Application Note
Assessment of photoreceptor functional activity in retinal organoids using calcium imaging
- Generate real-time functional evaluation of retinal organoids.
- Get deeper understanding of cellular function by mapping calcium oscillations across different organoid regions.
- Versatile application can be used for disease modeling and therapeutic high-throughput screening other models, including neural, cardiac, and sensory organoids.
Prathyushakrishna Macha, Oksana Sirenko | Molecular Devices
Hardeep Singh, Yi Ting Lai, Nalinda Wasala, Alexandra Collin de l’Hortet | EpiCrispr Biotechnologies, Inc.
Introduction
Three-dimensional (3D) retinal organoids from humaninduced pluripotent stem cells (hiPSCs) have the potential to help scientists better understand and model retinal diseases. These models mimic the retina’s spatial and temporal differentiation and form laminar structures with cell types found in the retina. These structures recapitulate the different retina cell types, including light-sensing photoreceptors that are usually found in the outer region of the organoids, retinal pigment epithelial (RPE) cells— usually dark, and ganglion cells.
In this study, retinal organoids were generated using wild-type (WT) or Rhodopsin gene heterozygous mutation containing iPSCs. The organoids were cultured for 150–200 days in a culture medium supplemented with growth factors promoting retina-like tissue development. The development of various retinal cell types was verified through visual inspection, noting the emergence of distinct structures, a phase-bright layer at the outer margins, and immunofluorescence analysis using markers specific to each cell type.
Photoreceptor functionality was tested by imaging and analyzing calcium fluctuations using an automated confocal imaging system. We developed a method to analyze spikes of calcium activity upon excitation with blue light (405/20nm). Calcium oscillations in various regions of the retinal organoids’ outer segments/photoreceptor layers were recorded as a time series and analyzed.
The region-specific calcium oscillation pattern was analyzed for metrics, including standard deviation of fluorescence intensity, peak count, and amplitude over time. Distinct differences in pattern and a substantial increase in intensity standard deviation were observed between the baseline and excited in the wild-type models. While within the group, the calcium kinetics and patterns were highly consistent but we did not discover patterns in disease models. This new method for functional characterization of calcium oscillations in human iPSCderived retinal organoids using high-content imaging and analysis is suitable for high-throughput assays and demonstrates a promising method for comparing various types of organoids with asymmetric/regional activity.
Methods
Generation and culture of retinal organoids
Retinal organoids were generated from wild-type (WT) hiPSCs using the STEMdiff™ Retinal Organoid Kit. The hiPSCs were cultured in a medium supplemented with growth factors to promote retinal tissue development. The iPSCs were seeded and organoids were generated via checkerboard scraping and cultured for a period of 150–200 days with medium changes every 2–3 days. Over this period, the organoids developed distinct laminar structures, including the formation of photoreceptors in the outer regions, RPE cells, and ganglion cells.
Retinal cell type verification
The development of retinal cell types was verified through visual inspection, focusing on the appearance of distinct retinal structures, such as a phase-bright layer at the outer margins. Immunofluorescence staining was performed to validate the presence of retinal cell types. Specific markers for rod photoreceptors, bipolar cells, and ganglion cells were used to confirm the differentiation of each retinal cell type. Image acquisition and analysis was conducted using the ImageXpress® Micro Confocal system and the MetaXpress® High-Content Image Acquisition and Analysis Software for the quantification of immunostaining and cell type-specific markers and to ensure consistency across samples. This high-throughput analysis enables scientists to get more detailed analysis of organoid morphology and better identification of differentiated cell types within the organoids.
Calcium imaging and functional assays
Calcium functional activity in retinal organoids was assessed by recording calcium spikes and oscillations in the photoreceptor layer, which serves as an indicator of cellular responses to light stimulation. These calcium dynamics were analyzed based on metrics such as peak count, amplitude, and fluorescence intensity variation. Before imaging, organoids were treated for 2 hours with a 50% media and 50% Calcium 6 dye solution, diluted to a final concentration of 5 µM, using the Calcium 6 Dye Kit (Molecular Devices). To prevent premature activation, the organoids were kept in the dark during incubation. After treatment, they were exposed to blue light excitation (405/20nm) to induce calcium fluctuations. Calcium imaging was then performed using an automated confocal imaging system to capture time-series data of the calcium activity and to precisely quantify calcium flux dynamics. This method allowed for high-throughput analysis and comparison of baseline and excited states, providing detailed insights into the organoids’ functional responsiveness to light stimulation.
Human iPSC-derived retinal organoids were generated using STEMdiff retinal organoid kit (Alpha testing). Retinal organoids cultured up to D150 started showing outer segments from rod photoreceptors. Mature retinal organoids with abundant outer segments around D200 in culture were used.
Figure 1. Schematic diagram of the retinal organoids’ growth and differentiation
Acquisition settings for optogenetics
In this optogenetics experiment, a multi-wavelength highcontent imaging system was used to stimulate and record activity in organoids. The experiment involved selective excitation using DAPI (405/20nm) and subsequent recording of the photoreceptor response using FITC (520/28nm).
