Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (<i>Oreochromis mossambicus</i>) Cell Lines

<div><p>Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (<i>Oreochromis mossambicus</i>) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, <i>myo</i>-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.</p></div

Heat map representing comparative proteomics data for newly derived OmB and OmL cell lines.

<p>Data presented are significantly different ratios of protein abundances (relative to OmB abundance) for four technical replicates of OmB and OmL generated with a proteomics software package (PEAKS suite 7, BSI, Inc.). Protein accession numbers and relationships are given on the left side of the heat map.</p

Salinity-induced changes in cell viability.

<p>Means ± SEM of number of live cells as a measure of cell viability for three cell lines (OmB, OmL, TmB) exposed to osmolalities ranging from 300–900 mOsm/kg for 24 hours. Cell numbers were measured using the Coulter Counter method. If standard errors are not visible then they are smaller than the size of the symbol for the corresponding data point. *, **, and *** indicates significant differences from control cells at p<0.05, p<0.01, and p<0.001, respectively.</p

Osmolality dependent MIB gene expression in OmB cell line.

<p>Means ± SEM of MIPS-160 (A), MIPS-250 (B), IMPA1 (C), and IMPA2 (D) mRNA abundance in the OmB cell line derived from brain after chronic hyperosmotic challenge (450, 600, 750 mOsm/kg). Tukey HSD post-hoc comparisons (p<0.05) are indicated with a letter assignments (only within gene comparisons). Groups that share the same letter are not significantly different.</p

Expression of MIB genes in OmL cell line.

<p>Means ± SEM of MIPS-160 (A+E), MIPS-250 (B+F), IMPA1 (C+G), IMPA2 (D+H) mRNA abundance in the OmL cell line derived from lip epithelium following acute hyperosmotic challenge to 450 mOsm/kg (A–D) and 700 mOsm/kg (E–F) for 4, 8, 16, and 24 hours. Tukey HSD post-hoc comparisons (p<0.05) are indicated with a letter assignments (only within gene comparisons). Groups that share the same letter are not significantly different.</p

Properties of all tilapia cell lines used in this study, including a previously derived line, TmB, and two newly derived lines, OmB and OmL.

<p>Properties of all tilapia cell lines used in this study, including a previously derived line, TmB, and two newly derived lines, OmB and OmL.</p

Expression of MIB genes in OmB cell line.

<p>Means ± SEM of MIPS-160 (A+E), MIPS-250 (B+F), IMPA1 (C+G), IMPA2 (D+H) mRNA abundance in the TmB cell line derived from bulbus arteriosus endothelium following acute hyperosmotic challenge to 450 mOsm/kg (A–D) and 700 mOsm/kg (E–H) for 4, 8, 16, and 24 hours. Tukey HSD post-hoc comparisons (p<0.05) are indicated with a letter assignments (only within gene comparisons). Groups that share the same letter are not significantly different.</p

Salinity-induced changes in morphology and confluency.

<p>Cell morphology and confluency changes observed after three cell lines (OmB, OmL, TmB) are exposed to acute hyperosmotic stress (450, 600, 750 mOsm/kg) for 24 hours. Images taken on an inverted microscope at 250X magnification.</p