1 Department of Health, Kinesiology, and Applied Physiology, Concordia University, Montréal, Quebec, Canada.
2 Metabolism, Obesity, and Nutrition Lab, School of Health, Concordia University, Montreal, Quebec, Canada.
3 Centre de Recherche-Axe Maladies Chroniques, CIUSSS-NIM, Hôpital du Sacré-Coeur de Montréal, Montreal, Quebec, Canada.
4 Academy of Wellness and Human Development, Faculty of Arts and Social Sciences, Hong Kong Baptist University, Hong Kong SAR, China.
5 Dr. Stephen Hui Research Centre for Physical Recreation and Wellness, Faculty of Arts and Social Sciences, Hong Kong Baptist University, Hong Kong SAR, China.
6 Département Du Chirurgie, Hôpital Du Sacré-Coeur de Montréal, Montréal, Quebec, Canada.
7 Division of Geriatric Medicine, Department of Medicine, McGill University, McGill University Health Centre (MUHC)-Montreal General Hospital, Montreal, Quebec, Canada.
8 Department of Biology, Concordia University, Montreal, Quebec, Canada.

Transwell co-culture systems are models for examining cellular crosstalk, but the impact of porous membrane material on myogenic outcomes remains uncharacterized. We investigated the influence of transwell membrane material on myoblast differentiation in co-culture with human primary adipocytes and in varying concentrations of insulin-transferrin-selenium (ITS). Primary human myoblasts were differentiated on 0.4 µm polycarbonate or polyester membranes, either alone (Myo-Only) or with adipocytes (Myo + Adipo) in two different ITS concentrations ([ITS]; 0.5× or 1×). Myogenesis was evaluated by immunofluorescence, using fusion index (%) and myotube thickness (µm), and adipokine levels were measured in each co-culture compartment. Polycarbonate membranes supported a greater fusion index and myotube thickness than polyester at both 1× (p < 0.001) and 0.5× ITS (p = 0.002). Across [ITS] (1× p = 0.035; 0.5× p = 0.006), the fusion index was higher in Myo + Adipo co-cultures than in Myo-Only conditions. Leptin diffusion was reduced across polyester compared to polycarbonate membranes (0.5× p = 0.036; 1× p = 0.091), whereas adiponectin diffusion was similar between materials. These findings underscore that membrane material and medium composition are critical variables in transwell adipocyte-myoblast co-culture systems. This study highlights the importance in the selection and reporting of these parameters to improve reproducibility across future studies.