Improving physiological relevance of cell culture: the possibilities, considerations, and future directions of the ex vivo coculture model

Abstract

In vitro models provide an important platform for the investigation of cellular growth and atrophy to inform, or extend mechanistic insights from, logistically challenging in vivo trials. Although these models allow for the identification of candidate mechanistic pathways, many models involve supraphysiological dosages, nonphysiological conditions, or experimental changes relating to individual proteins or receptors, all of which limit translation to human trials. To overcome these drawbacks, the use of ex vivo human plasma and serum has been used in cellular models to investigate changes in myotube hypertrophy, cellular protein synthesis, anabolic and catabolic markers in response to differing age, disease states, and nutrient status. However, there are currently no concurrent guidelines outlining the optimal methodology for this model. This review discusses the key methodological considerations surrounding the use of ex vivo plasma and serum with a focus in application to skeletal muscle cell lines (i.e., C2C12, L6, and LHCN-M2) and human primary skeletal muscle cells (HSMCs) as a means to investigate molecular signaling in models of atrophy and hypertrophy, alongside future directions.

Keywords: cell culture; ex vivo; in vitro; plasma; serum.

Graphical abstract

Figure 1.

Schematic overview of the experimental set up of ex vivo cell culture. Skeletal muscle cells (cell lines or primary human cells) maintained under normal growth conditions should be plated for experiments. To assess intracellular signaling in myotubes, cells should be allowed to differentiate for 5–7 days before treatment with human plasma/serum. To assess markers of cell proliferation and differentiation, incubation with human plasma/serum should be completed once plated for experimentation. FBS, fetal bovine serum; HS, horse serum.

Figure 2.

Recommended operational model for in vitro and in vivo work. We suggest that in vitro models should utilize human primary skeletal muscle cells or immortalized cell lines, e.g., C2C12s alongside human serum/plasma treatment. Results from in vitro studies may be used to inform in vivo human analysis. Further in vitro work may be conducted after human data collection to probe for further mechanistic data and the effectiveness of nutraceutical and pharmacological treatments to inform later in vivo work, based upon initial findings.