Long-Term Culture Of Murine Corneal Epithelial Cells And Reconstruction Of The Tissue Engineered Corneal Epithelium

PurposeMethods for culture and subculture of human corneal epithelial cells have been well documented.However,such has not been the case for mouse corneal epithelial cells. With explant culture method,Hazlett et al.cultured mouse corneal epithelial cells firstly,but failed in passaging the cells over three passages.The limited life span, insufficient cell yields and quick differentiation of cells greatly hindered the application of the method in research.Besides,only Kawakita et al.reported a method to successfully isolate viable mouse corneal/limbal epithelial sheets and culture in serum-free low-Ca²⁺ medium.Furthermore,by both prolonging the culturing time and lowering the seeding density they successfully established a mouse corneal epithelial line.However,they used more than 200 eye globes.To our knowledge,all the existing methods failed in reproducible the long-term culture of mouse corneal epithelial cells from a single mouse.The corneal epithelium is a rapidly regenerating stratified squamous epithelium.The epithelial progenitor cells including limbal stem cells(LSCs) and transient amplifying cells(TACs) with extensive proliferative potential are crucial for maintaining the homeostasis of corneal epithelium,which is an important prerequisite not only for the integrity of the ocular surface but also for visual function.It has been hypothesized that LSC may be maintained and controlled by intrinsic and extrinsic factors in their local microenvironment,the so-called SC niche.This SC niche hypothesis,first proposed by Schofield,suggested that SCs are maintained in an environment that prevents their differentiation and maintain the self-renewal ability,but detailed mechanism of niche is still unknown^1-3.The advent of genetic manipulation resulting in corneal phenotypes in both transgenic and knockout mice provides a powerful tool to study the molecular mechanism of both LSCs and SC niche.Therefore,to establish long-term culture of mouse corneal epithelial cells from a single mouse for in vitro studies becomes increasingly important and it is necessary to make further study about culture condition of mouse corneal epithelial cells.Materials and Method Sectionâ… Long-Term Culture of Murine Corneal Epithelial Cells in vitroCorneal limbal explants of C57BL6/J mice were cultured in SHEM medium by cell-suspending culture,KSFM medium by cell-suspending culture,SHEM medium by explant culture and KSFM medium by explant culture,respectively.Epithelial cells were subcultured at 1:3 split until passage 4(P4) and at low density after P4. Colony-forming efficiency(CFE),population doubling times,and population doublings (PDs) were determined.Sectionâ…¡Determination of phenotype and Induced Differentiation of Murine Corneal Epithelial CellsThe expression of p63,keratin 19(K19),keratin 12(K12) and involucrin was investigated by RT-PCR analysis,immunocytochemistry and Western blotting analysis. Differentiation potential was investigated by switching the medium to KSFM supplemented with 0.9 mM Ca²⁺,KSFM with 10%fetal bovine serum,KSFM with 0.9 mM Ca²⁺ and 10%FBS,or DMEM/F12 with 10%FBS,respectively.Sectionâ…¢Reconstruction of the Tissue Engineered Cornea EpitheliumUsing duplex culture,separated culture and culture without feeder cells,tissue engineered cornea epithelium was reconstructed.Results1,Long-Term Culture of Murine Corneal Epithelial Cells in vitro Nineteen of 20 cornea explants(95%) were successfully subcultured to P1,and the success rate decreased to 55%by P4.However,after P4,cells were stably subcultured though at least 25 passages.2,CFE and characteristics of growth kineticsCFE increased from 9.7±2.6%at P5 to 29.0±3.3%at P20(mean±SD,n=3).Tds increased with successive passages,and it seemed to reach plateau after P12,on an average of 45.9±2.4 hours(mean±SD,n=6.Fig.1E).Reaching the plateau,cells seeded at 700/cm² normally reached saturation density within 8 days after plating.They were stably subcultured though at least 25 passages for 98.8±1.0 PDs(mean±SD,n=3, Fig.1F),without showing signs of replicative senescence.3,Determination of phenotype of Murine Corneal Epithelial CellsRT-PCR analysis disclosed that p63,K19 and involucrin were expressed;however, K12 was not detectable cultured in KSFM.Western blotting confirmed the results. Immunostaining also showed about 70%cells were positive for p63 and K19, approximally 30%cells were positive for involucrin but no cell expressed K12.4,Induced Differentiation of Murine Corneal Epithelial CellsIn order to investigate the differentiation potential of the cells,we cultured the cells in KSFM supplemented with Ca²⁺ or/and FBS and in DMEM/F12 with FBS to induce differentiation.In morphology,the cells cultured in KSFM were homogeneous small cells,cells in KSFM with Ca²⁺ were a heterogeneous mix of small and large cells,and cells in media with FBS were homogeneous large and squamous cells.Both RT-PCR and Western blotting analysis showed that the expression of progenitor markers p63 and K19 and the differentiation marker involucrin did not changed significantly after high concentration of Ca²⁺ was supplemented.However,after medium was supplemented with serum,the expression of progenitor markers P63 and K19 were reduced significantly,while the differentiation marker involucrin was increased significantly.Moreover,the cells cultured in DMEM/F12 with FBS did not express p63 but expressed K12 in the level of mRNA.5,Reconstruction of the Tissue Engineered Cornea EpitheliumCells revealed marked stratification with 4-6 layers by exposure to the air-liquid interface.Immunostaining showed that cells were positive for p63,K19 and involucrin but negative for K12.Conclusion1,we report a reproducible procedure for the long-term culture of corneal epithelial cells from a single mouse using explant culture method and a serum-free low-Ca²⁺ medium without feeder cells.2,The cells obtained show the phenotype of corneal epithelial progenitor cell3,The cells obtained retain differentiation potential.4,The cells obtained maintain stratification ability and can reconstruct tissue engineered corneal epithelium.5,Duplex culture is the best option to reconstruct tissue engineered corneal epithelium.