast, easily causes the propagation of immune response and the derivative pathogen of source of mouse, exist
Pathogenic risk；In the selection of culture solution ingredient, wherein cholera toxin is the virulence factor that comma bacillus generates, and can be caused serious
Diarrhea and human body dehydration, and 3- iodine thyronine is the main active substances of thyroid hormone, but the first shape of high concentration
Glandular hormone can promote breaks down proteins, cause negative nitrogen balance.The corneal limbus that above-mentioned factor can influence primary culture in vitro is dry
The activity and biological safety of cell.In addition, the limbal stem cell that existing method obtains is easy to happen differentiation, from certain degree
On be lost the attribute of limbal stem cell and lead to graft after there are the risks of corneal limbus insufficiency.
Therefore, it is badly in need of establishing a kind of ideal limbal stem cell primary culture method, is guaranteeing limbal stem cell
On the basis of biological safety, its stem cell attribute can be maintained without breaking up, well to be used for clinical treatment.
Summary of the invention
The object of the present invention is to provide a kind of limbal stem cell primary culture method, with overcome the deficiencies in the prior art,
Realize the demand of clinical treatment.
Method of the invention:
Cleaning corneal limbal tissue with sterile 0.9%(w/v) physiological saline first, (region of wide about 2 mm, comes between cornea and sclera
The remaining leftover pieces after contributing corneal transplantation), then the corneal limbal tissue after cleaning is immersed in gentamycin solution, in
37 DEG C of 10 min of processing are then rinsed 3 times with sterile PBS balanced salt solution, DMEM/F12(1:1) culture medium rinsing 3 times；
Above-mentioned corneal limbal tissue is placed in 500 μ l tissue digestion liquid again, with eye scissors by tissue cut into size about 0.5 ~
1 mm3Fritter；
Then 4.5 ml tissue digestion liquid are added, are placed in 37 DEG C of constant-temperature tables, 60 rpm digest 2 ~ 4 h；It later, will be after digestion
Corneal limbal tissue suspension be filtered with 200 aim cell sieves, and collect filtrate and remaining tissue block respectively and (filter
Slag)；
Then above-mentioned filtrate is centrifuged 10 min in 500 rpm, the histocyte precipitating after removing supernatant carries out conventional of short duration jelly
It deposits；
Above-mentioned filter residue limbal stem cell trophoderm culture solution is resuspended later, and is equably layered on pretreated with coating buffer
In tissue culture plate, after cell is moved out and covers with single layer, conventional cell passage is carried out, obtains corneal limbus fibroblast simultaneously
It is inoculated in the pretreated tissue culture plate of coating buffer, it is long to logarithmic growth phase to cell, culture solution is sucked out, mitomycin is added
C solution after 37 DEG C are protected from light 2 h of incubation, discards mitomycin c solution therein, then make after being rinsed with sterile PBS balanced salt solution
For limbal stem cell trophoderm；
Then the above-mentioned of short duration histocyte frozen is subjected to conventional recovery, is centrifuged 10 min in 500 rpm, after removing supernatant, used
Histocyte precipitating is resuspended in limbal stem cell originally culture liquid, is inoculated in and is covered with the trophoblastic cell of above-mentioned limbal stem cell
In culture plate, it is placed in 37 DEG C, 5%CO2In vitro culture is carried out in incubator, uses limbal stem cell originally culture liquid within every 1 ~ 2 day
It carries out changing liquid processing, can be obtained the limbal stem cell of originally culture.
The formula of the above-mentioned gentamycin solution of the present invention: the DMEM/F12(1:1 containing 100 U/ml gentamicins) training
Support base.
The formula of above-mentioned tissue digestion liquid: the Dispase II enzyme of 2.4 ~ 4.8 U/ml is dissolved in containing 5% fetal calf serum
DMEM/F12(1:1) culture medium.
The formula of above-mentioned limbal stem cell trophoderm culture solution: containing 10% fetal calf serum and 10 ~ 20 ng/ml alkalinity at
The DMEM/F12(1:1 of fibroblast growth factor) culture medium.
