Christchurch Regenerative Medicine and Tissue Engineering (CReaTE) Group, Department of Orthopaedic Surgery and Musculoskeletal Medicine, University of Otago Christchurch, Christchurch 8011, New Zealand.
Biofabrication. 2018 Jan 12;10(2):024103. doi: 10.1088/1758-5090/aa9ef1.
Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.
基于挤出的热塑性聚合物支架的 3D 打印与微组织制造技术相结合的自下而上的生物制造方法是组织工程中的新兴策略。这些生物制造策略支持组织或类器官生长的发育阶段中观察到的天然自组装机制，同时促进细胞-细胞相互作用和细胞分化能力。已经开发出几种技术来自动精确组装微组织或组织模块到结构支架中。我们描述了一种自动化 3D 生物组装平台，该平台能够通过两步自下而上的生物组装策略制造简单的混合结构，以及通过多步自下而上的生物组装策略制造复杂的混合层次结构。生物组装系统由集成到商业 3D 打印机中的基于流体的单化和注射模块组成。单化模块将单个微组织输送到注射模块中，以便将其插入 3D 绘制支架内的精确位置。为了证明其在软骨组织工程中的适用性，分离出人软骨细胞，并利用高通量 96 孔板格式生成直径为 1 毫米的微组织。微组织的单化效率为 96.0±5.1%。自动单化和注射前后微组织的大小、形状或活力没有明显差异。通过交替 3D 绘制热塑性（PEGT/PBT）聚合物支架和插入预分化的软骨形成微组织或负载细胞的明胶基（GelMA）水凝胶微球，采用逐层或上述自下而上的生物组装策略来制造双层结构，这两种方法均通过高通量制造技术形成。利用自动化生物组装系统组装的构建体与手动组装的构建体之间的活力没有明显差异。预分化的微组织和负载软骨细胞的水凝胶微球的生物组装证明了该平台的灵活性，同时支持组织融合、长期细胞活力和软骨特异性细胞外基质蛋白的沉积。该技术为具有临床相关形状和尺寸的简单和复杂 3D 组织构建体的生物组装提供了一种自动化和可扩展的途径，并且具有促进微组织模块的直接空间组织和分层 3D 组装的能力，范围从无生物材料的细胞球到负载细胞的水凝胶制剂。
