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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.

近年来，三维（3D）细胞培养模型作为动物实验与二维（2D）细胞培养模型的替代方案，在药物测试与疾病建模领域的应用愈发广泛。聚二甲基硅氧烷（Polydimethylsiloxane, PDMS）是一种成本低廉、易于获取的3D培养成型材料，但常规PDMS成型工艺并不适用于高收缩性、高代谢活性组织的长期培养，常见问题包括细胞培养物与PDMS模具的黏附力丧失，以及用于营养物质与代谢废物扩散的培养表面积不足。
本研究针对高收缩性、高代谢活性的3D细胞培养模型，评估了多款PDMS成型材料及其表面处理方案。经聚多巴胺（polydopamine）功能化修饰的PDMS，其最长培养时长可达14.8 ± 3.97天，显著长于经聚乙烯亚胺/戊二醛（polyethylamine/glutaraldehyde）功能化修饰的PDMS（仅6.94 ± 2.74天）；此外，经TGF-β2处理以增强培养物收缩能力后，多孔PDMS的培养时长可达16.7 ± 3.51天，同样显著优于光滑PDMS（6.33 ± 2.05天）。
多孔PDMS还可支持大尺寸（高13 mm × 直径8 mm）细胞构建物通过模具完成扩散供氧供能，最终其平均细胞核占比可达0.0210 ± 0.0049，显著高于对照组的0.0045 ± 0.0016。作为多孔PDMS灵活性的实用验证，我们构建了血管生物人工肌肉模型（vascular bioartificial muscle model, VBAM），并实现了搭载多孔PDMS锚定柱的VBAM长期培养。
利用该模型，我们评估了换液频率对VBAM细胞丰度的影响：相较于每日换液2次的组别，每周换液3次的组别在多个组织深度下的细胞核占比均显著提升。同时，3次/周换液组的VBAM成熟度也得到改善，其细胞核排列角度为23.49° ± 3.644°、细胞核长宽比为2.274 ± 0.0643，均优于2次/天换液组（分别为35.93° ± 2.942°与1.371 ± 0.1127）。
本研究所描述的技术方案设计简便，仅需少量培训与低成本即可实施，有助于推广高密度和/或高代谢活性3D细胞培养模型的应用。