Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

J. Thomas Gebert; Francesca J. Scribano; Kristen A. Engevik; Joseph M. Hyser

doi:10.3791/66132

JoVE Journal > Immunology and Infection

Immunology and Infection

Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators

Published: January 19, 2024

doi:

10.3791/66132

J. Thomas Gebert¹, Francesca J. Scribano¹, Kristen A. Engevik¹, Joseph M. Hyser¹

¹Alkek Center for Metagenomics and Microbiome Research, Department of Molecular Virology and Microbiology,Baylor College of Medicine

Summary

This protocol describes an approach for performing calcium imaging in virus-infected human intestinal organoids and offers an approach to analysis.

Abstract

Calcium signaling is an integral regulator of nearly every tissue. Within the intestinal epithelium, calcium is involved in the regulation of secretory activity, actin dynamics, inflammatory responses, stem cell proliferation, and many other uncharacterized cellular functions. As such, mapping calcium signaling dynamics within the intestinal epithelium can provide insight into homeostatic cellular processes and unveil unique responses to various stimuli. Human intestinal organoids (HIOs) are a high-throughput, human-derived model to study the intestinal epithelium and thus represent a useful system to investigate calcium dynamics. This paper describes a protocol to stably transduce HIOs with genetically encoded calcium indicators (GECIs), perform live fluorescence microscopy, and analyze imaging data to meaningfully characterize calcium signals. As a representative example, 3-dimensional HIOs were transduced with lentivirus to stably express GCaMP6s, a green fluorescent protein-based cytosolic GECI. The engineered HIOs were then dispersed into a single-cell suspension and seeded as monolayers. After differentiation, the HIO monolayers were infected with rotavirus and/or treated with drugs known to stimulate a calcium response. An epifluorescence microscope fitted with a temperature-controlled, humidified live-imaging chamber allowed for long-term imaging of infected or drug-treated monolayers. Following imaging, acquired images were analyzed using the freely available analysis software, ImageJ. Overall, this work establishes an adaptable pipeline for characterizing cellular signaling in HIOs.

Introduction

Calcium is a widely conserved second messenger that plays a critical role in regulating cellular physiology¹. Given its strong charge, small size, and high solubility in physiological conditions, calcium is an ideal manipulator of protein conformation. This makes calcium a powerful means to transduce electrochemical signals into enzymatic, transcriptional, or post-transcriptional alterations. The strict calcium concentration gradients across the endoplasmic reticulum (ER) and plasma membranes create a high driving force that allows for rapid changes in cytosolic calcium concentration. Multiple mechanisms, including both buffering and active transport, tightly maintain this gradient. While necessary for normal cellular functions, this maintenance is energetically expensive, making it particularly susceptible in states of stress ².

As such, dysregulation of calcium within the cytosol is a near-universal signal of many kinds of cellular stress. Metabolic disturbances, toxins, pathogens, mechanical damage, and genetic perturbations can all disrupt calcium signaling. Regardless of the stimulus, on a whole-cell level, sustained, uncontrolled rises in cytosolic calcium can promote apoptosis and eventually necrosis³^,⁴. Alterations in cytosolic calcium levels of lower amplitude or higher frequency, however, have varying effects². Likewise, the outcomes of calcium fluctuations may differ based on the spatial microdomain in which they occur⁵. Monitoring calcium levels can therefore offer insight into dynamic signaling processes, but this requires sampling with relatively high temporal and spatial resolution.

Genetically encoded calcium indicators (GECIs) are powerful tools for continuous sampling in live-cell systems⁶. Some of the most widely used GECIs are GFP-based calcium-responsive fluorescent proteins known as GCaMPs⁷. The canonical GCaMP is a fusion of three distinct protein domains: a circularly permuted GFP (cpGFP), calmodulin, and M13⁶. The calmodulin domain undergoes a conformation change upon binding calcium, allowing its interaction with M13. The calmodulin-M13 interaction induces a conformational change in the cpGFP that increases its fluorescent emission upon excitation. As such, an increase in calcium concentration correlates with an increase in GCaMP fluorescence intensity. These sensors can be cytosolic or targeted to specific organelles⁸.

Similar to most tissues, calcium regulates a variety of functions within the gastrointestinal epithelium. The intestinal epithelium is integral for nutrient and fluid absorption but also must form a tight barrier and immune interface to avoid pathogen invasion or toxic insults. Calcium-dependent pathways influence nearly all of these vital functions⁹^,¹⁰^,¹¹. However, calcium signaling within the intestinal epithelium remains an underexplored frontier with promising potential as a therapeutic target. While monitoring calcium dynamics within the intestinal epithelium in vivo continues to present challenges, human intestinal organoids (HIOs) offer an adaptable ex vivo system for experimentation¹². HIOs are 3-dimensional (3D) spheroids derived from human intestinal stem cells and, upon differentiation, recapitulate much of the cellular diversity of the native intestinal epithelium¹².

