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Oksana Sirenko, Krishna Macha | Molecular Device

Introduction

The most common side effects of anti-cancer drugs is their toxic effect to intestinal cells which often limits the dose that can be administered to treat patients. In vitro assay using three-dimensional (3D) organoids can evaluate toxicity effects to the intestine and provide essential information in the process of drug development. Assay automation will greatly increase productivity and scale of these models, as well as the accuracy of assays that involve complex 3D biology.

Organoids are 3D multicellular tissue constructs that originate from human pluripotent stem cells (iPSCs) or adult stem cells.^1,2 They can recreate the physiological structure and function of human organs through selfassembly in matrix. Studies show that patients and their derived organoids respond similarly to drugs, suggesting the therapeutic value of using organoids to improve therapeutic outcomes. However, challenges commonly associated with using organoids, such as assay reproducibility, ability to scale up, and cost have limited their widespread adoption as a primary screening method.

The new CellXpress.ai™ Automated Cell Culture System can overcome these limitations. The system can automate the entire cell culture process, providing total control over media exchanges, compound additions, imaging, and passaging processes to reduce hands-on lab time while maintaining 24/7 operation for cell culture and scaling up various cell models, cell lines, iPSCs, spheroids, or organoids.

The system contains four essential components for cell culture workflow: liquid handling, incubation, imaging, and a unified software environment to manage complex protocols, scheduling, and image-based analysis. The software is one of the most powerful elements of the CellXpress.ai system. Not only does it manage scheduling and analysis, it also uses AI and machine learning to determine the optimal time for feeding and passaging events.

Here, we describe how to automate the compound testing assay for toxicity using 3D mouse intestinal organoids cultured in Matrigel domes.

Consumables and reagents

• Ibidi μ-Plate 96 Well Square, Cat No: 89626, IBIDI GmbH, Germany
• Corning 96-well plates, Corning, NY, USA
• Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, 10 mL, Cat # 356231, Corning, NY, USA.
• Gentle Cell Dissociation Reagent, enzyme-free cell dissociation reagent, #07174, STEMCELL Technologies Vancouver, Canada
• IntestiCult Organoid Growth Medium (Mouse), Catalog #06005, STEMCELL Technologies Vancouver, Canada
• Mouse Intestinal Organoids, cryopreserved, Catalog #70931, STEMCELL Technologies Vancouver, Canada
• Compounds: Cisapride, etoposide, tamoxifen, staurosporine, doxorubicin, Imatinib, all from Tocris, Minnesota, USA
• Hoechst 33342, trihydrochloride, trihydrate (Cat.No.H1399)
• Calcein AM, Thermo Fisher Scientific, USA
• Phalloidin, Thermo Fisher Scientific, USA
• EthD III, Thermo Fisher Scientific, USA

Methods

Assay automation

CellXpress.ai Automated Cell Culture System (Figure 1) was used to automate all processes involved in plating and culturing organoids, treatments, measurements, and data analysis. Automation included processing of organoid expansion, seeding into 96-well plates, culture and monitoring, media exchanges, compound addition, staining with viability markers, imaging, and image analysis.

Organoid culture

Intestinal organoids were cultured, passaged, and expanded in Matrigel domes in 24 wells using the CellXpress.ai system. During organoid culture, automated media exchanges and monitoring in transmitted light by imaging was done every 24h. Organoids self-organized and developed complex crypt structures and expected intestinal organoid phenotypes. After 4–5 days, organoids were automatically passaged: collected, purified from Matrigel and dispersed, then mixed with fresh Matrigel and re-plated.

Experimental setup and compound additions

For the endpoint assay, organoids were harvested from 24-well plates into deep-well 2 ml 96-well blocks (Hamilton) using 3D cell passaging steps described earlier (see application note: Automation of 3D intestinal organoids culture with CellXpress.ai Automated Cell Culture System), mixed with Matrigel to wield 50% Matrigel concentration,and then plated into the 96-well plate format (Ibidi plates). Importantly, during the seeding procedure, cells with Matrigel were kept cold at all times and mixed six times before the plating. 1 ml pipette tips were used for cell plating, 8 tips at a time (see Figure 2). We used consecutive dispensing of Matrigel domes by 1 ml tips (aspiration with one mix and dispensing into 6 domes), dispensing domes at 15 µl volume. Domes were left to solidify for 15 min on the deck, then media was added to wells, off center, to avoid breaking the domes. After that, plates were moved into the incubator and were continuously monitored every 12 hours for two days.

Compounds were then added on day 3 into the test plates from the pre-diluted compound plates. Organoid cultures were treated with several compounds known to cause toxicity to intestines. Compounds were applied in triplicate, in 7-point concentration dilution range for 72 hours. During those 72 hours, organoids were imaged every 24 hours. All compounds were used with 1/4 serial dilutions with 100 µM as the highest concentration; 10 µM was the highest concentration for Staurosporine.

Figure 1. CellXpress.ai Automated Imaging System and software.

