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Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies

Nature Protocols volume 13, pages 927–945 (2018)Cite this article

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Abstract

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

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Figure 1: Overview of the protocol.
Figure 2: Screening of AP propagation in Kir2.
Figure 3: Example of using hERG expression to vary the AP duration.
Figure 4: Stepwise procedures for the generation of monoclonal Ex-293 line and E-HDFs (Step 24).
Figure 5: Characterization of electrical coupling in engineered cells (Step 25C–E).
Figure 6: Characterization of ion currents and APs in engineered cells (Step 25A).
Figure 7: Characterization of AP propagation in engineered cells (Step 25B) and fine-tuning of conduction velocity with FACS.

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Acknowledgements

We thank M.R. Abraham (John Hopkins University) for Kir2.1 cDNA, A.O. Grant (Duke University) for Nav1.5 cDNA, and K. Irie (Kyoto University) for BacNav cDNA. This work was supported by predoctoral fellowships from the National Science Foundation and the American Heart Association to R.D.K. and H.X.N., respectively, as well as by research grants to N.B. from the American Heart Association (AHA 0530256N) and National Institutes of Health (HL106203, HL104326, HL083342, HL095069, HL104326, HL132389, HL126524, and HL126193).

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  1. Department of Biomedical Engineering, Duke University, Durham, North Carolina, USA

    Hung X Nguyen, Robert D Kirkton & Nenad Bursac

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  1. Hung X Nguyen
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Contributions

H.X.N., R.D.K., and N.B. jointly developed the protocol; H.X.N. and R.D.K. performed the experiments and generated the figures; and H.X.N. and N.B. co-wrote the manuscript.

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Correspondence to Nenad Bursac.

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Nguyen, H., Kirkton, R. & Bursac, N. Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies. Nat Protoc 13, 927–945 (2018). https://doi.org/10.1038/nprot.2018.016

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