Application Note
Automated culture and functional analysis of compound responses in human iPSC-derived 3D tri-culture cardiac spheroids
- Gain valuable insights into in vitro toxicity assessment using iPSC-derived 3d tri-culture cardiac spheroids.
- Discover a fully automated protocol to efficiently develop mature cardiac spheroids.
- Measure compound toxicity with precision using functional kinetic recording and in-depth analysis of spontaneous calcium oscillation activity
Oksana Sirenko, Krishna Macha, Angelina Chopoff | Molecular Devices
Ravi Vaidyanathan, Coby Carlson | FUJIFILM Cellular Dynamics, Inc.
Introduction
The use of human iPSC-derived cell types to model human tissues is a promising approach to facilitate drug development and safety/toxicity assessment. Individual and highly specialized cell types can be mixed and cultured together in 3D to generate cellular models that more closely resemble human tissues. For example, the heart is composed of cardiomyocytes, endothelial cells, fibroblasts, and other supporting cell types. FUJIFILM Cellular Dynamics, Inc. (FCDI) has developed a method to generate a 3D tri-culture model composed of iPSCderived cardiomyocytes (CM), endothelial cells (EC), and cardiac fibroblasts (CF) all derived from the same human-iPSC donor.
In this study, we automated the process for forming and maintaining these tri-culture cardiac spheroids (a.k.a. iCell® Cardiospheres) using the CellXpress.ai® Automated Cell Culture System. This platform is able to automate every step of cell culture including cell plating, periodic media exchanges, monitoring, and analyzing the size and morphology of the tri-culture cardiac spheroids in 3D over time. After two weeks in culture, the functional activity was tested by loading the cells with a calcium-sensitive dye (Calcium 6, Molecular Devices) and recording spontaneous calcium oscillations with the FLIPR® Penta High-Throughput Cellular Screening System, which is a fast kinetic fluorescence recording instrument. Cells were treated with a panel of 18 compounds, including modulators of cardiac activity, blockers of ion channels, and known cardiotoxic compounds. Analysis of waveform patterns was performed with ScreenWorks® Peak Pro 2 software. Additonal characterization of cell viability and morphology was done after the assay via imaging.
The combination of iPSC technology with cell culture automation and functional kinetic calcium imaging is essential to ensure robustness when using complex cell models for high-throughput compound profiling.
Materials and methods
Instruments and software
The CellXpress.ai system was used to plate and culture cells via automation with periodic media exchanges and regular monitoring of spheroids. The system contains four essential components for automated cell culture: a liquid handler, an automated incubator, an automated imager, and integrated AI-powered software that automates complex protocols, scheduling, image analysis, and decision-making.
The FLIPR Penta system was used to record and analyze calcium oscillations from 3D tri-culture cardiac spheroids. The high-speed EMCCD camera measured patterns and frequencies of spontaneous waveform oscillations corresponding to changes in intracellular Ca2+ levels from cells loaded with FLIPR Calcium 6 dye. The ScreenWorks Peak Pro 2 peak analysis software yielded multiple readouts, such as peak count or frequency, peak amplitude, peak prolongation, irregularity, the appearance of secondary peaks, and other measurements of complex oscillation patterns.
Imaging and image analysis. Live-dead analysis of the spheroids was performed after imaging with 10X magnification using the CellXpress.ai system and software. For live-cell staining, a mixture of 3 dyes was used: Calcein AM (1 μM), EtHD (2 μM), and Hoechst 33342 nuclear dye (2 μM) (Life Technologies). For high-content image analysis with higher resolution, the ImageXpress® Micro Confocal High-Content Imaging System was used to capture 3D images of whole microtissues stained with antibodies or dyes in different fluorescent channels. The custom module editor (CME) in MetaXpress® High-Content Image Acquisition and Analysis Software was used reconstruct the 3D structure of the microtissues and can be adjusted for tailored image analysis.
Cells and assay
Cryopreserved human iPSC-derived cell types, optimized culture media, and supplements are commercially available from FUJIFILM Cellular Dynamics, Inc. Protocol guidance can be found in the Application Protocol entitled: “Generation of 3D iCell Cardiospheres with iCell Products”( see schematic workflow). Ultra-low attachment (ULA) 3D spheroids forming plates (384-well format from Corning) were used to form tri-culture cardiac spheroids at a 65:15:20 ratio of CMEC. 3D spheroids formed within 48 hours and started to contract spontaneously and regularly on day 4 or 5. The presence of strong synchronous contractions in 3D cultures was confirmed visually prior to running experiments. On the day of assay (typically day 14), cells were loaded with 2X FLIPR Calcium 6 dye indicator and incubated for 2 hours. The FLIPR Penta detected spontaneous cardiac waveforms and baseline recordings were acquired for 1–2 min. Cells were dosed with drugs at the indicated final concentration and recordings were acquired again after 15–90 minutes.
Results
Automated formation, culture, and monitoring cardiac micro-tissues with the CellXpress.ai system
Since manual culture and organoid tracking is labor intensive, we developed protocols for automated cell plating, media exchanges, and cardiac spheroid imaging using the CellXpress.ai automated cell culture system. Tri-cultures were seeded into 96- or 384-well ULA plates and cultured for ~14 days with imaging and media exchange every 48 hours. 10X images were taken with transmitted light and the liquid handler replaced half of the media volumes automatically. Imaging and analysis for fluorescent markers was done using the CellXpress. ai system’s integrated imager (Figure 1). Cultured spheroids were consistent in size, and demonstrated strong, consistent spontaneous activity on the day of the assays (Figure 2).
