iPSC High-Throughput Screening

Pivot Park Screening Center is a CRO specializing in high-throughput screening and operating as a vital part of the European Lead Factory (ELF). ELF is a pan-European platform dedicated to drug discovery and supported by IMI. IMI was started three years ago to revive drug research in Europe.

The European Lead Factory has a repository of more than 400,000 unique proprietary compounds and an experienced screening center. It provides a unique opportunity to researchers in academia as well as SMEs to boost medical research and produce new medicines. The achievement of the European Lead Factory can be perceived by the completion of over 40 screening campaigns and by the effective transfer of over 20 Qualified Hit List reports to the target owners.

The more than 40 HTS campaigns conducted so far encompass an array of target classes, including more challenging cellular targets such as GPCRs and ion channels. For a cost-efficient performance, these cellular HTS assays have been effectively miniaturized to 384- and 1536-well format.

Induced pluripotent stem cells (iPSCs) are part of an increasing list of stem cell populations that show huge potential in the development and screening of cell-based assays. iPSC-derived cells such as neuronal cells and cardiomyocytes serve as a reliable source of cellular material with robust assay performance and scalability which is important to any HTS campaign and subsequent follow-up.

This article discusses the effect of iPSC-derived neurons on modern drug discovery. It provides instances of assay automation and miniaturization of cellular assays on the basis of high content imaging through the Ca-flux and Operetta using the FLIPR instruments.

The instances described in this article present the opportunities and advantages that might soon be provided by iPSC-derived cells in the quest for new chemical starting points for drug discovery.

Cellular Screening Assay: From 384- to 1536-Well Format

Homogeneous Ion Channel Assay with FLIPR Tetra (Molecular Devices)

Schematic overview of the potassium assay. Thallium sensitive dye enters the cells through passive diffusion (a). Upon activation of the potassium ion channel thallium enters the cell causing increased fluorescence which can be measured on the FLIPR (b) excitation: 470–495 nm; emission: 515–575 nm Adopted from product insert R8223, Molecular Devices, 2015.

Schematic overview of the potassium assay. Thallium sensitive dye enters the cells through passive diffusion (a). Upon activation of the potassium ion channel thallium enters the cell causing increased fluorescence which can be measured on the FLIPR (b) excitation: 470–495 nm; emission: 515–575 nm Adopted from product insert R8223, Molecular Devices, 2015.

Figure 1. Schematic overview of the potassium assay. Thallium sensitive dye enters the cells through passive diffusion (a). Upon activation of the potassium ion channel thallium enters the cell causing increased fluorescence which can be measured on the FLIPR (b) excitation: 470–495 nm; emission: 515–575 nm Adopted from product insert R8223, Molecular Devices, 2015.

Assay Transfer from 384- to 1536-Well Format

Potassium assay. HEK-IC cells were grown overnight and a potassium assay was performed. Cells were treated with the potassium channel activator as positive control and DMSO as negative control and the signals were measured during 90 seconds in 384-wells (a) and 1536-wells (b). The signal over background correction and negative control correction on the raw fluorescence data was applied and plotted vs time.

Potassium assay. HEK-IC cells were grown overnight and a potassium assay was performed. Cells were treated with the potassium channel activator as positive control and DMSO as negative control and the signals were measured during 90 seconds in 384-wells (a) and 1536-wells (b). The signal over background correction and negative control correction on the raw fluorescence data was applied and plotted vs time.

Figure 2. Potassium assay. HEK-IC cells were grown overnight and a potassium assay was performed. Cells were treated with the potassium channel activator as positive control and DMSO as negative control and the signals were measured during 90 seconds in 384-wells (a) and 1536-wells (b). The signal over background correction and negative control correction on the raw fluorescence data was applied and plotted vs time.

Full HTS in 1536-Well Format

HTS protocols and screening procedure. Plate preparation was done on the automated ASPIRE system (a). HTS on the assay plates was performed on the fully automated ultra-HTS (uHTS) system (b)

HTS protocols and screening procedure. Plate preparation was done on the automated ASPIRE system (a). HTS on the assay plates was performed on the fully automated ultra-HTS (uHTS) system (b)

Figure 3. HTS protocols and screening procedure. Plate preparation was done on the automated ASPIRE system (a). HTS on the assay plates was performed on the fully automated ultra-HTS (uHTS) system (b)

HTS screening results. Full HTS was performed in 4 days. The assay performance was determined by running QC-plates with a full dose-response of the agonist each day (a). The S/B and Z’ of the assay were calculated from Min and Max wells measured on each plate. The assay performed well, with average S/B = 25 (15 < S/B < 30) and average Z’= 0.65 (0.51 < Z’ < 0.75) (b).

HTS screening results. Full HTS was performed in 4 days. The assay performance was determined by running QC-plates with a full dose-response of the agonist each day (a). The S/B and Z’ of the assay were calculated from Min and Max wells measured on each plate. The assay performed well, with average S/B = 25 (15 < S/B < 30) and average Z’= 0.65 (0.51 < Z’ < 0.75) (b).