The formula of above-mentioned coating buffer: contain 10 ~ 30 μ g/ml fibronectins, 5 ~ 20 μ g/ml laminins 5 and 1 ~ 2
The DMEM/F12(1:1 of mg/ml gelatin) culture medium.
The formula of above-mentioned mitomycin c solution: the DMEM/F12(1:1 containing 10 μ g/ml mitomycin Cs) culture medium.
The formula of above-mentioned limbal stem cell originally culture liquid: raw containing 5 ~ 10% fetal calf serums, 10 ~ 40 ng/ml epidermises
The long factor, 10 ~ 20 ng/ml basic fibroblast growth factors, 1 ~ 10 μ g/ml insulin, 1 ~ 10 μ g/ml transferrins,
1 ~ 5 ng/ml sodium selenite, 0.2 ~ 1 μ g/ml hydrocortisone, 5 ~ 20 ng/ml LIF ELISAs, 5 ~ 10 ng/ml are white
Interleukin -6,5 ~ 20 μ g/ml fibronectins, 5 ~ 10 μ g/ml laminins 5 and 5 ~ 10% corneal limbus fibroblast (logarithms
Phase) culture supernatant DMEM/F12(1:1) culture medium.
The preparation method of above-mentioned coating buffer pretreatment cell culture plate: drawing coating buffer and add to the cell culture board bottom,
After the board bottom complete wetting, extra coating buffer is sucked out, then tissue culture plate is placed on superclean bench and air-dries 1 h.
Since the unused source of mouse cell of the present invention is as trophoderm, but the very strong cornea of proliferative capacity is dexterously utilized
On the one hand edge fibroblast and trophoderm as limbal stem cell after being handled by mitomycin C, eliminate different in this way
The possibility that the derivative pathogen in source is propagated, the component of another aspect corneal limbus fibroblast inherently corneal limbus microenvironment reach
To preferably simulated in vivo environment, limbal stem cell is made to keep natural attribute.In addition, limbal stem cell of the invention is primary
Also cholera toxin is not added in culture solution, therefore, the limbal stem cell for cultivating acquisition, which is not present, causes a disease risk, safety more
Height, and the ingredient in the factor and coating buffer added in the limbal stem cell originally culture liquid being directed to, can be fine
Guarantee limbal stem cell attribute stability, without break up.
Specific embodiment
The preparation method of various solution of the present invention is as follows:
1,10 ml DMEM/F12(1:1 the preparation method of above-mentioned gentamycin solution: are taken) culture medium, it is big mould that 10 mg celebrating is added
Plain (1000 U/mg of potency) uses 0.22 μm of syringe needle filter filtration sterilization, then uses DMEM/F12(1:1 after being completely dissolved) it trains
Feeding base is settled to 100 ml.
2,10 ml DMEM/F12(1:1 the preparation method of above-mentioned tissue digestion liquid: are taken) culture medium, 24 ~ 48 mg are added
Then Dispase II enzyme (10 U/mg of enzyme activity) adds 5 with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved
Ml fetal calf serum is finally settled to 100 ml with DMEM/F12(1:1) culture medium.
3,10 ml DMEM/F12(1:1 the preparation method of above-mentioned limbal stem cell trophoderm culture solution: are taken) culture medium,
1 ~ 2 μ g basic fibroblast growth factor is added, with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved, then again
10 ml fetal calf serums are added, are finally settled to 100 ml with DMEM/F12(1:1) culture medium.
4,10 ml DMEM/F12(1:1 the preparation method of above-mentioned coating buffer: are taken) culture medium, 1 ~ 3 mg fibre is added and connects egg
White, 0.5 ~ 2 g gelatin of mg laminin 5 and 0.1 ~ 0.2, with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved,
Finally 100 ml are settled to DMEM/F12(1:1) culture medium.
