CN110079500A - Limbal stem cell and preparation method thereof - Google Patents
Limbal stem cell and preparation method thereof Download PDFInfo
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Abstract
The present invention relates to a kind of high activity limbal stem cells and preparation method thereof, and preparation method comprises the steps of: acquisition cornea tissue, and stand-by cornea tissue is pre-processed；The cells frozen storing liquid containing cell induction agent is added into pretreated cornea tissue；Freeze the cornea tissue added with cells frozen storing liquid；The cell induction agent is that the cell that can induce in cornea tissue is locally inflamed or stress reaction, but does not cause the reagent of cell death directly.This method can realize the screening of all kinds of keratocytes of limbal stem cell and mature differentiation before cell culture by the operation of simple cryogenic freezing in cornea tissue, it can realize that the long-term frozen of high activity limbal stem cell saves simultaneously, this method is not necessarily to specific instrument and equipment, it is easy to operate, screening efficiency is high, it is high to screen cell purity, the ratio and activity of limbal stem cell can be improved.
Description
Technical field
The present invention relates to field of tissue engineering technology, more particularly to a kind of preparation method of limbal stem cell.
Background technique
Corneal limbus is the transitional areas of cornea and sclera, is embedded in opaque sclera due to transparent cornea, and gradually mistake
It crosses to sclera, there is no a specific line of demarcation on eyeball surface and histology.Corneal limbus is that natural reserves are responsible on cornea
The adult stem cell position of skin Regeneration and Repair, limbal stem cell are arranged in epithelial layer substrate at paliform in corneal limbal tissue
Portion, is the high-quality cell origin rebuild for ocular corneal epithelium ulcer and organizational project lamellar cornea, and limbal stem cell moves
Vegetation is considered to be directed to one of most promising treatment method of eye surface diseases at present.
Limbal stem cell plays an important role in cornea ocular surface reconstruction, but when being cultivated in vitro due to limbal stem cell
Easily differentiation, it is difficult to which massive amplification is needed to obtain primary limbal stem cell using the means of milder thus, and found more preferably
Method maintains the Stem Cell Activity of cell in vitro.In order to obtain limbal stem cell, existing corneal limbus from cornea tissue
The method of drawing material of stem cell mainly has: enzyme digestion and tissue block adherent method etc..The way of enzyme digestion mainly utilizes pancreas egg
White enzyme, clostridiopetidase A or neutral proteinase etc. handle digestion cornea tissue at 33 DEG C, are inoculated with after corneal epithelium is digested
In being cultivated on culture dish；The step of tissue block adherent method, is attached to culture dish ware bottom for a certain size corneal limbal tissue block of clip,
Culture solution, which is added, makes its move out cell and then culture.Although enzyme digestion can be obtained compared with many cells, limbal stem cell
Purity is lower；The cell that tissue block adherent method obtains is purer, but tissue block adherent time and cell are moved out, the time is longer.
Although the above method can isolated limbal stem cell, there are still corneal limbus in subsequent in vitro culture
The problems such as Stem Cell Activity is lower, easy differentiation.In order to maintain the stemness of limbal stem cell, it is often used cell co-cultivation, carrier
Culture and culture medium add the method amplification in vitro cell of growth factor etc..Cell co-culture method is that limbal stem cell is straight
Culture is connect on feeder cells, the growth factor and cell factor of feeder cells secretion are able to maintain the dry of limbal stem cell
Property, but it is easy to cause cell cross contamination using cell feeder layers, subsequent cell separating difficulty is larger.Amnion is that corneal limbus is dry thin
The good carrier of born of the same parents' culture, many studies have shown that the limbal stem cell stemness cultivated on amnion keeps good, survival rate is high,
Growth rapidly, but the limbal stem cell separated from amnion the problem of there is also cell cross contaminations.Add in the medium
The growth of limbal stem cell can be promoted by adding Porcine HGF also, as epidermal growth factor, hepatocyte growth factor, alkalinity are fine
Dimension mother cell growth factor and keratinocyte growth factor etc. can promote cell mitogen, cell migration and the increasing of stem cell
It grows, but the type of addition growth factor and ratio do not have determining saying at present.
Although a variety of methods has been used to maintain the activity of limbal stem cell in culture, inevitably still there is portion
Point cell is broken up, in order in practical applications as limbal stem cell transplantation operation, the building of organizational project lamellar cornea,
The acquisitions such as keratonosus Mechanism Study more preferably effect needs to carry out limbal stem cell screening and obtains the dry thin of wherein high activity
Born of the same parents, current screening technique have: airflow classification method, immunomagnetic beads method and collagen absorption separating method etc..Airflow classification method is to pass through
The method that flow cytometer screens the cell of label, immunomagnetic beads screening are according to immunology principle, using being coated with
The magnetic bead of antibody and the distinctive mark of stem cell surface are specifically bound, then by the magnetic field of some strength, by Cellular retention
The method of sorting, airflow classification method and immunomagnetic beads method are common cell screening methods, they the advantages of be cell
Purity is high needs to elute from medium, is easily lost a part of cornea the disadvantage is that cell is calibrated and combines with medium
Limbal stem cell, both methods also need specific instrument and equipment and antibody reagent, higher cost.It is root that collagen, which adsorbs separating method,
There is the ability of extracellular matrix quick adsorption according to epithelial stem cell, pass through the absorption of cells on extracellular matrix such as collagen etc.
Duration, the method for filtering out the higher limbal stem cell of Stem Cell Activity, this method separation velocity is fast, avoids because the time is long
Or particular matter toxicity and influence cell activity, however the method is a kind of relatively coarse screening technique, can not accurately be sieved
Choose limbal stem cell.
