title: N-isopropylacrylamide-co-glycidylmethacrylate as a thermoresponsive substrate for corneal endothelial cell sheet engineering.
abstract: Endothelial keratoplasty is a recent shift in the surgical treatment of corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using the thermoresponsive poly(N-isopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-to-cell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells in the cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na(+)-K(+) ATPase, and FLK-1. Na(+)-K(+) ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.
authors.affiliation: Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695012, India.
authors.forename: Bernadette K
authors.initials: BK
authors.lastname: Madathil
authors.affiliation: Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695012, India.
authors.forename: Pallickaveedu RajanAsari Anil
authors.initials: PR
authors.lastname: Kumar
authors.affiliation: Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695012, India.
authors.forename: Thrikkovil Variyath
authors.initials: TV
authors.lastname: Kumary
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D011109:Polymethacrylic Acids / Q000494:pharmacology*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
chemical_list: D011109:Polymethacrylic Acids