CN108277204A - A kind of method that bioengineering cultivates eye Full-thickness corneal - Google Patents
A kind of method that bioengineering cultivates eye Full-thickness corneal Download PDFInfo
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- CN108277204A CN108277204A CN201810023662.9A CN201810023662A CN108277204A CN 108277204 A CN108277204 A CN 108277204A CN 201810023662 A CN201810023662 A CN 201810023662A CN 108277204 A CN108277204 A CN 108277204A
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Abstract
The present invention relates to field of cell culture, more particularly to the method that a kind of bioengineering cultivates eye Full-thickness corneal, each layer of cornea is cultivated respectively, and advantage is when patient does not need Allograft rejection, it is only necessary to cultivate the vitreous layer of patient's needs.The seed cell of the preparation method of the total corneal provided in the present invention, corneal epidermal layer and interstitial lamella is autogenous cell, and endothelial cell comes from contributor, can obtain a large amount of endothelial cell by culture, avoid the finiteness in contributor source.The present invention can promote growth in the nerve of life object engineering cornea using collagen culture medium as the glued medium of epidermis and interstitial lamella.Total corneal provided by the invention or part cornea, all have good optical characteristics, after operation, patient's full recovery eyesight；And cornea has stable biochemical characteristic, no late period corneal solution, good biocompatibility, no rejection.
Description
Technical field
The present invention relates to field of cell culture, a kind of method for cultivating eye Full-thickness corneal in particular to bioengineering.
Background technology
Disease of cornea is that incidence is high in the world at present, since treatment means are limited, the main reason for being blinding it
One.The main method for the treatment of disease of cornea is total corneal or lamellar keratoplasty at present.Due to by cornea qualification donor source
The limitation of shortage, the overwhelming majority cannot be treated effectively because of the patient of disease of cornea blindness or some lost eyesight.
Nearly ten years, the external structure biology cornea that develops into of bioengineered tissue technology provides technology possibility, biology
Reactor preferably simulates the living environment in human body, can be with external preparation group in the way of seed cell and timbering material
Knit engineering cornea；Meanwhile many technologies can provide other selections after animal heterogenic cornea takes off cell processing to clinical.
What the country formed industrialization is exactly the production of corneal stroma at present, as pig cornea hypothallus takes off cell processing.
But this is a layer in the middle part of cornea and the most ripe part of bioengineered tissue technology.Most patients need angle
Film endodermis or the preceding deep plate layer of cornea (corneal epidermal layer and corneal stroma), therefore total corneal or corneal endothelial layer, cornea
The bioengineering culture of preceding deep plate layer (corneal epidermal layer and corneal stroma) is the weight treated because of cornea illness blindness patient
The culture of a ring, especially corneal endothelial layer is wanted, is built.
Other tissue engineering techniques cultivate holostrome biology cornea using foreign peoples or variant cell as seed cell on holder,
Which not only improves the risks of infection transfection disease, and there is also the danger of potential histogenic immunity rejection, lead to operative failure.
In view of this, special propose the present invention.
Invention content
The purpose of the present invention is to provide the preparation methods of a kind of each layer of cornea and full cornea and obtained each
Layer and full cornea, good basis is provided for the treatment of cornea.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
A kind of preparation method of collagen culture medium, collagen solution are mixed with buffer solution, and EDC solution is then added
With NHS solution, pH is adjusted as after 5.4-5.6, then solidification is afforded using 10 ± 2mM PBS；
Wherein, the mole ratio of the 3- amine groups of EDC, NHS and collagen is 3：2.8-3.5：2.8-3.5.
Solid collagen protein culture medium is made in the preparation method of collagen culture medium provided by the invention, is cornea
The culture of epidermis and total corneal provides good basis.
Wherein, the mole ratio of the 3- amine groups of EDC, NHS and collagen can be 3：2.8：2.8、3：3：3、3：
3.5：3.5、3：3.2：3.5 etc..
In the present invention, EDC is the abbreviation of 1- ethyls -3- (3- dimethylaminopropyls)-carbodiimide；NHS is N- hydroxyls
The abbreviation of succinimide.
Further, a concentration of 13.7w/w% ± 0.5w/w% of the collagen solution, the buffer solution are MES
Buffer solution.
Preferably, collagen solution is still people's recombination III-type collagen solution.
Preferably, a concentration of 0.625 ± 0.05M of the MES buffer solutions.
Further, the volume of the MES buffer solutions and the collagen solution is 1:0.8-1.5.Such as the body of the two
Product is than that can be 1:0.8、1:1、1:1.2、1:1.5 etc..
