The independence of cultured fat body was also indicated by an increase in Shannon��s entropy of RPKMs. This finding indicated that gene expression in the fat body that resulted from inter-tissue communications between the constituent tissues of the insect donor had become dysregulated in the cultured fat body. The expression of genes relating to a particular function or processes may have changed in a coordinated fashion during culture. To assess whether such changes occurred, RPKMs were compared for 147 immunity-related genes, 54 apoptosisrelated genes, and 10 juvenile hormone-related genes. Of the 147 immunity-related genes, expression of 60 was increased more than 5-fold during culture; in two extreme examples, expression of the genes encoding gloverins and a gene encoding lebocine increased more than 1000-fold. Expression of most of the 54 apoptosis-related genes did not change during culture. Expression of all 10 genes related to juvenile hormone synthesis increased during culture, and expression 6 of these genes increased more than 5-fold. Cultured tissues were derived from living donor organisms, which have circulating hormones, and were transferred into Benzyl alcohol Culture medium lacking hormones. To avoid exposing cultured explants to abrupt changes in insect hormone levels, 5th instar larva were harvested 3 days after the 4th ecdysis; these larva have juvenile hormone and 20-hydroxyecdysone levels that are lower than those of other developmental stages. Hence, cultured fat body would not progress according to the original program of development. A previous study on a Drosophila cell line demonstrated that expression of 5 characteristic genes was elevated in continuously cultured cell lines. However, in the current study, the silkworm homologs of these five genes were not upregulated in the cultured fat body, indicating that increased expression of these genes was not necessary for Timosaponin-BII survival in culture for silkworm cells or tissue explants. These five genes may be essential to maintenance of immortal cells, but not persistence in primary culture conditions. However, further study of these genes in silkworm cell line is warranted. Culture conditions are very important to cell culture; these conditions comprise the complete environment provided for survival of cultured cells.