In vitro production of amoebocytes from tachypleus gigas in leibovitz culture medium

摘要

The present invention provides a process for large scale in vitro production of amoebocytes of Indian Horseshoe Crab (Tachypleus gigas) (T. gigas) from dissected gill flaps of T. gigas, in Leibovitz L-15 culture medium concentration (2×), to provide enhanced generation of amoebocytes. The process comprises the steps of: dissecting gill flaps of T. gigas; washing the gill flaps with an antibiotic solution followed by alcohol; culturing the gill flaps in tissue culture plates of sterile saline on a Rocker platform; culturing further the gill flaps in Leibovitz L-15 culture medium (2×); purging the gill flaps with Tween 80 solution; and purging again the gill flaps with horseshoe crab serum, while keeping the gill flaps in the culture medium viable for 90 days by feeding with fresh medium at an interval of 10-15 days to enable the enhanced release of amoebocytes both within and outside the gill flaps.