5,10 ml DMEM/F12(1:1 the preparation method of above-mentioned mitomycin c solution: are taken) culture medium, it is added 1 mg
Rimocidin C is finally settled to DMEM/F12(1:1) culture medium with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved
100 ml。
6,10 ml DMEM/F12(1:1 the preparation method of above-mentioned limbal stem cell originally culture liquid: are taken) culture medium, add
Enter 1 ~ 4 μ g epidermal growth factor, 1 ~ 2 μ g basic fibroblast growth factor, 0.1 ~ 1 mg insulin, 0.1 ~ 1 mg and turns iron
Albumen, 0.1 ~ 0.5 mg sodium selenite, 20 ~ 100 μ g hydrocortisones, 0.5 ~ 2 μ g LIF ELISA, 0.5 ~ 1 μ g are white
Interleukin -6,0.5 ~ 2 mg fibronectin, 0.5 ~ 1 mg laminin 5 are filtered after being completely dissolved with 0.22 μm of syringe needle filter
Then degerming adds 5 ~ 10 ml fetal calf serums and 5 ~ 10 ml with the corneal limbus of 0.22 μm of syringe needle filter filtering into fiber
Cell (logarithmic phase) culture supernatant, is finally settled to 100 ml with DMEM/F12(1:1) culture medium.
Specific implementation step of the invention:
1, the processing of corneal limbal tissue: corneal limbal tissue is cleaned with sterile 0.9%(w/v) physiological saline until without impurity, then
The corneal limbal tissue cleaned up is immersed in gentamycin solution, in 37 DEG C of 10 min of processing, is then balanced with sterile PBS
Flushed 3 times, DMEM/F12(1:1) culture medium rinsing 3 times；
2, the digestion of corneal limbal tissue: corneal limbal tissue is placed in 500 μ l tissue digestion liquid, is sheared tissue with eye scissors
At about 0.5 ~ 1 mm of size3Fritter, then add 4.5 ml tissue digestion liquid, be placed in 37 DEG C of constant-temperature tables, 60 rpm digestion 2
~4 h；
By 200 aim cell the screen to filtrates of postdigestive corneal limbal tissue suspension, then 3, corneal limbal tissue cell freezes:
Filtrate is centrifuged 10 min in 500 rpm, after removing supernatant, carries out conventional of short duration freeze；
4, the pretreatment of tissue culture plate: drawing coating buffer and add to cell culture board bottom, will be extra after board bottom complete wetting
Coating buffer is sucked out, then tissue culture plate is placed on superclean bench and air-dries 1 h；
5, it the trophoblastic preparation of limbal stem cell: (is filtered above-mentioned with remaining tissue block after 200 aim cell the screen to filtrates
Slag) it is resuspended with limbal stem cell trophoderm culture solution, it is equably layered on in the pretreated tissue culture plate of coating buffer, to thin
After born of the same parents move out and cover with single layer, conventional cell passage is carried out, obtains corneal limbus fibroblast, then by corneal limbus at fiber finer
Born of the same parents are inoculated in the pretreated tissue culture plate of coating buffer, long to logarithmic growth phase to cell, and culture solution is sucked out, it is mould that mitogen is added
Plain C solution discards mitomycin c solution after 37 DEG C are protected from light and are incubated for 2 h, then use after the rinsing of sterile PBS balanced salt solution as angle
Film limbal stem cell trophoderm；
6, the originally culture of the limbal stem cell carried out later: carrying out conventional recovery for the above-mentioned of short duration histocyte frozen,
10 min are centrifuged in 500 rpm, after removing supernatant, histocyte precipitating is resuspended with limbal stem cell originally culture liquid, is inoculated in
It is covered in the trophoblastic tissue culture plate of limbal stem cell, is placed in 37 DEG C, 5%CO2Carry out in vitro culture in incubator, every 1 ~ 2
It carries out changing liquid processing using limbal stem cell originally culture liquid, can be obtained the limbal stem cell of originally culture.