Staining and imaging organoids

After 72 hours, half of the media volume was removed (150 µl) and 150 µl of the staining solution was added to the cultures at 2X concentration. Media removal and addition of dye mix was performed using “feeding” (media exchange) phase. Dye solution was placed into the 4-well deep-well reservoir positioned in the media area. Staining solution contained a mix of three dyes: Calcein AM 1 µM, Hoechst 1 µM, and EtHD 2 µM diluted in PBS (all dyes were from Thermofisher Scientific). After media addition, the “Incubation” phase held the process for one hour of incubation with the dyes. After incubation, the dye solution was removed and replaced with PBS using media exchange (“Feeding”) routine. Then organoids were imaged using three fluorescent channels DAPI, FITC, and TexasRed with 4X magnification. Each Matrigel dome was imaged using four 4X tiled sites. Z-stack of 10–15 images was taken, images 8 µm apart, and Best Focus projection images were used for analysis.

Image analysis

Image analysis was performed using built in IN Carta® Image Analysis Software. For image analysis, a custom module editor was created that defined organoids using DAPI channel (nuclear stain) and then measured numbers of organoids, average organoid area, average fluorescent intensities of organoids for FITC (Calcein AM), and Texas Red (EtHD). The ratio of average fluorescent intensity of organoids in FITC (live stain) and average fluorescent intensity of organoids in Texas Red (EtHD, stained dead cells) were calculated for all wells. Then, concentration dependencies of the ratios were used to estimate effective compound toxicity concentrations.

Results

The CellXpress.ai automated cell culture system was used to expand organoids and set up the end point assay in a 96-well format. Plating organoid domes was performed using the “Seeding” step. Figure 2A shows plating organoids mixed with Matrigel (50% Matrigel) from 96-deep well containers into the 96-well Ibidi plates. Figure 2B shows a screenshot of organoid domes in 96-well plate. Then, media exchanges and staining steps were done by using the “feeding” phases to enable media exchanges and to monitor organoids in transmitted light. After compound treatment for 72 hours, phenotypic effects were analyzed using fluorescent imaging after staining organoids with viability markers Calcein AM, EtHD, and nuclear marker Hoechst.

Image analysis determined the number of organoids in the dome, measured organoid size (diameter, area), and fluorescent intensities with different markers. Figure 3 shows a screenshot of 4X tiled images of organoids in Matrigel domes stained with Hoechst nuclear dye, and a graph representing average intensities of organoids in FITC and TexasRed channels. Increased cell death resulted in decreased Calcein AM signals and increased intensity staining with EtHD. Image analysis was done using IN Carta software. The custom module editor was created to find organoids using Hoechst stain, then organoid numbers, organoid areas, diameters, and average intensities for different colors, which were evaluated by analysis. The bar graph shows concentration-dependent decreases of the ratios of average intensities for live and dead stains (Calcein AM and EtHD respectively). Ratios of average fluorescent intensities for live and dead stains were used to demonstrate concentration-dependent compound effects indicating toxicity to intestinal organoids.

Figure 2. A. Seeding organoid domes in 96-well format with the CellXpress.ai system. Process was automated using 8 channels simultaneously. The mixture of organoids in Matrigel was dispensed into 96-well plates from the chilled 96-well deep-well reservoir. Reservoir was placed on chilled platform. B. Screenshot of 96-well plate shows images of organoid domes in transmitted light taken using 4X magnification. C. Image of the organoid dome in transmitted light.

Figure 4A shows composite tiled images, best focus projections of fluorescent images of control well and well treated with 10 µM of doxorubicin. The bar graph (Figure 4B) shows decrease of average fluorescent intensities ratios of live/dead stains. Data shows that doxorubicin causes the greatest toxicity effect, while etoposide and imatinib caused toxicity only in the highest concentrations tested.

Figure 3. A screenshot of 4X tiled images of organoids in Matrigel domes stained with Hoechst nuclear dye (blue) and a graph representing average intensities of organoids. Note decrease of nuclear staining observed in samples treated with staurosporine and doxorubicin (shown with arrows).

Figure 4. Intestinal organoids were expanded by the CellXpress.ai system and placed into the multi-well plate format. Organoids were treated with anticancer drugs with seven concentrations and then intestinal toxicity was evaluated by imaging. A. Composite Best Focus projection images of organoids, 4X magnification, four sites tiled. B. Image analysis was done using IN Carta analysis software. A custom module editor was created to find organoids using Noechst stain, then organoid numbers, organoid areas, diameters, and average intensities for different colors evaluated by analysis. The bar graph shows concentration-dependent decreases of average intensities ratios for live and dead stains (Calcein AM and EtHD respectively).

Conclusion

This method shows an automated protocol for compound testing using intestinal organoids in a 96-well format. This method is suitable for toxicity assessment studies and can greatly reduce manual processing steps. Automating the cell culture process when powered by imaging and culture control has great potential to increase productivity and throughput.

References

1. Kim J, Koo BK, KnoblichJA. Human organoids: model systems for human biology and medicine. Nat Rev Mol Cell Biol. 2020; 21:571–584.
2. Sato T, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 2009 459(7244):262–5.

*HUB Organoid Technology used herein was used under license from HUB Organoids. To use HUB Organoid Technology for commercial purposes, please contact bd@huborganoids.nl for a commercial use license.

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