This protocol describes comprehensive methods to engineer HIOs that express GECIs and then prepare engineered HIOs as monolayers for live-cell calcium imaging. It offers viral infection as an example of a pathologic manipulation that disrupts calcium signaling and provides an analytic approach to quantify these changes.

Protocol

All of the human intestinal organoids (HIOs) used in this protocol and the representative experiments were derived from human tissue obtained and maintained by the Texas Medical Center Digestive Diseases Enteroid Core. All samples were collected in accordance with a protocol approved by the Institutional Review Board at Baylor College of Medicine.

1. Preparation of materials and reagents

For organoid maintenance, gather cell-culture treated 24-well plates, basement membrane matrix (BMM), 15 mL conical tubes, and 1.5 mL conical tubes.
1. To prepare complete media without growth factors (CMGF-), to 500 mL of advanced DMEM F12 add 5 mL of 1M HEPES, 5 mL of 100x antibiotic-antimycotic, 5 mL of 100x glutamine supplement.
2. To prepare Wnt-, R-spondin-, and Noggin-containing (WRNE) media, mix equal parts CMGF- and Wnt-conditioned media, add Noggin-conditioned medium, 10% by volume, R-spondin conditioned medium, 20% by volume, 50 ng/mL human epidermal growth factor, 10 mM nicotinamide, 10 nM [Leu15]-Gastrin I, 500 nM A-83-01, 10 µM SB202190, 1x B27 supplement, 1x N2 supplement, and 1 mM N-acetylcysteine.
For lentiviral transduction, prepare Fetal Bovine Serum (FBS), 0.05% Trypsin-EDTA in 1x phosphate buffered saline (PBS), CMGF- with 10% FBS, Sterile 1x PBS, Polybrene, 10 µM Y-27632, Lentivirus, High Wnt WRNE + 10 µM Y-27632
For generating organoid monolayers, prepare glass bottom 10-well cell culture slide, FBS, Collagen IV (1 mg/mL in de-ionized (di)H₂O), basement membrane matrix, 0.5 mM EDTA in 1x PBS, 5 mM EDTA in 1x PBS, enzymatic dissociation buffer, CMGF- with 10% FBS, WRNE + 10 µM Y-27632.
For viral infection of organoid monolayers, prepare trypsin, rotavirus stock, CMGF-, 25G needle, Sterile 1x PBS, and phenol red-free differentiation media.
1. To prepare phenol red-free differentiation media, take 500 mL of phenol red-free cell culture media, add 5 mL of 100x MEM non-essential amino acids, 5 mL of 100x L-Glutamine, 5 mL of 100 nM sodium pyruvate, and 7.5 mL of 1M HEPES.
For immunofluorescence staining of organoids, prepare 4% formaldehyde (16% formaldehyde diluted in 1x PBS), Triton X-100 (0.1% Triton X-100 in 1x PBS), Bovine serum albumin (3% bovine serum albumin in 1x PBS), NH₄Cl solution (50 mM), DAPI (1 µg/mL DAPI solution in 1x PBS).

2. Engineering organoids to express genetically encoded calcium sensors

NOTE: This protocol describes the steps to transduce a single well of 3-dimensional human intestinal organoids plated in 30 μL of Basement Membrane Matrix (BMM) on a 24-well plate¹³. Most lines will contain around 400,000 cells per well. A second, non-transduced well should be included as a control. Keep all reagents and cell suspensions on ice.

