CN1580248A - Method for configurating feeder-layer-free corneal eipithelium for frost self-corneal limbus stem cell - Google Patents
Method for configurating feeder-layer-free corneal eipithelium for frost self-corneal limbus stem cell Download PDFInfo
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- CN1580248A CN1580248A CN 200410026154 CN200410026154A CN1580248A CN 1580248 A CN1580248 A CN 1580248A CN 200410026154 CN200410026154 CN 200410026154 CN 200410026154 A CN200410026154 A CN 200410026154A CN 1580248 A CN1580248 A CN 1580248A
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Abstract
The invention publishes a kind of method to stores the stem cell of corneal limbus by constructing planting piece of corneal epithelium without feeder layer. stores the stem cell of corneal limbus by constructing planting piece of corneal epithelium without feeder layer. It can stores self stem cell of corneal limbus for long time in order to satisfy the request of transporting, storing and proliferating stem cell of corneal limbus under different experimental conditions. The invention stores the stem cell of corneal limbus by constructing planting piece of corneal epithelium without feeder layer, to make that corneal epithelium rebuild self when stem cell of corneal limbus colobomas come true in clinic. The invention can successfully form planting piece of artificial corneal epithelium which can autoplastic graft to misshapen surface of cornea of corneal limbus stem cell and the effect to get eyesight back is obvious. So it has a wide application potency in clinic.
Description
Technical field
The invention belongs to stem cell and tissue engineering technique field, be specifically related to in-vitro separation clone limbal stem cell, and under external different condition, preserve, be used for the method for the no feeder layer structure of corneal epithelium and artificial cornea after thawing.
Background technology
Normal eyeball surface is coated with cornea, corneal limbus, three kinds of epitheliums of conjunctiva, and they are different fully on cell phenotype, has constituted complete eye table with the tear film of its front surface.Wherein, the limbal stem cell of limbal epithelium stratum basale is not only keeping having important effect aspect the corneal epithelium integrity, and has following feature: (1) but proliferative; (2) self ability; (3) can produce a large amount of whole undifferentiated states and the daughter cell of function; (4) after tissue damaged, have regeneration updating ability (1.CotsarelisG et al.Cell, 1989; 57:201.2.Kruse FE, et al.Eye.1994; 8:170.3.Schemer, et al.J.Cell Biol, 1986; 103:49-62).
Clinically, various chemistry or thermal burn, the stevens-Johnson syndromes, eye table pemphigoid is worn contact lens and serious infected by microbes etc. and all can be caused limbal stem cell to lack fully, destroys the corneal epithelium integrity and blinding.Present treatment measure is according to the limbal stem cell guide of theory, and the tissue that operation transplantation has a limbal stem cell replenishes and upgrades limbal stem cell (4.Kenyon, KR.Et al.ophthalmology.1989; 96:709. 5.Tseng, SC.Mol.Biol, Rep, 1996; 23:47-58.6.Dua HS.Et al.Br, J, ophthalmology2000; 84:273.).Require to cut the corneal limbus tissue of 2～3 hour position spans of donor's cornea during operation, just can have the limbal stem cell of sufficient amount to be used for the reconstruction of corneal epithelium.Drawing materials like this caused the danger of donor cornea limbal stem cell disappearance, in the transplanting of allosome corneal limbus tissue, requires the immunological rejection after the application immunosuppressive drug prevents to transplant in addition, and patient is had to a certain degree toxic side effect.Make the ophthalmologist sight be turned to the research that is used to transplant after the external a large amount of amplification cultivation of corneal limbal stem cell autograft.Purpose is to realize to obtain small amounts of cells obtains a large amount of cells behind cultured and amplified in vitro hope from a small pieces live cornea edge organization material, and it and timbering material one homologous transplantation are used for corneal epithelium reconstruction and treatment.(7.Friend?J，et?al.Invest?ophthalmol?Vis?Sci，1982；23：41-49.8.He?YG.Et?al.Curr?Eye?Res.1991；9：85-63.9.Lindberg?K，et?al.Invest?ophthalmol?Vis?Sci，1993；34：2672-2679.10.Koizumi?N，et?al.Archophthalmol?2001；119：298-300.11.Tsai?RT，et?al.N.Engl?J.Med.2000；343：86-93.12.Koizumi?N，et?al.Invest?ophthalmol?Vis?Sci，2002；43：2114-2121.)。