The effects of three-dimensional cell culture on single myoblasts

Identification of cellular and morphological changes in myoblasts during three-dimensional (3D) culture may provide novel insight into skeletal muscle morphogenesis. One particular morphological change that occurs during the transition from monolayer culture to the 3D environment is the appearance of cytoplasmic projections (podia). The purpose of these studies was to determine if: (1) 3D culture increased podia formation in single cells, and (2) podia were F-actin dependent. C2C12 cells were grown in 3D conditions using a rotary cell culture system (RCCS) for 3, 6, and 9 h, fixed, and stained. Analysis of confocal images revealed that podia were significantly more numerous on RCCS cultured cells than those on suspension controls. Further, the podia of RCCS cultured cells decreased in number and increased in length during the time intervals examined. RCCS cultured cells showed no significant changes in viability, Annexin V staining, and activated Caspase 3 expression over time. In contrast, significant decreases in viability of suspension controls occurred. The application of 2 μM Latrunculin A (Lat A), an actin depolymerizing agent, significantly reduced the number of cells with podia. The number of cells with podia recovered with Lat A removal. Changes in viability and apoptosis markers were not significant during Lat A application or washout experiments. These observations reveal that: (1) culture conditions in the RCCS increase the quantity of podia formation; (2) these podia increase in length with time; and (3) F-actin plays a role in podia formation.