Figure 1. The CellXpress.ai system in action. (A) View of the CellXpress.ai system performing media exchanges in a 384-well plate. Hands-free and ratecontrolled liquid handling reduces error rates and saves time for other tasks in the lab. (B) Transmitted light image (10X magnification) of iPSC-derived 3D tri-culture cardiac spheroids cultured in a 96-well plate. (C) CME was used to perform segmentation and automated analysis during 3D cell culture.
Figure 2. Reproducible formation of 3D spheroids.Diameters of formed 3D tri-culture cardiac spheroids were measured every second day during the experiment. Diameter measurements were consistent within wells, between plates, and across experiments (%CV 20% or less). Additionally, functional analysis (i.e., oscillation frequencies in peaks per min) were measured for untreated cells / control samples on each plate with a %CV of 25% or less.
Figure 3. 3D spheroid images with CellXpress.ai. (A) Cardiac microtissues stained with Calcium 6 dye for FLIPR assay were imaged with the CellXpress.ai system (FITC, 10X). Best-focus projection from Z-stack of images was taken in 10 µm increments. (B) Best focus projection 10X images shown for cells stained with dyes for cell viability, including Hoechst (blue), Calcein AM (green), and EtHD (red). Best focus projections of 8 images were taken 10 µm apart. Cells were treated with cardiotoxic compounds, including 100 μM Doxorubicin and 10 μM Staurosporine. The 3D cardiac spheroids were imaged with the CellXpress.ai system (DAPI, FITC, and Texas Red at 10X). Image analysis evaluated the spheroid area and average intensities for different fluorophores. Cell viability can be presented as a normalized ratio of live/dead average fluorescence intensities.
Functional analysis of cardiac activity and compound effects by measuring calcium oscillation patterns with the FLIPR Penta system
Spontaneously contracting 3D tri-culture cardiac spheroids generate calcium oscillations that correspond with contracting activity and can be visualized with calciumsensitive dyes. Oscillations were recorded by the FLIPR Penta system after loading microtissues with Calcium 6 dye. Spheroids were then treated with a panel of different compounds in either triplicates (n=3) or quadruplicates (n=4) with a 7-point concentration response. Typically, assay recordings were captured 30 minutes after dosing.
To investigate the cardiac activity of the iCell Cardiospheres, we treated spheroids with a panel of 18 compounds, including modulators of cardiac activity, blockers of ion channels, and known cardiotoxic compounds. All selected compounds, including hERG inhibitors, ion channel blockers, or beta-blockers demonstrated marked changes in the Ca2+ oscillation patterns consistent with the expected mode of action (Figure 4). For example, isoproterenol caused increase on oscillation frequency; compounds known as hERG blockers, i.e. E-4031, cisapride, and flecainide caused marked peak prolongation; digoxin caused stopping of oscillation activity.
Waveform analysis of patterns was performed with ScreenWorks® Peak Pro 2 software which yielded multiple readouts, such as peak count or frequency, peak amplitude, peak prolongation, irregularity, the appearance of secondary peaks, and other measurements characterizing modulations of oscillation patterns. Compound EC50 values or effective concentrations for modulations of oscillation frequency were calculated and are presented in Table 2.
Table 2.
Figure 4. Calcium assay with 3D spheroids. Calcium oscillations measured after treatment of 3D cardiac spheroids with various compounds (concentrations indicated in [microM]. Ca2+ waveforms were recorded by kinetic calcium imaging using the FLIPR Penta system and analyzed using Peak Pro 2 software. After the addition of indicated compounds, the resulting patterns (that differed strongly from control signals) are shown in the representative traces. Peak frequencies, amplitudes, peak prolongations, and other measurements were evaluated for different compounds and concentrations.
Evaluation of compound effects on cell viability and morphology using high-content imaging
Additionally, we characterized the morphology and viability of 3D microtissues with image analysis after staining microtissues with cell viability dyes. 3D tri-culture cardiac spheroids were treated with compounds for 24 hours and then morphological changes were evaluated using automation and the ImageXpress® Micro Confocal High-Content Imaging System. Analysis of confocal images allowed cell segmentation using nuclei stain followed by evaluation of number and percent of live cells by Calcein AM and EtHD stains. Doxorubicin and staurosporine caused marked decrease in cell viability after 24 hours of treatment, as shown in the Figure 5. Other compounds did not cause detectable effect on cell viability.
Figure 5. End point assay: Evaluation morphological changes using high-content imaging. Composite confocal images shown for Hoechst, Calcein AM, and EthD in DAPI, FITC, and Texas Red channels, respectively. 10X magnification and maximum projections of 15 images were taken at 8 µm apart. Compound treatments in this example were 100 μM of doxorubicin and 10 μM of staurosporine. Percent of viable cells is indicated below each image.
This study demonstrates the automation of a cell culture workflow using the CellXpress.ai system with endpoint assays analyzing compound effects through functional and morphological readouts from a complex biological system. The data demonstrates the usefulness and biological relevance of using iPSC-derived cell types in 3D microtissues as a model for assessing potential cardiotoxic effects on human cardiac tissues. The combination of iPSC technology with automated cell culture and imaging is essential to ensure consistency and throughput when using complex cell models for compound profiling.
Summary
- 3D tri-culture cardiac spheroids generated from human iPSC-derived cell types relevant to heart function present a useful in vitro cell model to assess cardiotoxic and cardio-active drug efficacy.
- Analysis of kinetic calcium oscillations demonstrates functional activity of cardiac microtissues and provides readouts for drug-induced changes in oscillation patterns and for toxicity evaluation.
- A fully automated method for the formation and culture of cardiac microtissues including automated media exchange and imaging enhances the robustness and throughput of a complex biological system.
- This assay platform shows promise for the evaluation of compound effects and early detection of cardiotoxicity.