The treatment of the eye surface diseases that causes for those limbal stem cells disappearance of more than working provides a new measure, but, just be applied to clinical after limbal stem cell of cultivating and graft thereof can only be cultivated and finish immediately, need patient's body situation and the optimum growh state of cultivating end back cell to match, otherwise, will cause entire operation and operative failure.If can be, and guarantee that its multiplication capacity does not fail, in needs, can continue to cultivate the reconstruction that is used for corneal epithelium, will greatly help its clinical application with patient's corneal limbal stem cell autograft at external preservation certain hour.In addition, what be used for the cultivation of limbal stem cell amplification in vitro at present is a culture systems that is called as 3T3, in this system, can keep limbal stem cell cloning growth and be in a kind of not (13.Pellegrini G.et al.J cell Biol, 1999 in the differentiation state relatively; 145:769-782.14.TsengSC, et al, Curr Eye Res 1996; 15:973-984.).But, when adopting the 3T3 cell to do feeder layer to cultivate and make up limbal epithelium and plant sheet, because the 3T3 cell is a inoblast from the tire mouse, it has in clinical application gives human danger with the heterogenous animal pathophoresis, and it is widely-used because patient's misgivings limit greatly.Therefore, if external can preserve corneal limbal stem cell autograft and the corneal limbal stem cell autograft of external preservation do not had feeder layer make up corneal epithelium and plant sheet, can solve above-mentioned contradiction well, in the clinical application of tissue engineering comea epithelium, have great importance.
Summary of the invention
The objective of the invention is to, provide a kind of frozen corneal limbal stem cell autograft not have the method that feeder layer makes up corneal epithelium, with the external prolonged preservation of corneal limbal stem cell autograft, and adopt the method structure corneal epithelium of no feeder layer to plant sheet with the limbal stem cell of preserving, realize that corneal epithelium behind the limbal stem cell deficiency clinically is from volume reconstruction.
The technical scheme that realizes the foregoing invention purpose is, the no feeder layer of frozen corneal limbal stem cell autograft makes up the corneal epithelium method, it is characterized in that, with the external prolonged preservation of corneal limbal stem cell autograft, and adopt the method structure corneal epithelium of no feeder layer to plant sheet with the limbal stem cell of preserving, realize that corneal epithelium behind the limbal stem cell deficiency clinically is from volume reconstruction; May further comprise the steps:
1) at first adopts the cold digestion limbal epithelium of dispase tissue, make it to separate well with basement membrane of epithelium, obtain the corneal epithelial cell that contains more limbal stem cell, cultivation amplifies limbal stem cell on the six hole plastic culture plates of gelatin being covered with, the molecule marker of corneal limbal stem cell carries out immunohistochemical methods and detects: when CK19, P63 and the PCNA positive, when CK3/K12 is negative, prove that the cell that is obtained is a limbal stem cell;
2) with limbal stem cell with enzymic digestion after, adding frozen solution is placed in the different envrionment temperatures frozen, protection liquid formula: DMEM/F12+10%DMSO+10%FBS, the source of record limbal stem cell when frozen, so that carry out corresponding cornea autotransplantation, limbal stem cell after frozen still has the limbal stem cell characteristic, and external the continuation increased;
3) will go people's amnion of epithelium to adhere on the special support, and put into six orifice plates, and behind 37 ℃ of dry 30min～60min, make at the bottom of amnion and six orifice plates and support adheres to firmly, getting and being prepared into concentration after frozen limbal stem cell thaws is 1 * 10
6The corneal limbal epithelial cell suspension of individual cell/ml, carefully be added drop-wise on the amnion basement membrane of epithelium with micropipet, the corneal epithelium nutrient solution that adds no feeder layer, corneal epithelium nutrient solution prescription is: the DMEM/F12 basic culture solution, add 20% FBS, 20ng/mlEGF, 5ug/ml insulin, 0.5%DMSO and 100Iu/ml penicillin 100ug/ml Streptomycin sulphate move into the CO2 incubator and cultivate, and are cultured to 8～10 days, support is peeled off at the bottom of culture plate, continued suspension culture 5～7 days, and treated that cell became multiple layer growth promptly to stop to cultivate, the artificial cornea epithelium that promptly obtains preparing to transplant is planted sheet.
The frozen limbal stem cell shelf time is under the above-mentioned different envrionment temperature: the refrigerator shelf time is 12-48h under 4 ℃ of conditions; The refrigerator shelf time is 7d under-20 ℃ of conditions, and refrigerator shelf time 1-1.5 is individual month under-80 ℃ of conditions, and-196 ℃ of liquid following shelf times of condition were greater than 6 months.