After 2-5 days from the last passage, remove maintenance WRNE medium from two wells of HIOs. Replace with 300 μL of 0.05% Trypsin-EDTA per well and gently pipette up and down 5x to detach the BMM from the plate. Place in a 37 °C incubator for 4 min.
Add 500 μL of CMGF- + 10% FBS per well. Pre-coat a 1 mL low-bind pipette tip with FBS by pipetting 1 mL of FBS up and down 2x. FBS can be re-used on multiple tips. With the pre-coated tip, pipette the HIOs up and down 10x.
Transfer the contents of each well to its own pre-coated 1.5 mL microcentrifuge tube. Rinse each well with an additional 500 μL of CMGF- and add the wash to the respective tube.
Centrifuge the tubes at 100 x g in a swinging bucket centrifuge for 5 min at 4 °C. Remove supernatant and any residual BMM.
Resuspend in 1 mL of 1x PBS. Split each tube into two microcentrifuge tubes for 4 total tubes. Centrifuge the tubes at 100 x g for 5 min at 4 °C. Remove supernatant.
Resuspend each tube one additional time in 1x PBS and centrifuge at 100 x g for 5 min at 4 °C.
Prepare 400 µL of transduction medium and control medium (Table 1). Resuspend 2 tubes in 200 µL of the control medium and 2 tubes in 200 µL of the transduction medium.
Incubate in a 37 °C cell culture incubator for 24 h. Resuspend by pipetting up and down with a coated tip periodically (e.g., 2 h post transduction (hpt), 12 hpt, 18 hpt) to encourage uniform transduction.
After 24 h, centrifuge tubes at 100 x g for 5 min at 4 °C. Remove supernatant.
Resuspend pellet in 500 µL of 1x PBS to wash. Centrifuge at 100 x g for 5 min at 4 °C. Remove supernatant.
Using an ice-cold 200 µL pipette tip, resuspend each of the 4 pellets in 30 µL of BMM. Gently pipette up and down to disperse evenly.
Plate the contents of each tube into its own well on a 24-well plate. Incubate for 10 min at 37 °C to allow BMM to solidify before adding 500 μL of HighWnt WRNE + 10μM Y-27632.
Allow transduced HIOs to grow for 1 week, refreshing the media (HighWnt WRNE + 10 μM Y-27632) every other day. After 1 week, check for the expression of the fluorescent indicator by microscopy. If the signal is strong, begin drug selection. If the signal is weak, repeat the transduction as described above.
Once the line is established, verify the function of the fluorescence indicator via agonist treatment. 100nM ADP is a reliable agonist for GCaMP validation. Test 3D organoids for initial validation as described below.
1. After a passage, plate a well (or multiple) of organoids in BMM on a separate imaging-bottom plate. Plate the organoids at about 1/3 of the normal density to avoid excess overlap when imaging. Do not continue to passage these HIOs following agonist treatment, as it is difficult to ensure sterility.
2. Allow 2-3 days for the HIOs to recover post-passage. Before imaging, switch the media to phenol red-free differentiation media.
3. Using a fluorescent microscope, set up a 3 min run with images acquired every 5 s using 488 nm excitation and a FITC/GFP filter set. After 30 s of imaging, add 100 nM ADP or vehicle control. Continue imaging until the signal returns to near-baseline, ~2 min. A transient, ~2-fold increase in GCaMP fluorescence with ADP treatment indicates successful transduction and biosensor function. For more precise estimate of transduction efficiency, repeat agonist test with imaging using monolayers generated via the process described in part 3.

3. Preparation of HIO monolayers for live fluorescence imaging

Coat all wells of a 10-well imaging bottom chamber slide with Collagen IV. To do this, mix 34 μL of 1 mg/mL Collagen IV with 960 μL of sterile deionized water. Add 95 μL of diluted collagen IV solution to each well and incubate at 37 °C for 0.5-2 h.
Remove the WRNE maintenance medium from 4 wells of 3D HIOs. HIOs should be 5-7 days from their last passage.
NOTE: 1 10-well plate typically requires ~1.25 x 10⁶ cells. This typically requires 2-4 wells of 3D HIOs plated in 30 μL of BMM each, but will vary based on density.
Add 500 μL of 1x PBS + 0.5 mM EDTA per well. With a pre-coated 1 mL tip, pipette up and down gently to detach BMM from the plate. Transfer the suspension to a pre-coated 15 mL conical tube, combining like wells into the same tube.
Rinse each well with an additional 500 μL of PBS + 0.5 mM EDTA. Centrifuge at 300 x g for 5 min at 4 °C. Remove the supernatant and residual BMM.
Using a pre-coated tip, resuspend the remaining pellet in 3 mL of PBS + 5 mM EDTA (note that this is 10x more EDTA than the first wash).
Centrifuge at 300 x g for 5 min at 4 °C. Remove the supernatant and residual BMM. Resuspend the pellet in 2 mL of enzymatic dissociation buffer.
Incubate in 37 °C bead/water bath for 5 min. Add 3 mL of CMGF- + 10% FBS and pipette gently to mix.
Centrifuge at 300 x g for 5 min at 4 °C. Remove the supernatant. Add 1 mL of CMGF-.
Vigorously pipette up and down with a pre-coated tip 80x-100x to mechanically break HIOs into single cells. Centrifuge at 300 x g for 5 min at 4 °C. Remove supernatant.
Resuspend in 1 mL of WRNE + 10 µM Y-27632. Obtain a cell count for the 1 mL suspension.
Dilute the cell suspension with WRNE + 10µM Y-27632 to achieve a concentration of 1.25 x 10⁵ cells/100 μL (1.25 x 10⁶ cells/mL).
Using a 200 μL pipette, remove the collagen solution from the plate prepared in step 1, as the collagen has now settled to the bottom of the well. Avoid touching the bottom of the well with the pipette tip.
Using a pre-coated 200 μL pipette tip, add 100 μL of the cell solution from step 3.11 (1.25 x 10⁵ cells) per well.
Incubate for 24 h in a 37 °C cell culture incubator. After 24 h, remove the culture medium from all wells and replace it with 100 μL of differentiation medium per well.
NOTE: By this point, the cells should be adhering to the plate. Note that the monolayer likely will not be confluent or fully planar.
Place the slide back into the 37 °C cell culture incubator. Refresh the differentiation medium every 24 h until the monolayer is confluent. This usually requires 3-5 days from plating. After this point, the monolayers are ready for downstream applications.

