Merging organoid and organ-on-a-chip technology to generate complex multi-layer tissue models in a human retina-on-a-chip platform

The RoC reproduces the retinopathic side-effects of chloroquine and gentamicin.

“In addition, we demonstrate the applicability of the RoC for drug testing, by reproducing the retinopathic side-effects of the anti-malaria drug chloroquine and the antibiotic gentamicin.”

Polarized expression of ezrin, an apical microvilli marker, was observed in RPE in the RoC.

“Further, we observed the polarized expression of ezrin, an apical microvilli marker (Kivelä et al., 2000) (Figure 4f).”

• cites Kivelä et al., 2000 sourceNot checked yet

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PNA lectin labels not only cone but also rod segments in ROs.

“Co-staining of rhodopsin with PNA lectin exhibited that not only cone but also rod segments in ROs are labeled with PNA lectin (Figure 2e).”

Day 180 ROs harbor matured photoreceptors with simple forms of inner and outer segments situated on the surface of the ROs.

“ROs of that age harbor matured photoreceptors which have simple forms of inner and outer segments, situated on the surface of the ROs, visible in bright field microscopy or by immunostaining of respective markers (Figure 2b–f).”

The interaction between mature photoreceptor segments and RPE in the RoC enhances the formation of outer segment-like structures.

“We show that this interaction enhances the formation of outer segment-like structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics.”

Subsequent injection of a colorless liquid demonstrated a washout in the same time frame.

“Subsequent injection of a colorless liquid again demonstrated a washout in the same time frame (see Video 1 and 2).”

Direct contact and uncontrolled outgrowth of cells from the ROs during culture was successfully avoided.

“Thereby, a direct contact and, thus, an uncontrolled outgrowth of cells from the ROs during culture was successfully avoided.”

ROs derived from hiPSCs harbor all known major retinal subtypes such as ganglion cells, bipolar cells, horizontal cells, amacrine cells, Müller glia and photoreceptors.

“ROs derived from hiPSCs harbor all known major retinal subtypes such as ganglion cells, bipolar cells, horizontal cells, amacrine cells, Müller glia and photoreceptors (Zhong et al., 2014; Figure 2—figure supplement 1).”

• cites Zhong et al., 2014 sourceNot checked yet

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The hydrogel layer generated a defined distance between RPE cells and the outer limiting membrane of the ROs.

“This led to the formation of a thin hydrogel layer, generating a defined distance between RPE cells and the outer limiting membrane of the ROs.”

• AssumptionRests on an assumption: The hyaluronic acid-based hydrogel represents the major component of the interphotoreceptor matrix between RPE and PRC.

Day 180 ROs contain a mixed population of rods and cones.

“Immunostaining of the respective organoids demonstrates the presence of a mixed population of rods and cones, identified by specific markers (rhodopsin for rods and Arrestin-3 for cones, Figure 2c–e, Figure 2—figure supplement 1).”

The RoC integrates more than seven different essential retinal cell types derived from hiPSCs.

“In this study, we present the retina-on-a-chip (RoC), a novel microphysiological model of the human retina integrating more than seven different essential retinal cell types derived from hiPSCs.”

Nutrients, compounds, and further dissolved molecules are transported to the tissue chamber via diffusion.

“At the same time, nutrients, compounds, and further dissolved molecules are transported to the tissue chamber via diffusion.”

The interaction between mature photoreceptor segments and RPE in the RoC establishes in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics.

“We show that this interaction enhances the formation of outer segment-like structures and the establishment of in vivo-like physiological processes such as outer segment phagocytosis and calcium dynamics.”

The RoC provides vasculature-like perfusion.

“It provides vasculature-like perfusion and enables, for the first time, the recapitulation of the interaction of mature photoreceptor segments with RPE in vitro.”

Melanoma gp100 is highly expressed in chip-cultured RPE, indicating strong pigmentation.

“The melanosome and pigmentation marker Melanoma gp100 (also called PMEL17 or Silver locus protein) (Berson et al., 2001; Theos et al., 2005) was highly expressed in chip-cultured RPE (Figure 4d) indicating a strong pigmentation.”

