Marine sponges (Porifera) are producers of the largest variety of bioactive compounds among benthic marine organisms. In vitro culture of marine sponge cells has been proposed for the sustainable production of these pharmacologically interesting compounds from marine sponges but with limited success. The development of a suitable growth medium is an essential prerequisite for sponge cells grown in vitro. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was adapted to screen for potential nutritional factors in formulating a growth medium for primary cell culture of Suberites domuncula. In 96-well plates, the effects of nutritional factors including glutamine, pyruvate, iron citrate, silicon, RPMI 1640, and Marine Broth 2216 on the viable cell density were examined in primary cell culture of S. domuncula 36 h after inoculation. Ferric iron (Fe(3+)) and pyruvate were found to significantly improve cell viability in a dose-dependent manner. Silicon and glutamine showed limited improvements at certain concentrations. The supplement of RPMI 1640 and Marine Broth 2216 did not increase cell viability. As a result, several improved media able to maintain higher cell viability in a short-term culture of primary sponge cells could be formulated.