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A controllable perfusion microfluidic chip with the advantages of precisely controlling fluidic shear stress and oxygen concentration distribution is designed to promote the development of retinal organoids.

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PSCs (iPSCs) and embryonic stem cells (ESCs), or adult stem cells (ASCs). Pioneered in the Yoshiki Sasai lab, where cortical tissues and optic-cuplike structures were firstly induced from ESCs in vitro, [1,2] organoid technology is regarded as a milestone in the stem cell field because it cast light upon the path to stably growing self-aggregating and selfrenewing stem cells into native tissues by mimicking developmental processes. [3] Subsequently, the rapidly developing field of organoid technology is advancing the maturity of technology for developing 3D macrostructures of organs such as the retina, [4] heart, [5] liver, [6] kidney, [7] and intestines. [8] In marked contrast to traditional 2D monocultures of cell lines losing cell-cell and cell-matrix interactions, organoids maintain and define in situ phenotypes fairly well. Despite organoids having shown great progress in tissue morphogenesis and organogenesis, the lack of organ-specific biochemical and physical microenvironments and nonphysiological shape and size have limited the function and real-life applications of organoids. Therefore, innovative strategies are urgently needed to precisely control perfusion conditions, nutrient supply, and mechanical cues in order to establish an optimal microenvironment for organoid cultures.The recently developed organs-on-a-chip (OOC) technology provides an in vivo-like microenvironment, showing great potential to enhance our understanding of organ development, physiology, and pathology. [9] Besides, the application of dynamic flow conditions or mechanical stimuli provided by OOCs can improve the terminal maturation of cells. [10,11] OOCs usually are comprised of specialized cell types and tissue elements (biochemical and biophysical cues of in vivo microenvironment), aiming to capture key functions of a specific organ. [12] Organoids rely on the self-organization of growing stem cell aggregates, whereas OOC is based on a reductionist engineering approach. In addition, organoids are multicellular 3D tissue with an architecture resembling some aspects of original tissues, which are not classified as OOC because of their production through stochastic self-organization from PSCs or ASCs and lack of microenvironment elements. [13] However, organoid and OOC techniques each have their own advantages, and they can be appropriately combined to enable highfidelity human modeling from stem cells. [14] Such synergistic Organoids are 3D multicellular structures derived from pluripotent stem cells (PSCs) or adult stem cells (ASCs), which have attracted increasing interest in the fields of drug screening, cell therapy, and regenerative medicine. Despite considerable success in culturing organoids with native microanatomy, challenges to achieving a physiologically relevant microenvironment remain. Complex dynamic feedback between cells and the extracellular matrix and uncontrollable mechano-physiological cues hamper the further study of organoid systems. Innovative engineering approaches are needed to produce, control, and analy...

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Background: Over 90% of children with fetal alcohol syndrome live with ocular aberration due to the susceptible and intricate human eye development process. Initial neurogenesis of the neural retina around six-week gestation is the critical period of human eye development while sustaining the highest risk of prenatal ethanol exposure because of ignorance of early pregnancy. However, the influence and mechanism of short-term ethanol exposure on this developmental process of the human neural retina remain largely unknown. Methods: To faithfully recapitulate the initial retinal neurogenesis of human neural retina, human embryonic stem cell derived retinal organoids (hROs) were induced and identified by immunostaining. Morphological measurement was performed to primarily assess the influence of short-term ethanol exposure on the growth of neural retina. TUNNEL assay, immunostaining, and flow cytometry were utilized to detect cell death, retinal ganglion cell differentiation, and cell cycle progression in hROs. Bulk RNA-seq analysis and cnet plotting were performed to screen signaling pathway and regulated genes of ethanol treatment. GCaMP5G-expressing human embryonic stem cells were constructed by transduction of pLOV-CMV-GCaMP5G and fluorescence-activated cell sorting. Two-photon microscope live calcium imaging were utilized to reveal altered calcium signaling dynamics after ethanol treatment. Quantitative RT-PCR was performed to verify the expression of screened potential targeted genes of ethanol treatment. Results: The hROs from D24 to D30 well recapitulate the initial neurogenesis of the human neural retina around six-week gestation in vivo at the histological, cellular, and molecular level. 1% (v/v) ethanol slowed the growth of hROs by inducing robust cell death and retinal ganglion cell differentiation defect. Calcium signaling dynamics was proved significantly altered and derived from ethanol-induced down-regulation of RYR1and CACNA1S. Moreover, the calcium-binding protein RET, one of the downstream effector genes of the calcium signaling pathway, synergistically integrates ethanol and calcium signals to abort neuron differentiation and cause cell death. Conclusion: Our study demonstrated that short-term ethanol exposure greatly impaired the initial neurogenesis of hROs by disrupting the RYR1 related calcium signaling. These results may help us elaborate on more detailed principles of ethanol-induced teratogenesis and instruct the rational application of alcohol and ethanol-contained drugs during pregnancy.

Recent advances in stem cell technologies have enabled the application of three-dimensional neural organoids for exploring the mechanisms of neurodevelopment and regenerative medicine. Over the past decade, series of studies have been carried out to investigate the cellular and molecular events of human neurogenesis using animal models, while the species differences between animal models and human being prevent a full understanding of human neurogenesis. Human neural organoids provide a new model system for gaining a more complete understanding of human neural development and their applications in regenerative medicine. In this chapter, the recent advances of the neural organoids of the brain and retina as well as their applications in neurodevelopment and regenerative medicine are reviewed.