4. Viral infection of HIO monolayers

Prepare the virus inoculum. If necessary, trypsin activate the virus stock. For rotavirus, add 10 µg/mL Worthington's Trypsin to the virus stocks and incubate at 37 °C for 1 h.
1. Dilute the activated virus stock using one of the two following methods.
  1. If aiming for the maximum number of infected cells using only one viral strain, mix 50 μL of the activated viral stock with 50 μL CMGF-.
  2. If comparing multiple strains, prepare the inoculums to achieve equivalent multiplicities of infection (MOIs). Use the following formula to determine the volume of virus stock per well necessary for the desired MOI. Add CMGF- to the volume of virus stock calculated to achieve a final volume of 100 μL per well.
    
    NOTE: A single HIO monolayer plated in a well with a diameter of 5.46 mm (96-well plate) contains about 200,000 cells. Because of the poor efficiency of infection in monolayers, a high MOI (100-1 million viral particles per cell) is preferred. The optimal MOI must be determined empirically.
2. Infect the monolayers by gently removing the media from the HIO monolayer using a 200 mL pipette. Replace with 100 mL of virus inoculum or mock inoculum.
  1. Since most rotavirus stocks are propagated in MA104 cells, use an uninfected MA104 cell lysate for the mock inoculum. Use a volume equivalent to the volume of virus stock used for the infected wells and add CMGF- for a final volume of 100 mL per well.
  2. For viruses that infect cells basolaterally, such as rotavirus¹⁴, use a 25G needle to score the monolayer. It is easiest to make a single score across the length of the monolayer, from the bottom to the top of the well. Gently press the needle into the monolayer at an angle with the bevel side up, opposite the direction of motion, and drag it across the length of the monolayer to create a scar (Figure 2A).
3. Incubate in a 37 °C incubator for 2 h. Remove the virus and mock inoculum. Wash monolayers once with 1x PBS.
4. Add 100 μL of phenol red-free differentiation media. Place slide in a 37 °C incubator until ready to image. The optimal imaging window will vary based on the kinetics of the viral infection. For rotavirus, begin imaging at 6-8 h post infection.

5. Ca²⁺ Imaging of infected monolayers

Pre-heat the stage-top incubator to 37 °C. Run humidified CO₂ at 0.02 L/min. Place slide with the infected monolayers into the incubator chamber and seal the lid.
Using brightfield (BF) illumination and a 20x objective, select the X and Y coordinates of the fields of view within the monolayer that will be imaged. It is best to select points with the scored region in view, as most infected cells will be near the scratch.
Optimize imaging parameters and acquire an image using 488 nm excitation. To minimize phototoxicity, set the light source to 50% power with a 50 ms exposure time. The appropriate acquisition parameters may vary and should be optimized by acquiring multiple acquisitions. Ensure no pixels are saturated. Adjust exposure time and light power as needed to ensure the entire dynamic range of the fluorescent calcium sensor can be detected.
If using other fluorophores (e.g., fluorescently tagged viruses), repeat optimization on these channels. Set up the imaging loop and acquire a 488 nm image at every selected X, Y coordinate in a 1 min loop. Image this loop continuously. When using a fluorescently tagged virus, acquire an image on the appropriate channel every 10^th loop (i.e., 1 image/10 min).
Collect images for ~18 h. If using viruses without fluorescent tags, fix monolayers and perform immunofluorescence staining to identify infected cells. Perform this after completing the imaging run as described below.
1. Remove imaging media and replace with 100 μL of 4% formaldehyde in 1x PBS. Incubate at room temperature for 30 min.
2. Remove fixative and quench with 100 μL of 50mM NH₄Cl for 10 min. Remove NH₄Cl. Permeabilize the monolayers with 100 μL of 0.1% Triton X-100 in 1x PBS for 1 h at room temperature or overnight at 4 °C.
3. Remove permeabilization buffer. Block with 100 μL of 3% bovine serum albumin in 1x PBS for 1 h.
4. Remove the blocking solution. Add primary antibodies diluted to the recommended/optimized concentration in 1x PBS. Add 100 μL of antibody solution per well.
5. Incubate overnight at 4 °C with gentle rocking. Remove primary antibodies. Wash 3x with 1x PBS.
6. Add fluorescent-conjugated secondary antibodies diluted to the recommended/optimized concentration in 1x PBS. Add 100 μL per well. Incubate for 2 h at room temperature.
  NOTE: FPBase¹⁵ is a great resource to help select secondaries that will avoid bleed-through when multiplexing. Most secondaries are effective at a 1:1000 dilution in 1x PBS.
7. Remove secondary antibodies. Make a 1 µg/mL DAPI solution in 1x PBS and add 100 μL per well. Incubate for 20 min. Remove DAPI. Wash 3x with 1x PBS.
8. Keep fixed monolayers in 100 μL of 1x PBS for imaging. Place the slide back onto the microscope stage, reload the X and Y coordinates from the live imaging run, and image each multi-point on the channel that corresponds to the secondary antibody that was used for staining. Reference the images from the live imaging run to ensure the same points are captured.