Figure 4. HTS screening results. Full HTS was performed in 4 days. The assay performance was determined by running QC-plates with a full dose-response of the agonist each day (a). The S/B and Z’ of the assay were calculated from Min and Max wells measured on each plate. The assay performed well, with average S/B = 25 (15 < S/B < 30) and average Z’= 0.65 (0.51 < Z’ < 0.75) (b).

iPS Cells in Assay Development: Neuronal Cytotox Assay

Culturing and Differentiation of Axol Human iPSC-Derived Neural Stem Cells

Neurite outgrowth. Axol Human iPSC-Derived Neural Stem Cells (ax0016) were seeded in 384-well plates (10.000 cells/well) with the Multidrop combi dispenser (a) in Axol Neural Expansion-XF Medium (ax0030) followed by synchronous differentiation in Axol Neural Differentiation-XF Medium (ax0034) and Axol Neural Maintenance-XF Medium (ax0032). Growth of cells was monitored using the IncuCyte Live-Cell Imaging System from Essen Bioscience (b). The differentiation of the cells was demonstrated in phase contrast images after 6 days (neurites are shown in pink; c, d). Cell proliferation and neurite length were plotted against time (e, f).

Neurite outgrowth. Axol Human iPSC-Derived Neural Stem Cells (ax0016) were seeded in 384-well plates (10.000 cells/well) with the Multidrop combi dispenser (a) in Axol Neural Expansion-XF Medium (ax0030) followed by synchronous differentiation in Axol Neural Differentiation-XF Medium (ax0034) and Axol Neural Maintenance-XF Medium (ax0032). Growth of cells was monitored using the IncuCyte Live-Cell Imaging System from Essen Bioscience (b). The differentiation of the cells was demonstrated in phase contrast images after 6 days (neurites are shown in pink; c, d). Cell proliferation and neurite length were plotted against time (e, f).

Neurite outgrowth. Axol Human iPSC-Derived Neural Stem Cells (ax0016) were seeded in 384-well plates (10.000 cells/well) with the Multidrop combi dispenser (a) in Axol Neural Expansion-XF Medium (ax0030) followed by synchronous differentiation in Axol Neural Differentiation-XF Medium (ax0034) and Axol Neural Maintenance-XF Medium (ax0032). Growth of cells was monitored using the IncuCyte Live-Cell Imaging System from Essen Bioscience (b). The differentiation of the cells was demonstrated in phase contrast images after 6 days (neurites are shown in pink; c, d). Cell proliferation and neurite length were plotted against time (e, f).

Neurite outgrowth. Axol Human iPSC-Derived Neural Stem Cells (ax0016) were seeded in 384-well plates (10.000 cells/well) with the Multidrop combi dispenser (a) in Axol Neural Expansion-XF Medium (ax0030) followed by synchronous differentiation in Axol Neural Differentiation-XF Medium (ax0034) and Axol Neural Maintenance-XF Medium (ax0032). Growth of cells was monitored using the IncuCyte Live-Cell Imaging System from Essen Bioscience (b). The differentiation of the cells was demonstrated in phase contrast images after 6 days (neurites are shown in pink; c, d). Cell proliferation and neurite length were plotted against time (e, f).

Neurite outgrowth. Axol Human iPSC-Derived Neural Stem Cells (ax0016) were seeded in 384-well plates (10.000 cells/well) with the Multidrop combi dispenser (a) in Axol Neural Expansion-XF Medium (ax0030) followed by synchronous differentiation in Axol Neural Differentiation-XF Medium (ax0034) and Axol Neural Maintenance-XF Medium (ax0032). Growth of cells was monitored using the IncuCyte Live-Cell Imaging System from Essen Bioscience (b). The differentiation of the cells was demonstrated in phase contrast images after 6 days (neurites are shown in pink; c, d). Cell proliferation and neurite length were plotted against time (e, f).

Neurite outgrowth. Axol Human iPSC-Derived Neural Stem Cells (ax0016) were seeded in 384-well plates (10.000 cells/well) with the Multidrop combi dispenser (a) in Axol Neural Expansion-XF Medium (ax0030) followed by synchronous differentiation in Axol Neural Differentiation-XF Medium (ax0034) and Axol Neural Maintenance-XF Medium (ax0032). Growth of cells was monitored using the IncuCyte Live-Cell Imaging System from Essen Bioscience (b). The differentiation of the cells was demonstrated in phase contrast images after 6 days (neurites are shown in pink; c, d). Cell proliferation and neurite length were plotted against time (e, f).

Figure 5. Neurite outgrowth. Axol Human iPSC-Derived Neural Stem Cells (ax0016) were seeded in 384-well plates (10.000 cells/well) with the Multidrop combi dispenser (a) in Axol Neural Expansion-XF Medium (ax0030) followed by synchronous differentiation in Axol Neural Differentiation-XF Medium (ax0034) and Axol Neural Maintenance-XF Medium (ax0032). Growth of cells was monitored using the IncuCyte Live-Cell Imaging System from Essen Bioscience (b). The differentiation of the cells was demonstrated in phase contrast images after 6 days (neurites are shown in pink; c, d). Cell proliferation and neurite length were plotted against time (e, f).