• cites Berson et al., 2001 sourceNot checked yet

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• cites Theos et al., 2005 sourceNot checked yet

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After 24 hr, a second peak of VEGF-A was reached.

“Finally, after 24 hr a second peak was reached.”

RPE in the RoC expresses RPE markers PAX6 and MITF after 7-14 days in the chip.

“Expression of RPE markers PAX6 and MITF can be observed after 7–14 days in the chip (Figure 4a–b).”

After the initial peak, VEGF-A levels decreased over time resting above baseline level.

“After this initial peak, the VEGF-A levels decreased over time resting above the baseline level.”

Due to fluidic resistance of the porous membrane, convective fluid flow is confined to the media channels.

“Due to the fluidic resistance of the porous membrane, the convective fluid flow is confined to the media channels (Figure 5a).”

Controlled culture conditions enabled a stable culture of the RoC for at least 21 days.

“The controlled culture conditions enabled a stable culture of the RoC for at least 21 days (Figure 3—figure supplement 1).”

A proof of concept experiment showed complete distribution of a dye within 300 s at a flow rate of 20 μl/hr.

“In order to verify this, we conducted a proof of concept experiment during which we switched from a colorless liquid to a colored one and observed a complete distribution of the dye within 300 s (flow rate of 20 µl/hr) (Figure 5—figure supplement 1).”

The RoC is a microfluidic platform that enables culture of hiPSC-derived RPE and ROs in a defined physiological structure.

“To recapitulate the complex in vivo anatomy of the human retina in vitro (Figure 3a), we developed a microfluidic platform that enables the culture of hiPSC-derived RPE and ROs in a defined physiological structure (Figure 2b).”

• AssumptionRests on an assumption: No MPS has successfully recapitulated the complex 3D architecture of the human retina.
• AssumptionRests on an assumption: The porous membrane mimics the endothelial barrier.

VEGF-A concentration was higher in the basal channel than in the apical channel (70 vs 40 pg per chip in 24 hr).

“The VEGF-A concentration was higher in the basal channel than in the apical (70 vs 40 pg per chip in 24 hr).”

After 2 hr of TGF-β1 stimulation, VEGF-A levels in effluent medium increased 2-fold relative to baseline at 0 hr.

“Already after 2 hr, we could observe a 2-fold increase of VEGF-A levels in the effluent medium relative to the baseline level at 0 hr.”

Many of the lectin-positive particles found in RPE cells were positive for rhodopsin.

“This revealed that many of the lectin-positive particles found in the RPE cells were positive for rhodopsin.”

The distance between lectin-marked segment tips and GFP-labeled RPE was approximately 5 ± 3.19 μm over different experiments.

“By measuring the distance between lectin-marked segment tips and GFP-labeled RPE, we found a distance of approximately 5 ± 3.19 µm over different experiments (Figure 6c).”

The distance of outer segment tips and RPE microvilli in an exemplary image is around 5 μm, in accordance with the data above.

“The distance of outer segment tips and RPE microvilli in this exemplary image is around 5 µm (Figure 6e), which is in accordance with the data shown above.”

The RoC contains a complex multi-layer structure that includes all cell types and layers present in neuroretinal ROs, connected to an RPE layer.

“This combination of interdisciplinary approaches enabled us to successfully create a complex multi-layer structure that includes all cell types and layers present in the neuroretinal ROs, connected to an RPE layer.”

In the RoC, numerous outer segments facing towards the RPE were formed.

“In the RoC, hence, numerous outer segments facing towards the RPE were formed.”

The increase in LAMP2 after 80 μg/ml CQ was not limited to the RPE but was also very pronounced in the RO.

“The increase in LAMP2 was not limited to the RPE but was also very pronounced in the RO (Figure 8c).”

The general capability of hiPSC-RPE to perform phagocytosis was verified using bovine retinal outer segments.

