Abstract
Cellular therapies have the potential to advance treatment for a broad array of diseases but rely on viruses for genetic reprogramming. The time and cost required to produce viruses has created a bottleneck that constricts development of and access to cellular therapies. Electroporation is a non-viral alternative for genetic reprogramming that bypasses these bottlenecks, but current electroporation technology suffers from low throughput, tedious optimization, and difficulty scaling to large-scale cell manufacturing. Here, we present an adaptable microfluidic electroporation platform with the capability for rapid, multiplexed optimization with 96-well plates. Once parameters are optimized using small volumes of cells, transfection can be seamlessly scaled to high-volume cell manufacturing without re-optimization. We demonstrate optimizing transfection of plasmid DNA to Jurkat cells, screening hundreds of different electrical waveforms of varying shapes at a speed of ~3 s per waveform using ~20 µL of cells per waveform. We selected an optimal set of transfection parameters using a low-volume flow cell. These parameters were then used in a separate high-volume flow cell where we obtained similar transfection performance by design. This demonstrates an alternative non-viral and economical transfection method for scaling to the volume required for producing a cell therapy without sacrificing performance. Importantly, this transfection method is disease-agnostic with broad applications beyond cell therapy.
Graphical abstract
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The data that support the findings of this study are available from the corresponding author, HGC, upon reasonable request.
References
M. Bozza et al., A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells. Sci. Adv. 7(16), 1–14 (2021)
J.P. Brody, P. Yager, R.E. Goldstein, R.H. Austin, Biotechnology at low Reynolds numbers. Biophys. J. 71(6), 3430–3441 (1996)
E. Buzhor et al., Cell-based therapy approaches: the hope for incurable diseases. Regen. Med. 9(5), 14–35 (2014)
B. Byhansonwade, Beacon adoptive cell: The current landscale (2023)
E. Capra, A. Gennari, C. Temps, viral-vector therapies at scale: Today’s challenges and future opportunities. (2022)
M. Cerrano et al., The advent of CAR T-cell therapy for lymphoproliferative neoplasms: integrating research into clinical practice. Front. Immunol. 11, 1–25 (2020)
S. Depil, P. Duchateau, S.A. Grupp, G. Mufti, L. Poirot, ‘Off-the-shelf’ allogeneic CAR T cells: development and challenges. Nat. Rev. Drug Discov. (Nature Research) 19(3), 185–199 (2020)
S. Grupp et al., Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N. Engl. J. Med. 368, 1509–1518 (2013)
T. Ingegnere et al., Human CAR NK cells: A new non-viral method allowing high efficient transfection and strong tumor cell killing. Front. Immunol. 10(APR), 1–10 (2019)
C.H. June, R.S. O’Connor, O.U. Kawalekar, S. Ghassemi, M.C. Milone, CAR T cell immunotherapy for human cancer. Science (American Association for the Advancement of Science) 359(6382), 1361–1365 (2018)
M. Kanduser, D. Miklavcic, M. Pavlin, Mechanisms involved in gene electrotransfer using high- and low-voltage pulses — An in vitro study. Bioelectrochemistry 74(2), 265–271 (2009)
T. Kotnik, G. Pucihar, M. Reberšek, D. Miklavčič, L.M. Mir, Role of pulse shape in cell membrane electropermeabilization. Biochim. Biophys. Acta Biomembr. 1614(2), 193–200 (2003)
T. Kotnik, L. Rems, M. Tarek, D. Miklavcic, Membrane electroporation and electropermeabilization: mechanisms and models. Annu. Rev. Biophys. 48, 63–91 (2019)
B.L. Levine, J. Miskin, K. Wonnacott, C. Keir, Global manufacturing of CAR T cell therapy. Mol. Ther. Methods Clin. Dev. 4(March), 92–101 (2017)
X. Liu et al., Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation. Protein Cell 8(7), 514–526 (2017)
C.A. Lissandrello, et al., High‑throughput continuous‑flow microfluidic electroporation of MRNA into primary human T cells for applications in cellular therapy manufacturing. Sci. Rep. 10, 1–16 (2020)