It for the limbal stem cell not used temporarily, is often saved, the corneal limbus being widely used at present is dry thin
The store method of born of the same parents is cryogenic freezing preservation method, and substantially process is as follows: after cell dissociation dispersion, (containing 10% with cryoprotector
The culture medium of serum adds 10% glycerol or 3.5%~10% dimethyl sulfoxide cell-protecting) it is cooling, while cell suspension being diluted
At (2~5) × 106/ ml dispenses cell suspension by the amount of 1ml/ ampoule, the ampoule of sealing is put into slow jelly machine or program cools down
In instrument, is slowly freezed by the speed of 1 DEG C of decline per minute, add protectant cell suspension slowly to be freezed, temperature is down to -25
DEG C, the ampoule freezed is put into low temperature refrigerator or liquid nitrogen container.Existing cell-preservation liquid, nutritional ingredient is simple, does not fix
Proportion, store method is single, causes the cell survival rate of stem cell low, and cell stability is poor, cannot keep stem cell very well
Activity.And the cornea tissue for not using in short term, it often puts it into corneal storage medium under the conditions of being placed in 2~2 DEG C and is protected
Deposit, the holding time is 2 weeks, when saved between more than 2 weeks when the method that uses have organ culture preservation method and deep-bed drying method,
Two methods are required to special preservation liquid and save equipment, and because of the growth with the holding time, cornea apoptotic cell quantity is not
It is disconnected to increase, structure gradually disorder, therefore long-term preservation is carried out compared with major general's cornea in practical applications.
Culture, screening and the store method of above-mentioned limbal stem cell, common feature are from fresh cornea tissue sampling
The culture, screening and preservation of limbal stem cell are carried out after cell again, it is necessary to the step of passing through traditionally cell screening, operate
Journey is cumbersome, and each step is likely to cause the loss of limbal stem cell, it is difficult to successfully obtain a fairly large number of high activity
Limbal stem cell.
Summary of the invention
Based on this, a kind of preparation method for being designed to provide limbal stem cell of the application, this method passes through at angle
Cell induction agent is added in membrane tissue, can be realized in cornea tissue before cell culture by the operation of simple cryogenic freezing
The screening of limbal stem cell and all kinds of keratocytes of mature differentiation, while can realize the long-term of high activity limbal stem cell
Freezen protective, this method is not necessarily to specific instrument and equipment, easy to operate, and screening efficiency is high, and screening cell purity is high, can larger journey
The ratio and activity of the raising limbal stem cell of degree.
Realize that the specific technical solution of above-mentioned technical purpose is as follows:
A kind of preparation method of high activity limbal stem cell comprising the steps of:
Cornea tissue is obtained, stand-by cornea tissue is pre-processed；
The cells frozen storing liquid containing cell induction agent is added into pretreated cornea tissue；
Freeze the cornea tissue added with cells frozen storing liquid；
The cell induction agent is that the cell that can induce in cornea tissue is locally inflamed or stress reaction, but not straight
Connect the reagent for causing cell death.
In wherein some embodiments, the cell induction agent is selected from inflammatory factor, hormone, intracellular signal molecular regulation agent
With one of Cycle Regulation agent or a variety of.
In wherein some embodiments, the inflammatory factor in interleukin, interferon and tumor necrosis factor one
Kind is a variety of；The hormone is selected from one of glucocorticoid, mineralocorticoid and sex hormone or a variety of；The intracellular signal
Molecular regulation agent is in actinomyces, protein kinase C activators, inhibitors of protein kinase C and DNA topoisomerase enzyme inhibitor
It is one or more；The Cycle Regulation agent is selected from cell cycle protein dependent kinase inhibitor, period element, cell cycle
Protein dependent kinase inhibiting factor, G1 specific cell cyclin, G2/ mitosis specific cell cyclin and its
One of transcription or degradation factor are a variety of.
In wherein some embodiments, the cell induction agent is selected from glucocorticoid, inhibitors of protein kinase C and tumour
One of necrosin is a variety of.
In wherein some embodiments, it is bad that the cell induction agent is selected from Econopred, Staurosporine and tumour
One of necrosis factor α or a variety of.
In wherein some embodiments, the cells frozen storing liquid is the cell culture fluid containing the cell induction agent and cold
Freeze protectant mixed liquor；The volume ratio of the cryoprotector and the cell culture fluid containing the cell induction agent is 1:2-
20。
In wherein some embodiments, the body of the cryoprotector and the cell culture fluid containing the cell induction agent
Product is than being 1:2-10.
In wherein some embodiments, the cryoprotector is selected from dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, second
In amide, methanol, polyvinylpyrrolidone, fructose, sucrose, glucan, albumin, polyethylene glycol, hydroxyethyl starch and yolk
It is one or more.
In wherein some embodiments, concentration of the cell induction agent in the cell culture fluid is 1ng/ml-100
μg/ml。
In wherein some embodiments, concentration of the cell induction agent in the cell culture fluid is 1ng/ml-10 μ
g/ml。
In wherein some embodiments, concentration of the cell induction agent in the cell culture fluid is 10ng/ml-5 μ
g/ml。
In wherein some embodiments, concentration of the cell induction agent in the cell culture fluid is 4-4.5 μ g/
ml。
In wherein some embodiments, the cell culture fluid is the high glycoform DMEM:0- added with following component
50wt% fetal calf serum, 0-20wt% glutamine, 0-20wt% nonessential amino acid, 0-20wt% Pen .- Strep are molten
Liquid, 0-1000ng/ml epidermal growth factor, 0-1000 μ g/ml transferrins, 0-1000 μ g/ml insulin, 1ng/ml-10 μ g/
Cell induction agent described in ml.
