Key Laboratory for Advanced Materials Processing Technology of Ministry of Education, Biomanufacturing and Rapid Forming Technology Key Laboratory of Beijing, Department of Mechanical Engineering, Tsinghua University, Beijing 100084, China.
Key Laboratory of Medical Information and 3D Bioprinting of Zhejiang Province, Hangzhou Dianzi University, Hangzhou 310018, China.
ACS Biomater Sci Eng. 2020 May 11;6(5):2995-3004. doi: 10.1021/acsbiomaterials.9b01825. Epub 2020 Apr 29.
The expansion and harvest of stem cells at clinically relevant scales is critical for cell-based therapies. These approaches need to be robust and cost-effective, support the functional maintenance of desired cell behaviors, and allow for simple harvest. Here, we introduce a real-time monitoring 3D printing approach to fabricate scaffolds with quadruple hierarchical structure that meet these design goals for stem cell expansion. Specifically, a versatile strategy was developed to produce scaffolds from alginate and gelatin with approximately 102 μm interconnected macropores, 300 μm microfilaments, 1.3 mm hollow channels, and centimeter-scale overall dimensions. The scaffolds exhibited good pattern fidelity and stable mechanical properties (compressive modulus value was 22-fold that of hydrogels from the same materials), facilitating uniform and efficient cell seeding with high viability (98.9%). The utility of the scaffold was shown with the 3D culture of HepaRG cells and embryonic stem cells (ESCs) with aggregated morphology, and significantly enhanced cell proliferation was observed compared to those of cultures on flat surfaces, obtaining approximately 2 × 10 cells within a single culture. Interestingly, the functional behavior of the cells was dependent on the cell type, as ESCs maintained their pluripotency, while HepaRG cells improved their hepatic differentiation. Cells were harmlessly harvested through chelating the calcium ions in the cross-linked alginate and de-cross-linking the scaffolds, indicating the potential of this study for scalable stem cell culture for numerous downstream applications.
在临床上相关的规模上扩展和收获干细胞对于基于细胞的治疗至关重要。这些方法需要强大且具有成本效益，支持所需细胞行为的功能维持，并允许简单收获。在这里，我们介绍了一种实时监测 3D 打印方法，用于制造满足这些干细胞扩展设计目标的具有四重层次结构的支架。具体来说，开发了一种从海藻酸钠和明胶生产支架的通用策略，这些支架具有约 102 μm 相互连通的大孔、300 μm 微丝、1.3 mm 空心通道和厘米级的整体尺寸。支架表现出良好的图案保真度和稳定的机械性能（压缩模量值是相同材料水凝胶的 22 倍），有利于均匀高效的细胞接种和高存活率（98.9%）。通过 HepaRG 细胞和胚胎干细胞（ESCs）的 3D 培养证明了支架的实用性，与在平面上培养相比，观察到细胞增殖显著增强，在单个培养中获得了大约 2 × 10 个细胞。有趣的是，细胞的功能行为取决于细胞类型，因为 ESCs 保持其多能性，而 HepaRG 细胞则改善其肝分化。通过螯合交联海藻酸中的钙离子和去交联支架来无害地收获细胞，这表明该研究具有用于众多下游应用的可扩展干细胞培养的潜力。
