Limbal corneal epithelial stem cells (LSCs) are responsible for maintaining the corneal epithelial cell mass. These cells undergo proliferation, migration and differentiation under both normal conditions as well as following injury. They can be expanded ex-vivo for intracorneal autologous transplantation.
Limbal biopsies, of 2x1mm2 in size, were obtained from patients who underwent pterygium excision. Amniotic membranes (AM) were procured, as per protocol, from donated placenta after screening the donor for the human immunodeficiency virus (HIV), hepatitis B surface antigen (HBs Ag) and syphilis.
The limbal explants were placed on de-epithelialized amniotic membranes (3 x 3 cm2). For de-epithelialization, the AM was spread in a Petri dish and 1 mL trypsin-EDTA was added on the surface and incubated for 25 min at 37 °C. The trypsin solution was then discarded and 2 mL of human corneal epithelium medium (HCEM), comprised of DMEM/F12, epidermal growth factor, insulin, hydrocortisone, antibiotics-antimycotics and fetal bovine serum (10%) was added to the AM. The AM was mechanically scraped with a cell scraper, to achieve complete de-epithelialization and washed three times with phosphate buffered saline (PBS). After washing, the AM was spread taut on to a sterile grass slide (2.5 x 2.5 cm2) and carefully placed in a 35 mm culture dish. Fresh limbal biopsy tissue maintained in HCEM, was minced into 4-6 tiny bits and explanted onto two denuded AM. The explants were allowed to adhere to the AM for 30 min before 4 mL of HCEM was added to the culture dishes. The medium was changed every other day. Cells were cultured for 2-3 weeks before being subjected to various tests.
EMBRYONIC DEVELOPMENT & STEM CELL COMPENDIUM
