Retinal organoids on-a-chip: a micro-millifluidic bioreactor for long-term organoid maintenance

Xue, Yuntian; Seiler, Magdalene J.; Tang, William C.; Wang, Jasmine Y.; Delgado, Jeffrey; McLelland, Bryce T.; Nistor, Gabriel; Keirstead, Hans S.; Browne, Andrew

doi:10.1039/d1lc00011j

Cited by 39 publications

(34 citation statements)

References 71 publications

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“…Differentiation into retinal progenitor cells was indicated by strong expression of genes of primitive retina (VSX2, CRX, PAX6, RAX, SIX3, and SIX6) (Figure 1C) and terminally differentiated retinal cell populations (PRKCB, RCVRN, MAP1A, MAP2, POU4F2, POU4F1, and GFAP) (Figure 1D). These include expression of markers for progenitors of photoreceptors, glia, ganglion cells, bipolar, horizontal, amacrine, and Müller cells, similar to what we have shown in previous publications for the CSC-14 cell line (McLelland et al, 2018;Lin et al, 2020) and for the CRX-GFP cell line (Xue et al, 2021). Flow analysis showed that GFP expression was absent in the undifferentiated stem cells, early embryoid bodies, and young ROs.…”

Section: Generation and Characterization Of Retina Organoids And Human Embryonic Stem Cell-retinal Pigment Epithelium Sheetssupporting

confidence: 85%

Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats

et al. 2021

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End-stage age-related macular degeneration (AMD) and retinitis pigmentosa (RP) are two major retinal degenerative (RD) conditions that result in irreversible vision loss. Permanent eye damage can also occur in battlefields or due to accidents. This suggests there is an unmet need for developing effective strategies for treating permanent retinal damages. In previous studies, co-grafted sheets of fetal retina with its retinal pigment epithelium (RPE) have demonstrated vision improvement in rat retinal disease models and in patients, but this has not yet been attempted with stem-cell derived tissue. Here we demonstrate a cellular therapy for irreversible retinal eye injuries using a “total retina patch” consisting of retinal photoreceptor progenitor sheets and healthy RPE cells on an artificial Bruch’s membrane (BM). For this, retina organoids (ROs) (cultured in suspension) and polarized RPE sheets (cultured on an ultrathin parylene substrate) were made into a co-graft using bio-adhesives [gelatin, growth factor-reduced matrigel, and medium viscosity (MVG) alginate]. In vivo transplantation experiments were conducted in immunodeficient Royal College of Surgeons (RCS) rats at advanced stages of retinal degeneration. Structural reconstruction of the severely damaged retina was observed based on histological assessments and optical coherence tomography (OCT) imaging. Visual functional assessments were conducted by optokinetic behavioral testing and superior colliculus electrophysiology. Long-term survival of the co-graft in the rat subretinal space and improvement in visual function were observed. Immunohistochemistry showed that co-grafts grew, generated new photoreceptors and developed neuronal processes that were integrated into the host retina. This novel approach can be considered as a new therapy for complete replacement of a degenerated retina.

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Section: Generation and Characterization Of Retina Organoids And Human Embryonic Stem Cell-retinal Pigment Epithelium Sheetssupporting

confidence: 85%

Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats

et al. 2021

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“…Current methods for generating hPSC retinal organoids are both time and labor-intensive, limiting their utility in clinical production pipelines. The use of bioreactors, 172 microfluidics, 166 , 173 and automated culture systems 27 are all promising approaches currently under investigation for scaling clinical-grade organoid-based technologies.…”

Section: Current Status and Remaining Questions For Retinal Cell Therapiesmentioning

confidence: 99%

Outer Retinal Cell Replacement: Putting the Pieces Together

Ludwig

Gamm

2021

Trans. Vis. Sci. Tech.

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“…The stem cell culture and RtOg initiation procedures were detailed in our previous publication (Xue et al, 2021), which were based on RtOgs differentiated from two hESC lines [cell line CSC14 with NIH registration no. 0284 and H9 (WA09) CRX-GFP with NIH registration no.…”

Section: Stem Cell Culture and Retinal Organoid Differentiationmentioning

confidence: 99%

“…Fluorescence lifetime imaging microscopy imaging settings used in this study were the same as in our previous publication (Xue et al, 2021). Before imaging, the system was calibrated on frequency factor and lifetime with coumarin 6 solution, which has the known lifetime of 2.5 ns.…”

Section: Two-photon Fluorescence Lifetime Microscopy and Hyperspectral Imagingmentioning

confidence: 99%

“…In this study we focused on the lifetime of free (0.4 ns) and lactate bound (3.4 ns) NADH (Supplementary Figure 1D). Additional details for FLIM imaging and data and data analysis using the phasor approach with SimFCS software (Globals Software G-SOFT Inc., Champaign, IL, United States) have been published previously (Digman et al, 2008;Xue et al, 2021). The fraction of free and bound NADH was normalized to the orthogonal intersection value on the metabolic NADH trajectory line by mapping the phasor plot center of mass directly to the free-bound NADH axis.…”

Section: Two-photon Fluorescence Lifetime Microscopy and Hyperspectral Imagingmentioning

confidence: 99%

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Retinal Organoids Long-Term Functional Characterization Using Two-Photon Fluorescence Lifetime and Hyperspectral Microscopy

Xue

Browne

Tang

et al. 2021

Front. Cell. Neurosci.

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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.

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Retinal organoids on-a-chip: a micro-millifluidic bioreactor for long-term organoid maintenance

Abstract: 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.

Cited by 39 publications

References 71 publications

Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats

Co-grafts of Human Embryonic Stem Cell Derived Retina Organoids and Retinal Pigment Epithelium for Retinal Reconstruction in Immunodeficient Retinal Degenerate Royal College of Surgeons Rats

Outer Retinal Cell Replacement: Putting the Pieces Together

Retinal Organoids Long-Term Functional Characterization Using Two-Photon Fluorescence Lifetime and Hyperspectral Microscopy

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