We described an automated microfluidic bioreactor manufactured using soft lithography from 3D printed molds, and optimized for long-term retinal organoid maintenance with functional imaging.
Pluripotent stem cell-derived organoid technologies have opened avenues to preclinical basic science research, drug discovery, and transplantation therapy in organ systems. Stem cell-derived organoids follow a time course similar to species-specific organ gestation in vivo. However, heterogeneous tissue yields, and subjective tissue selection reduce the repeatability of organoid-based scientific experiments and clinical studies. To improve the quality control of organoids, we introduced a live imaging technique based on two-photon microscopy to non-invasively monitor and characterize retinal organoids’ (RtOgs’) long-term development. Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the metabolic trajectory, and hyperspectral imaging was applied to characterize structural and molecular changes. We further validated the live imaging experimental results with endpoint biological tests, including quantitative polymerase chain reaction (qPCR), single-cell RNA sequencing, and immunohistochemistry. With FLIM results, we analyzed the free/bound nicotinamide adenine dinucleotide (f/b NADH) ratio of the imaged regions and found that there was a metabolic shift from glycolysis to oxidative phosphorylation. This shift occurred between the second and third months of differentiation. The total metabolic activity shifted slightly back toward glycolysis between the third and fourth months and stayed relatively stable between the fourth and sixth months. Consistency in organoid development among cell lines and production lots was examined. Molecular analysis showed that retinal progenitor genes were expressed in all groups between days 51 and 159. Photoreceptor gene expression emerged around the second month of differentiation, which corresponded to the shift in the f/b NADH ratio. RtOgs between 3 and 6 months of differentiation exhibited photoreceptor gene expression levels that were between the native human fetal and adult retina gene expression levels. The occurrence of cone opsin expression (OPN1 SW and OPN1 LW) indicated the maturation of photoreceptors in the fourth month of differentiation, which was consistent with the stabilized level of f/b NADH ratio starting from 4 months. Endpoint single-cell RNA and immunohistology data showed that the cellular compositions and lamination of RtOgs at different developmental stages followed those in vivo.
Retinal degeneration (RD) is a significant cause of incurable blindness worldwide. Photoreceptors and retinal pigmented epithelium are irreversibly damaged in advanced RD. Functional replacement of photoreceptors and/or retinal pigmented epithelium cells is a promising approach to restoring vision. This paper reviews the current status and explores future prospects of the transplantation therapy provided by pluripotent stem cell-derived retinal organoids (ROs). This review summarizes the status of rodent RD disease models and discusses RO culture and analytical tools to evaluate RO quality and function. Finally, we review and discuss the studies in which ROderived cells or sheets were transplanted. In conclusion, methods to derive ROs from pluripotent stem cells have significantly improved and become more efficient in recent years. Meanwhile, more novel technologies are applied to characterize and validate RO quality. However, opportunity remains to optimize tissue differentiation protocols and achieve better RO reproducibility. In order to screen high-quality ROs for downstream applications, approaches such as noninvasive and label-free imaging and electrophysiological functional testing are promising and worth further investigation. Lastly, transplanted RO-derived tissues have allowed improvements in visual function in several RD models, showing promises for clinical applications in the future.
<div>Retinal degeneration is a leading cause of vision impairment and blindness worldwide and
medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs)
became an emerging tool for tissue replacement therapy. However, existing RtOg production
methods are highly heterogeneous. Controlled and predictable methodology and tools are needed
to standardize RtOg production and maintenance. In this study, we designed a shear stress-free
micro-millifluidic bioreactor. We used a stereolithography (SLA) 3D printer to fabricate a mold
from which Polydimethylsiloxane (PDMS) was cast. The multi-chamber bioreactor design and
fabrications methods easily combined micro and millimeter features with very low cost and short
manufacturing time. We optimized the chip design using in silico simulations and in vitro
evaluation to optimize mass transfer efficiency and concentration uniformity in each culture
chamber. We successfully cultured RtOgs on an optimized bioreactor chip for 37 days. We also
characterized the RtOgs produced by static dish culture and chip culture methods using
qualitative and quantitative techniques. Phase contrast imaging showed that both conventional
and chip-cultured RtOgs developed a transparent outermost surface structure. Fluorescence
lifetime imaging (FLIM) showed that RtOgs on the chip had significantly lower long lifetime species
(LLS) ratio than static cultured ones, which demonstrated that bioreactor cultured RtOgs exhibited
less oxidative stress. RtOgs in bioreactor culture demonstrated higher NADH signal overall, but
both bioreactor and conventional cultures showed similar free/bound NADH ratio over time, which
indicated normal differentiation time course. RtOg gene expression was examined by
fluorescence imaging and quantitative polymerase chain reaction (qPCR) analyses. RtOgs in both
groups showed thick nuclear outer layers expressing CRX on day 120 of differentiation. The gene
profiling showed both groups expressed retinal progenitor genes and most of the tested
photoreceptor markers. We, therefore, validated an autonomous micro-millifluidic device with
significantly reduced shear stress and lower oxidative stress to produce RtOgs of equal or greater
quality than those maintained in conventional static culture. </div>
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