Calkins, O. P. | Search bioRxiv

bioRxiv2024

Establishment & Characterization of a Non-Adherent Insect Cell Line for Cultivated Meat

This study presents a blueprint for developing, scaling, and analyzing novel insect cell lines for food. The large-scale production of cultivated meat requires the development and analysis of cell lines that are simple to grow and easy to scale. Insect cells may be a favorable cell source due to their robust growth properties, adaptability to different culture conditions, and resiliency in culture. Cells were isolated from Tobacco hornworm (Manduca sexta) embryos and subsequently adapted to single-cell suspension culture in animal-free growth media. Cells were able to reach relatively high cell densities of over 20 million cells per mL in shake flasks. Cell growth data is presented in various culture vessels and spent media analysis was performed to better understand cell metabolic processes. Finally, a preliminary nutritional profile consisting of proximate, amino acid, mineral, and fatty acid analysis is reported. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/618906v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@ef6bbborg.highwire.dtl.DTLVardef@89c37eorg.highwire.dtl.DTLVardef@81a024org.highwire.dtl.DTLVardef@38bc91_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Continuous Fish Muscle Cell Line with Capacity for Myogenic and Adipogenic-like Phenotypes

Cell-cultivated fish offers the potential for a more ethical, sustainable, and safe seafood system. However, fish cell culture is relatively understudied in comparison to mammalian cells. Here, we established and characterized a continuous Atlantic mackerel (Scomber scombrus) skeletal muscle cell line ("Mack" cells). The cells were isolated from muscle biopsies of fresh-caught fish, with separate isolations performed from two distinct fish. Mack1 cells (cells from the first isolation) were cultured for over a year and subcultured over 130 times. The cells proliferated at initial doubling times of 63.9 hr ({+/-} 19.1 SD). After a spontaneous immortalization crisis from passages 37-43, the cells proliferated at doubling times of 24.3 hr ({+/-} 4.91 SD). A muscle phenotype was confirmed through characterization of muscle stemness and differentiation via paired-box protein 7 (PAX7) and myosin heavy chain (MHC) immunostaining, respectively. An adipocyte-like phenotype was also demonstrated for the cells through lipid accumulation, confirmed via Oil Red O (ORO) staining and quantification of neutral lipids. New qPCR primers (HPRT, PAX3B, MYOD1, MYOG, TNNT3A, and PPARG) were tailored to the mackerel genome and used to characterize mackerel cell genotypes. This work provides the first spontaneously immortalized fish muscle cell line for research, ideally serving as a reference for subsequent investigation.

bioengineering↗