High-throughput Spheroid Bioprinting Technology for Scalable Fabrication of Tissues

研究概要/摘要 以高通量方式将细胞聚集体（如球状体）生物打印成所需图案的能力 或细胞微环境是至关重要的，以促进制造可扩展的构建体， 即天然组织和器官。尽管球状体生物打印技术取得了进展，但主要的 与它们相关的缺点，例如球状体的定位差，存活力的显著损失， 结构完整性，当使用不均匀尺寸的球状体时，工艺的可重复性差，不能形成 复杂的3D形状，最重要的是，缺乏可扩展性，限制了它们的转换。在这个项目中，我们建议 一种高度独特的技术，此后被称为“高通量球体（HTS）生物打印”， 同时生物打印具有数量级尺寸范围和最小细胞密度的几个球状体 以高定位精度和前所未有的速度进行破坏。所提出的技术是高度通用的 并且能够将复杂结构生物打印到（1）凝胶基质的表面上（即，水凝胶）中 基于支架的方式或（2）在支撑槽内（即，牺牲微凝胶）以无支架的方式用于 组织的可扩展制造。在具体目标1中，我们建议开发HTS生物打印，它具有以下能力： 在3D凝胶基质上同时沉积几个球状体，从而生物打印出一个完整的3D层。 组织以快速的方式一次（即，100个球状体可以在<20秒内生物打印）。我们将把HTS生物打印 与基于挤出的凝胶基质生物打印，并探索球状体-凝胶相互作用，在很宽的范围内， 在生物打印的过程中。为了验证这项技术，我们将展示生物打印 术中沉积成骨定向骨球修复颅颌面缺损 大鼠模型中的骨缺损。在具体目标2中，我们将重新配置HTS生物打印技术， 将球状体定位在牺牲载体浴内。在这里，我们将生物打印球体顺序（一个后 另一个）以快速的方式并根据目标设计图案化它们。我们将探索凝胶球体 生物打印过程的相互作用，其中该技术的有效性将受到多重支持的测试 浴，包括藻酸盐微凝胶，其使用空气喷射辅助同轴流动技术沿着制造， 商用基准。我们将研究利用该技术制造 解剖学相关的复杂形状的人支气管肺段。在这方面，我们已成立了一个 互补合作，将生物打印、生物打印过程和 仪器开发，生物材料，颅面外科，骨和肺组织工程与深度 因此，必须采取必要措施，推动拟议的工作取得有意义的进展，否则就不可能取得这些进展。 拟议工作的成功完成预计将产生先进的生物打印技术， HTS生物打印，从而为制造可扩展的组织和器官提供了一种新的工具。

ROJECT SUMMARY/ABSTRACT The ability to bioprint cellular aggregates, such as spheroids, in a high-throughput manner into desired patterns or cellular microenvironments is crucial to facilitate fabrication of scalable constructs with cell densities similar to that of native tissues and organs. Despite the progress in spheroid bioprinting technologies, the major shortcomings associated with them, such as poor positioning of spheroids, significant loss of viability and structural integrity, poor repeatability of the process when using non-uniform size spheroids, inability to form complex 3D shapes, and most importantly, the lack of scalability, limit their translation. In this project, we propose a highly unique technology, henceforth referred as “high-throughput spheroid (HTS) bioprinting,” that enables simultaneous bioprinting of several spheroids with an order of magnitude size range and minimal cellular damage, at a high positional precision and an unprecedented speed. The proposed technology is highly versatile and enables the bioprinting of complex structures either (1) onto the surface of gel substrates (i.e., hydrogels) in a scaffold-based manner or (2) within support baths (i.e., sacrificial microgels) in a scaffold-free manner for scalable fabrication of tissues. In Specific Aim 1, we propose to develop HTS bioprinting, which has the capability of depositing several spheroids simultaneously on 3D gel substrates, thus bioprinting a complete layer of the 3D tissue at once in a rapid fashion (i.e., 100 spheroids can be bioprinted in <20 sec). We will couple HTS bioprinting with extrusion-based bioprinting of gel substrates and explore the spheroid-gel interactions, across a wide range of hydrogels, during the bioprinting process. To exemplify the technology, we will demonstrate bioprinting intraoperatively via depositing osteogenically-committed bone spheroids for the repair of craniomaxillofacial bone defects in a rat model. In Specific Aim 2, we will reconfigure the HTS bioprinting technology for freeform positioning of spheroids within sacrificial support baths. Here, we will bioprint spheroids sequentially (one after the other) in a rapid manner and pattern them according to the target design. We will explore the gel-spheroid- bioprinting process interactions, where the effectiveness of the technology will be tested for multiple support baths, including alginate microgels to be fabricated using the air-jet assisted coaxial flow technique along with a commercially available benchmark. We will exemplify the utilization of the technology for fabrication of anatomically-relevant complex-shaped human bronchopulmonary segments. In this regard, we have formed a complementary collaboration that merges essential domain knowledge in bioprinting, bioprinting process and instrument development, biomaterials, craniofacial surgery, and bone and lung tissue engineering with the depth necessary to propel the proposed work towards meaningful advances that would otherwise not be possible. Successful completion of the proposed work is anticipated to give rise to an advanced bioprinting technology for HTS bioprinting and thereby provide a novel tool for fabrication of scalable tissues and organs.