In wherein some embodiments, the cell culture fluid is the high glycoform DMEM:2- added with following component
12wt% fetal calf serum, 0.2-1.2wt% glutamine, 0.2-1.2wt% nonessential amino acid, 0.2-1.2wt% penicillin-
Streptomycin Solution, 2-12ng/ml epidermal growth factor, 4-6 μ g/ml transferrins, 4-6 μ g/ml insulin, 10ng/ml-5 μ g/
Cell induction agent described in ml.
In wherein some embodiments, the cell culture fluid is the high glycoform DMEM:10wt% added with following component
Fetal calf serum, 1wt% glutamine, 1wt% nonessential amino acid, 1wt% Pen .- Strep solution, 10ng/ml epidermis are raw
The long factor, 5 μ g/ml transferrins, 5 μ g/ml insulin, cell induction agent described in 4-4.5 μ g/ml.
In wherein some embodiments, the temperature of the freezing is 0 DEG C~-200 DEG C.
In wherein some embodiments, the temperature of the freezing is -20 DEG C~-196 DEG C.
In wherein some embodiments, the time of the freezing is 1-120 months.
In wherein some embodiments, the time of the freezing is 1-24 months.
In wherein some embodiments, the time of the freezing is 3-6 months.
In wherein some embodiments, the preparation method of the limbal stem cell, which also comprises the steps of:, to be separately cultured
Cornea tissue after freezing is to get the limbal stem cell.
In wherein some embodiments, for being added with 1ng/ml-100 μ g/ml institute in the cell culture fluid of the culture
State cell induction agent.
In wherein some embodiments, for being added with described in 1ng/ml-10 μ g/ml in the cell culture fluid of the culture
Cell induction agent.
In wherein some embodiments, for being added with described in 1ng/ml-5 μ g/ml in the cell culture fluid of the culture
Cell induction agent.
In wherein some embodiments, for being added with cell described in 4-4.5 μ g/ml in the cell culture fluid of the culture
Inducer.
In wherein some embodiments, the cell culture fluid for the culture is the high glycoform added with following component
DMEM:0-50wt% fetal calf serum, 0-20wt% glutamine, 0-20wt% nonessential amino acid, 0-20wt% penicillin-chain
Mycin solution, 0-1000ng/ml epidermal growth factor, 0-1000 μ g/ml transferrins, 0-1000 μ g/ml insulin, 1ng/
Cell induction agent described in ml-10 μ g/ml.
In wherein some embodiments, the cell culture fluid is the high glycoform DMEM:2- added with following component
12wt% fetal calf serum, 0.2-1.2wt% glutamine, 0.2-1.2wt% nonessential amino acid, 0.2-1.2wt% penicillin-
Streptomycin Solution, 2-12ng/ml epidermal growth factor, 4-6 μ g/ml transferrins, 4-6 μ g/ml insulin, 10ng/ml-5 μ g/
Cell induction agent described in ml.
In wherein some embodiments, the cell culture fluid for the culture is the high glycoform added with following component
DMEM:10wt% fetal calf serum, 1wt% glutamine, 1wt% nonessential amino acid, 1wt% Pen .- Strep solution,
10ng/ml epidermal growth factor, 5 μ g/ml transferrins, 5 μ g/ml insulin, cell induction agent described in 4-4.5 μ g/ml.
In wherein some embodiments, the method being separately cultured be selected from one of tissue adherent method and digestion method or
It is a variety of.
In wherein some embodiments, the tissue adherent method includes the following steps: corneal limbus group from cornea tissue
After knitting separation, corneal limbal tissue epithelial layer note is attached in culture vessel and is cultivated, makes limbal stem cell from corneal limbal tissue
It moves out, obtains limbal stem cell.Preferably, the size of isolated corneal limbal tissue is 0.1mm~20mm, with a thickness of 0.01mm
~3mm.
In wherein some embodiments, the digestion method includes the following steps: will be dry thin comprising corneal limbus using digestive ferment
The corneal epithelium of born of the same parents digests separation from cornea tissue, and then corneal epithelium is inoculated in culture vessel and is cultivated, and obtains
Limbal stem cell.Preferably, the digestive ferment is any one of clostridiopetidase A, neutral proteinase and trypsase or more
Kind.
In wherein some embodiments, the pretreatment includes: using the physiological buffer comprising or not comprising antibiotic
Conventional cleaning, disinfection, separation are carried out to stand-by cornea tissue.
In wherein some embodiments, the cornea tissue is selected from the cornea of people, rodent and other mammals
Any one or more in tissue.
In wherein some embodiments, the cornea tissue is fresh cornea tissue or the cornea group by short-term preservation
Any one of knit, wherein the time of the short-term preservation is 1 day~20 days.
Another object of the present invention is to provide a kind of high activity limbal stem cells.
Specific technical solution is as follows:
The limbal stem cell being prepared by the preparation method of above-mentioned limbal stem cell.
The preparation method of limbal stem cell provided by the invention contains cell induction agent by being added into cornea tissue
Frozen stock solution after carry out cryogenic freezing to be screened and be saved to cell, the cell induction agent being added can induce cornea tissue
Middle cell is locally inflamed or Apoptosis, and cryogenic freezing condition can induce the adult cell in cornea tissue and cell occurs
Apoptosis or meronecrosis, while maintaining to can reach corneal limbus with the activity of the limbal stem cell in cornea preservation tissue with this
The screening of stem cell and the purpose of long-term preservation, therefore, this method can get the limbal stem cell of high activity；Again to freezing after
Cornea tissue be separately cultured, can further obtain a high proportion of limbal stem cell.