Embodiment 1
Corneal limbal tissue first is cleaned with sterile 0.9%(w/v) physiological saline until not having impurity, then take 10 ml DMEM/F12(1:
1) culture medium is added 10 mg gentamicins (1000 U/mg of potency), is filtered after being completely dissolved with 0.22 μm of syringe needle filter
Then degerming is settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to gentamycin solution.Then it will clean up
Corneal limbal tissue afterwards is immersed in gentamycin solution, in 37 DEG C of 10 min of processing, is then rushed with sterile PBS balanced salt solution
3 times are washed, DMEM/F12(1:1) culture medium rinsing 3 times；
10 ml DMEM/F12(1:1 are taken later) culture medium, 24 mg Dispase II enzymes (10 U/mg of enzyme activity) are added, completely
With 0.22 μm of syringe needle filter filtration sterilization after dissolution, 5 ml fetal calf serums are then added, finally use DMEM/F12(1:1)
Culture medium is settled to 100 ml, is configured to tissue digestion liquid.Corneal limbal tissue is placed in 500 μ l tissue digestion liquid, ophthalmology is used
It cuts and tissue is cut into about 1 mm of size3Fritter, then add 4.5 ml tissue digestion liquid, be placed in 37 DEG C of constant-temperature tables, 60
Rpm digests 4 h；
By 200 aim cell the screen to filtrates of postdigestive corneal limbal tissue suspension, filtrate is then centrifuged 10 in 500 rpm
Min after removing supernatant, carries out conventional of short duration freeze；
10 ml DMEM/F12(1:1 are taken again) culture medium, it is bright that 1 mg fibronectin, 0.5 g of mg laminin 5 and 0.1 is added
Glue is finally settled to 100 with DMEM/F12(1:1) culture medium with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved
Ml is configured to coating buffer.Coating buffer is drawn later and adds to cell culture board bottom, after board bottom complete wetting, by extra coating
Liquid is sucked out, then tissue culture plate is placed on superclean bench and air-dries 1 h, and it is spare to be prepared into pretreated tissue culture plate；
Then 10 ml DMEM/F12(1:1 are taken) culture medium, 1 μ g basic fibroblast growth factor is added, after being completely dissolved
With 0.22 μm of syringe needle filter filtration sterilization, 10 ml fetal calf serums are added, finally use DMEM/F12(1:1) culture medium constant volume
To 100 ml, it is configured to limbal stem cell trophoderm culture solution.Later by above-mentioned with remaining after 200 aim cell the screen to filtrates
Tissue block (i.e. filter residue) with limbal stem cell trophoderm culture solution be resuspended, be equably layered on the pretreated cell of coating buffer
In culture plate, after cell is moved out and covers with single layer, conventional cell passage is carried out, corneal limbus fibroblast is obtained；
10 ml DMEM/F12(1:1 are taken again) culture medium, 1 mg mitomycin C is added, with 0.22 μm of syringe needle after being completely dissolved
Filter filtration sterilization is finally settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to mitomycin c solution.Then again
Corneal limbus fibroblast is inoculated in the pretreated tissue culture plate of coating buffer, it is long to logarithmic growth phase to cell, it is sucked out
Mitomycin c solution is added in culture solution, after 37 DEG C are protected from light 2 h of incubation, discards mitomycin c solution, then balanced with sterile PBS
After salting liquid rinsing, limbal stem cell trophoderm is obtained；
Then 10 ml DMEM/F12(1:1 are taken) culture medium, it is raw that 1 μ g epidermal growth factor, 1 μ g basic fibroblast is added
The long factor, 0.5 mg insulin, 0.1 mg transferrins, 0.1 mg sodium selenite, 20 μ g hydrocortisones, the 0.5 white blood of μ g
Sick inhibiting factor, 0.5 μ g interleukin-6,0.5 mg fibronectin and 0.5 mg laminin 5, with 0.22 after being completely dissolved
μm syringe needle filter filtration sterilization, then add the angle that 10 ml fetal calf serums and 5 ml are filtered with 0.22 μm of syringe needle filter
Film edge fibroblast (logarithmic phase) culture supernatant, is finally settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to
Limbal stem cell originally culture liquid.The above-mentioned of short duration histocyte frozen is finally subjected to conventional recovery, is centrifuged in 500 rpm
After removing supernatant, histocyte precipitating is resuspended with limbal stem cell originally culture liquid in 10 min, is inoculated in that be covered with corneal limbus dry thin
In the trophoblastic tissue culture plate of born of the same parents, it is placed in 37 DEG C, 5%CO2In vitro culture is carried out in incubator, it is dry thin using corneal limbus daily
Born of the same parents' originally culture liquid carries out changing liquid processing, can be obtained the limbal stem cell of originally culture.
