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Summer Research Fellowship Programme of India's Science Academies

Culturing human limbal stromal stem cells and determining their doubling time and cell proliferation under in-vitro condition

R Reshma

RV College of Engineering, Mysuru Road, Bengaluru, Karnataka 560059

Dr. Vivek Singh

LV Prasad Eye Institute, Banjara Hills, Hyderbad, Telangana 500034

Abstract

Eye is the most developed sensory organ which provides us a binocular vision. The light rays entering both the eyes are converted to neuro-electrical impulses by retina, and carried to the brain by optic nerve to form a single three-dimensional image. The retina receives the light that the cornea focuses through the eye’s lens. Hence, cornea is imperative for a clear vision, its transparency and shape provides a better and sharp vision. Transparency of the cornea is a vital importance and it is maintained by the regular arrangement of the collagen. The cornea is made up of three layers, the outermost epithelial layer, the middle stromal layer and the innermost endothelial layer. The corneal epithelial and the corneal stromal layers are separated by a thin layer called bowman’s layer, whereas the corneal stromal and corneal endothelial layers are separated by descemet’s layer. Corneal diseases like scarring, ulceration, keratoconus, fuch’s endothelial dystrophy, and corneal injuries, can damage the corneal transparency leading to a blurred vision or complete loss in vision. Epithelial and stromal injuries can be cured by the eye itself by regenerating new cells or by the process of corneal wound healing. The limbus present between the cornea and conjunctiva consists of highly proliferative limbal stem cells (LSCs), which regenerates the epithelium and stroma. LSCs differentiate into two different types of stem cells that are, limbal epithelial stem cells (LESCs) and limbal stromal stem cells (LSSCs). LESCs regenerates corneal epithelial cells, whereas LSSCs differentiate into keratocytes, which participate in wound healing. These stem cells are cultured in-vitro and transplanted to the patient to cure or reduce corneal damages. Stroma covers almost 90% of the corneal thickness, Hence, study of corneal stroma is necessary in order to understand the functioning of cornea more clearly. In this experiment LSCs are cultured in-vitro in 2% (serum) DMEM-F12 complete media to obtain limbal stromal stem cells. The doubling time and cell proliferation of human LSSCs under in-vitro condition are determined by counting the cells using hemocytometer using the dye exclusion method (by using the reagent trypan blue), and MTT assay. The study is done on sub-cultured LSSCs under two different conditions, i.e in 2% and 0% (serum-free) DMEM-F12 complete media.

Keywords: cornea, limbal stem cells, cell culture

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