scholarly journals A Temperature-Controlled Patch Clamp Platform Demonstrated on Jurkat T Lymphocytes and Human Induced Pluripotent Stem Cell-Derived Neurons

2020 ◽  
Vol 7 (2) ◽  
pp. 46 ◽  
Author(s):  
Jann Harberts ◽  
Max Kusch ◽  
John O’Sullivan ◽  
Robert Zierold ◽  
Robert H. Blick

Though patch clamping at room temperature is a widely disseminated standard procedure in the electrophysiological community, it does not represent the biological system in mammals at around 37 °C. In order to better mimic the natural environment in electrophysiological studies, we present a custom-built, temperature-controlled patch clamp platform for upright microscopes, which can easily be adapted to any upright patch clamp setup independently, whether commercially available or home built. Our setup can both cool and heat the platform having only small temperature variations of less than 0.5 °C. We demonstrate our setup with patch clamp measurements at 36 °C on Jurkat T lymphocytes and human induced pluripotent stem cell-derived neurons. Passive membrane parameters and characteristic electrophysiological properties, such as the gating properties of voltage-gated ion channels and the firing of action potentials, are compared to measurements at room temperature. We observe that many processes that are not explicitly considered as temperature dependent show changes with temperature. Thus, we believe in the need of a temperature control in patch clamp measurements if improved physiological conditions are required. Furthermore, we advise researchers to only compare electrophysiological results directly that have been measured at similar temperatures since small variations in cellular properties might be caused by temperature alterations.

2012 ◽  
Vol 17 (9) ◽  
pp. 1264-1272 ◽  
Author(s):  
Alison Haythornthwaite ◽  
Sonja Stoelzle ◽  
Alexander Hasler ◽  
Andrea Kiss ◽  
Johannes Mosbacher ◽  
...  

Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na+ (Nav), Ca2+ (Cav), and K+ (Kv) channels as expected from excitable cells. The Nav current was TTX sensitive, IC50 = 12 ± 6 nM ( n = 5). About 50% of the Cav current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the Kv channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC50 = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABAA receptor antagonist bicuculline, IC50 = 632 ± 149 nM ( n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Nav currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching “excitable cells.” These results show high potential for pluripotent stem cell–derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.


2015 ◽  
Vol 5 (4) ◽  
pp. 660-671 ◽  
Author(s):  
Chukwuma A. Agu ◽  
Filipa A.C. Soares ◽  
Alex Alderton ◽  
Minal Patel ◽  
Rizwan Ansari ◽  
...  

2019 ◽  
Vol 39 ◽  
pp. 101487
Author(s):  
Ratchapong Netsrithong ◽  
Nutchanawan Promnakhon ◽  
Bootsakorn Boonkaew ◽  
Chinnavuth Vatanashevanopakorn ◽  
Kovit Pattanapanyasat ◽  
...  

2012 ◽  
Vol 17 (9) ◽  
pp. 1192-1203 ◽  
Author(s):  
Tadahiro Shinozawa ◽  
Kenichi Imahashi ◽  
Hiroshi Sawada ◽  
Hatsue Furukawa ◽  
Kenji Takami

Human-induced pluripotent stem cell–derived cardiomyocytes (hiPS-CMs) at different stages (approximate days 30, 60, and 90) were used to determine the appropriate stage for functional and morphological assessment of drug effects in vitro. The hiPS-CMs had spontaneous beating activity, and β-adrenergic function was comparable in all stages of differentiation. Microelectrode array analyses using ion channel blockers indicated that the electrophysiological properties of these ion channels were comparable at all differentiation stages. Ultrastructural analysis using electron microscopy showed that myofibrillar structures at days 60 and 90 were similarly distributed and more mature than that at day 30. Analysis of motion vectors in contracting cells showed that the velocity of contraction was the highest at day 90 and was the most mature among the three stages. Gene expression analysis demonstrated that expression of some genes related to myofilament and sarcoplasmic reticulum increased with maturation of morphological and contractile properties. In conclusion, day 30 cardiomyocytes are useful for basic screening such as the assessment of electrophysiological properties, and days 60 and 90 are the appropriate differentiation stage for morphological assays. For the assay of contractile function associated with subcellular components such as sarcoplasmic reticulum, day 90 cardiomyocytes are the most suitable.


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