Embodiment 2
Corneal limbal tissue first is cleaned with sterile 0.9%(w/v) physiological saline until not having impurity, then take 10 ml DMEM/F12(1:
1) culture medium is added 10 mg gentamicins (1000 U/mg of potency), is filtered after being completely dissolved with 0.22 μm of syringe needle filter
Then degerming is settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to gentamycin solution.Then it will clean up
Corneal limbal tissue afterwards is immersed in gentamycin solution, in 37 DEG C of 10 min of processing, is then rushed with sterile PBS balanced salt solution
3 times are washed, DMEM/F12(1:1) culture medium rinsing 3 times；
10 ml DMEM/F12(1:1 are taken later) culture medium, 48 mg Dispase II enzymes (10 U/mg of enzyme activity) are added, completely
With 0.22 μm of syringe needle filter filtration sterilization after dissolution, 5 ml fetal calf serums are then added, finally use DMEM/F12(1:1)
Culture medium is settled to 100 ml, is configured to tissue digestion liquid.Corneal limbal tissue is placed in 500 μ l tissue digestion liquid, ophthalmology is used
It cuts and tissue is cut into about 0.5 mm of size3Fritter, then add 4.5 ml tissue digestion liquid, be placed in 37 DEG C of constant-temperature tables,
60 rpm digest 2 h；
By 200 aim cell the screen to filtrates of postdigestive corneal limbal tissue suspension, filtrate is then centrifuged 10 in 500 rpm
Min after removing supernatant, carries out conventional of short duration freeze；
10 ml DMEM/F12(1:1 are taken again) culture medium, it is bright that 2 mg fibronectins, 2 g of mg laminin 5 and 0.15 are added
Glue is finally settled to 100 with DMEM/F12(1:1) culture medium with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved
Ml is configured to coating buffer.Coating buffer is drawn later and adds to cell culture board bottom, after board bottom complete wetting, by extra coating
Liquid is sucked out, then tissue culture plate is placed on superclean bench and air-dries 1 h, and it is spare to be prepared into pretreated tissue culture plate；
Then 10 ml DMEM/F12(1:1 are taken) culture medium, 1.5 μ g basic fibroblast growth factors are added, are completely dissolved
Afterwards with 0.22 μm of syringe needle filter filtration sterilization, 10 ml fetal calf serums are added, it is finally fixed with DMEM/F12(1:1) culture medium
Hold to 100 ml, is configured to limbal stem cell trophoderm culture solution.It is remained later by above-mentioned with after 200 aim cell the screen to filtrates
Remaining tissue block (i.e. filter residue) is resuspended with limbal stem cell trophoderm culture solution, is equably layered on pretreated thin with coating buffer
In born of the same parents' culture plate, after cell is moved out and covers with single layer, conventional cell passage is carried out, corneal limbus fibroblast is obtained；
10 ml DMEM/F12(1:1 are taken again) culture medium, 1 mg mitomycin C is added, with 0.22 μm of syringe needle after being completely dissolved
Filter filtration sterilization is finally settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to mitomycin c solution.Then again
Corneal limbus fibroblast is inoculated in the pretreated tissue culture plate of coating buffer, it is long to logarithmic growth phase to cell, it is sucked out
Mitomycin c solution is added in culture solution, after 37 DEG C are protected from light 2 h of incubation, discards mitomycin c solution, then balanced with sterile PBS
After salting liquid rinsing, limbal stem cell trophoderm is obtained；
Then 10 ml DMEM/F12(1:1 are taken) culture medium, it is thin that 2.5 μ g epidermal growth factor, 1.5 μ g basic fibroblasts are added
The intracellular growth factor, 0.2 mg insulin, 0.5 mg transferrins, 0.2 mg sodium selenite, 50 μ g hydrocortisones, 1 μ g are white
Blood disease inhibiting factor, 0.75 μ g interleukin-6,1 mg fibronectin and 1 mg laminin 5, with 0.22 μ after being completely dissolved
Then the syringe needle filter filtration sterilization of m adds what 7.5 ml fetal calf serums and 7.5 ml were filtered with 0.22 μm of syringe needle filter
