Neurobiology, Neurodegeneration & Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, MD.
Mol Vis. 2020 Mar 3;26:97-105. eCollection 2020.
Retinal organoids (ROs) derived from human pluripotent stem cells largely recapitulate key features of in vivo retinal development, thus permitting the study of retinogenesis, disease modeling, and therapeutic development. However, the complexities of current protocols limit the use of this in vitro system in applications requiring large-scale production of organoids. Currently, widely used methods require the isolation of presumed optic vesicle-like structures from adherent cultures by dissection, a labor-intensive and time-consuming step that involves extensive practice and training.
We report a simple and efficient method for generating ROs by scraping the entire adherent culture and growing the resulting cell aggregates in a free-floating condition.
Within 1 to 7 days following the procedure, emerging morphologically well-defined optic vesicles can be identified and harvested with ease. The transition from two-dimensional (2D) to 3D culture condition favored the formation of ROs from areas devoid of typical optic vesicle-like structures, thus increasing the RO yield. Moreover, ROs generated by this approach were more often associated with the pigment epithelium.
This improved, robust, and efficient protocol should facilitate large-scale differentiation of pluripotent stem cells into retinal organoids in support of human disease modeling and therapy development.
源自人类多能干细胞的视网膜类器官（RO）在很大程度上再现了体内视网膜发育的关键特征，从而允许对视网膜发生、疾病建模和治疗开发进行研究。然而，当前方案的复杂性限制了该体外系统在需要大规模生产类器官的应用中的使用。目前，广泛使用的方法需要通过解剖从贴壁培养物中分离假定的视囊样结构，这是一个劳动强度大且耗时的步骤，需要广泛的实践和培训。
我们报告了一种简单有效的方法，通过刮取整个贴壁培养物并在自由悬浮条件下培养所得细胞聚集体来生成 RO。
在进行该程序后 1 至 7 天内，可以轻松识别和收获形态上定义明确的新兴视囊。从缺乏典型视囊样结构的区域形成 RO 的二维（2D）到三维（3D）培养条件的转变有利于提高 RO 的产量。此外，通过这种方法生成的 RO 更常与色素上皮有关。
这种改进、稳健且高效的方案应有助于大规模地将多能干细胞分化为视网膜类器官，以支持人类疾病建模和治疗开发。