For optogenetic stimulation, the DAPI channel was used for excitation with an illumination power of 25% and an exposure time of 30,000 ms. Autofocus was configured using a laser with a Z-offset of -16.02 µm. A Z-stack was implemented with a step size of 2.47 µm over a range of 61.73 µm, ensuring accurate depth capture. Timelapse acquisition was set to record images only at the start of the experiment to capture baseline responses to excitation.
For response recording, the FITC channel was used for emission detection, with an exposure time of 100 ms. The illumination power was set to 50% to ensure clear fluorescence detection. Autofocus was adjusted using a Z-offset from W1 (DAPI) with a 0 µm offset. Z-stacking was maintained with the same step and range settings as in the DAPI channel. Recording was configured to capture images at all time points, allowing continuous monitoring of fluorescence changes.
These settings allowed precise tracking of optogenetic activation and subsequent fluorescence response, facilitating robust analysis of neural activity in organoids.
Results
Retinal organoids replicate key features of the human retina, particularly the photoreceptor layer, which consists of rods and cones that form organized outer segments for light detection. These photoreceptors extend toward the apical side, where they are involved in capturing light and initiating visual signaling. However, not all retinal organoids develop a distinct RPE layer, and its presence can be variable, especially in more mature organoids. Despite the absence of a well-defined RPE in some organoids, the photoreceptors can still exhibit functional activity.
The immunofluorescence (IF) staining highlights the layered architecture of the retinal organoids and identifies key retinal cell types, such as rod photoreceptors and bipolar cells, which are characteristic of the human retina.
These results show the development and organization of retinal cell types within the organoid, resembling the structure of the human retina.
Post-acquisition, we used MetaXpress software to select specific regions within the photoreceptor layer to analyze activity. By measuring time vs. intensity standard deviation, it quantifies the variability in photoreceptor response. This data can be plotted to compare activity across different regions and provides insight into photoreceptor function over time.
Figure 2. The figure presents separate images of a retinal organoid using different staining techniques. DAPI (blue) stains the nuclei, Phalloidin (green) highlights actin filaments, and transmitted light (TL) shows the overall structure. The outer segments of photoreceptors, visible in the TL image, display hair-like structures, indicating maturation.
Figure 3. A and B, hiPSC-derived retinal organoid at day 200 with an abundant outer segment from photoreceptors (TL microscopy image). C. This image was obtained from a wild-type hiPSC-derived retinal organoid at Day 222, after cryosectioning and IF staining. The staining reveals: Blue indicates the nuclei of the cells, Green marks rhodopsin in the outer segments of rod photoreceptors, Red labels PKCα, a specific marker for bipolar cells.
Figure 4. MetaXpress software settings for region selection and time vs. intensity standard deviation plots of photoreceptor activity.
Figure 5. A 4x cross-section image of a retinal organoid captured using the ImageXpress Micro Confocal system (left), followed by 20x magnification images of the same region. Time-series images taken over 100 seconds track changes in photoreceptor activity and reveal dynamic responses within the organoid’s structure.
Figure 6. The 20x images highlight specific regions of the retinal organoid that show Calcium 6 fluorescence activity. Plots of time vs. standard deviation of intensity reveal the most significant intensity change (indicated by an arrow). Calcium 6 activity regulates cellular gates which control calcium influx and photoreceptor activation.
Analysis of organoids (excited and baseline activities)
Figure 7. Representative calcium-flux signal traces over time (intensity standard deviation) for optogenetics using blue light excitation (405/20nm). BR1–BR4 shows baseline readouts from regions 1 through 4 of the retinal organoids, while ER1-ER4 shows excited readouts from the same regions following blue light stimulation (405/20nm).
Analysis using organoids without baseline activity
Figure 8. A. The TL image of a representative wild-type retinal organoid was captured using a confocal microscope, highlighting the hairy photoreceptor regions selected for further analysis. B. Calcium 6 image of the retinal organoid in FITC (emission – 520/28nm), with four regions (R1, R2, R3, R4) selected post-acquisition. This type of analysis included two batches with three wild-type organoids per batch and four regions per organoid. C. Peaks per organoid across the two batches of wild-type organoids show the number of peaks observed in each region for every organoid. D. Plots of intensity standard deviation over time, recorded in the FITC channel (emission – 520/28nm), for the four regions (R1, R2, R3, R4) of the same organoid. These plots represent the activity of each region following optogenetic stimulation and capture the calcium flux dynamics over time.
Conclusion
We developed a high-content imaging method to assess photoreceptor functionality in hiPSC-derived retinal organoids by analyzing region-specific calcium oscillations in response to blue light stimulation (405/20nm). Using automated confocal imaging and high-content assay analysis, we quantified intensity fluctuations and revealed asymmetric activity across organoid regions. This approach enables the detection of functional differences between wild-type and disease-model organoids to provide a robust, high-throughput tool for retinal research. Our method offers a valuable framework for studying disease-related dysfunctions and to screen potential therapeutics, advancing the use of retinal organoids as models for understanding human retinal physiology and pathology. Additionally, this imaging and analysis workflow can be adapted to study asymmetric activity in other organoid systems, broadening its applicability to other complex tissue models.