Collagen solution is sufficiently mixed with MES buffer solutions under ice water environment, such as can be in syringe mixed stocker
System mixing.
Preferably, the EDC solution and NHS solution are the mixed liquor of EDC and NHS, in the mixed liquor, the concentration of EDC
For 200 ± 5mg/ml, a concentration of 120 ± 5mg/ml of NHS, the MES that the solvent of the mixed liquor is 0.625 ± 0.05M is buffered
Liquid.
The solution of debita spissitudo, the ingredient mixing between more conducively each ingredient is uniform, also, conducive to the mutual friendship of each ingredient
Connection reaction provides good basis for subsequent solidification.
Further, pH is adjusted to adjust using lye.
Further, pH is adjusted to adjust using 2N sodium hydroxide solutions.
Further, further include before the solidification：Mixed liquor is put into mold, is subsequently placed in the room temperature of 95%-100% humidity
Lower placement 15-18h.Wherein, room temperature refers to 15-25 DEG C.
It by being put into mold, is then placed under conditions of certain humidity so that surface is not done when follow-up mixed liquor solidification
It is dry.
Further, the cured temperature is 37 ± 2 DEG C.
Further, the cured time is 4.5-5.5h.
Solidification carries out in the incubator.
After solidification, 10 ± 2mM PBS are soaked into, in favor of demoulding, obtain hydrogel sample culture medium, it is then placed in 10 ±
2mM PBS elutions, a liquid are replaced per 5-10h, until using.
Further, the hydrogel sample culture medium obtained after the elution, which immerses, contains penicillin and streptomysin and 1%
In 10 ± 1mM PBS of ± 0.1% chloroform, 2-10 DEG C of preservation.
Further, the present invention also provides collagen culture mediums made from above-mentioned preparation method.
Further, amnion is spread on the surface of the collagen culture medium, then coats N- on the surface of the amnion
Isopropyl based polyalcohol.Obtained collagen culture medium can be used.
The present invention also provides a kind of cultural methods of cornea epidermis, include the following steps：
The single cell suspension of oral mucosa epithelial cell is made in oral mucosas tissue；
The single cell suspension accesses on above-mentioned collagen culture medium, and is put into the single celled periphery of inoculation
T3T feeder cells and mitomycin C, to secrete growth hormone and cell factor；
Culture obtains corneal epithelium.
The cultural method of cornea epidermis provided by the invention is using Buccal mucosa cell through induction differentiation system
At.
Further, the oral mucosas tissue uses dispase II processing, and after removing epithelial layer, it is thin that collection obtains epithelium
Born of the same parents are handled with digestive juice, obtain the single cell suspension of the oral mucosa epithelial cell.
Further, a concentration of 2.8-3.5mg/ml of the dispase II, using the dispase II at 37 ± 2 DEG C
Handle 50-70min.
A concentration of 2.8mg/ml, 3.0mg/ml, 3.2mg/ml, 3.5mg/ml of such as dispase II.
The purpose of digestive juice processing is to decompose epithelial cell, to obtain single celled epithelial cell.
Further, the digestive juice is that the trypsase that mass concentration is 0.2%-0.3% is with mass concentration
The time of the mixed solution of the EDTA of 0.005%-0.015%, the digestive juice processing is 12-18min.
Preferably, the temperature of the culture is 37 ± 2 DEG C, and the time of the culture is 12-15 days.
Further, temperature can be removed after being down to 20 DEG C, 30 minutes to patient do corneal epithelium transplanting or and with
Interstitial lamella is glued, continues to cultivate.
The characteristic of temperature sense makes N- isopropyls based polyalcohol at 30 DEG C or more, its hydrability of reversible change, generates
Different characteristics.At 30 DEG C or more, can chemistry the amnion for being fixed on cell culturing surfaces, promote cell adherence and normal
Growth.Cultivation temperature, which is reduced to 30 DEG C or less, can make the surface hydration and expand rapidly, and attached cell is promoted to be kept completely separate, without
It needs using typical proteolytic enzyme or with trypsase EDTA processing.
The present invention also provides a kind of cultural methods of cornea interstitial lamella, include the following steps：
By the skin corium of thigh skin, it is unicellular that fibroblast is made；
The fibrocyte is unicellular to be planted in different transparent fibroin protein films, and the transparent fibroin albumen enzyme impregnates
In the DMEM in high glucose culture solution containing penicillin and streptomysin, two-dimentional culture is carried out；
Different transparent fibroin protein films overlap, and carry out dimensional culture, obtain corneal stroma layer.
