Development of a reliable in vitro cell culture system for sea cucumber (Apostichopus japonicus) and its use in viral susceptibility and heat shock response

Holothurian cell culture remains unsuccessful due to the lacking of suitable medium and tissues. Here, a reliable in vitro cell culture system has been developed for Apostichopus japonicus by optimizing the basal and growth media as well as the sourced tissues. Blood vessel and longitudinal muscle tissues were first found to be the best two sourced tissues. Subsequently, the composition and content of free amino acids and carbohydrates in holothurian coelomic fluid were analyzed and the obtained data were used to design and obtain an optimal basal medium (SCBM-4). Next, an optimal growth medium (SCGM-24) was further developed by supplementing 1 % FBS, 25 % coelomic fluid and 20 % body wall enzymatic hydrolysate of sea cucumbers into SCBM-4. Primary blood vessel (SCBV) cells could be efficiently isolated by mechanical dissociation in tissue dissociation apparatus and survive healthily for 22 d in SCBM-4, and 58 d in SCGM-24 with a gradually decreased proliferation activity. Primary longitudinal muscle (SCLM) cells could only be efficiently isolated by explant culture and healthily survive for 52 d in SCBM-4 but with no proliferation activity, whereas their viabilities decreased sharply in SCGM-24. The epithelial-shaped SCBV cells could be successfully sub-cultured for 3 times using 4 °C PE solution, whereas the loosely attached round-shaped SCBV and SCLM cells could be passaged for 6 and 4 times by pipetting, respectively. Additionally, shrimp covert mortality nodavirus (CMNV) could cross-species infect and multiply in SCBV cells, and acute heat shock treatment could significantly upregulate the transcriptional expression of heat shock protein genes.