Corneal limbus fibroblast (logarithmic phase) culture supernatant, is finally settled to 100 ml with DMEM/F12(1:1) culture medium, prepares
At limbal stem cell originally culture liquid.The above-mentioned of short duration histocyte that freezes finally is subjected to conventional recovery, in 500 rpm from
After removing supernatant, histocyte precipitating is resuspended with limbal stem cell originally culture liquid in 10 min of the heart, is inoculated in that be covered with corneal limbus dry
In the tissue culture plate of cytotrophoblast, it is placed in 37 DEG C, 5%CO2In vitro culture is carried out in incubator, uses corneal limbus within every 1.5 days
Stem cell primary culture solution carries out changing liquid processing, can be obtained the limbal stem cell of originally culture.
Embodiment 3
Corneal limbal tissue first is cleaned with sterile 0.9%(w/v) physiological saline until not having impurity, then take 10 ml DMEM/F12(1:
1) culture medium is added 10 mg gentamicins (1000 U/mg of potency), is filtered after being completely dissolved with 0.22 μm of syringe needle filter
Then degerming is settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to gentamycin solution.Then it will clean up
Corneal limbal tissue afterwards is immersed in gentamycin solution, in 37 DEG C of 10 min of processing, is then rushed with sterile PBS balanced salt solution
3 times are washed, DMEM/F12(1:1) culture medium rinsing 3 times；
10 ml DMEM/F12(1:1 are taken later) culture medium, 36 mg Dispase II enzymes (10 U/mg of enzyme activity) are added, completely
With 0.22 μm of syringe needle filter filtration sterilization after dissolution, 5 ml fetal calf serums are then added, finally use DMEM/F12(1:1)
Culture medium is settled to 100 ml, is configured to tissue digestion liquid.Corneal limbal tissue is placed in 500 μ l tissue digestion liquid, ophthalmology is used
It cuts and tissue is cut into about 1 mm of size3Fritter, then add 4.5 ml tissue digestion liquid, be placed in 37 DEG C of constant-temperature tables, 60
Rpm digests 3 h；
By 200 aim cell the screen to filtrates of postdigestive corneal limbal tissue suspension, filtrate is then centrifuged 10 in 500 rpm
Min after removing supernatant, carries out conventional of short duration freeze；
10 ml DMEM/F12(1:1 are taken again) culture medium, it is bright that 3 mg fibronectins, 2 g of mg laminin 5 and 0.2 are added
Glue is finally settled to 100 with DMEM/F12(1:1) culture medium with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved
Ml is configured to coating buffer.Coating buffer is drawn later and adds to cell culture board bottom, after board bottom complete wetting, by extra coating
Liquid is sucked out, then tissue culture plate is placed on superclean bench and air-dries 1 h, and it is spare to be prepared into pretreated tissue culture plate；
Then 10 ml DMEM/F12(1:1 are taken) culture medium, 2 μ g basic fibroblast growth factors are added, after being completely dissolved
With 0.22 μm of syringe needle filter filtration sterilization, 10 ml fetal calf serums are added, finally use DMEM/F12(1:1) culture medium constant volume
To 100 ml, it is configured to limbal stem cell trophoderm culture solution.Later by above-mentioned with remaining after 200 aim cell the screen to filtrates
Tissue block (i.e. filter residue) with limbal stem cell trophoderm culture solution be resuspended, be equably layered on the pretreated cell of coating buffer
In culture plate, after cell is moved out and covers with single layer, conventional cell passage is carried out, corneal limbus fibroblast is obtained；
10 ml DMEM/F12(1:1 are taken again) culture medium, 1 mg mitomycin C is added, with 0.22 μm of syringe needle after being completely dissolved
Filter filtration sterilization is finally settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to mitomycin c solution.Then again
Corneal limbus fibroblast is inoculated in the pretreated tissue culture plate of coating buffer, it is long to logarithmic growth phase to cell, it is sucked out
Mitomycin c solution is added in culture solution, after 37 DEG C are protected from light 2 h of incubation, discards mitomycin c solution, then balanced with sterile PBS