The cultural method of cornea interstitial lamella provided by the invention is that fibrocyte is made using the skin corium of thigh skin
It is unicellular, then two and three dimensions culture is carried out, obtain corneal stroma layer.
Further, the thigh skin is medial thigh skin.
Preferably, the thigh skin is first pre-processed, and the pretreatment is removal blood stains after cleaning and wipes out connective
Tissue.
Further, after the pretreatment, digestion process is carried out, after cleaning, epidermis is removed, corium is shredded, clostridiopetidase A
Processing is incubated, is filtered after piping and druming, it is unicellular to obtain fibroblast.
Further, the digestion process is：Skin is immersed in the mixed liquor containing penicillin, streptomysin and digestive juice,
Wherein, a concentration of 18-25U/ml of penicillin, a concentration of 18-25 μ g/ml of streptomysin are dense containing quality in the digestive juice
The EDTA that the trypsase and mass concentration that degree is 0.25% are 0.01%, the volume ratio of the digestive juice account for the mixed liquor
8%-15% keeps 2-10 DEG C of temperature, places 8-14h.
Further, described clean is：It is rinsed repeatedly with the phosphate buffered saline containing penicillin and streptomysin
3-5 times.
Further, the collagen enzymatic treatment is：Mixed liquor containing penicillin, streptomysin and clostridiopetidase A is incubated at 37 DEG C
Educate 4-8h, wherein a concentration of 0.8-1.5mg/ml of the clostridiopetidase A.
Further, PBS mixing is added, blows and beats repeatedly, filters, the DMEM trainings containing 10% fetal calf serum are resuspended in after centrifugation
Nutrient solution obtains fibroblast single cell suspension.
Further, the fibroblast single cell suspension is by 3500-4500/cm2Be inoculated in containing penicillin and
In the DMEM in high glucose culture solution of streptomysin, in 37 ± 2 DEG C and 5% ± 0.2%CO2Lower culture cell, after 24-48h for the first time more
Culture solution is changed, was replaced later per 2-3 days primary.
Further, the unicellular density for being seeded in transparent fibroin protein film of the fibrocyte is 4 × 104-6×
104cells/cm2, condition of culture is：In 37 ± 2 DEG C and 5% ± 0.2%CO2, the time of culture is preferably 40-55h.
Further, the dimensional culture is that 5-10 transparent fibroin protein films overlap.Such as can be 5,6
It is a, 7,8,9 or 10 transparent fibroin protein films overlap.
Further, condition of culture is：In 37 ± 2 DEG C and 5% ± 0.2%CO2。
Further, the culture solution used in the dimensional culture is the DMEM in high glucose culture solution containing penicillin and streptomysin,
A culture solution was replaced per 2-3 days.
Further, the time of the dimensional culture is 55-70 days.As dimensional culture time can be 55 days, 58 days,
60 days, 62 days, 65 days, 68 days, 70 days etc..
The present invention also provides a kind of cultural methods of endothelial cell, include the following steps：
By canthus Membrane cleaning, after removing blood stains, Descemet films and endodermis are removed；
The endodermis is further processed, and obtains single corneal endothelium confluent monolayer cells；
The corneal endothelial layer cell culture, obtains more endothelial cells.
A kind of cultural method of endothelial cell provided by the invention is using cornea as raw material, through detaching and cultivating
Endothelial cell is obtained, obtained endothelial cell can further pass on or expand culture, obtain a large amount of corneal endothelium
Cell.
Further, it is used in ten days after the cornea is in vitro.
Further, the cleaning is given birth to using containing the phosphate-buffered of 100U/ml penicillin and 100 μ g/ml streptomysins
Brine is managed, is cleaned 2-5 times, each 10-18min.
Further, the endodermis of stripping uses collagen enzymatic treatment, it is preferred to use the collagen of a concentration of 0.8-1.51mg/ml
Enzymatic treatment obtains the endothelial cell of clustering, is then handled, is obtained with TryPLE Express Human Umbilical Vein Endothelial Cells clusters again
The endothelial cell of smaller agglomerate or individual cells, separation covers the primary training of the new culture medium progress of MEM of FNC mixtures on surface
It supports, obtains two generation cells；
The FNC mixtures are the mixed liquor of fibronectin and collagen, wherein containing 10 μ g/ml of fibronectin and glue
35 μ g/ml of former albumen, solvent is fetal calf serum；
The new culture mediums of MEM are：On MEM medium bases, also contain following component：Fetal calf serum 8% ±
0.2%, 20 ± 1ng/ml of nerve growth factor, 5 ± 0.2ng/ml of epithelical cell growth factor, 20 ± 1mg/ml of ascorbic acid,
200 ± 2mg/L of calcium chloride, 100 ± 2 μ g/ml of pituitary extract, gentamicin 50 ± 2 μ g/ml, 100 ± 2U/ml of penicillin,
Streptomysin 100 ± 2 μ g/ml, chondroitin sulfate 0.08w/w% ± 0.01w/w%.