It the principle of the preparation method of the limbal stem cell of high activity of the invention and has the beneficial effect that:
(1) screening of all kinds of keratocytes of limbal stem cell and mature differentiation is effectively realized in cornea tissue:
Under the action of cell induction agent, cooperation cryogenic freezing condition processing, cornea tissue can generate inflammation stress.Due in cornea tissue
Endothelial cell, stroma cell and epithelial cell be mature noble cells, do not have resist this inflammation stress ability, because
Apoptosis or necrosis can gradually occurs in this under conditions of cryogenic freezing.And because limbal stem cell to have a nucleocytoplasmic ratio big, update
The features such as period is slow can resist these stimulations, and apoptosis does not occur, and the limbal stem cell in tissue is maintained and saved.
Therefore, by simple freezing processing, other mature noble cells of removal can be realized in cornea tissue, it is dry to retain corneal limbus
The purpose of cell, and screening efficiency is high, and screening cell purity is high.
(2) it by natural stem cell alcove advantage, maintains and cornea preservation limbal stem cell and its Stem Cell Activity: corneal limbus
In palisade tissue be limbal stem cell storage alcove.In the natural alcove of corneal limbus, there is extracellular bases abundant
The favorable factors such as matter, growth factor, special three dimensional matrix structure can protect limbal stem cell in inflammation or stress situation
Lower maintenance stem cell properties.Therefore, by these advantages, the limbal stem cell in cornea tissue is under conditions of cryogenic freezing
Its Stem Cell Activity can be kept, while achieving the purpose that limbal stem cell long term storage.
(3) in carrying out screening process, inflammation stress situation in analogue body can accelerate limbal stem cell and cultivate
The activation of journey: limbal stem cell is responsible for the update and reparation of corneal epithelium in vivo.When in corneal epithelium, matrix at
When Apoptosis or necrosis gradually occur for ripe noble cells, these signals can be transmitted to corneal limbus position.Limbal stem cell connects
After receiving damage signal, it can start to be repaired under the suitable conditions.Therefore, by cornea tissue from cryogenic freezing condition take out into
When row primitive cell culture, limbal stem cell can rapidly be activated and be proliferated from tissue and move out.
(4) method of the invention is easy to operate, low production cost: inhaling with airflow classification method, immunomagnetic beads method and collagen
The screening methods such as attached separating method are compared, and the method for the present invention need to only be added cell induction agent and carry out cryogenic freezing processing can be to cornea
Cell in tissue is screened, and specific instrument and equipment is not necessarily to, and greatly reduces reagent consumptive material, instrument use and human resources
Etc. cost.
(5) method of the invention can largely improve the ratio and activity of limbal stem cell, through the invention
Method can get the higher limbal stem cell of activity, can be used for basic research such as cell model in the Mechanism Study of keratonosus
Building, can also provide good seed cell source for field of tissue engineering technology, or limbal stem cell transplantation can be pushed to treat eye
The development of surface diseases.
Detailed description of the invention
Fig. 1 is the fresh cornea in embodiment 1 and the caspase 1 through cell induction agent and cryogenic freezing processing cornea
(Caspase 1) coloration result figure；
Fig. 2 is fresh cornea and processing group cornea tissue originally culture limbal stem cell fluidic cell in embodiment 1
Art testing result figure；
Fig. 3 is 2 (ABCG2) immunofluorescence dyeing of atriphos binding transport Protein G superfamily member in embodiment 1
With western blot result figure；
Fig. 4 is tumor necrosis factor α, interleukin 6 and the interleukin-11 that cornea is detected by RT-qPCR method in embodiment 2
The relative expression quantity result figure of β；
Fig. 5 is that the cornea tissue in embodiment 2 is sliced K3, P63 coloration result figure；
Fig. 6 is fresh cornea and processing group cornea tissue originally culture limbal stem cell fluidic cell in embodiment 2
Art testing result figure.
Specific embodiment
To facilitate the understanding of the present invention, it below with reference to embodiment to invention is more fully described, is given below
Presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to described herein
Embodiment.Purpose of providing these embodiments is makes the disclosure of the present invention more thorough and comprehensive.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Used term is intended merely to describe specific reality in the description of the invention
Apply the purpose of example, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more relevant institutes
Any and all combinations of list of items.
A kind of preparation method of limbal stem cell is provided in one embodiment of this invention comprising the steps of:
Cornea tissue is obtained, stand-by cornea tissue is pre-processed；
The cells frozen storing liquid containing cell induction agent is added into pretreated cornea tissue；
Freeze the cornea tissue added with cells frozen storing liquid；
The cell induction agent is that the cell that can induce in cornea tissue is locally inflamed or stress reaction, but not straight
Connect the reagent for causing cell death.
The above method carries out cryogenic freezing by being added after the frozen stock solution containing cell induction agent into cornea tissue with right
Cell is screened and is saved, and cell is locally inflamed in the inducible cornea tissue of the cell induction agent being added or cell withers
Die, and cryogenic freezing condition can induce the adult cell in cornea tissue and Apoptosis or meronecrosis occurs, at the same maintain and
The activity of limbal stem cell in cornea preservation tissue can reach screening and the mesh of long-term preservation of limbal stem cell with this
, therefore, this method can get the limbal stem cell of high activity.
In some of these embodiments, cell induction agent is preferably that inflammation locally occurs for the cell that can induce in cornea tissue
Disease or stress reaction, but do not cause inflammatory factor, hormone, the agent of intracellular signal molecular regulation and the cell cycle of cell death directly
One of regulator is a variety of.
