Artificial biological scaffolds made of extracellular matrix (ECM) components, such as type I collagen, provide ideal physicochemical cues to various cell culture platforms. However, it remains a challenge to fabricate micrometer-sized ECM materials with precisely controlled morphologies that could reconstitute the 3-dimensional (3D) microenvironments surrounding cells. In the present study, we proposed a unique process to fabricate fragmented collagen microfibers using a microfluidic laminar-flow system. The continuous flow of an acidic collagen solution was neutralized to generate solid fibers, which were subsequently fragmented by applying a gentle shear stress in a polyanion-containing phosphate buffer. The morphology of the fiber fragment was controllable in a wide range by changing the type and/or concentration of the polyanion and by tuning the applied shear stress. The biological benefits of the fragmented fibers were investigated through the formation of multicellular spheroids composed of primary rat hepatocytes and microfibers on non-cell-adhesive micro-vessels. The microfibers enhanced the survival and functions of the hepatocytes and reproduced proper cell polarity, because the fibers facilitated the formation of cell−cell and cell−matrix interactions while modulating the close packing of cells. These results clearly indicated that the microengineered fragmented collagen fibers have great potential to reconstitute extracellular microenvironments for hepatocytes in 3D culture, which will be of significant benefit for cell-based drug testing and bottom-up tissue engineering.

由细胞外基质 (ECM) 成分（如 I 型胶原蛋白）制成的人工生物支架为各种细胞培养平台提供了理想的理化线索。然而，制造具有精确控制形态的微米级 ECM 材料仍然是一个挑战，这些材料可以重建细胞周围的 3D (3D) 微环境。在本研究中，我们提出了一种使用微流体层流系统制造碎片化胶原微纤维的独特工艺。酸性胶原溶液的连续流动被中和以产生固体纤维，随后通过在含有聚阴离子的磷酸盐缓冲液中施加温和的剪切应力将其破碎。通过改变聚阴离子的类型和/或浓度以及调整施加的剪切应力，纤维碎片的形态可以在很宽的范围内控制。通过在非细胞粘附微血管上形成由原代大鼠肝细胞和微纤维组成的多细胞球体，研究了碎片纤维的生物学益处。微纤维增强了肝细胞的存活和功能，并再现了适当的细胞极性，因为这些纤维促进了细胞-细胞和细胞-基质相互作用的形成，同时调节了细胞的紧密堆积。这些结果清楚地表明，微工程化的碎片化胶原纤维具有在 3D 培养中重建肝细胞细胞外微环境的巨大潜力，