Further, FNC mixtures are 0.15-0.25ml/cm in the additive amount of the new media surfaces of the MEM2。
Further, the two generations cell carries out secondary culture after pancreatin digests.
Preferably, the density of the secondary culture is 4000-6000 cell/cm2。
The present invention also provides a kind of preparation methods of total corneal, include the following steps：Above-mentioned cultural method is obtained
Cornea interstitial be placed on above-mentioned collagen culture medium；
The cornea epidermis that above-mentioned cultural method obtains is fitted in the cornea interstitial layer surface, in the cornea
Chrotoplast injects between the cornea interstitial lamella and iris the endothelial cell layer for forming single layer, obtains the total corneal.
Rejection in order to prevent, the present invention in cornea interstitial lamella and cornea epidermis raw material be taken from from
Body, endothelial cell are provided by donor, then can get a large amount of endothelial cell through cultivating, in this way, different patients
Cornea interstitial lamella and cornea epidermis between injection endothelial cell can be obtained total corneal, therefore, this is with regard to effective
Avoid the limited problem of rejection and contributor's quantity.
Further, the cornea interstitial lamella is all from autogenous cell with the cornea epidermis
Further, the cornea epidermis is fitted in the interstitial layer surface in 37 ± 2 DEG C and 5%CO2Lower culture
3-5h。
Compared with prior art, beneficial effects of the present invention are：
(1) existing cornea technology is first to do interstitial lamella holder, then the one piece of culture of epithelial layer endodermis interstitial lamella；This hair
The advantages of bright cultural method for each providing each layer of cornea obtains each layer of cornea, and each layer of cornea is cultivated respectively, which also resides in, works as
When patient does not need Allograft rejection, it is only necessary to cultivate the vitreous layer of patient's needs.
(2) rejection cause the incidence of operative failure in total keratoplasty postoperative 2 years be about 13%, plate layer
Postoperative 2 years of corneal transplantation is 7.5%, and the seed cell of corneal epidermal layer and interstitial lamella of the present invention is autogenous cell, non-xenogenesis
Or variant cell avoids tissue rejection reaction in the case of preceding deep lamellar keratoplasty.
(3) seed cell of the preparation method of the total corneal provided in the present invention, corneal epidermal layer and interstitial lamella is self
Cell, and endothelial cell comes from contributor, can obtain a large amount of endothelial cell by culture, avoid contributor source
Finiteness.
(4) present invention can promote life object using collagen culture medium as the glued medium of epidermis and interstitial lamella
Growth in the nerve of engineering cornea.
(5) total corneal provided by the invention or part cornea, all have good optical characteristics, and after operation, patient is whole
Restore eyesight；And cornea has stable biochemical characteristic, and no late period corneal solution, good biocompatibility, no repulsion is instead
It answers.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 is the corneal epidermal layer and normal cornea table for the method culture provided by the invention that the embodiment of the present invention 1 provides
The Electronic Speculum observation chart of cortex；
Fig. 2 is the plan view of the corneal stroma layer for the method culture provided by the invention that the embodiment of the present invention 2 provides；
Fig. 3 is the cross-sectional view of the corneal stroma layer for the method culture provided by the invention that the embodiment of the present invention 2 provides；
Fig. 4 be the corneal stroma layer of method culture provided by the invention that provides of the embodiment of the present invention 2 with other products into
The curve graph that capable transparency compares.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Collagen culture medium：By the still people's recombination III-type collagen solutions of the 13.7%w/w of 0.3ml and 0.3ml
MES buffer solutions are sufficiently stirred in syringe hybrid system under ice water bubble；After stirring, the EDC/NHS solution of 57 μ l is injected, this is molten
In liquid, the solvent of a concentration of 120 ± 5mg/ml of a concentration of 200 ± 5mg/ml of EDC, NHS, the solution are 0.625 ± 0.05M
MES buffer solutions.
