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Oksana Sirenko, Carole Crittenden, Krishna Macha, Angeline Lim | Molecular Devices, LLC

Rebecca Fiene, Scott Schachtele, Coby Carlson | FUJIFILM Cellular Dynamics, Inc.

Introduction

Neural organoids and 3D spheroids are a rapidly developing technology with great potential for understanding brain development and neuronal diseases. They provide a more advanced and biologically relevant system for basic research and high-throughput drug discovery, including compound profiling and toxicity testing. Here, we describe methods for assembling normal human iPSC-derived cell types, including glutamatergic neurons, GABAergic neurons, and astrocytes into 3D neurospheres for compound profiling.

We monitored the formation, morphology, and functional activity (Ca2+ oscillations) of the neurospheres after 3 weeks in culture. Spheroid formation occurred within 24–28 hours and cells were analyzed by confocal fluorescence imaging for cell organization and expression of neuronal markers (TUJ1) and astrocytes (GFAP). Cellular and spheroid morphology was characterized by using highcontent imaging. The calcium assay was performed on a FLIPR Penta instrument capable of fast kinetic recordings using a calcium-sensitive dye and oscillation patterns were analyzed for peak frequency, amplitude, width, & spacing. For pharmacological characterization, we used a panel of 14 compounds, including selected molecules that affect GABA, AMPA, NMDA, sodium and potassium channels, dopamine receptors, as well as select neuroactive and neurotoxic substances. The functional responses demonstrated the predicted effects based on mode of action. This biological system of 3D neurospheres paired with high-content imaging and detailed analysis of calcium oscillations on the FLIPR Penta demonstrates a promising tool for neurotoxicity testing and compound screening.

Materials and methods

3D spheroids were formed using human isogenic iPSCderived cell types from FUJIFILM Cellular Dynamics, including iCell® GlutaNeurons, iCell GABANeurons, and iCell Astrocytes 2.0. Briefly, cryopreserved vials were thawed and mixed in desired ratios following the iCell NeuroSpheres protocol FUJIFILM CDI (e.g., 90% neurons (70:30 Gluta:GABA) and 10% astrocytes), and then 25K cells/well in complete BrainPhys™ (StemCell Technologies) medium were plated into 384-well ULA spheroid plates (Corning). After 2 days, cells formed compact spheroids and then maintained until at least day 21. On the day of assay, cell spheroids were loaded with 2X concentration of FLIPR Calcium 6 dye (Molecular Devices), incubated for 2 hours, then dosed with the indicated compounds. After drug exposure for 30 min and 60 min, calcium oscillations were recorded for 10 min using FLIPR Penta instrument (Molecular Devices). High content imaging was done by automated imager instrument (ImageXpress Micro Confocal System, Molecular Devices).

• We used a high-speed EMCCD camera on the FLIPR Penta cellular screening system to measure the patterns and frequencies of the Ca2+ oscillations of 3D neurospheres through changes in intracellular Ca2+ levels with the FLIPR Calcium 6 dye. The instrument equipped with Screen Works PeakPro2 peak analysis software allowing analysis and characterization of the primary and secondary peaks and complex oscillation patterns.
• ImageXpress Micro Confocal High-Content Imaging System was used to capture the 3D structures of the spheroids and viability evaluation.

Figure 1. Schematic diagram of the process workflow

Results

Recording and analysis of Ca²⁺ oscillation patterns

Figure 2. Calcium oscillations of 3D neurosphers were recorded over 600 seconds by kinetic calcium imaging using FLIPR Penta instrument. Traces represent fluctuations in fluorescent intensities recorded with calcium sensitive dye. The oscillation patterns affected by neuroactive compounds. The representative time-lapse images recorded by automated images shown below the traces (green fluorescence). Images were captured using the ImageXpress Micro Confocal system with an interval of 0.4 sec per image..

Figure 3. Ca²⁺ oscillations of neural spheroids determined by kinetic calcium imaging using FLIPR instrument and analyzed using PeakPro2 software. Measurements were derived from recordings from samples treated with different compounds and different concentrations. Peak count, peak amplitude, peak width, distance between peaks, and several other metrics were derived from the analysis. Concentration-response curves were generated via GraphPad Prism and concentration dependencies were evaluated. Interestingly, an increase in peak frequency was observed with increasing concentration, which then halted oscillations and activities at the highest concentrations tested. A. Bar graphs represent changes in selected readouts for several compounds tested (20 µM). B. Concentration curves shown for DNQX and Lidocaine.

Summary of parametric effects for select compounds

Summary

• 3D spheroids are a complementary approach to neural organoids. They can be generated in vitro using terminallydifferentiated iPSC-derived cell types from the same human donor (isogenic). The resulting products present a useful cell-based assay for assessment of neurotoxicity, functional effects of neuro-active modulators, and disease modelling.
• 3D neural organoids paired with FLIPR Penta show promise for the evaluation of compound effects and early detection of neurotoxicity in vitro due to its easy of use, consistency across wells and assay plates, compatibility with high-throughput screening and 384-well platforms, and ultimate biological relevance.
• Analysis of kinetic calcium imaging provides reliable and accurate read-outs for the functional neural activity and can be used for evaluation of phenotypic changes and compound effects.
• These technologies open the door for future experiments, including disease modeling, assessment of other iPSC-derived neuronal cell types (e.g., dopaminergic or motor neurons), and incorporation of additional cell types, such as microglia.

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