6. Quantitation of intercellular calcium waves

Ensure Fiji is Just ImageJ (FIJI) is installed with the following plug-ins: Bio-Formats (should be pre-installed in FIJI but will not be in ImageJ); Cookbook: To install, from the FIJI menu, go to Help > Updates > Manage update sites. Check Cookbook. Restart FIJI.
Split the data file from the live imaging run into multiple files, separating each X, Y coordinate. In Nikon NES Elements, select File > Import/Export > Split Multipoints. Select a folder for export and an appropriate prefix.
Open FIJI. Load a single file from the split multipoints. If using a multi-channel image, split the channels. Select the window with the images from the 488 nm channel (GCaMP).
Run the DeltaF Up function to determine the change in each pixel value from one timepoint to the next. To do this, select Cookbook > T-functions > Delta F Up.
Scroll through the stack until an image with a calcium wave is found. With the calcium wave in view, set a threshold by clicking Image > Adjust > Threshold. Adjust the lower limit to minimize the amount of signal that is not part of the wave that makes it past the threshold. For 16-bit images acquired using the parameters described above, start with a lower threshold of 600 and adjust as needed.
Run the particle analyzer to segment waves by clicking Analyze > Analyze Particles. Set the minimum size of wave in µm² by adjusting the range. It is set to 0-infinity at baseline. For images of MA104 cells acquired using a 20x objective, start by increasing the lower limit to 10,000 µm² and adjust as appropriate.
Check the boxes for Display results, and Clear results. From the "Show" dropdown menu, select Outlines, then click OK. This should result in 2 outputs: One window, Results, will list each wave detected, the area of the wave, and its mean, minimum, and maximum intensity values (based on delta F). The other window, titled Drawing of [file name], will include a new stack with the outlines of segmented waves. Use this to verify wave detection. Adjust threshold values and size range if needed, and re-check the wave calls.
For batch processing, use the macro script included (Supplementary Coding File 1). To utilize this, follow the steps described below.
1. Split multi-points into single files as described in step 6.2. Open FIJI. Select Process > Batch > Macro. In the "input" box, provide the map to the folder containing the split multipoint files. Leave the "output" box empty.
2. Paste the script from Supplementary Coding File 1 into the box. Adjust the intensity threshold and size threshold in the script to reflect the optimized parameters from steps 6.5 and 6.6.
3. Click Process. Depending on the number of multi-points and processing speed of the computer, it can take 30 min or longer to process all images. Once complete, the data will be written into a new spreadsheet file named "Rename me after writing" on the Desktop. The output file should contain, for each multipoint, a wave count and wave metrics (area, mean intensity, minimum and maximum intensity, and intensity density).

Representative Results

Figure 1A shows a BMM dome containing 3-dimensional human intestinal organoids that have been transduced to stably express GCaMP6s. Figure 1B shows the same line of organoid replated as a monolayer at 24, 48, and 72 h post-seeding. To validate the function of GCaMP6s, the monolayer was imaged by fluorescence microscopy every 2 s for 4 min, and 100 nM ADP was added to the media after ~20 s. ADP elicits calcium release from the endoplasmic reticulum, increasing cytosolic calcium. The fluorescence intensity detected on the 488 nm channel at each exposure is plotted in Figure 1C. The trace shows a steady baseline fluorescence intensity followed by a rapid increase upon ADP addition, and a gradual return toward baseline. A vehicle control is included to verify that the response is a true signaling event, and not merely a change in fluorescence intensity that may arise from the addition of media.

Scoring an HIO monolayer increases the efficiency of rotavirus infection. While techniques for scoring vary, it is easiest to get consistent infection using a single score along the length of the monolayer, as shown in Figure 2A. When using recombinant strains of rotavirus that express fluorescent proteins, infection can be verified during live imaging (Figure 2B). When using a strain that does not express a marker, infection can be detected by immunofluorescence after imaging. Given the motility of cells in HIO monolayers, it is often difficult to track a single infected cell over time. Instead, use the endpoint staining as a method to verify infection and confirm the multiplicity of infection is matched between fields-of-view (Figure 2C).

Intercellular calcium waves in HIOs can be observed qualitatively, as depicted in Figure 3A. However, determining the frequency of calcium waves in a given field-of-view requires quantitative analysis. The steps of successful segmentation, as described in step 6 of the protocol above, are illustrated in Figure 3A. The figure shows the raw image (Figure 3Ai), the image after the pixel intensities from the previous frame have been subtracted (Figure 3Aii), then the segmented calcium wave (Figure 3Aiii).