Wherein, feasible inflammatory factor include but is not limited to interleukin, interferon, tumor necrosis factor, conversion growth because
Son, chemotactic factor (CF) etc.；Feasible hormone include but is not limited to cortin, sex hormone, growth hormone, thyroxine, insulin,
Adrenal hormone etc.；Feasible intracellular signal molecular regulation agent includes but is not limited to actinomyces, protein kinase C activators, albumen
Kinase C inhibitors, cytoplasmic tyrosine kinase inhibitor, phosphatidyl inositol kinase inhibitor, wither at DNA topoisomerase enzyme inhibitor
Die Signal regulated kinase inhibitor etc.；Feasible Cycle Regulation agent includes but is not limited to cell cycle protein dependent kinase
Inhibitor, period element, Cyclin kinase inhibitor, G1 specific cell cyclin, G2/ mitosis
Specific cell cyclin and its transcription or degradation factor, histone deacetylase inhibitors, Janus kinase inhibitor
Deng.
It is further preferred that the inflammatory factor is selected from one of interleukin, interferon and tumor necrosis factor or more
Kind；The hormone is selected from one of glucocorticoid, mineralocorticoid and sex hormone or a variety of；The intracellular signal molecule tune
Save agent be selected from one of actinomyces, protein kinase C activators, inhibitors of protein kinase C and DNA topoisomerase enzyme inhibitor or
It is a variety of；The Cycle Regulation agent be selected from cell cycle protein dependent kinase inhibitor, the period element, cyclin according to
Rely property kinase inhibitor, G1 specific cell cyclin, G2/ mitosis specific cell cyclin and its transcription or
One of degradation factor is a variety of.
It is further preferred that the cell induction agent is selected from glucocorticoid (such as can be Econopred), egg
White kinase C inhibitors (such as can be Staurosporine) and tumor necrosis factor (such as can be tumor necrosis factor α
(TNF-α)) one of or it is a variety of.
In some of these embodiments, the cell induction agent is worked by being added in cells frozen storing liquid,
The cells frozen storing liquid can be existing conventional cryopreservation liquid, the preferably cell culture fluid containing the cell induction agent and freezing
Protectant mixed liquor；The preferably volume ratio of the cryoprotector and the cell culture fluid containing the cell induction agent is
1:2-20, preferred volume ratio are 1:2-10.Wherein the cryoprotector is selected from dimethyl sulfoxide, glycerol, ethylene glycol, third
Glycol, acetamide, methanol, polyvinylpyrrolidone, fructose, sucrose, glucan, albumin, polyethylene glycol, hydroxyethyl starch and ovum
One of Huang is a variety of；Preferably dimethyl sulfoxide.
Cell culture fluid in frozen stock solution can be the routine culture liquid of culture limbal stem cell.It preferably adds
State high glycoform DMEM:0-50wt% fetal calf serum, the 0-20wt% glutamine, 0-20wt% of cell induction agent and following component
Nonessential amino acid, 0-20wt% Pen .- Strep solution, 0-1000ng/ml epidermal growth factor (EGF), 40-1000 μ
G/ml transferrins, 0-1000 μ g/ml insulin.More preferably it is added with the high glycoform of the cell induction agent and following component
DMEM:2-12wt% fetal calf serum, 0.2-1.2wt% glutamine, 0.2-1.2wt% nonessential amino acid, 0.2-1.2wt%
Pen .- Strep solution, 2-12ng/ml epidermal growth factor, 4-6 μ g/ml transferrins, 4-6 μ g/ml insulin.It is optimal
Be selected as high glycoform DMEM:10wt% fetal calf serum added with the cell induction agent and following component, 1wt% glutamine,
1wt% nonessential amino acid, 1wt% Pen .- Strep solution, 10ng/ml epidermal growth factor, 5 μ g/ml transferrins, 5
μ g/ml insulin.Wherein the cell induction agent preferably additive amount is 1ng/ml-100 μ g/ml, the addition of cell induction agent
When measuring in the range, the inflammation or stress reaction that the cell in cornea tissue can be made locally to occur are more moderate, so that angle
Endothelial cell, stroma cell and epithelial cell in membrane tissue etc. maturation noble cells under conditions of subsequent cryogenic freezing most
The generation apoptosis of big degree or necrosis, at the same make limbal stem cell withstand to the greatest extent the stimulation of cell induction agent without
Apoptosis occurs, to maintain ratio and activity with cornea preservation limbal stem cell to the greatest extent.Preferred additive amount is 10ng/
Ml-10 μ g/ml, preferred additive amount are 10ng/ml-5 μ g/ml, and more preferably additive amount is 4-4.5 μ g/ml.
In some of these embodiments, the temperature of the freezing is 0 DEG C~-200 DEG C.Preferably -20 DEG C~-196 DEG C,
It can preferably induce the adult cell in cornea tissue that Apoptosis or meronecrosis occurs under the conditions of the temperature, while more
The activity of good maintenance and the limbal stem cell in cornea preservation tissue, so that it is dry thin to further increase corneal limbus obtained
The ratio and activity of born of the same parents.
In some of these embodiments, the time of the freezing is 1-120 months.Within this time range, Ke Yichong
Divide the adult cell in induction cornea tissue that Apoptosis or meronecrosis occurs, while the activity of limbal stem cell can be made
Obtain the preservation of higher degree.The time is more preferably 1-24 months, more preferably 3-6 months.
Further, the preparation method of limbal stem cell of the invention further includes the cornea tissue being separately cultured after freezing
The step of.The cornea tissue after freezing is separately cultured by conventional method, can further be obtained at high proportion and high
Active limbal stem cell.
