Primary Cells Transfected with a Calcium Biosensor and Reactivated from Frozen State Provide a Ready-to-Use Platform for Cell-Based Ca2+- Assays in High Throughput Format Silke Valentin1, Bodo Ortmann1, Kristin Atze1, Thomas Koblizek1, Nadine Breuer1, Steffi Franke1, Mathias Kühn1, Daniela Litzenberger1, Yvonne Strübing1, Leon de Bruin1, Sabrina Corazza2 and Nicole Faust1

1Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany; 2Axxam, San Raffaele, Biomedical Science Park, via Olgettina 58, 20132 Milan, Italy.

1. Abstract

In drug discovery research cell-based assays are a widely accepted tool for high throughput screening of potential drug candidates. To date usually immortalized cell lines are employed in cell-based assays. They bear some limitations as they are (1) often not originated from the actual tissue or native cell of interest, (2) sometimes non-human (e.g. hamster derived CHO cell line), and (3) often expressing the transfected targets at non-physiological levels.In contrast primary cell types can be derived from the tissue of interest. They allow for a higher predictability of the drug reaction in humans. The cells express relevant drug targets at physiological level and carry all the components necessary for specific signal transduction.There is a growing demand for primary cells in secondary and even primary drug screens, although the major draw-back has been the unreliable availability of high quality primary cells.Here we show that Clonetics® primary endothelial cells of human umbilical vein (HUVEC) and microvasculature of human lung (HMVEC-L) can be used in high throughput formats (i.e. 96-well or 384-well plate) to monitor intracellular Ca2+ fluxes. The cells were transiently transfected with the i-Photina® luminescent calcium biosensor using the Amaxa® 96-well Shuttle® Nucleofector® Technology. After subsequent loading with the biosensor’s substrate coelenterazine the cells could be used either directly or they could be cryopreserved and reactivated as needed. Stimulation through agonists of endogenously expressed surface receptors lead to dose-dependent Ca2+ responses at a very high signal to background ratio.This ready-to-use cell-based assay system represents an excellent tool to study agonist effects on calcium signaling in primary cells and will open new roads for more predictable drug screenings.

2. Introduction

In spite of the growing demands for primary cells in drug screenings, they have rarely been used for high throughput. This may be mainly because the label-free methods currently used for their analysis cannot properly address signal transduction at the molecular level. A further aspect is the limited availability of reliable high quality primary cells.

The data presented here demonstrate that Clonetics® primary cells transiently expressing a calcium dependent photoprotein can be employed for detecting intracellular Ca2+ release upon stimulation of different signaling pathways.Monitoring changes in intracellular calcium concentrations through calcium-binding bioluminescent proteins offers the advantage of an instant signal after stimulus. The i-Photina® apo-protein used in this study covalently binds coelenterazine as substrate which is converted to coelenteramide upon binding of Ca2+ to the protein accompanied by emission of a light flash (Fig. 1).The method is non-toxic and, unlike with fluorescent dye-based Ca2+ assays, there is virtually no background signal and no interference from fluorescent compounds.

3. Materials and Methods

Transfection of primary cellsClonetics® and Poietics® primary cells (human umbilical vein endothelial cells (HUVEC), human microvascular endothelial cells of the lung (HMVEC-L), aortic smooth muscle cells (AoSMC), human mesenchymal stem cells (hMSC), normal human dermal fibroblasts adult (NHDF-ad)) were transiently transfected with an expression plasmid encoding i-Photina® using the appropriate Amaxa® 96-well Nucleofector® Kits and the Amaxa® 96-well Shuttle® Nucleofector®.