Once the calcium waves have been segmented and quantified for each field-of-view over the course of the imaging run, it is often informative to compare the frequency of waves in monolayers exposed to various conditions. The strip chart in Figure 3B shows the total number of calcium waves per field-of-view in mock and rotavirus-inoculated monolayers. This example includes two strains of RV: a strain of human rotavirus (Ito HRV) and a recombinant strain of simian rotavirus SA11 that expresses mRuby (SA11-mRuby). The mock-infected control is taken as the baseline calcium wave frequency, the untreated rotavirus-infected monolayers serve as positive controls, and the rotavirus-infected, treated monolayers are the experimental conditions. Mock-infected and RV-infected monolayers treated with drugs, including the ADP receptor inhibitor, BPTU, can then be compared to both control conditions by one-way ANOVA. The data suggests that BPTU effectively decreases the frequency of intercellular calcium waves.

Figure 1: Validation of GCaMP6s expression in jejunal human intestinal organoids. (A) After lentivirus transduction, the GCaMP-expressing HIOs underwent selection and multiple passages, shown here at 7 days since the most recent passage. Upon stimulation with the 488 nm laser, GCaMP6s expression is evident in the transduced HIOs plated in 15 μL basement membrane matrix as 3-dimensional spheroids (Ai). 50 ms exposures were acquired at 50% laser power using a 20x objective, then stitched together to visualize the entire basement membrane matrix dome. A similar process was performed using 4 ms brightfield exposures (Aii). In the brightfield image, the dark HIOs in the center of the dome are an indication that the cultures are ready for passage. (B) HIOs were enzymatically digested into a single-cell suspension, then re-plated at a density of 150,000 cells per well (5mm in diameter) of an imaging-bottom plate pre-coated with collagen IV. By 72 h post-seeding, the monolayer was confluent and ready for downstream applications. To validate the function of GCaMP, images were acquired using the 488 nm filter set with 50 ms exposures at 50% laser power every 2 s for 4 min. (C) The traces shown here represent the whole-field fluorescence intensity normalized to the first acquisition over the course of imaging. At around 20 s (arrow), after the tenth exposure, 100 nM adenosine diphosphate (ADP) was added to elicit a spike in cytosolic calcium (red). The addition of an equal volume of 1x PBS did not elicit a meaningful response (blue). Please click here to view a larger version of this figure.

Figure 2: Scoring an HIO monolayer to facilitate RV infection. (A) The figure illustrates a suitable technique for scoring a monolayer using a 25G needle. (B) An HIO monolayer was infected with a recombinant strain of RV that expresses the mRuby fluorescent protein (magenta, arrow), allowing for the identification of infected cells. Alternatively, when using a naturally occurring strain of RV, infected cells can be identified using immunofluorescence. (C) The images shown are from 4 different HIO monolayers after fixation, DAPI staining (blue), and antibody labeling of rotavirus antigen (magenta). Both techniques illustrate the preferential RV infection near the scored portion of the monolayer. Please click here to view a larger version of this figure.

Figure 3: Segmentation and quantitation of RV-induced intercellular calcium waves. The 488 nm exposures of RV-infected HIOs expressing GCaMP6s were acquired every 1 min for 16 h using the protocol. (A) The figure illustrates the process of intercellular calcium wave segmentation. First, the pixel values from the previous acquisition (t_-1) are subtracted from each raw image (Ai) to quantify the change in fluorescence intensity (Aii). A threshold based on an experimentally optimized minimum increase in intensity from the previous frame is then applied. The images are then filtered for a minimum contiguous signal area to select for intercellular calcium waves (Aiii) and exclude single-cell calcium signals. This processing allows for the quantification of calcium waves at each field-of-view. In this experiment, monolayers were either mock-infected, infected with the ITO strain of human rotavirus (HRV), or infected with a recombinant strain of simian rotavirus encoding mRuby on gene segment 7, downstream of non-structural protein 3 (SA11-mRuby). Infected monolayers were either treated with the ADP receptor inhibitor BPTU or left untreated. Monolayers were then imaged at 8 different positions, and calcium waves in each field-of-view were quantified using the pipeline described in this protocol. The points on the strip chart indicate the number of calcium waves detected at each position superimposed on a bar chart marking the mean and SD. One-way ANOVA followed by Dunnett's tests show that while either strain of RV increases the frequency of intercellular calcium waves above that of the uninfected group, BPTU treatment prevents the RV-mediated increase in calcium wave frequency. **p<0.01, ***p<0.001. Please click here to view a larger version of this figure.