The artificial cornea epithelium that adopts the present invention to make up is planted sheet, autotransplantation is to the anterior corneal surface of limbal stem cell disappearance, can successfully rebuild the corneal epithelium of limbal stem cell deficiency, be used for corneal epithelial defect clinically and rebuild and rehabilitation, have application potential widely.
Description of drawings
Fig. 1 is goat limbal stem cell figure;
Fig. 2 is the goat limbal stem cell figure that cultivates after thawing;
Fig. 3 is the human limbal stem cell figure that cultivates after thawing;
To be cell forming 3～5 similar at body time confluent monolayer cells structure iron to Fig. 4;
Fig. 5 forms complete desmosome structure iron between the limbal stem cell on the amnion;
Fig. 6 plants sheet figure for artificial corneal epithelium;
Fig. 7 is the pathological model figure of limbal stem cell disappearance;
Fig. 8 plants the outstanding effect picture of recovering lost eyesight after sheet is transplanted for corneal epithelium.
Embodiment
The present invention is described in further detail according to embodiment that technique scheme is finished below in conjunction with accompanying drawing and contriver.
Embodiment 1:
The preservation of limbal stem cell makes up corneal epithelium (is example with Central Shanxi Plain milk goat) with no feeder layer
Central Shanxi Plain milk goat 846 mixture for animals (animal pharmaceutical factory of agriculture and animal husbandry university of PLA) 2.4ml anesthesia, two antibiosis reason salt solution (self-control) flushing conjunctival sac and anterior corneal surface, Xylotox 5ml (Shanghai Hefeng Pharmaceutical Co., Ltd.) 5ml, bupivacaine (Shanghai Hefeng Pharmaceutical Co., Ltd.) 5ml retrobulbar anaesthesia.The shop aseptic hole-towel, eyelid left by eye speculum (Jiangsu six or six vision company limiteds), under ophthalmic operating microscope (Jiangsu six or six vision company limiteds), the aseptic upside limbal epithelium (being with a small amount of corneal limbus matrix) that cuts, put cold digestion 14-16h among the 1.2IU dispase (Gibco Lot No.1126857), move in DMEM/F12 (Gibco Lot No.1107011)+10%NBS (Beijing Heng Shengma of unit biotechnology research institute product) nutrient solution and stop digestion, peel off digesting good epithelial lining and stratum basale, epithelial lining is blown and beaten discrete cell repeatedly with suction pipe, centrifugal twice of 1000r/min, each 5min, collecting cell.
Cell is with 1 * 10
5Individual cell/ml is inoculated in and is covered with 0.1% gelatin (Sigma, Lot50K02505) in the six hole plastic plates (CELLSTAR Lot02030151), add and contain 20%FBS, 20ng/mlEGF (epithelical cell growth factor, Sigma, Number E 9644), (Regular Insulin is available from Beijing ancient cooking vessel state biotechnology limited liability company, Sigma packing for 5ug/ml insulin, original product is numbered 15500), the DMEM/F12 nutrient solution of 0.5%DMSO and 100Iu/ml penicillin (HARBIN PHARMACEUTICAL GROUP CO., LTD. General Pharm. Factory, lot number B01124412) 100ug/ml Streptomycin sulphate (Huabei Pharmaceutic Co., Ltd, lot number 01072), 5%CO2, saturated humidity CO
2Incubator (WTB CO
2Incubator, U.S.) 37 ℃ of cultivations were changed nutrient solution one time in per 2 days, were cultured to 7～9 days, formed intensive individual layer (Fig. 1).Go down to posterity and should when 80%～90% cell begins to converge, carry out, with 0.125% trypsin 1: 250, Huamei Bio-Engrg Co.)+(LOT1120193) peptic cell is with 1 * 10 for ethylenediamine tetraacetic acid (EDTA), Invitrogen for 0.01%EDTA
5The concentration of individual cell/ml goes down to posterity.The used freeze-stored cell of the present invention be 2～4 generation limbal stem cell, add frozen solution (DMEM/F12+10%DMSO+10% FBS), respectively at 4 ℃, preserve in-20 ℃ ,-80 ℃ refrigerators and-196 ℃ of liquid nitrogens.Write down the source of limbal stem cell when frozen, so that carry out corresponding cornea autotransplantation later on.