M.V. Maus, J.A. Fraietta, B.L. Levine, M. Kalos, Y. Zhao, C.H. June, Adoptive immunotherapy for cancer or viruses. Annu. Rev. Immunol. 32, 189–225 (2014)
N.M. Mount, S.J. Ward, P. Kefalas, J. Hyllner, N.M. Mount, Cell-based therapy technology classifications and translational challenges. Philos. Trans. B 370(20150017) (2015)
D. Porter, B. Levine, M. Kalos, A. Bagg, C.H. June, Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365, 725–733 (2011)
S. Rafiq, C.S. Hackett, R.J. Brentjens, Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat. Rev. Clin. Oncol. 17(March), 147–167 (2020)
R.P.T. Somerville, L. Devillier, M.R. Parkhurst, S.A. Rosenberg, M.E. Dudley, Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE ®bioreactor. J. Transl. Med. 10(1), 69 (2012)
M.P. Stewart, R. Langer, K.F. Jensen, Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts. Chem. Rev. 118(16), 7409–7531 (2018)
S.I. Sukharev, V.A. Klenchin, S.M. Serov, L.V. Chernomordik, A. Chizmadzhev Yu, Electroporation and electrophoretic DNA transfer into cells. The effect of DNA interaction with electropores. Biophys. J. 63(5), 1320–1327 (1992)
S. Sun, H. Hao, G. Yang, Y. Zhang, Y. Fu, Immunotherapy with CAR-modified T cells: toxicities and overcoming strategies. J. Immunol. Res. 2018, 1–10 (2018)
J. Teissie, M. Golzio, M.P. Rols, Mechanisms of cell membrane electropermeabilization: A minireview of our present (lack of ?) knowledge. Biochim. Biophys. Acta 1724, 270–280 (2005)
J. VanderBurgh, T. Corso, S. Levy, H. Craighead, Scalable continuous-flow electroporation platform enabling T cell transfection for cellular therapy manufacturing. Sci. Rep. 13 (2023)
J.C.M. Van Der Loo, J.F. Wright, Progress and challenges in viral vector manufacturing. Hum. Mol. Genet. 25, 42–52 (2016)
D.L. Wagner et al., Review: Sustainable clinical development of CAR-T cells – switching from viral transduction towards CRISPR-Cas gene editing. Front. Immunol. 13(June), 1–13 (2022)
J.C. Weaver, Y.A. Chizmadzhev, Theory of electroporation: A review. Bioelectrochemistry Bioenerg. 41, 135–160 (1996)
L. Wong, C. Brampton, D. Woo, E. Dreskin, Optimizing electroporation conditions for high-efficiency mRNA transfection of CD8+ T cells with the gene pulser xcell electroporation system mRNA using the gene pulser xcell electroporation system and gene pulser electroporation. (2020)
Y. Zhan et al., Low-frequency ac electroporation shows strong frequency dependence and yields comparable transfection results to dc electroporation. J. Control. Release 160(3), 570–576 (2012)
Z. Zhang, S. Qiu, X. Zhang, W. Chen, Optimized DNA electroporation for primary human T cell engineering. BMC Biotechnol. 18(1), 1–9 (2018)
Funding
This work was performed in part at the Cornell NanoScale Facility (CNF), a member of the National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation (Grant NNCI-2025233). Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R43GM148147. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Author information
Authors and Affiliations
Contributions
Conceptualization: JAV, GTC, SLL, HGC. Methodology: JAV, GTC, SLL, HGC. Validation: JAV, GTC. Formal analysis: JAV. Investigation: JAV. Writing – original draft: JAV. Writing – review & editing: JAV, GTC, SLL, HGC. Visualization: JAV. Supervision: JAV, SLL, HGC. Project administration: JAV, SLL, HGC. Funding acquisition: JAV, SLL, HGC.
Corresponding author
Ethics declarations
Competing interests
JAV, GTC, and HGC are listed as inventors on patent applications related to the technology presented and JAV, GTC, SLL, and HGC have a financial interest in CyteQuest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
VanderBurgh, J.A., Corso, G.T., Levy, S.L. et al. A multiplexed microfluidic continuous-flow electroporation system for efficient cell transfection. Biomed Microdevices 26, 10 (2024). https://doi.org/10.1007/s10544-023-00692-w
Accepted:
Published:
DOI: https://doi.org/10.1007/s10544-023-00692-w