The cell culture fluid that limbal stem cell is cultivated in method of the invention can be existing routine culture liquid, preferably
For high glycoform DMEM:0-50wt% fetal calf serum, 0-20wt% glutamy added with the cell induction agent and following component
Amine, 0-20wt% nonessential amino acid, 0-20wt% Pen .- Strep solution, 0-1000ng/ml epidermal growth factor, 0-
1000 μ g/ml transferrins, 0-1000 μ g/ml insulin.More preferably added with the cell induction agent and following component
High glycoform DMEM:2-12wt% fetal calf serum, 0.2-1.2wt% glutamine, 0.2-1.2wt% nonessential amino acid, 0.2-
1.2wt% Pen .- Strep solution, 2-12ng/ml epidermal growth factor, 4-6 μ g/ml transferrins, 4-6 μ g/ml pancreas islet
Element.Most more preferably added with high glycoform DMEM:10wt% fetal calf serum, the 1wt% of the cell induction agent and following component
Glutamine, 1wt% nonessential amino acid, 1wt% Pen .- Strep solution, 10ng/ml epidermal growth factor, 5 μ g/ml
Transferrins, 5 μ g/ml insulin.Wherein the cell induction agent preferably additive amount is 1ng/ml-100 μ g/ml, more preferably
Additive amount be 10ng/ml-10 μ g/ml, preferred additive amount be 10ng/ml-5 μ g/ml, more preferably additive amount be 4-
4.5μg/ml。
In method provided by the invention, the method being separately cultured can be conventional cell culture processes, such as can
To be one of tissue adherent method and digestion method or a variety of.The tissue adherent method includes the following steps: from cornea tissue
After corneal limbal tissue is separated, corneal limbal tissue epithelial layer note is attached in culture vessel and is cultivated, makes limbal stem cell from angle
It moves out in film edge tissue, obtains limbal stem cell.Preferably, the size of isolated corneal limbal tissue is 0.1mm~20mm, thick
Degree is 0.01mm~3mm.The digestion method includes the following steps: to utilize digestive ferment by the corneal epithelium comprising limbal stem cell
Layer digests separation from cornea tissue, and then corneal epithelium is inoculated in culture vessel and is cultivated, and obtains limbal stem cell.
Preferably, the digestive ferment is any one or more of clostridiopetidase A, neutral proteinase and trypsase.
In some of these embodiments, the pretreatment includes: using the physiological buffer comprising or not comprising antibiotic
Liquid carries out conventional cleaning, disinfection, separation to stand-by cornea tissue.
Cornea tissue used in method of the invention can be the cornea group of people, rodent and other mammals
Any one or more in knitting.Cornea tissue is preferably fresh cornea tissue or passes through in the cornea tissue of short-term preservation
Any one, wherein the time of the short-term preservation is 1 day~20 days.The corneal limbus met in the cornea tissue of the condition is dry thin
Born of the same parents have higher activity, are conducive to subsequent screening, save and cultivate.
The high activity corneal limbus being prepared the present invention also provides the preparation method by above-mentioned limbal stem cell is dry thin
Born of the same parents can be used for the building of cell model in the Mechanism Study of basic research such as keratonosus, can also provide for field of tissue engineering technology excellent
The seed cell source of matter, or the development of limbal stem cell transplantation treatment eye surface diseases can be pushed.
Below in conjunction with specific embodiment, the present invention is described in further detail.
The percentage being not particularly illustrated in following embodiment refers both to mass percent.
Embodiment 1: the preparation of Rabbit Corneal Limbal Stem Cells
(1) cornea tissue is obtained, stand-by cornea tissue is pre-processed:
The eyeball removed from new zealand rabbit is clear with the phosphate buffer (PBS) of 1% Pen .- Strep solution of addition
After washing 3 times, eyeball exterior circumferential excess tissue removed with tissue shear it is clean, along corneal limbus retain 1~5mm sclera and by its
Remaining part sub-cut is gone, and is exenterated simultaneously, and is cleaned 3 times by the parts such as iris scraped clean, then with PBS.
(2) cells frozen storing liquid containing cell induction agent is added into pretreated cornea tissue:
The cell culture fluid KCM :+10% fetal calf serum+1% of DMEM in high glucose of in vitro culture limbal stem cell is prepared first
+ 1% nonessential amino acid of glutamine+1% Pen .- Strep solution+10ng/ml epidermal growth factor (EGF)+5 μ g/ml
+ 5 μ g/ml insulin of transferrins, and add 4 μ g/ml Econopreds and 20ng/ml Staurosporine (STS), wherein vinegar
Sour prednisolone is glucocorticoid, and Staurosporine is inhibitors of protein kinase C, and the two together functions as cell induction
Agent, then prepare cells frozen storing liquid: 10%DMSO adds 90%KCM culture solution (percent by volume).By step (1) pretreated angle
Membrane tissue is put into 1.5ml cryopreservation tube, and the prepared cells frozen storing liquid of 1ml is added.
(3) cryogenic freezing will be carried out to be screened and be protected to limbal stem cell added with the cornea tissue of cells frozen storing liquid
It deposits:
Cryopreservation tube in step (2) is put into program temperature reduction box and is placed in -20 DEG C of refrigerators, shifts cryopreservation tube after freezing
Into liquid nitrogen container.
(4) it is separately cultured to obtain limbal stem cell from the cornea tissue after step (3) freezing:
The cornea tissue frozen is taken out after six months from liquid nitrogen container, the quick-thawing in 1 minute in 33 DEG C of water-baths is used
PBS is cleaned 3 times.Entire cornea (containing a small amount of sclera) epithelial layer is placed on upward in clean big ware and is spread out, with 3mm trepan with
Corneal limbus is that center line drills through tissue block (will be comprising transparent part and opaque section), obtains corneal limbal tissue block.Corneal limbus
Tissue block takes out after first digesting 15 minutes in 0.1%IV Collagenase Type, and epithelial layer is attached to downward after dipping a small amount of fetal calf serum
In six orifice plates, it is put into drying 30 minutes in incubator later, then 1.5ml KCM culture solution stationary culture is added in every hole respectively,
Cell is moved out after 2~4 days, and the high activity limbal stem cell after freezing screening can be obtained.
