Table_1_Mouse Retinal Organoid Growth and Maintenance in Longer-Term Culture.XLSX

Using retinal organoid systems, organ-like 3D tissues, relies implicitly on their robustness. However, essential key parameters, particularly retinal growth and longer-term culture, are still insufficiently defined. Here, we hypothesize that a previously optimized protocol for high yield of evenly-sized mouse retinal organoids with low variability facilitates assessment of such parameters. We demonstrate that these organoids reliably complete retinogenesis, and can be maintained at least up to 60 days in culture. During this time, the organoids continue to mature on a molecular and (ultra)structural level: They develop photoreceptor outer segments and synapses, transiently maintain its cell composition for about 5–10 days after completing retinogenesis, and subsequently develop pathologic changes – mainly of the inner but also outer retina and reactive gliosis. To test whether this organoid system provides experimental access to the retina during and upon completion of development, we defined and stimulated organoid growth by activating sonic hedgehog signaling, which in patients and mice in vivo with a congenital defect leads to enlarged eyes. Here, a sonic hedgehog signaling activator increased retinal epithelia length in the organoid system when applied during but not after completion of development. This experimentally supports organoid maturation, stability, and experimental reproducibility in this organoid system, and provides a potential enlarged retina pathology model, as well as a protocol for producing larger organoids. Together, our study advances the understanding of retinal growth, maturation, and maintenance, and further optimizes the organoid system for future utilization.

本研究依托视网膜类器官（retinal organoid）系统——一类模拟器官结构的三维组织——其应用的核心前提是该系统具备足够的健壮性。然而，诸多核心关键参数仍未得到充分阐明，尤其是视网膜生长与长期培养相关的环节。本研究提出假说：此前优化得到的低变异、高产量且尺寸均一的小鼠视网膜类器官培养方案，可为这类参数的探究提供便利。我们证实，此类类器官可稳定完成视网膜发生（retinogenesis）过程，且至少可在体外培养至60天。在此培养周期内，类器官在分子与（超）结构层面持续成熟：其可发育出光感受器外节（photoreceptor outer segments）与突触结构，在完成视网膜发生后会短暂维持约5~10天的细胞组成，随后逐步出现病理改变——主要累及内层视网膜，亦可累及外层视网膜，并伴随反应性胶质增生（reactive gliosis）。为验证该类器官系统能否为发育中及发育完成后的视网膜研究提供实验模型，我们通过激活音猬因子信号通路（sonic hedgehog signaling）调控并促进类器官生长。音猬因子信号通路异常激活在先天性缺陷患者与活体小鼠中可引发眼球增大，本研究中，若在类器官发育阶段而非发育完成后施加音猬因子信号通路激活剂，可显著增加视网膜上皮的长度。上述实验结果证实了该类器官系统的成熟性、稳定性与实验可重复性，同时提供了一种潜在的视网膜增大病理模型，以及一套可制备更大尺寸类器官的培养方案。综上，本研究加深了我们对视网膜生长、成熟与维持机制的理解，并进一步优化了该类器官系统，为其后续应用奠定了基础。