Effects of three-dimensional culture conditions on skeletal muscle myoblasts

Effects of Three-dimensional Culture Conditions on Skeletal Muscle Myoblasts\r\n\r\nPublication No._____________\r\n\r\n\r\nMichele Lynn Marquette, Ph.D.\r\nThe University of Texas Medical Branch, 2007\r\n\r\nSupervisor: Marguerite A. Sognier\r\n\r\nThe objectives of this research were to: 1) develop a three-dimensional in vitro model; and 2) subsequently, utilize this model to investigate mechanisms of myoblast adhesion, fusion, and differentiation. C2C12 cells were examined as pre-aggregated single cells and multicellular aggregates in the Rotary Cell Culture System (RCCS). At the time intervals tested, RCCS cultured cells maintained viability and did not exhibit increased apoptosis markers such as Caspase 3 (activated) and phosphatydylserine. In contrast, increases in cell death and apoptotic markers were noted in suspension culture (SC) control cells. RCCS cultured cells fused to form multinucleated syncitia and expressed sarcomeric myosin heavy chain (MHC) in significantly higher levels than SC aggregates after cultivation for 3 and 6 days. This occurred in the presence of mitogens without exogenous matrix or support structures. Myoblast fusion was inhibited by exposure to soluble anti-Neural-cadherin antibody, but this treatment increased MHC levels assessed using immunohistochemistry.\r\nDuring early RCCS culture, myoblasts exhibited numerous cytoplasmic protrusions (podia). Microscopic examination of cells cultured in RCCS and SC revealed significantly more and slightly longer podia in the RCCS at 3, 6, and 9-hours. Podia were F-actin dependent as shown by exposure to an F-actin depolymerizing agent, Latrunculin A. Podia were inhibited, but recovered upon Latrunculin A removal. \r\nPodia were postulated to play a role in cell-cell adhesion in conjunction with Neural Cadherin (N-cadherin), an adhesion molecule important in myoblast differentiation. To determine if N-cadherin was critical to cell-cell adhesion, RCCS cultured cells were examined for the presence of N-cadherin at both the podia and membrane using confocal microscopy. N-cadherin levels decreased at the podia and membrane of RCCS cultured cells but not in SC cells at 3, 6, and 9-hours. \r\nIn summary, these results revealed: 1) podia formation is F-actin dependent but N-cadherin independent; 2) N-cadherin is critical for myoblast maturation; 3) synctia formation and differentiation can occur with mitogens present, without exogenous substrates in the RCCS; 4) this novel myoblast model test system is suitable for defining muscle development/regeneration processes, identification of molecular targets for development of therapies, and potential regenerative medicine applications.\r\n \r\n