To being added cell induction agent and cryogenic freezing treated cornea (processing group cornea) and fresh cornea carries out respectively
Paraffin embedding, slice and immunofluorescence dyeing processing, as a result as shown in Figure 1.The results show that two groups of cornea tissue epithelial structures are complete
Whole preservation, hypothallus arrangement is not close, and obscission occurs in endodermis.In fresh cornea, central cornea and corneal limbus position
Have no that Caspase 1 is expressed.In processing group, central cornea holostrome expresses Caspase 1, and corneal limbus surface layer is visible a small amount of
Caspase 1 is expressed, and basal part position has no the expression of Caspase 1 (white arrow instruction expression position in Fig. 1).In cornea group
In knitting, central cornea position is the corneal epithelial cell of mature differentiation, and corneal limbus basal part is limbal stem cell.At cell
When stress situation, cell can express Caspase 1 and cause cellular inflammation and apoptosis.Therefore, above-mentioned testing result illustrates to pass through
The corneal epithelial cell of mature differentiation, which is inflamed, after cell induction agent and cryogenic freezing processing, in processing group cornea tissue withers
It dies, and the limbal stem cell in organizing is uninfluenced.
Primary training to being added cell induction agent and cryogenic freezing treated cornea (processing group cornea) and fresh cornea
Feeding limbal stem cell carries out FCM analysis respectively, observes P63 positive cell ratio, as a result as shown in Figure 2.As a result it shows
Show, the ratio of processing group cell P63 is 95%, much higher than the 32% of untreated fresh cornea group.P63 is that corneal limbus is dry thin
One of born of the same parents' marker is expressed closely related with the proliferation activity of stem cell.Testing result explanation is by cell induction agent and low
The cornea tissue of warm freezing processing can get the better limbal stem cell of more proliferation activities.
To being added cell induction agent and cryogenic freezing treated cornea (processing group cornea) and fresh cornea originally culture
Limbal stem cell carries out immunofluorescence dyeing and western blot detection, and (atriphos combines observation stem cell ABCG2
Transport protein G superfamily member 2) expression, as a result as shown in Figure 3.The results show that ABCG2 positive expression in processing group
Cell quantity far more than fresh cornea group, processing group cell ABCG2 is expressed western blot half-quantitative detection as the result is shown
It is higher to measure comparatively fresh cornea group.ABCG2 is one of marker of limbal stem cell, and testing result explanation passes through cell induction agent
It can get more better limbal stem cells of activity with the cornea tissue of cryogenic freezing processing.
The above result shows that the corneal limbus that limbal stem cell screening, preservation and cultural method provided by the invention obtain is dry
Cell proportion is high, is able to maintain good Stem Cell Activity.
Embodiment 2: the preparation of human limbal stem cell
(1) cornea tissue is obtained, stand-by cornea tissue is pre-processed:
Human eye is cleaned 5~6 times with Cravit eye drops, later respectively with III type ANER DIAN and addition gentamicin
Normal saline flushing removes eyeball tissue shear and removes eyeball exterior circumferential excess tissue clean, retain 1 along corneal limbus~
5mm sclera simultaneously cuts off rest part, exenterates simultaneously, and by the parts such as iris scraped clean, cornea is added later
The phosphate buffer (PBS) of 1% Pen .- Strep solution cleans 3 times.
(2) cells frozen storing liquid containing cell induction agent is added into pretreated cornea tissue:
The cell culture fluid KCM :+10% fetal calf serum+1% of DMEM in high glucose of in vitro culture limbal stem cell is prepared first
+ 1% nonessential amino acid of glutamine+1% Pen .- Strep solution+10ng/ml epidermal growth factor (EGF)+5 μ g/ml
+ 5 μ g/ml insulin of transferrins, and 4 μ g/ml Econopreds and 10ng/ml tumor necrosis factor α (TNF-α) are added,
Econopred is glucocorticoid, and tumor necrosis factor α is inflammatory factor, and the two together functions as cell induction agent,
Prepare cells frozen storing liquid again: 10% dimethyl sulfoxide adds 90%KCM culture solution.Step (1) pretreated cornea tissue is put
Enter in 1.5ml cryopreservation tube, the prepared cells frozen storing liquid of 1ml is added.
(3) cryogenic freezing will be carried out to be screened and be protected to limbal stem cell added with the cornea tissue of cells frozen storing liquid
It deposits:
Cryopreservation tube in step (2) is put into program temperature reduction box and is placed in -20 DEG C of refrigerators, by cryopreservation tube after being frozen overnight
It is transferred in liquid nitrogen container.
(4) it is separately cultured to obtain limbal stem cell from the cornea tissue after step (3) freezing:
The cornea tissue frozen is taken out after three months from liquid nitrogen container, the quick-thawing in 1 minute in 33 DEG C of water-baths is used
PBS is cleaned 3 times.Entire cornea (containing a small amount of sclera) epithelial layer is placed on upward in clean big ware and is spread out, with scissors clip
The corneal limbal tissue block of 5mm size.Corneal limbal tissue block takes out after first digesting 15 minutes in 0.1%IV Collagenase Type, epithelium
Layer is attached in six orifice plates downward after dipping a small amount of fetal calf serum, is put into drying 30 minutes in incubator later, then every hole difference
1.5ml KCM culture solution stationary culture is added, cell is moved out after 2~4 days, and the high activity cornea after freezing screening can be obtained
Limbal stem cell.