After salting liquid rinsing, limbal stem cell trophoderm is obtained；
Then 10 ml DMEM/F12(1:1 are taken) culture medium, it is raw that 4 μ g epidermal growth factor, 2 μ g basic fibroblasts are added
The long factor, 0.1 mg insulin, 1 mg transferrins, 0.5 mg sodium selenite, 100 μ g hydrocortisones, the suppression of 2 μ g leukaemia
The factor, 1 μ g interleukin-6,2 mg fibronectins and 0.75 mg laminin 5 processed, with 0.22 μm of needle after being completely dissolved
Head filter filtration sterilization, then add 5 ml fetal calf serums and 10 ml with the corneal limbus of 0.22 μm of syringe needle filter filtering at
Fibrocyte (logarithmic phase) culture supernatant, is finally settled to 100 ml with DMEM/F12(1:1) culture medium, is configured to corneal limbus
Stem cell primary culture solution.The above-mentioned of short duration histocyte frozen is finally subjected to conventional recovery, is centrifuged 10 in 500 rpm
Min after removing supernatant, is resuspended histocyte precipitating with limbal stem cell originally culture liquid, is inoculated in and is covered with limbal stem cell
In trophoblastic tissue culture plate, it is placed in 37 DEG C, 5%CO2In vitro culture is carried out in incubator, uses limbal stem cell within every 2 days
Originally culture liquid carries out changing liquid processing, can be obtained the limbal stem cell of originally culture.
Limited 3 embodiments for the limbal stem cell that originally culture of the invention obtains can maintain it dry well
Cellular attributes keep undifferentiated state, and the high level for reaching clinical treatment requires.
Claims (7)
1. a kind of limbal stem cell primary culture method, it is characterised in that be first immersed in the corneal limbal tissue cleaned up
It carries out disinfection and rinses in gentamycin solution, then corneal limbal tissue is placed in tissue digestion liquid, mended after cutting into fritter
Add tissue digestion liquid to be digested, then filter, the corneal limbal tissue cell precipitation obtained after filtrate is centrifuged carries out of short duration jelly
It deposits, then remaining tissue block limbal stem cell trophoderm culture solution will be filtered and be resuspended, be inoculated in pretreated with coating buffer
Tissue culture plate is cultivated, and corneal limbus fibroblast is obtained, and it is dry thin then to prepare corneal limbus with mitomycin c solution processing
Born of the same parents' trophoderm, then the of short duration histocyte frozen of recovering are seeded in after being resuspended with limbal stem cell originally culture liquid and are covered with angle
Originally culture is carried out in the trophoblastic tissue culture plate of film limbal stem cell, can be obtained limbal stem cell；
Above-mentioned gentamycin solution is the DMEM/F12 culture medium containing 100 U/ml gentamicins；
Above-mentioned tissue digestion liquid is that the Dispase II enzyme of 2.4 ~ 4.8 U/ml is dissolved in the DMEM/F12 containing 5% fetal calf serum
Culture medium；
Above-mentioned limbal stem cell trophoderm culture solution is containing 10% fetal calf serum and 10 ~ 20 ng/ml basic fibroblasts
The DMEM/F12 culture medium of growth factor；
The formula of above-mentioned coating buffer is containing 10 ~ 30 μ g/ml fibronectins, 5 ~ 20 μ g/ml laminins 5 and 1 ~ 2 mg/
The DMEM/F12 culture medium of ml gelatin；
Above-mentioned mitomycin c solution is the DMEM/F12 culture medium containing 10 μ g/ml mitomycin Cs；
The formula of above-mentioned limbal stem cell originally culture liquid is containing 5 ~ 10% fetal calf serums, 10 ~ 40 ng/ml epidermal growth factors
Son, 10 ~ 20 ng/ml basic fibroblast growth factors, 1 ~ 10 μ g/ml insulin, 1 ~ 10 μ g/ml transferrins, 1 ~ 5
Ng/ml sodium selenite, 0.2 ~ 1 μ g/ml hydrocortisone, 5 ~ 20 ng/ml LIF ELISAs, white Jie of 5 ~ 10 ng/ml
The corneal limbus that plain -6,5 ~ 20 μ g/ml fibronectins, 5 ~ 10 μ g/ml laminins 5 and 5 ~ 10% are in growth logarithmic phase is dry
The DMEM/F12 culture medium of cytotrophoblast culture supernatant.
