Biofabrication. 2017 Sep 1;9(4):044102. doi: 10.1088/1758-5090/aa869f.
Recent advancements in 3D bioprinting have led to the fabrication of more complex, more precise, and larger printed tissue constructs. As the field continues to advance, it is critical to develop quantitative benchmarks to compare different bio-inks for key cell-biomaterial interactions, including (1) cell sedimentation within the ink cartridge, (2) cell viability during extrusion, and (3) cell viability after ink curing. Here we develop three simple protocols for quantitative analysis of bio-ink performance. These methods are used to benchmark the performance of two commonly used bio-inks, poly(ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), against three formulations of a novel bio-ink, Recombinant-protein Alginate Platform for Injectable Dual-crosslinked ink (RAPID ink). RAPID inks undergo peptide-self-assembly to form weak, shear-thinning gels in the ink cartridge and undergo electrostatic crosslinking with divalent cations during curing. In the one hour cell sedimentation assay, GelMA, the RAPID inks, and PEGDA with xanthan gum prevented appreciable cell sedimentation, while PEGDA alone or PEGDA with alginate experienced significant cell settling. To quantify cell viability during printing, 3T3 fibroblasts were printed at a constant flow rate of 75 μl min and immediately tested for cell membrane integrity. Less than 10% of cells were damaged using the PEGDA and GelMA bio-inks, while less than 4% of cells were damaged using the RAPID inks. Finally, to evaluate cell viability after curing, cells were exposed to ink-specific curing conditions for five minutes and tested for membrane integrity. After exposure to light with photoinitiator at ambient conditions, over 50% of cells near the edges of printed PEGDA and GelMA droplets were damaged. In contrast, fewer than 20% of cells found near the edges of RAPID inks were damaged after a 5 min exposure to curing in a 10 mM CaCl solution. As new bio-inks continue to be developed, these protocols offer a convenient means to quantitatively benchmark their performance against existing inks.
最近 3D 生物打印技术的进步使得更复杂、更精确和更大的打印组织构建成为可能。随着该领域的不断发展，开发定量基准来比较不同生物墨水对于关键细胞-生物材料相互作用至关重要，包括（1）墨水盒内细胞沉降，（2）挤出过程中的细胞活力，以及（3）墨水固化后的细胞活力。在这里，我们开发了三种用于定量分析生物墨水性能的简单方案。这些方法用于基准测试两种常用生物墨水，聚乙二醇二丙烯酸酯（PEGDA）和明胶甲基丙烯酰胺（GelMA），以及三种新型生物墨水，重组蛋白藻酸盐平台可注射双交联墨水（RAPID 墨水）的性能。RAPID 墨水通过肽自组装在墨盒中形成弱剪切稀化凝胶，并在固化过程中与二价阳离子发生静电交联。在一小时细胞沉降测定中，GelMA、RAPID 墨水和添加黄原胶的 PEGDA 阻止了明显的细胞沉降，而单独的 PEGDA 或添加藻酸盐的 PEGDA 则经历了明显的细胞沉降。为了量化打印过程中的细胞活力，3T3 成纤维细胞以 75 μl min 的恒定流速打印，并立即测试细胞膜完整性。使用 PEGDA 和 GelMA 生物墨水，不到 10%的细胞受损，而使用 RAPID 墨水，不到 4%的细胞受损。最后，为了评估固化后的细胞活力，将细胞暴露于特定于墨水的固化条件下 5 分钟，然后测试细胞膜完整性。在环境条件下用光引发剂照射后，打印 PEGDA 和 GelMA 液滴边缘附近超过 50%的细胞受损。相比之下，在 10 mM CaCl2 溶液中暴露于固化 5 分钟后，RAPID 墨水边缘附近的细胞受损率不到 20%。随着新的生物墨水的不断发展，这些方案为定量基准测试提供了一种方便的方法，以评估它们与现有墨水的性能。