The cell of preserving under the above-mentioned condition of in different time, thawing, trypan blue dyeing detects the vigor of cell, those have 85% cell still to survive, increase through external the continuation, cellular form to frozen before similar substantially cell (Fig. 2), immunohistochemical methods detects limbal stem cell molecule marker CK19 (Keratin sulfate 19, Sigma, sigma-aldrich.com, Product Number C 6930), P63 (Santa CruzBiotechnology, Inc.4A4:sc-8431), PCNA (SA2024 of Wuhan Boster Biological Technology Co., Ltd.), CK3/K12 (United States Biological Inc.Lot No 3051563), concrete operations are undertaken by Beijing Zhong Shan biological reagent company limited immunohistochemical staining test kit (SP-9000/9001/9002) specification sheets.Those cellular elements marks still are CK19, P63 and the PCNA positive, and the cell of CK3/K12 feminine gender can think still have the limbal stem cell characteristic.Can preserve 12-48h ,-20 ℃ of preservation 1-7d, 1-1.5 month ,-196 ℃ liquid nitrogens preservations of-80 ℃ of preservations more than at least 6 months at 4 ℃, but the imagination prolonged preservation.Can satisfy under different experiment conditions the requirement of the transportation of corneal limbal stem cell, preservation and propagation.
The amnion that removes epithelium is adhered to a special support (nitrocellulose filter Sigma, the sigma-aldrich.com transformation of the way forms) on, put into six orifice plates, 37 ℃ of dry 30min～60min, make adhere at the bottom of amnion and support and six orifice plates firmly after, getting the limbal stem cell of preserving under above-mentioned arbitrary condition, to be prepared into concentration be 1 * 10
6Individual cell/ml corneal limbal epithelial cell suspension, carefully be added drop-wise on the amnion basement membrane of epithelium with micropipet, do not make cell flow out the amnion surface, static 20min on 37 ℃ of constant temperature method platforms, (the DMEM/F12 basic culture solution adds 20%FBS, 20ng/mlEGF, 5ug/ml insulin to add a small amount of nutrient solution, 0.5%DMSO and 100Iu/ml penicillin 100ug/ml Streptomycin sulphate), move into CO
2Spend the night in the incubator, added the 2ml nutrient solution to every hole again and cultivate when treating that cell adhesion begins to breed on amnion in second day.The next day change nutrient solution one time, be cultured to 8～10 days, gently support is peeled off at the bottom of culture plate, add nutrient solution amnion and support be suspended in the nutrient solution.Continue to cultivate 5～7 days, cell becomes multiple layer growth, when the surface has cell to begin to come off, stops to cultivate.After fixing, be used for histology and electron microscopic examination, cell forms and 3～5 similar when body confluent monolayer cells structures (as Fig. 4) on the visible amnion of histological examination as a result, goblet cell is not seen in PAS dyeing, form more complete desmosome structure (as Fig. 5) between the transmission electron microscope observing visible cell, form the hemidesmosome structure between cell and amnion, illustrate that cell set up basilar membrane on amnion.
Cell after will thawing is transplanted the artificial cornea epithelium that makes up and is planted sheet (as Fig. 6) behind pathological model (as Fig. 7) the goat anterior corneal surface of limbal stem cell disappearance after amplification cultivation on people's amnion support, and the effect of recovering lost eyesight is (as Fig. 8) obviously.Illustrate that the constructed tissue engineering comea epithelium of the present invention has application potential widely clinically.
Embodiment 2:
The preservation of people's limbal stem cell makes up corneal epithelium with no feeder layer
Get and suffer from limbal deficiency patient homonymy or offside 2-4mm
2The limbal epithelium of size is 200 * 10
-3U/L penicillin and mass concentration are to soak 15-20min in the 200mg/L Streptomycin sulphate physiological saline, bring sterilisable chamber into.PBS (-) flushing several times.Under the stereoscopic microscope (Leica company), further behind the flaggy separation of corneal epithelium, put cold digestion 12-14h among the 1.2IU dispase, putting into PBS (-) washes one time, move into and stop digestion in the DMEM/F12+10%NBS nutrient solution, scrape gently with cell scraper (Nunc.Inc.USA) and to have digested good epithelial lining, suction pipe is blown and beaten discrete cell repeatedly.1000r/min is centrifugal twice, each 5min, collecting cell.
Later implementation method is identical with enforcement 1.Fig. 3 is the human limbal stem cell after frozen.