Detection of intracellular calcium releaseThe transfected cells were loaded with 10 µM native coelenterazine 20 hours after transfection for 4 hours. Measurement of luminescence was carried out in 96-well format with a micro-plate reader equipped with injection devices. The agonists (ATP, histamine, 2-pyridylethylamine, thrombin; all from Sigma-Aldrich) were injected into the wells. The luminescence signals were recorded at 25°C every second for a total of 30 to 60 seconds.For freezing experiments cells were incubated after Nucleofection® for 4 hours. The loading with 10 µM native coelenterazine was done for another 2 hours right before freezing. The cells were frozen in vials in cryoprotective agent. In order to perform the Ca2+-assay cryopreserved cells were thawed, seeded on a 96-well-plate, and were allowed to recover for 18 hours. 4 hours after thawing medium was exchanged to remove the cryoprotective agent. NHDF, hMSC and AoSMC were loaded with coelenterazine after reactivation for 4 hours right before stimulation.Dose-dependent responses were calculated using area under the curve (AUC) integration.

4. Results

Figure 2. Signal kinetics of HUVEC and HMVEC-L transfected with i-Photina® upon stimulation with 100 µM ATP. Cells were stimulated 24 hours after transfection with the agonist ATP, which is a ligand for the P2Y family of purinergic receptors. These receptors modulate mainly intracellular calcium levels. Luminescence signal was recorded every second for a total of 60 seconds.

i-Photina® is a trademark of Axxam. Unless otherwise noted, all other trademarks herein are marks of the Lonza Group or its affiliates. The Nucleofector® Technology, comprising Nucleofection® Process, Nucleofector® Device, Nucleofector® Solutions, Nucleofector® 96-well Shuttle® System and Nucleocuvette® Plates and Modules is covered by patent and/or patent pending rights owned by Lonza Cologne AG. The i-Photina® luminescent calcium biosensor is covered by patent and patent pending rights owned by Axxam.

Figure 5. Dose-dependent response to thrombin in HUVEC transfected with i-Photina®. After thawing and recovery over night cells were stimulated with various aconcentrations of thrombin. Thrombin interacts with protease-activated receptors (PAR1, PAR3, PAR4) and unmasks through proteolysis the actual ligand of the receptor.

5. Conclusions

Primary cells transiently transfected with a calcium biosensor can be employed for monitoring intracellular Ca2+ release in high throughput formats. The cells expressing the biosensor and loaded with the substrate coelenterazine are provided frozen ready-to-use. They can be seeded directly into micro plate formats and can be used for cell-based assays. Lonza’s Clonetics® Primary Sensors represent a groundbreaking system to study agonist triggered calcium signaling in primary cells. This will open new roads for more predictable drug screenings.

Figure 3. Dose-dependent responses to histamine in HUVEC and HMVEC-L transfected with i-Photina®. After thawing and recovery over night cells were stimulated with various concentrations of histamine. Histamine binds to the H1 receptor which is expressed throughout the whole body, specifically e.g. on endothelial cells, in the central nervous system.

Figure 7. Histamine induced signal kinetic of i-Photina® in transiently transfected hMSC after reactivation from frozen state. Arrow indicates time point of injection.

Figure 1. Mechanism of Calcium Biosensor. Primary cells transiently transfected with the Calcium Biosensor express the protein in an inactive state called apo-photoprotein. Upon incubation of the cells with the substrate coelenterazine in the presence of oxygen a stable complex of coelenterazine and the active photoprotein is build up. Stimulation of cells with agonists regulating calcium signaling via Gαq-Protein coupled receptor induces calcium release from internal stores. Binding of calcium to the complex causes a conformational change to an excited state. The following rapid reaction results in a blue luminescence light flash which can be detected by a photo-multiplier in a plate reader.

Figure 8. Signal kinetic of i-Photina® in transiently transfected AoSMC (A) and NHDF-adult (B) upon stimulation with histamine. After thawing and recovery over night cells were stimulated with histamine. Luminescence signal was recorded every second for a total of 35 seconds starting with a 5 second pre-run for baseline recording.

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World Pharmaceutical Congress.15 – 17 June 2010, Philadelphia, PA, USA

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Figure 6. Dose-dependent response to histamine in hMSC transiently transfected with i-Photina®. Signal to background ratio could be improved to 180 (calculated from the areas under the curves) through optimizing the injection parameter (300 µl/sec to 100 µl/sec).

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