Novo Nordisk Foundation Center for Stem Cell Medicine (reNEW), University of Copenhagen;
Department of Health Technology (DTU Health Tech), Technical University of Denmark.
J Vis Exp. 2023 May 5(195). doi: 10.3791/65158.
The embedded 3D printing of cells inside a granular support medium has emerged in the past decade as a powerful approach for the freeform biofabrication of soft tissue constructs. However, granular gel formulations have been restricted to a limited number of biomaterials that allow for the cost-effective generation of large amounts of hydrogel microparticles. Therefore, granular gel support media have generally lacked the cell-adhesive and cell-instructive functions found in the native extracellular matrix (ECM). To address this, a methodology has been developed for the generation of self-healing annealable particle-extracellular matrix (SHAPE) composites. SHAPE composites consist of a granular phase (microgels) and a continuous phase (viscous ECM solution) that, together, allow for both programmable high-fidelity printing and an adjustable biofunctional extracellular environment. This work describes how the developed methodology can be utilized for the precise biofabrication of human neural constructs. First, alginate microparticles, which serve as the granular component in the SHAPE composites, are fabricated and combined with a collagen-based continuous component. Then, human neural stem cells are printed inside the support material, followed by the annealing of the support. The printed constructs can be maintained for weeks to allow the differentiation of the printed cells into neurons. Simultaneously, the collagen continuous phase allows for axonal outgrowth and the interconnection of regions. Finally, this works provides information on how to perform live-cell fluorescence imaging and immunocytochemistry to characterize the 3D-printed human neural constructs.
在过去的十年中，嵌入式 3D 打印技术在颗粒状支撑介质中的细胞已成为自由成型软组织构建的强大方法。然而，颗粒凝胶配方已经受到限制，只能使用少数几种允许以具有成本效益的方式生成大量水凝胶微球的生物材料。因此，颗粒凝胶支撑介质通常缺乏天然细胞外基质（ECM）中存在的细胞黏附和细胞指示功能。为了解决这个问题，已经开发出一种用于生成自修复可退火颗粒-细胞外基质（SHAPE）复合材料的方法。SHAPE 复合材料由颗粒相（微凝胶）和连续相（粘性 ECM 溶液）组成，它们共同允许进行可编程的高保真打印和可调节的生物功能细胞外环境。这项工作描述了如何利用所开发的方法精确地生物制造人类神经结构。首先，藻酸盐微球被制造出来，作为 SHAPE 复合材料中的颗粒成分，并与基于胶原蛋白的连续成分结合。然后，将人类神经干细胞打印到支撑材料内部，然后对支撑材料进行退火。可以将打印的结构维持数周，以允许打印的细胞分化为神经元。同时，胶原蛋白连续相允许轴突生长和区域的互连。最后，本工作提供了如何进行活细胞荧光成像和免疫细胞化学以表征 3D 打印的人类神经结构的信息。