Reagent	Transduction	Control
Y-27632 (1 mM stock)	4 μL	4 μL
Polybrene (1 mg/μL stock)	2 μL	2 μL
Lentivirus	variable*
HighWnt-WRNE *see notes	to 400 μL	to 400 μL

Table 1: Lentivirus transduction medium. Prepare the transduction media and control media by combining the reagents in the volumes indicated in the second and third columns, respectively. Aim for a multiplicity of infection (MOI) of 10 lentivirus transduction units per cell. For LV packaged in-house, this usually requires ~25-50 μL of concentrated LV. The titer of most commercially packaged LV will be provided with the Certificate of Analysis.

Supplementary Coding File 1: ImageJ macro script to allow for calcium wave quantitation by batch processing. Please click here to download this File.

Discussion

Alterations in cytosolic Ca²⁺ levels can be both a cause and effect of pathologies within the epithelium¹⁰^,¹⁶^,¹⁷. Increases in cytosolic calcium can directly drive secretion via activation of the calcium-dependent chloride channel TMEM16A¹⁸^,¹⁹. Activation of TMEM16A in response to Ca²⁺ allows for the apical efflux of chloride, establishing an osmotic gradient that promotes fluid secretion²⁰^,²¹. Furthermore, increases in Ca²⁺ within enteroendocrine cells trigger the release of serotonin, which stimulates afferent sensory neurons that can modulate activity within the enteric nervous system²². Ca²⁺ signals can also modulate tight junctions to influence barrier function²³^,²⁴, and regulate proliferation in intestinal stem cells²⁵. Thus, dysregulation of Ca²⁺ can have broad and far-reaching effects.

Many viruses manipulate cytosolic Ca²⁺ to create a cellular environment conducive to replication²⁶. Rotavirus is a prime example of this within the intestinal epithelium²⁷. Rotavirus non-structural protein 4 (NSP4) is a Ca²⁺-conducting channel that allows for the efflux of Ca²⁺ from the endoplasmic reticulum into the cytosol²⁸^,²⁹. This stimulates autophagy and aids the assembly of the outer capsid³⁰^,³¹^,³². Rotavirus infection also induces the release of ADP, triggering intercellular Ca²⁺ waves that contribute to pathogenesis¹⁷. Other enteric viruses, including both caliciviruses and enteroviruses, encode Ca²⁺-conducting viroporins that trigger similar dysregulation of Ca²⁺ within infected cells³³^,³⁴.

Live-cell Ca²⁺ imaging of human intestinal organoids offers a versatile platform to study virus-induced signaling within the epithelium. However, the strength of the conclusions drawn from the imaging data depends, inextricably, on the health and quality of the HIOs, appropriate imaging parameters, and a sound analytical approach.

Efficient lentiviral transduction is often the limiting step when establishing new HIO lines, but high-titer lentivirus and multiple transductions help to ensure sufficient transgene expression. Importantly, this will have to be balanced with the potential for significant cellular stress upon transduction. This is most often temporary and subsides after selection and multiple passages. Nonetheless, it is critical that HIOs engineered to express GECIs display normal growth and proliferation before beginning experiments. Likewise, dispersing 3-dimensional HIOs and reforming them as monolayers induces cellular stress. It is best practice to allow three or more days for monolayers to adapt after plating. Coating the plates with collagen IV is critical for monolayer integrity³⁵^,³⁶. Aliquoting collagen IV and storing at -80 °C between uses helps to ensure monolayer adherence. Determining the optimal seeding density for each HIO line can also help to improve consistency. Finally, including the proper controls, and comparing them across experiments, is indispensable to ensure any observation represents a response to the variable in question, rather than a technical confounder.

While scoring a monolayer can cause unwanted cell damage, it greatly enhances RV infection of 2D HIOs. Figure 2 illustrates this, as almost all infected cells are directly adjacent to the scratch. Experimental evidence suggests that RV infects most efficiently at the basolateral surface of intestinal epithelial cells³⁷. Thus, it is predicted that scoring a monolayer enhances infectivity by exposing the basolateral surface. Efficient monolayer infection can also be achieved by plating HIOs on polycarbonate membranes suspended in wells containing culture medium, which allow access to the basolateral surface. However, the relatively long distance between the plate and the polycarbonate membrane, the polycarbonate membrane itself, and the plastic surface at the base of the well render them inappropriate for epifluorescence imaging. Thus, the scoring method described in this protocol remains the favored approach. Given that scoring itself may lead to signaling perturbations, the inclusion of the uninfected, scored control is critical for interpretation. For viruses, such as human norovirus, that efficiently infect via the apical membrane, scoring the monolayer is not required³⁸.

As with any live imaging system, phototoxicity can easily confound experiments utilizing HIO monolayers. Throughout an imaging experiment, it is important to monitor for unexpected vacuolization, membrane blebbing, or other morphological indicators of stress³⁹. If any of these signs are detected, the duration, frequency, or power of excitation must be reduced. Increasing the signal-to-noise ratio often comes at the expense of long-term cell health thus, empirically optimizing parameters is crucial. Photobleaching, as indicated by a decrease in the baseline fluorescence intensity over time, is a harbinger of cell stress and should be avoided.

