Investigating non-lethal tissue sources for the development of cultivated crustacean meat

Lisa Musgrove

doi:10.25907/01126

Cultivated crustacean meat (CCM) is a way to produce highly valued shrimp, lobster, and crab products by utilising stem cell technology instead of live animals. CCM can thus be a means to help meet growing demand for crustacean foods while avoiding some of the challenges that currently face conventional fishing and farming practices, including climate change impacts, pollution, disease threats, and animal welfare concerns. To support the development of this emerging field, the work presented here includes a comprehensive literature review and several investigations into the utility of specific tissues as potential stem cell sources for CCM. Designed to assist future CCM researchers, the literature review covers the current state of research relating to crustacean cell culture, cell line development and characterisation tools, while introducing relevant aspects of crustacean muscle biology and limb regeneration, and presenting strategies to assist with cell line immortalisation and myogenic differentiation. Additionally, in speaking to the ethical drivers behind cultivated meat, it introduces three non- lethal stem cell sources investigated in the remainder of the thesis: mature limbs, regenerating limbs and cells within circulating hemolymph. A transcriptomic analysis of stem cell and myogenic gene expression in crayfish claw regeneration is presented to evaluate the regenerating limb model as a stem cell source for CCM. Differential expression analysis of RNA-Seq data obtained from six stages of regeneration revealed expression patterns of significantly up- or down-regulated genes in 14 functional categories. Relative expression of targeted stem cell genes highlighted two peaks of stem cell activity during pre-moult regeneration, with limited stem cell activity after the moult. Histology and qPCR results supported these findings suggesting that pre-moult Stage 4 tissues may be a better source of stem cells than post-moult Stage 6 tissues. Methods were explored to isolate putative adult muscle stem cells (AMSCs) from mature limb tissues (Stage 6) and regenerating tissues (Stage 4), including enzymatic digestions and tissue explants. Uncertainty around the expected in vitro morphology and behaviour of AMSCs resulted in misidentification of some proliferating cultures, later suspected to be microbial species. Putative AMSCs were observed in situ in the tissue explant cultures, however, cells that appeared as ‘free’ and unattached in the cultures remain unidentified. Subsequently, to enable better characterisation of the AMSCs, commercial antibodies against various muscle and stem cell related proteins were screened and tested, resulting in the identification of four antibodies (against MHC, Pax3, Pax7 and Mef2) that produced biologically relevant staining patterns in crayfish claw tissues. This staining shed light on the expected morphology of AMSCs and suggested that both tissue types (Stage 4 and Stage 6) could be valuable cell sources for CCM. In highlighting the suitability of these antibodies for crustacean muscle tissue, they can now be presented as four readily accessible and cost-effective tools available for future CCM research. Cells derived from circulating hemolymph were investigated as another stem cell source for CCM. Comparison of different culture conditions showed hemocytes maintained in commercial media formulations displayed signs of over-activation and earlier fragmentation, compared to cells maintained in hemolymph and anticoagulant only. These results highlighted the need to find a balance between encouraging sufficient activation (for proliferation) and limiting over-activation. Co-cultures with hemocytes and claw muscle tissue explants demonstrated cross-talk between the cell types as well as evidence of contamination mitigation, both of which can be leveraged for CCM research. Finally, timed hemolymph withdrawals were conducted to investigate methods to induce putative stem cells into circulation. Numbers of the putative stem cells increased 5 and 96 hours after withdrawal, but not after 24 or 48 hours. qPCR amplification results showed that the cell cycle gene PCNA is detectable in hemocytes under all test conditions. Together, these results suggest that hemolymph withdrawal may be a relatively simple and harmless way to obtain circulating stem cells. This thesis contributes foundational knowledge to the emerging field of CCM and reinforces the need for more open-source, crustacean-specific cell culture research. While many challenges remain unresolved, the work presented here helps establish a clearer pathway toward the future development of cultivated crustacean meat.

Investigating non-lethal tissue sources for the development of cultivated crustacean meat

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