So that molar equivalent ratio EDC：NHS (quantity of the 3- amine groups of collagen) is 3：3：3.With 2N sodium hydroxides by pH value
5.5 are adjusted to, then, is injected the mixture into the plastic mould of bending (thickness 500mm, diameter 12mm).Mold is placed in 100%
16 hours at room temperature of humidity, solidification 5 hours after being then transferred into incubator at 37 ± 2 DEG C.After solidification, mold is immersed
30 minutes or so in 10 ± 1mM PBS, it is then carefully removed from the molds hydrogel.
The hydrogel of obtained bending is eluted in PBS, is replaced per 8h primary.Then hydrogel is immersed containing dual anti-
And 1% chloroform PBS in, 4 DEG C of conditions preserve.
Amnion is spread on the surface of collagen culture medium, then coats N- isopropyl based polyalcohols on the surface of amnion, i.e.,
It can be used for the culture application of cell.
Embodiment 1
Cornea epidermis culture
Patient's lidocaine local anaesthesia, patient's oral cavity partial povidone iodine disinfection, patient is taken from internal buccal mucosa epithelium
Oral mucosas tissue's about 3 × 3mm sizes.
Oral mucosas tissue is handled with 5ml dispases II (3mg/ml, Roche) at 37 DEG C to remove on all for 1 hour or so
After cortex, it is collected into oral mucosa epithelial cell, is subsequently placed in digestive juice 15 minutes, to form single cell suspension.
Isolated cell is put into collagen culture medium, and media surface is amnion, and N- isopropyls are coated on amnion
Polymer.
T3T feeder cells and mitomycin C are put into periphery simultaneously, to secrete growth hormone and cell factor.
At 37 DEG C, 2 weeks or so time is cultivated, you can the cuticular cellulose of macroscopic active 3-5 layers is obtained,
Through hematoxylin eosin staining, micro- sem observation, optical characteristics and structure and the normal cornea of the corneal epithelium cultivated
Epithelial layer indistinction.The results are shown in Figure 1, and in Fig. 1, A is the corneal epidermal layer that present invention culture obtains, and B is normal cornea table
Cortex.
Temperature can be removed after being down to 20 DEG C, 30 minutes to patient do corneal epithelium transplanting or and with interstitial lamella glue
It closes, continues to cultivate.
Embodiment 2
Corneal stroma layer culture
1. autologous skin fibroblast detaches
Under aseptic condition, limb femoribus internus partial skin tissue 3 × 2mm sizes are removed, are placed in containing 100U/ml penicillin
With cleaning 3-5 times in the phosphate buffered saline (PBS) of 100ug/ml streptomysins, removes blood stains and wipe out connective tissue.
Skin is immersed in 10ml and contains dual anti-dose of 2ml (100U/ml penicillin and 100 μ g/ml streptomysins) and 1ml digestive juices
Mixed liquor, keep 4 DEG C of temperature, stand overnight.
Next day is continuing with the phosphate buffered saline containing penicillin and streptomysin (dual anti-) and rinses 3-5 repeatedly
It is secondary, then detach corium and epidermis, corium be cut into the fragment of very little using new aseptic operation knife, by fragment be transferred to containing
In the 50ml centrifuge tubes of dual anti-dose of 0.5ml and 5ml clostridiopetidase As (1mg/ml).
It is incubated 4-8 hours at 37 DEG C, it is appropriate to shake.
2ml PBS are added, blow and beat repeatedly, then by 70 μm of cell filters, obtain fibroblast single cell suspension.
Single cell suspension 1000r/min centrifuges 5min, abandons supernatant, is resuspended in DMEMs of the 2ml containing 10% fetal calf serum and trains
Nutrient solution.
Cell count, and with 4000/cm2Be inoculated in the DMEM culture solutions containing dual anti-and high sugar (10% fetal calf serum,
100U/ml penicillin, 100ug/ml streptomysins) in culture dish, in 37 DEG C and 5% ± 0.2%CO2Lower culture cell, 24-48 are small
When after the first subculture replace, then per 2-3 days replace once.
2. single layer two dimension Fibroblast cell-culture
Obtained human fibroblasts, in 37 DEG C and 5% ± 0.2%CO2Under be planted in single-layer and transparent fibroin protein film (12
× 12mm) on, density is 5 × 104cells/cm2, cultivate in the DMEM culture solutions containing dual anti-and high sugar (10% fetal calf serum,
100U/ml penicillin, 100ug/ml streptomysins).Similar culture is done simultaneously plants piece 7.