The visuality of imaging data can lead investigators to favor qualitative, rather than quantitative, analyses. However, as with any modality, reproducible conclusions require quantitation. Quantitative image analysis contains far more complexity than can be covered in a single manuscript, but it is worthwhile to highlight a few key points. First, consistent quantitation requires consistent acquisition. An analysis pipeline developed for a given set of acquisition parameters may not be effective for images acquired at different frequencies, exposure times, laser powers, or wavelengths. Second, the multiplicity of infection has an outsized influence on the frequency of virally induced calcium signals. As such, it is important to evaluate the number of infected cells per field-of-view and ensure consistency across treatment conditions. Alternatively, normalizing signal frequency to the number of infected cells per field-of-view can help reduce error. Finally, while the recently developed mNG-based calcium indicator (NEMO) offers a promising approach for determining absolute Ca²⁺ concentrations in cells⁴⁰, most biosensors convey only relative information about biological systems. Thus, background subtraction, normalization, and comparison to a control are critical.

While this protocol focuses on calcium imaging during viral infection, the general approach is readily adaptable. Viral infection can easily be substituted for an alternative treatment condition, and there is an ever-growing litany of biosensors to monitor many different cellular pathways and processes. With any protocol adaptation, investigators must keep analysis top-of-mind, designing experiments with clear, quantifiable outcomes.

Overall, live imaging provides unrivaled temporal resolution, enhancing the characterization of highly dynamic processes. Incorporating organoids into live imaging experiments allows for the observation of these processes in a true-to-tissue, physiologically relevant model. We hope that the protocol described here facilitates experimental design and optimization, empowering researchers to uncover novel aspects of cell and tissue signaling.

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by grants R01DK115507 and R01AI158683 (PI: J. M. Hyser) from the National Institutes of Health (NIH). Trainee support was provided by NIH grants F30DK131828 (PI: J.T. Gebert), F31DK132942 (PI: F. J. Scribano), and F32DK130288 (PI: K.A. Engevik). We would like to thank the Texas Medical Center Digestive Diseases Enteroid Core for providing the organoid maintenance media.

Materials

Advanced DMEM F12	Gibco	12634028
[Leu15]-Gastrin I	Sigma-Aldrich	G9145
0.05% Trypsin EDTA	Gibco	25300054
0.05% Trypsin EDTA	Gibco	25300054
1.5mL microcentrifuge tubes	Fisherbrand	5408137
15mL conical tubes	Thermofisher Scientific	0553859A
16% formaldehyde	Thermofisher Scientific	28906
1M HEPES	Gibco	15630080
1M HEPES	Gibco	15630080
1X PBS	Corning	21-040-CV
25 gauge needle	Thermofisher Scientific	1482113D
A-83-01	Tocris	2939
ADP	Sigma-Aldrich	A2754
Advanced DMEM F12	Gibco	12634028
Antibiotic-antimycocytic	Gibco	15240062
Antibiotic-antimycotic	Gibco	15240062
B27 Supplement	Gibco	17504-044
Bovine serum albumin	FisherScientific	BP1600100
CellView Cell Culture Slide, PS, 75/25 MM, Glass Bottom, 10 compartments	Greiner	543979
Collagen IV	Sigma Aldrich	C5533
DAPI	Thermofisher Scientific	D1306
EDTA	Corning	46-034-CI
Fetal bovine serum	Corning	35010CV
Fetal bovine serum	Corning	35010CV
Fluorobrite	Gibco	A1896701
GlutaMAX	Gibco	35050079
GlutaMAX	Gibco	35050079
Human epidermal growth factor	ProteinTech	HZ-1326
Lentivirus	VectorBuilder	(variable)
Matrigel	BD Biosceicen	356231/CB40230C
N2 Supplement	Gibco	17502-048
N-acetylcysteine	Sigma-Aldrich	A9165-5G
NH4Cl	Sigma-Aldrich	A9434
Nicotinamide	Sigma-Aldrich	N0636
Nunc Cell Culture Treated 24-well Plates	Thermofisher Scientific	142475
Polybrene	MilliporeSigma	TR1003G
SB202190	Sigma-Aldrich	S70767
Triton X-100	Fisher BioReagents	BP151100
TrypLE Express Enzyme, no phenol red	Thermofisher Scientific	12604013
Trypsin	Worthington Biochemical	NC9811754
Y-27632	Tocris	1254

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Gebert, J. T., Scribano, F. J., Engevik, K. A., Hyser, J. M. Live Calcium Imaging of Virus-Infected Human Intestinal Organoid Monolayers Using Genetically Encoded Calcium Indicators. J. Vis. Exp. (203), e66132, doi:10.3791/66132 (2024).

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