Zebrafish skeletal muscle cell cultures: Monolayer to three-dimensional tissue-engineered collagen constructs

Zebrafish (Danio rerio) are a commonly used model organism to study human muscular myopathies and dystrophies. To date, much of the work has been conducted in vivo due to limitations surrounding the consistent isolation and culture of zebrafish muscle progenitor cells (MPCs) in vitro and the lack of physiologically relevant models. Here we report a robust, repeatable, and cost-effective protocol for the isolation and culture of zebrafish MPCs in conventional monolayer (2D) and have successfully transferred these cells to 3D culture in collagen based three-dimensional (3D) tissue-engineered constructs. Zebrafish MPCs cultured in 2D were consistently reported to be Desmin positive reflecting their muscle specificity, with those demonstrating Desmin positivity in the 3D cultures. In addition, mRNA expression of muscle markers specific for proliferation, differentiation and maturation measured from both monolayer and 3D cultures at appropriate developmental stages were found consistent with previously published from other species in vitro and in vivo muscle data. Collagen constructs seeded with zebrafish MPCs were initially characterised for optimal seeding density, followed by macroscopic characterisation (three-fold contraction) of the matrix. Direct comparison between the morphological characteristics (proportion of cells) and gene expression profiles of cells cultured in collagen constructs revealed higher maturation and differentiation compared to monolayer cultures. In this regard, cells embedded in 3D collagen constructs revealed higher fusion index, Desmin positivity, hypertrophic growth, myotube maturity and myogenic mRNA expression when compared to in monolayer. In conclusion, these methods and models developed herein will facilitate in vitro experiments, which would complement in vivo zebrafish studies used to investigate the basic developmental, myopathies and dystrophies in skeletal muscle cells.