3. three-dimensional cornea interstitial lamella culture
After 48 hours, the fibroblastic fibroin protein film of 7 kinds of someone is planted piece, overlaps, does dimensional culture.
Culture solution is updated, is kept for 37 DEG C and 5% ± 0.2%CO2, then change within every 3 days 1 DMEM training containing dual anti-and high sugar
Nutrient solution (10% fetal calf serum, 100U/ml penicillin, 100 μ g/ml streptomysins).Culture about 60 days, you can obtain transparent angle
Film interstitial lamella.
The plan view of obtained corneal stroma layer is as shown in Figure 2.The cross section of corneal stroma layer is through hematoxylin eosin staining
Afterwards as shown in Figure 3.
Obtained corneal stroma layer carries out transparency comparison with other products, and the results are shown in Figure 4.
Embodiment 3
The culture of endothelial cell
The culture seed cell of endothelial cell is obtained by the cornea of other donors's (age is the smaller the better),
It is used in 10 days after donations.
Cornea is placed in the phosphate buffered saline containing 100U/ml penicillin and 100 μ g/ml streptomysins and cleans 2-
5 times, each 10-18min, after removing blood stains, by means of the careful stripping posterior elastic membrane of vacuum aspirator (Descemet films) and
Endodermis, the posterior elastic membrane and endodermis of stripping can be rolled along endodermis automatically.
The posterior elastic membrane and endodermis being collected into are handled using clostridiopetidase A (1mg/ml), after 2 hours, it can be seen that corneal endothelium
Layer segment detaches.Endothelial cell is kept completely separate, need collagen enzymatic treatment about 6 hours (according to contributor's difference, when
Between distinguish it is very big) after, endothelial cell can clustering.
It usesTryPLE Express Human Umbilical Vein Endothelial Cells clusters carry out the processing of 5min, can be allowed to become smaller
Agglomerate or individual cells.
Moderate centrifuges the endothelial cell same day after (800g, 5 minutes) separation in FNC coverings (0.2ml NFC/cm2) MEM
New culture medium does original cuiture.FNC mixtures are the mixed liquor of fibronectin and collagen, wherein containing 10 μ of fibronectin
35 μ g/ml of g/ml and collagen, solvent is fetal calf serum.The new culture mediums of MEM are：On MEM medium bases, also contain with
Lower ingredient：Fetal calf serum 8%, nerve growth factor 20ng/ml, epithelical cell growth factor 5ng/ml, ascorbic acid 20mg/
Ml, calcium chloride 200mg/L, 100 μ g/ml of pituitary extract, gentamicin 50 μ g/ml, penicillin 100U/ml, streptomysin 100
μ g/ml, chondroitin sulfate 0.08w/w%.
After 2 weeks, it can be seen that the second subtituted culturing cell.Fused cell is usedTryPLE Express carry out pancreas egg
White enzymic digestion and with 5,000 cell/cm2Density continue to sow.
Embodiment 4
The structure of total corneal equivalent
It is maintained at 37 DEG C and 5% ± 0.2%CO2Under, cultured interstitial lamella is put into collagen culture medium, culture medium
Surface is amnion, N- isopropyl based polyalcohols is coated on amnion, between the cornea epidermis of bioengineered tissue culture is fitted in
Matter layer surface can take out use in 4 hours.
The endothelial cell of culture injects between interstitial lamella and iris the cornea that can form single layer in the course of surgery
Endothelial layer.
The present invention can promote people's bioengineering using collagen culture medium as the glued medium of epidermis and interstitial lamella
Growth in the nerve of cornea.
Through lot of experiment validation, different vitreous layers and the equal corresponding disease of successful treatment cornea of total corneal obtained.
Total corneal provided by the invention or part cornea, all have good optical characteristics, after operation, patient's full recovery eyesight；And
And cornea has stable biochemical characteristic, no late period corneal solution, good biocompatibility, no rejection.
In the embodiment of the present invention, digestive juice used is that the trypsase that mass concentration is 0.2%-0.3% and quality are dense
Degree is the mixed solution of the EDTA of 0.005%-0.015%.
Although illustrate and describing the present invention with specific embodiment, it will be appreciated that without departing substantially from the present invention's
Many other change and modification can be made in the case of spirit and scope.It is, therefore, intended that in the following claims
Including belonging to all such changes and modifications in the scope of the invention.