“The general capability of the hiPSC-RPE to perform phagocytosis was initially verified using bovine retinal outer segments (Figure 7—figure supplement 2).”

All integrated cell types in the RoC were derived from the same hiPSCs.

“All integrated cell types were thereby derived from the same hiPSCs.”

After exposure to 80 μg/ml CQ, a strong LAMP2 signal was visible.

“Furthermore, after exposure to 80 µg/ml CQ, a strong LAMP2 signal was visible (Figure 8c)”

Specific co-localization of GFP-labeled early endosomes with rhodopsin-positive fragments was detected in RPE cells in the RoC.

“After on-chip immunostaining of rhodopsin, specific co-localization of GFP-labeled early endosomes with rhodopsin-positive fragments was detected (Figure 7c).”

Already after 1 day in RoC culture, PNA lectin-positive structures were found within RPE cell bodies, indicating ongoing digestion of segment particles.

“Already after 1 day in culture, PNA lectin-positive structures were found within the RPE cell bodies, indicating ongoing digestion of segment particles (Figure 7a).”

In the RoC system, the number of outer segment structures was about three times higher than in RO chips without RPE and in dish cultured ROs.

“However, in the RoC system (RoC), the number of outer segment structures was about three times higher than in RO chips without RPE (RoC w/o RPE) and in dish cultured ROs (Figure 6h).”

The hydrogel-filled space between the RPE monolayer and the RO was strongly invaded by rhodopsin and ROM1-positive clusters, indicating an increased and cumulated presence of inner and outer segment-like structures.

“Further, the hydrogel-filled space between the RPE monolayer and the RO was strongly invaded by rhodopsin and ROM1 (outer segment marker)-positive clusters indicating an increased and cumulated presence of inner and outer segment-like structures (Figure 6d).”

There was no difference in outer segment number between the RoC chip without RPE and dish culture.

“Interestingly, there was no difference observed in the RoC chip without RPE and dish culture, indicating a positive effect of the RPE on outer segment formation and preservation.”

Immunostaining revealed that segment structures and RPE cells are in close apposition on-chip.

“Subsequently, immunostaining using rhodopsin (rod outer segments) and phalloidin (cytoskeleton of the RPE and the RO including tight junctions) revealed that the segment structures and RPE cells are in close apposition on-chip (Figure 6b).”

By loading RO and RPE on-chip with fura-2-AM, calcium dynamics for individual photoreceptors or RPE cell ROIs could be imaged over an extended timespan.

“By loading RO and RPE on-chip with the calcium dye fura-2-AM, we were able to image calcium dynamics for individual photoreceptors or RPE cell ROIs over an extended timespan (Figure 7—figure supplement 1).”

In the RPE below the RO, membrane stack-structures displayed multi-membrane formation and small round membrane structures, reminiscent of outer segment-like structures in ROs.

“Here, we found several membrane stack-structures in the RPE below the RO. These structures displayed multi-membrane formation (red arrow) as well as small round membrane structures, both strongly reminiscent of similar features found in outer segment-like structures in ROs (Figure 7d, right).”

Outer segment-like structures were found in all conditions (RoC, RoC without RPE, dish-cultured ROs), displaying distinct disk formation.

“Using electron microscopy, we could find outer segment-like structures in all conditions (Figure 6f), displaying distinct disk formation (Figure 6g, exemplarily).”

Exposure of RoCs to 80 μg/ml CQ for 3 days significantly increased PI staining compared to controls without CQ, indicating clearly impacted cell viability.

“However, at a concentration of 80 µg/ml, cell viability was clearly impacted as shown by a significantly stronger PI staining (Figure 8a,b) compared to controls without CQ treatment.”

In day 7 RoCs, outer segment-like structures were found in the RPE.

“We examined day 7 RoCs, identifying indigested outer segment-like structures in the RPE (Figure 7d).”

ROs contain most relevant retinal cell types in a physiological layering such as ganglion cells, amacrine cells, horizontal cells, bipolar cells, Müller glia as well as rods and cones.