The tumor necrosis factor α of cornea, the relative expression quantity of interleukin 6 and interleukin-11 β, knot are detected by RT-qPCR method
Fruit is as shown in Figure 4: compared with fresh cornea, after cell induction agent and cryogenic freezing processing is added, the cornea tissue of processing group is swollen
Tumor necrosis factor α, tri- kinds of inflammatory factors of interleukin 6 and interleukin-11 β transcriptional level significantly raise.It is damaged in cornea tissue body
After wound, tumor necrosis factor α, interleukin 6 and interleukin-11 β expression can be raised.Testing result illustrates through cell induction agent and low temperature
After freezing processing, the inflammation stress reaction of processing group cornea tissue can be caused, thus reach corneal injury signal in analogue body, it is real
The activation of existing limbal stem cell.
It carries out paraffin embedding, slice and immunofluorescence dyeing respectively to fresh cornea and processing group cornea to handle, as a result such as
Shown in Fig. 5.The results show that two groups of cornea tissue epithelial structures completely save, in fresh cornea and in processing group cornea tissue,
The visible P63 positive expression of corneal limbus position basal part, the limbal stem cell of K3 feminine gender expression.The result shows that being induced through cell
Agent will not have an impact the Stem Cell Activity of the limbal stem cell in tissue with cryogenic freezing processing.
FCM analysis, flow cytomery result such as Fig. 6 institute are carried out to two groups of originally culture limbal stem cells
Show.The positive cell rate of the stem cell markers P63 of fresh cornea group originally culture limbal stem cell is 62% as the result is shown,
It is 32% that corneal epithelial cell, which breaks up marker K3 positive cell rate,.In processing group, the positive cell rate of P63 is increased to 91%,
K3 positive cell rate is reduced to 20%.Testing result shows that the cornea tissue handled by cell induction agent and cryogenic freezing can
It is more preferable to obtain more proliferation activities, the lower limbal stem cell of differentiation degree.
The above result shows that the corneal limbus that limbal stem cell screening, preservation and cultural method provided by the invention obtain is dry
Cell proportion is high, and Stem Cell Activity is good.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (10)
1. a kind of preparation method of limbal stem cell, which is characterized in that comprise the steps of:
Cornea tissue is obtained, stand-by cornea tissue is pre-processed；
The cells frozen storing liquid containing cell induction agent is added into pretreated cornea tissue；
Freeze the cornea tissue added with cells frozen storing liquid；
The cell induction agent is that the cell that can induce in cornea tissue is locally inflamed or stress reaction, but does not draw directly
Play the reagent of cell death.
2. the preparation method of limbal stem cell according to claim 1, which is characterized in that the cell induction agent is selected from
One of inflammatory factor, hormone, the agent of intracellular signal molecular regulation and Cycle Regulation agent are a variety of.
3. the preparation method of limbal stem cell according to claim 2, which is characterized in that the inflammatory factor is selected from white
One of interleukin, interferon and tumor necrosis factor are a variety of；The hormone is selected from glucocorticoid, mineralocorticoid and property
One of hormone is a variety of；The intracellular signal molecular regulation agent is selected from actinomyces, protein kinase C activators, protein kinase C
One of inhibitor and DNA topoisomerase enzyme inhibitor are a variety of；The Cycle Regulation agent is selected from cyclin
Dependant kinase inhibitors, period element, Cyclin kinase inhibitor, G1 specific cell cyclin,
G2/ mitosis specific cell cyclin and its one of transcription or degradation factor or a variety of.
4. the preparation method of limbal stem cell according to claim 3, which is characterized in that the cell induction agent is selected from
One of Econopred, Staurosporine and tumor necrosis factor α are a variety of.
5. the preparation method of limbal stem cell according to claim 1, which is characterized in that the cells frozen storing liquid be containing
There are the cell culture fluid of the cell induction agent and the mixed liquor of cryoprotector；The cryoprotector and contain the cell
The volume ratio of the cell culture fluid of inducer is 1:2-20.
6. the preparation method of limbal stem cell according to claim 5, which is characterized in that the cell induction agent is in institute
Stating the concentration in cell culture fluid is 1ng/ml-100 μ g/ml；And/or
The cryoprotector is selected from dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, acetamide, methanol, polyvinylpyrrolidine
One of ketone, fructose, sucrose, glucan, albumin, polyethylene glycol, hydroxyethyl starch and yolk are a variety of.
7. the preparation method of limbal stem cell according to claim 1-6, which is characterized in that the freezing
Temperature is 0 DEG C~-200 DEG C；And/or
The time of the freezing is 1-120 months；And/or
The pretreatment includes: conventional to the progress of stand-by cornea tissue clear using the physiological buffer comprising or not comprising antibiotic
It washes, sterilize, separate；And/or
Any one or more in cornea tissue of the cornea tissue selected from people, rodent and other mammals,
The cornea tissue is fresh cornea tissue or any one of the cornea tissue by short-term preservation, wherein described short-term
The time of preservation is 1 day~20 days.
8. the preparation method of limbal stem cell according to claim 1-6, which is characterized in that also comprising following
Step: the cornea tissue after being separately cultured freezing is to get the limbal stem cell.
9. the preparation method of limbal stem cell according to claim 8, which is characterized in that the cell for the culture
Added with cell induction agent described in 1ng/ml-100 μ g/ml in culture solution.
10. the limbal stem cell being prepared by the preparation method of the described in any item limbal stem cells of claim 1-9.
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