Claims (10)
1. a kind of preparation method of collagen culture medium, which is characterized in that collagen solution is mixed with buffer solution, is then added
Enter EDC solution and NHS solution, adjusts pH as after 5.4-5.6, then solidification is afforded using 10 ± 2mM PBS；
Wherein, the mole ratio of the 3- amine groups of EDC, NHS and collagen is 3：2.8-3.5：2.8-3.5.
2. the preparation method of collagen culture medium according to claim 1, which is characterized in that the collagen solution
A concentration of 13.7w/w% ± 0.5w/w%, the buffer solution be MES buffer solutions, it is preferable that the concentration of the MES buffer solutions
For 0.625 ± 0.05M；
Further, the volume of the MES buffer solutions and the collagen solution is 1:0.8-1.5；
Preferably, the EDC solution and NHS solution are the mixed liquor of EDC and NHS, in the mixed liquor, a concentration of the 200 of EDC
A concentration of 120 ± 5mg/ml of ± 5mg/ml, NHS, the solvent of the mixed liquor are the MES buffer solutions of 0.625 ± 0.05M；
Further, further include before the solidification：Mixed liquor is put into mold, is subsequently placed in the room temperature decentralization of 95%-100% humidity
Set 15-18h；
Further, the cured temperature is 37 ± 2 DEG C；
Further, the hydrogel sample culture medium that is obtained after the elution immerses containing penicillin and streptomysin and 1% ±
In 10 ± 1mM PBS of 0.1% chloroform, 2-10 DEG C of preservation.
3. collagen culture medium made from the preparation method of collagen culture medium as claimed in claim 1 or 2；
Further, amnion is spread on the surface of the collagen culture medium, then coats N- isopropyls on the surface of the amnion
Based polyalcohol.
4. a kind of cultural method of cornea epidermis, which is characterized in that include the following steps：
The single cell suspension of oral mucosa epithelial cell is made in oral mucosas tissue；
On collagen culture medium described in the single cell suspension access claim 3, and in the single celled outer of inoculation
It encloses and is put into T3T feeder cells and mitomycin C, to secrete growth hormone and cell factor；
Culture obtains corneal epithelium.
5. the cultural method of cornea epidermis according to claim 4, which is characterized in that the oral mucosas tissue adopts
With dispase II processing, after removing epithelial layer, collection obtains epithelial cell, is handled, is obtained on the oral mucosa with digestive juice
The single cell suspension of chrotoplast；
Further, a concentration of 2.8-3.5mg/ml of the dispase II is handled using the dispase II at 37 ± 2 DEG C
50-70min；
Further, it is 0.005%- that the digestive juice, which is trypsase and mass concentration that mass concentration is 0.2%-0.3%,
The time of the mixed solution of 0.015% EDTA, the digestive juice processing is 12-18min；
Preferably, the temperature of the culture is 37 ± 2 DEG C, and the time of the culture is 12-15 days.
6. a kind of cultural method of cornea interstitial lamella, which is characterized in that include the following steps：
By the skin corium of thigh skin, it is unicellular that fibroblast is made；
The fibrocyte is unicellular to be planted in different transparent fibroin protein films, and the transparent fibroin albumen enzyme, which is immersed in, to be contained
In the DMEM in high glucose culture solution of penicillin and streptomysin, two-dimentional culture is carried out；
Different transparent fibroin protein films overlap, and carry out dimensional culture, obtain corneal stroma layer.
7. the cultural method of cornea interstitial lamella according to claim 6, which is characterized in that the thigh skin is thigh
Inside skin；
Preferably, the thigh skin is first pre-processed, and the pretreatment is removal blood stains after cleaning and wipes out connective tissue；
Further, after the pretreatment, digestion process is carried out, after cleaning, epidermis is removed, corium is shredded, collagen enzymatic treatment,
It is incubated, is filtered after piping and druming, it is unicellular to obtain fibroblast；
Further, the digestion process is：Skin is immersed in the mixed liquor containing penicillin, streptomysin and digestive juice, wherein
A concentration of 18-25U/ml of penicillin, a concentration of 18-25 μ g/ml of streptomysin are containing mass concentration in the digestive juice
The EDTA that 0.25% trypsase and mass concentration is 0.01%, the volume ratio of the digestive juice account for the 8%- of the mixed liquor
15%, 2-10 DEG C of temperature is kept, 8-14h is placed；
Further, described clean is：3-5 is rinsed repeatedly with the phosphate buffered saline containing penicillin and streptomysin
It is secondary；
Further, the collagen enzymatic treatment is：Mixed liquor containing penicillin, streptomysin and clostridiopetidase A is incubated 4- at 37 DEG C
8h, wherein a concentration of 0.8-1.5mg/ml of the clostridiopetidase A；
Further, PBS mixing is added, blows and beats repeatedly, filters, the DMEM culture solutions containing 10% fetal calf serum is resuspended in after centrifugation
Obtain fibroblast single cell suspension；
Further, the fibroblast single cell suspension is by 3500-4500/cm2It is inoculated in and contains penicillin and streptomysin
DMEM in high glucose culture solution in, in 37 ± 2 DEG C and 5% ± 0.2%CO2Lower culture cell replaces culture after 24-48h for the first time
Liquid was replaced per 2-3 days primary later.