“These ROs contain most relevant retinal cell types in a physiological layering such as ganglion cells, amacrine cells, horizontal cells, bipolar cells, Müller glia as well as rods and cones.”

Animal models used to explore new therapeutic options and assess retinal toxicities fail to reflect fundamental characteristics of human vision including trichromacy and a fovea centralis.

“Although animal models that are used to explore new therapeutic options and assess retinal toxicities resemble the human (patho-)physiology of vision in certain aspects, they fail to reflect fundamental characteristics including trichromacy or a fovea centralis, responsible for high visual acuity (Figure 1).”

Due to lack of physiological perfusion, delivery of compounds to ROs is uncontrolled and entirely artificial.

“Due to the lack of a physiological perfusion, the delivery of compounds to ROs is uncontrolled and entirely artificial.”

Lysosomal dysfunction is involved in the pathophysiology of chloroquine.

“since lysosomal dysfunction is involved in the pathophysiology of CQ (Chen et al., 2011; Mahon et al., 2004; Rosenthal et al., 1978).”

• cites Chen et al., 2011 sourceNot checked yet

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• cites Mahon et al., 2004 sourceNot checked yet

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• cites Rosenthal et al., 1978Not checked yet

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Chloroquine and gentamicin have previously been shown to have pathological side effects on the retina.

“the anti-malaria drug chloroquine (CQ) and the antibiotic gentamicin (GM), which both were previously shown to have pathological side effects on the retina (Elman et al., 1976; Ding et al., 2016; Yusuf et al., 2017; Zemel et al., 1995; McDonald et al., 1986).”

• cites Elman et al., 1976 sourceNot checked yet

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• cites Ding et al., 2016 sourceNot checked yet

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• cites Yusuf et al., 2017 sourceNot checked yet

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The RPE has a positive effect on outer segment formation and preservation.

“Interestingly, there was no difference observed in the RoC chip without RPE and dish culture, indicating a positive effect of the RPE on outer segment formation and preservation.”

The RPE may have a barrier or protective function, shielding the organoid from gentamicin and decreasing the toxic effects of the drug on the organoid.

“This could indicate a barrier or even protective function of the RPE, shielding the organoid from the drug and decreasing the toxic effects of the drug on the organoid.”

The human RoC system provides a physiologically relevant model system that recapitulates key functionalities of the human retina, which are impaired in patients suffering from retinal degeneration.

“By combining hiPSC-ROs and -RPE cells in a microphysiological environment, the introduced human RoC system provides a physiologically relevant model system that recapitulates key functionalities of the human retina, which are impaired in patients suffering from retinal degeneration.”

The RoC is a retina model that successfully recapitulates the interaction between photoreceptors and RPE in vitro.

“For the first time, we demonstrate a retina model successfully recapitulating the precisely orchestrated interaction between photoreceptors and RPE in vitro.”

The interphotoreceptor matrix in the RoC provides mechanical stability for developing photoreceptor outer segments, which would otherwise be damaged or prevented from outgrowth in conventional suspension cultures.

“This interphotoreceptor matrix in the RoC provides mechanical stability for the small and fragile developing POS, which would otherwise either be damaged or prevented from outgrowth as in conventional suspension cultures.”

• AssumptionRests on an assumption: Attempts in recreating the retinal niche in the past have failed to recapitulate the precise RPE-PR arrangement and did not yield matured photoreceptors with large formations of membrane disk-containing outer segments.

The RoC arrangement is more physiological and controlled than previous approaches that employed unpredictable and unorganized RPE formation during RO generation.

“This arrangement is much more physiological and controlled than previous approaches employing an unpredictable and unorganized RPE formation during RO generation.”

• AssumptionRests on an assumption: Traditional monolayer cell culture assays have been of limited value in retinal research because they solely include certain cell subtypes, providing restricted physiological relevance.
• AssumptionRests on an assumption: Attempts in recreating the retinal niche in the past have failed to recapitulate the precise RPE-PR arrangement and did not yield matured photoreceptors with large formations of membrane disk-containing outer segments.