Optimization of culture conditions for an efficient xeno-feeder free limbal cell culture system towards ocular surface regeneration.
Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever-increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno-diseases to human system. This study was aimed to establish an efficient xeno-feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using "interactive measurements" of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno-feeder free epithelial equivalents towards ocular surface reconstruction.
Varghese
Viji Mary
VM
Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India.
Prasad
Tilak
T
Kumary
T V
TV
D016475:3T3 Cells; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D004847:Epithelial Cells; D005455:Fluorescent Antibody Technique; D016850:Limbus Corneae / Q000166:cytology*; D051379:Mice; D008855:Microscopy, Electron, Scanning; D008858:Microscopy, Phase-Contrast; D009363:Neoplasm Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011817:Rabbits; D044967:Serum; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D046509:Tissue Culture Techniques; D015534:Trans-Activators / Q000378:metabolism
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D003470:Culture Media; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D015534:Trans-Activators; D010450:Endopeptidases; C023139:dispase