8. the cultural method of cornea interstitial lamella according to claim 6, which is characterized in that the fibrocyte is unicellular
The density for being seeded in transparent fibroin protein film is 4 × 104-6×104cells/cm2, condition of culture is：37 ± 2 DEG C and 5% ±
0.2%CO2, the time of culture is preferably 40-55h；
Further, the dimensional culture is that 5-10 transparent fibroin protein films overlap；
Further, condition of culture is：In 37 ± 2 DEG C and 5% ± 0.2%CO2；
Further, the culture solution used in the dimensional culture is the DMEM in high glucose culture solution containing penicillin and streptomysin, per 2-3
It replaces a culture solution；
Further, the time of the dimensional culture is 55-70 days.
9. a kind of cultural method of endothelial cell, which is characterized in that include the following steps：
By canthus Membrane cleaning, after removing blood stains, Descemet films and endodermis are removed；
The endodermis is further processed, and obtains single corneal endothelium confluent monolayer cells；
The corneal endothelial layer cell culture, obtains more endothelial cells；
Further, it is used in ten days after the cornea is in vitro；
Further, the cleaning uses the phosphate-buffered physiology salt containing 100U/ml penicillin and 100 μ g/ml streptomysins
Water cleans 2-5 times, each 10-18min；
Further, the endodermis of stripping uses collagen enzymatic treatment, it is preferred to use at the clostridiopetidase A of a concentration of 0.8-1.51mg/ml
Reason, obtains the endothelial cell of clustering, is then handled again with TryPLEExpress Human Umbilical Vein Endothelial Cells clusters, obtain smaller
Agglomerate or individual cells, the new culture mediums of MEM that the endothelial cell of separation covers FNC mixtures on surface carry out original cuiture,
Obtain two generation cells；
The FNC mixtures are the mixed liquor of fibronectin and collagen, wherein containing 10 μ g/ml of fibronectin and collagen egg
White 35 μ g/ml, solvent are fetal calf serum；
The new culture mediums of MEM are：On MEM medium bases, also contain following component：Fetal calf serum 8% ± 0.2%, god
Through 0 ± 1ng/ml of growth factor-2,5 ± 0.2ng/ml of epithelical cell growth factor, 20 ± 1mg/ml of ascorbic acid, calcium chloride 200
± 2mg/L, 100 ± 2 μ g/ml of pituitary extract, gentamicin 50 ± 2 μ g/ml, 100 ± 2U/ml of penicillin, streptomysin 100
± 2 μ g/ml, chondroitin sulfate 0.08w/w% ± 0.01w/w%；
Further, the two generations cell carries out secondary culture after pancreatin digests；
Preferably, the density of the secondary culture is 4000-6000 cell/cm2。
10. a kind of preparation method of total corneal, which is characterized in that include the following steps：By claim 6-8 any one of them
The cornea interstitial that cultural method obtains is placed on the collagen culture medium described in claim 3；
The cornea epidermis that claim 4-5 any one of them cultural methods obtain is fitted in the cornea interstitial lamella,
The endothelial cell injects between the cornea interstitial lamella and iris the endothelial cell layer for forming single layer, obtains institute
State total corneal；
Further, the cornea interstitial lamella is all from autogenous cell with the cornea epidermis；
Further, the cornea epidermis is fitted in the interstitial layer surface in 37 ± 2 DEG C and 5%CO2Lower culture 3-5h.
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| CN111235109B (en) * | 2019-12-26 | 2022-01-11 | 爱尔眼科医院集团股份有限公司 | Culture medium, corneal stroma tablet prepared from culture medium and preparation method |
| CN113773380A (en) * | 2021-09-28 | 2021-12-10 | 山东汉肽医美生物科技有限公司 | Recombinant human collagen, encoding gene and application thereof in preparation of biodegradable collagen-based cornea substitute |
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