Automated Cytotox Assay on Differentiated iPSC-Derived Neural Stem Cells

Automated neuronal apoptosis assay. Red phase contrast imaging demonstrated cytotoxicity on differentiated Human iPSC-Derived Neural Stem Cells after 6-hour treatment with 0.5% DMSO (a) or 20 μM trifluoperazine in 0.5% DMSO (b) in the presence of IncuCyte Cytotox Red Reagent. The proliferation of differentiated Human iPSC-Derived Neural Stem Cells treated with different compounds was plotted against time (c). Compound effects were tested in dose response on differentiated Human iPSC-Derived Neural Stem Cells (d).

Automated neuronal apoptosis assay. Red phase contrast imaging demonstrated cytotoxicity on differentiated Human iPSC-Derived Neural Stem Cells after 6-hour treatment with 0.5% DMSO (a) or 20 μM trifluoperazine in 0.5% DMSO (b) in the presence of IncuCyte Cytotox Red Reagent. The proliferation of differentiated Human iPSC-Derived Neural Stem Cells treated with different compounds was plotted against time (c). Compound effects were tested in dose response on differentiated Human iPSC-Derived Neural Stem Cells (d).

Automated neuronal apoptosis assay. Red phase contrast imaging demonstrated cytotoxicity on differentiated Human iPSC-Derived Neural Stem Cells after 6-hour treatment with 0.5% DMSO (a) or 20 μM trifluoperazine in 0.5% DMSO (b) in the presence of IncuCyte Cytotox Red Reagent. The proliferation of differentiated Human iPSC-Derived Neural Stem Cells treated with different compounds was plotted against time (c). Compound effects were tested in dose response on differentiated Human iPSC-Derived Neural Stem Cells (d).

Automated neuronal apoptosis assay. Red phase contrast imaging demonstrated cytotoxicity on differentiated Human iPSC-Derived Neural Stem Cells after 6-hour treatment with 0.5% DMSO (a) or 20 μM trifluoperazine in 0.5% DMSO (b) in the presence of IncuCyte Cytotox Red Reagent. The proliferation of differentiated Human iPSC-Derived Neural Stem Cells treated with different compounds was plotted against time (c). Compound effects were tested in dose response on differentiated Human iPSC-Derived Neural Stem Cells (d).

Figure 6. Automated neuronal apoptosis assay. Red phase contrast imaging demonstrated cytotoxicity on differentiated Human iPSC-Derived Neural Stem Cells after 6-hour treatment with 0.5% DMSO (a) or 20 μM trifluoperazine in 0.5% DMSO (b) in the presence of IncuCyte Cytotox Red Reagent. The proliferation of differentiated Human iPSC-Derived Neural Stem Cells treated with different compounds was plotted against time (c). Compound effects were tested in dose response on differentiated Human iPSC-Derived Neural Stem Cells (d).

Conclusion

Cellular Screening Assay: From 384- to 1536-Well Format

  • The HEK-IC assay can be transferred from 384- to 1536-well
  • A complete HTS of more than 350,000 compounds was carried out in four days by using highly advanced dispensing and measuring equipment
  • The assay was extremely reproducible as illustrated by the IC50-values of the agonist on the QC-plates, and the S/B and Z’ of the control wells on the assay plates
  • The uHTS system can be employed for quick and cost-effective HTS of cellular assays in 1536-well format

Assay Development and Testing of iPS Cells in 384-Well Format

  • Neurite outgrowth and differentiation was shown to be viable in 384-well format using automated dispensing and measuring equipment
  • Cells cultured and differentiated in 384-well plates treated with trifluoperazine exhibited a concentration-dependent cell death, while other compounds exhibited little or no effect
  • Axol Human iPSC-Derived Neural Stem Cells can be employed for compound testing in an automated system

About AXOL Biosciences

Axol specializes in human cell culture.

Axol produces high quality human cell products and critical reagents such as media and growth supplements. We have a passion for great science, delivering epic support and innovating future products to help our customers advance faster in their research.

Our expertise includes reprogramming cells to iPSCs and then differentiating to various cell types. We supply differentiated cells derived from healthy donors and patients of specific disease backgrounds. As a service, we also take cells provided by customers (primary or iPSC) and then do the reprogramming (when necessary) and differentiation. Clearly, by offloading the burden of generating cells, your time is freed up to focus on the research. Axol holds the necessary licenses that are required to do iPSC work.

The package wouldn't be complete without optimized media, coating solutions and other reagents. Our in-house R&D team works hard to improve on existing media and reagents as well as innovate new products for human cell culture. We also supply a growing range of human primary cells; making Axol your first port of call for your human cell culture needs.


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Last updated: Nov 4, 2019 at 8:22 AM

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