ZEM2S

Primary Screening of Algal/Cyanobacterial Extracts in ZEM2S Fibroblasts-Testing Extracts' Cytotoxicity

Cultured meat production, currently dependent on expensive and environmentally harmful fetal bovine serum (FBS), needs sustainable alternatives for commercial viability. Sibinčić et al. evaluated twenty-six water-based algal and cyanobacterial extracts for their ability to support cell growth in reduced serum conditions. The extracts were initially tested in zebrafish cells (ZEM2S) with 1% FBS, and ten promising ones were further assessed in Japanese quail myoblasts.

The primary screening aimed to identify which algae/cyanobacteria and concentrations promote ZEM2S cell viability. They focused on the protein component of the extracts and used protein concentration to quantify the amount added to the cells. ZEM2S cells were maintained in 10% FBS-supplemented media. For the experiment, cells were seeded in 96-well plates in 1% FBS media with or without extracts. The cells were incubated with the extracts for 72 hours to assess long-term tolerance. They used the resazurin reduction (Alamar Blue) assay due to its higher sensitivity and because ZEM2S cells did not metabolize tetrazolium salts. Twenty-six extracts were tested: seven from cyanobacteria, four from Rhodophyta, eight from Chlorophyta, five from Heterokontophyta, and two from Haptophyta. Figure 1 shows the results: cell viability generally decreased at concentrations ≥ 10 μg/mL, especially at 50 and 100 μg/mL. However, Arthrospira platensis and Dunaliella tertiolecta showed better viability even at higher concentrations. The phycocyanin-enriched blue spirulina extract was better tolerated at 100 μg/mL than the full Arthrospira platensis extract. Dunaliella tertiolecta was particularly interesting as higher concentrations enhanced cell viability.

Prediction of Acute Fish Toxicity and Fish Embryo Toxicity Tests by Cytotoxicity Assays Using Liver and Embryo Zebrafish Cell Lines (ZFL And ZEM2S)

Fish cell-based assays offer potential alternatives to using vertebrates in ecotoxicology. This study assessed the cytotoxicity of thirteen chemicals in two zebrafish cell lines (ZEM2S and ZFL) to determine if IC₅₀ values from viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict LC₅₀ values of Acute Fish Toxicity (AFT) and Fish Embryo Toxicity (FET) tests.

IC₅₀ values showed both cell lines were significantly less sensitive (IC₅₀ > 10-fold higher than the LC₅₀) than AFT test in up to seven substances (i.e., PCZ, LUV, DNP, TCL, OCT, DEP (except in MTT assay), and DCA (except in NR assay)) (Figs. 2A and B). When assessed by ZFL cell line, the same substances, and additionally SDS, had IC₅₀ values 10-fold higher than the LC₅₀ of FET (48 h) (Fig. 2C). ZEM2S cell line was also significantly less sensitive than the FET (48 h) to the same substances (Fig. 2D). The fold differences of LUV, DEP and MQ were not calculated because no data is available for these chemicals in FET test (48 h). Considering the FET test (96 h), both cell lines were significantly less sensitive to PCZ, TCL, DNP, OCT LUV (except in MTT assay in ZEM2S), and DCA (except in NR in ZFL) (Figs. 2E and F). The fold differences of DEP and SDS were not calculated because there is no available data (LC₅₀) for these substances in the FET test (96 h). On the other hand, ZEM2S was 10-fold more sensitive to TEG than AFT and FET (48 and 96 h) tests (Figs. 2B, D, and F). ZFL was also 10-fold more sensitive to TEG than the FET test (48 h) (Fig. 2C).