Establishment, Long‐Term Maintenance, and Characterization of Primary Liver Cells from Astyanax mexicanus

Abstract

The tetra fish species Astyanax mexicanus comprises two morphotypes: cavefish that live in caves and surface fish that inhabit rivers and lakes. Because cavefish have adapted to the nutrient‐poor conditions in their habitat whereas the surface fish populations can be used as a proxy for the ancestral condition, this species has become a powerful model system for understanding genetic variation underlying metabolic adaptation. The liver plays a critical role in glucose and fat metabolism in the body and hence is an important tissue for studying altered metabolism in health and disease. Cavefish morphs of A. mexicanus have been shown to develop fatty livers and exhibit massive differences in gene expression and chromatin architecture. Primary cell lines from various tissues have become invaluable tools for biochemical, toxicology, and cell biology experiments, as well as genetic and genomic analyses. To enhance the utility of the model system by enabling an expanded set of biochemical and in vitro experiments, we developed protocols for the isolation and maintenance of primary liver cells from A. mexicanus surface fish and cavefish. We also describe methods that can be used for primary cell characterization, including cloning, characterization of cell growth pattern, and lentivirus transduction. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Primary culture of liver cells Support Protocol 1: Maintenance of A. mexicanus primary liver cells Support Protocol 2: Banking of A. mexicanus primary liver cells Support Protocol 3: Recovery of A. mexicanus primary liver cells Support Protocol 4: Primary liver cell cloning Support Protocol 5: Characterization of A. mexicanus primary liver cell growth pattern Basic Protocol 2: Lentiviral transduction of A. mexicanus primary liver cells

3D spheroid culturing of Astyanax mexicanus liver‐derived cell lines recapitulates distinct transcriptomic and metabolic states of in vivo tissue environment

“…The disassociated cells were suspended with complete media and centrifuged at 1000 rpm for 5 min. The composition of the complete media for Astyanax liverderived cells was defined previously (Rajendran et al, 2023). The pelleted cells were re-suspended in complete media and counted using 0.4% trypan blue (1:1) and an automatic hemocytometer.…”

“…Nevertheless, the relatively lengthy generation time of at least 5 months (Kozol et al, 2023) and other husbandry constraints have made high-throughput approaches challenging. To overcome these challenges and to provide a more focused attempt at dissecting metabolic pathways associated with specific cell types, we realized the importance of in vitro and explant based systems (Pagano et al, 2018;Torres-Paz & Rétaux, 2021), and recently generated liverderived stable cell lines from both the cave and surface morphs (Krishnan, Wang, et al, 2022;Rajendran et al, 2023). These cell lines, termed as cavefish liver (CFL)-derived and surface fish liver (SFL)derived, express liver-specific enzymes and efficiently capture key metabolic traits of cavefish adaptation (Krishnan, Wang, et al, 2022).…”

Advancements of 3D bioprinting in regenerative medicine: Exploring cell sources for organ fabrication

“…Nonetheless, significant progress has been made in recent years with continuous in vitro expansion and culture techniques for various primary cell types. Hepatocytes, muscle satellite cells, small intestine stem cells, and lung stem cells have seen breakthroughs in large-scale expansion in vitro [ [195] , [196] , [197] , [198] , [199] ]. These in vitro expanded primary cells demonstrate improved adaptability to the 3D bioprinting process and exhibit relatively higher cell survival rates when subjected to in vitro conditioning [ 192 , 200 ].…”