Claims (2)
1. the no feeder layer of frozen corneal limbal stem cell autograft makes up the corneal epithelium method, it is characterized in that, with the external prolonged preservation of corneal limbal stem cell autograft, and adopt the method structure corneal epithelium of no feeder layer to plant sheet with the limbal stem cell of preserving, realize that corneal epithelium behind the limbal stem cell deficiency clinically is from volume reconstruction; May further comprise the steps:
1) at first adopts the cold digestion limbal epithelium of dispase tissue, make it to separate well with basement membrane of epithelium, obtain the corneal epithelial cell that contains more limbal stem cell, cultivation amplifies limbal stem cell on the six hole plastic culture plates of gelatin being covered with, the molecule marker of corneal limbal stem cell carries out immunohistochemical methods and detects: when CK19, P63 and the PCNA positive, when CK3/K12 is negative, prove that the cell that is obtained is a limbal stem cell;
2) with limbal stem cell with enzymic digestion after, adding frozen solution is placed in the different envrionment temperatures frozen, protection liquid formula: DMEM/F12+10%DMSO+10%FBS, the source of record limbal stem cell when frozen, so that carry out corresponding cornea autotransplantation, limbal stem cell after frozen still has the limbal stem cell characteristic, and external the continuation increased;
3) will go people's amnion of epithelium to adhere on the special support, and put into six orifice plates, and behind 37 ℃ of dry 30min～60min, make at the bottom of amnion and six orifice plates and support adheres to firmly, getting and being prepared into concentration after frozen limbal stem cell thaws is 1 * 10
6The corneal limbal epithelial cell suspension of individual cell/ml, carefully be added drop-wise on the amnion basement membrane of epithelium with micropipet, the corneal epithelium nutrient solution that adds no feeder layer, corneal epithelium nutrient solution prescription is: the DMEM/F12 basic culture solution, add 20%FBS, 20ng/mlEGF, 5ug/ml insulin, 0.5%DMSO and 100Iu/ml penicillin 100ug/ml Streptomycin sulphate move into the CO2 incubator and cultivate, and are cultured to 8～10 days, support is peeled off at the bottom of culture plate, continued suspension culture 5～7 days, and treated that cell became multiple layer growth promptly to stop to cultivate, the artificial cornea epithelium that promptly obtains preparing to transplant is planted sheet.
2. the no feeder layer of frozen corneal limbal stem cell autograft as claimed in claim 1 makes up the corneal epithelium method, and it is characterized in that the varying environment temperature shelf time of described frozen limbal stem cell is: the refrigerator shelf time is 12-48h under 4 ℃ of conditions; The refrigerator shelf time is 7d under-20 ℃ of conditions, and refrigerator shelf time 1-1.5 is individual month under-80 ℃ of conditions, and-196 ℃ of liquid following shelf times of condition were greater than 6 months.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726370A (en) * | 2012-06-29 | 2012-10-17 | 厦门大学附属厦门眼科中心 | Preservation method for corneal limbus tissue |
| CN105850980A (en) * | 2016-04-14 | 2016-08-17 | 广州赛莱拉干细胞科技股份有限公司 | Corneal limbal stem cell cryopreservation liquid and cryopreservation method |
| CN109321527A (en) * | 2018-10-10 | 2019-02-12 | 中国海洋大学 | The extracorporeal culturing method of limbal stem cell stability |
| CN115068504A (en) * | 2021-03-12 | 2022-09-20 | 广州康睿生物医药科技股份有限公司 | Cornea repairing material and preparation method and application thereof |
-
2004
- 2004-05-20 CN CN 200410026154 patent/CN1255531C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102726370A (en) * | 2012-06-29 | 2012-10-17 | 厦门大学附属厦门眼科中心 | Preservation method for corneal limbus tissue |
| CN105850980A (en) * | 2016-04-14 | 2016-08-17 | 广州赛莱拉干细胞科技股份有限公司 | Corneal limbal stem cell cryopreservation liquid and cryopreservation method |
| CN105850980B (en) * | 2016-04-14 | 2018-06-12 | 广州赛莱拉干细胞科技股份有限公司 | The frozen stock solution and cryopreservation methods of a kind of limbal stem cell |
| CN109321527A (en) * | 2018-10-10 | 2019-02-12 | 中国海洋大学 | The extracorporeal culturing method of limbal stem cell stability |
| CN115068504A (en) * | 2021-03-12 | 2022-09-20 | 广州康睿生物医药科技股份有限公司 | Cornea repairing material and preparation method and application thereof |
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