In vitro formation and extended culture of highly metabolically active and contractile tissues

3D cell culture models have gained popularity in recent years as an alternative to animal and 2D cell culture models for pharmaceutical testing and disease modeling. Polydimethylsiloxane (PDMS) is a cost-effective and accessible molding material for 3D cultures; however, routine PDMS molding may not be appropriate for extended culture of contractile and metabolically active tissues. Failures can include loss of culture adhesion to the PDMS mold and limited culture surfaces for nutrient and waste diffusion. In this study, we evaluated PDMS molding materials and surface treatments for highly contractile and metabolically active 3D cell cultures. PDMS functionalized with polydopamine allowed for extended culture duration (14.8 ± 3.97 days) when compared to polyethylamine/glutaraldehyde functionalization (6.94 ± 2.74 days); Additionally, porous PDMS extended culture duration (16.7 ± 3.51 days) compared to smooth PDMS (6.33 ± 2.05 days) after treatment with TGF-β2 to increase culture contraction. Porous PDMS additionally allowed for large (13 mm tall × 8 mm diameter) constructs to be fed by diffusion through the mold, resulting in increased cell density (0.0210 ± 0.0049 mean nuclear fraction) compared to controls (0.0045 ± 0.0016 mean nuclear fraction). As a practical demonstration of the flexibility of porous PDMS, we engineered a vascular bioartificial muscle model (VBAM) and demonstrated extended culture of VBAMs anchored with porous PDMS posts. Using this model, we assessed the effect of feeding frequency on VBAM cellularity. Feeding 3×/week significantly increased nuclear fraction at multiple tissue depths relative to 2×/day. VBAM maturation was similarly improved in 3×/week feeding as measured by nuclear alignment (23.49° ± 3.644) and nuclear aspect ratio (2.274 ± 0.0643) relative to 2x/day (35.93° ± 2.942) and (1.371 ± 0.1127), respectively. The described techniques are designed to be simple and easy to implement with minimal training or expense, improving access to dense and/or metabolically active 3D cell culture models.