First continuous marine sponge cell line established

Abstract

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Figure 1

Growth curves of G. barretti cells of 3 individuals, cultured in M1 and OpM1 media. Error bars indicate the standard deviation from the average of biological (N = 3) and technical (n = 3) replicates.

Figure 2

Impact of the additional components in OpM1 on the growth curve of G. barretti cells from 1 individual. Cells were cultured in different OpM1 media compositions, in each of which one component was replaced by sterile dH₂O. (A) OpM1-PFTα, -PDGF, or -GF1, (B) OpM1- ITS-X, -LM-1 or -FBS, (C) OpM1-Vit1, -TE or -RPMI. OpM1 and M1 were used as controls. Error bars indicate the standard deviation from the average of technical replicates (n = 3).

Figure 3

Passaging of G. barretti cells cultured in M1 and OpM1 media. OpM1_P16 represents cell cultures restarted from cells cryopreserved at passage 16. Error bars indicate the standard deviation from the average of biological (N = 3) and technical (n = 3) replicates.

Figure 4

Cumulative population doublings (N_d) of cells from 3 individuals of G. barretti cultured in M1 and OpM1 over 5 and 25 passages, respectively. OpM1_P16 shows doublings made by cells cryopreserved after passage 16 and used to start a new culture in OpM1, which continued for at least 30 more passages (46 passages total). Error bars indicate the standard deviation from the average of biological (N = 3) and technical (n = 3) replicates.

Figure 5

Eukaryotic community composition of G. barretti cell cultures. (A) Distribution of 18S rRNA genes in G. barretti cell cultures from the cell banks (t0) and after passage 19 (P19) separated by the dashed line. (B) Distribution of 18S rRNA gene sequences and other sequences that were generated from the cell banks (t0) and after passage 19 (P19). The color legend indicates the best Blastn hit obtained and in parentheses the percentage of identity.