2. limbal stem cell primary culture method as described in claim 1, it is characterized in that above-mentioned tissue culture plate is located in advance
The method of reason: drawing coating buffer and add to cell culture board bottom, and after board bottom complete wetting, extra coating buffer is sucked out, then will
Tissue culture plate, which is placed on superclean bench, air-dries 1 h.
3. limbal stem cell primary culture method as described in claim 1, it is characterized in that at above-mentioned gentamycin solution
The method of reason and rinsing: first being rinsed 3 times with sterile PBS balanced salt solution, then is rinsed 3 times with DMEM/F12 culture medium.
4. limbal stem cell primary culture method as described in claim 1, it is characterized in that above-mentioned corneal limbal tissue digests
Method: corneal limbal tissue is placed in 500 μ l tissue digestion liquid, corneal limbal tissue is cut into size 0.5 ~ 1 with eye scissors
mm3Fritter, then add 4.5 ml tissue digestion liquid, be placed in 37 DEG C of constant-temperature tables, 60 rpm digest 2 ~ 4 h.
5. limbal stem cell primary culture method as described in claim 1, it is characterized in that the side filtered after above-mentioned digestion
Method: postdigestive suspension is filtered with 200 aim cell sieves.
6. limbal stem cell primary culture method as described in claim 1, it is characterized in that above-mentioned limbal stem cell is grown
It supports the preparation method of layer: after corneal limbus fibroblast moves out and covers with single layer, carrying out conventional cell passage, obtain cornea
Edge fibroblast, then corneal limbus fibroblast is inoculated in the pretreated tissue culture plate of coating buffer, it is long extremely in cell
When logarithmic growth phase, culture solution is sucked out, mitomycin c solution is added, it is molten to discard mitomycin C after 37 DEG C are protected from light 2 h of incubation
Liquid, then with after the rinsing of sterile PBS balanced salt solution up to limbal stem cell trophoderm.
7. limbal stem cell primary culture method as described in claim 1, it is characterized in that above-mentioned limbal stem cell is former
The preparation method for nutrient solution of being commissioned to train: 10 ml DMEM/F12(1:1 are taken) culture medium, 1 ~ 4 μ g epidermal growth factor, 1 ~ 2 μ g is added
Basic fibroblast growth factor, 0.1 ~ 1 mg insulin, 0.1 ~ 1 mg transferrins, 0.1 ~ 0.5 mg sodium selenite, 20 ~
100 μ g hydrocortisones, 0.5 ~ 2 μ g LIF ELISA, 0.5 ~ 1 μ g interleukin-6,0.5 ~ 2 mg fibronectin, 0.5
Then ~ 1 mg laminin 5 adds 5 ~ 10 ml tire oxen with 0.22 μm of syringe needle filter filtration sterilization after being completely dissolved
Corneal limbus fibroblast (logarithmic phase) culture supernatant that serum and 5 ~ 10 ml are filtered with 0.22 μm of syringe needle filter, finally
100 ml are settled to DMEM/F12(1:1) culture medium.
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