Ready-to-use CHO-NaV1.5 And Hek-hERG Instant Cells: Study On Frozen Cells Thawed And Immediately Patched At Manual And Automatic Patch Clamp Devices

Abstract

Well characterised cell lines and constant high cell quality are prerequisites for reliable data in electrophysiological studies. We developed the cell culture system “Instant Cells” that enables quality control of frozen cell batches and guarantees a constant cell quality. To show that the Instant Cells are suited for pharmacological studies, we have adapted CHO-K1 cells stably expressing hNaV1.5 and HEK 293 cells stably expressing hERG to the Instant Cells system.The frozen Instant Cells were thawed, spun down and resuspended in a physiological buffer. Afterwards, the cell suspension was kept for four hours in the Cell Reservoir, a bench-top cell storage device. During this time span the cells were taken from the Cell Reservoir to be evaluated on a conventional and on an automated patch clamp device, the CytoPatchTM instrument.We show that both types of Instant Cells have the same characteristics in terms of electrophysiological and pharmacological properties compared to permanently cultured cells:- Trypan-blue tests showed 95 % vital cells after preparation.- The mean peak current of the NaV1.5 Instant Cells was 12.5 nA, the mean tail current of the hERG Instant Cells was 1.2 nA.- More than 80 % of the cells sealed (above 1 GOhm), more than 60 % were stable for 15-25 min (Rm was above 500 MOhm).- The isochronal I/V relationship of the hERG activation and tail current and the NaV1.5 activation current were similar to freshly prepared cultured cells.- No difference was observed in the dose-response relationship for blocking compounds between the Instant cells and the running culture of both cell types.This proves that the Instant Cells are well suited to investigate ion channel pharmacology.