In-vitro characterization of serially expanded porcine oesophageal epithelial cells in feeder layer-free culture system for tissue engineering application.
Thi, Thi Sein.
Date of Issue2009
School of Mechanical and Aerospace Engineering
The objectives of the study are to isolate and serially expand the porcine oesophageal epithelial cells (PEECs) in serum-free, feeder layer-free culture system, to monitor the growth kinetics in in-vitro cultures and to characterize the stem-like nature, proliferative and differentiation potential of cultured PEECs in-vitro with the use of immunocytochemical method. The porcine oesophageal epithelial cells were isolated by two-step enzymatic methods using dispase digestion at physiological temperature for earlier establishment of primary cultures. The primary cultures of PEECs were established from porcine oesophageal tissue collected directly from the abattoir. The cells could be cultured in serum-free, feeder layer-free culture medium with reasonable proliferation up to passage 6 – 8 although optimization of culture medium was needed for extended in-vitro growth. Sorting the cells depending on the rapid adhesiveness to extracellular matrix proteins coated on the culture surface could enrich highly proliferative cells with extended culture lifespan in-vitro. The morphology of the cultured cells monitored under light microscope confirmed their morphological stability in in-vitro cultures. The growth kinetics of the cultured cells was assessed by population doubling time and serial MTS assays.