Department of NanoEngineering, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0448, USA.
Lab Chip. 2015 Jun 7;15(11):2412-8. doi: 10.1039/c5lc00159e. Epub 2015 Apr 22.
Long-term culture and monitoring of individual multicellular spheroids and embryoid bodies (EBs) remains a challenge for in vitro cell propagation. Here, we used a continuous 3D projection printing approach - with an important modification of nonlinear exposure - to generate concave hydrogel microstructures that permit spheroid growth and long-term maintenance, without the need for spheroid transfer. Breast cancer spheroids grown to 10 d in the concave structures showed hypoxic cores and signs of necrosis using immunofluorescent and histochemical staining, key features of the tumor microenvironment in vivo. EBs consisting of induced pluripotent stem cells (iPSCs) grown on the hydrogels demonstrated narrow size distribution and undifferentiated markers at 3 d, followed by signs of differentiation by the presence of cavities and staining of the three germ layers at 10 d. These findings demonstrate a new method for long-term (e.g. beyond spheroid formation at day 2, and with media exchange) 3D cell culture that should be able to assist in cancer spheroid studies as well as embryogenesis and patient-derived disease modeling with iPSC EBs.
长期培养和监测单个多细胞球体和类胚体 (EBs) 仍然是体外细胞繁殖的一个挑战。在这里，我们使用了连续的 3D 投影打印方法——对非线性曝光进行了重要修改——来生成允许球体生长和长期维持的凹形水凝胶微结构，而无需球体转移。在凹形结构中生长至 10 天的乳腺癌球体通过免疫荧光和组织化学染色显示出缺氧核心和坏死迹象，这是体内肿瘤微环境的关键特征。在水凝胶上生长的诱导多能干细胞 (iPSC) 组成的 EBs 在 3 天时表现出狭窄的尺寸分布和未分化标记物，随后在 10 天时出现空腔和三个胚层染色的分化迹象。这些发现展示了一种新的长期（例如，超过第 2 天的球体形成，以及进行介质交换）3D 细胞培养方法，该方法应该能够辅助癌症球体研究以及胚胎发生和患者来源的疾病建模与 iPSC EBs。
