Abstract
We have recently reported on the high practical utility of using electroporation of clinically relevant cells with sugars for their xeno-free cryopreservation in suspension. This paper extends our earlier approach to in situ electroporating attached cells for their robust cryopreservation on artificial scaffolds. Using CAD modelling, a two-electrode setup has been designed and in-house constructed allowing for simultaneous electroporation in multi-well cell culture plates. Blend electrospinning process has been optimized in order to manufacture porous fibrous mats made of polycaprolactone (PCL, 100 mg/ml) and polylactide (PLA, 50 mg/ml) with an average thickness of 100 µm. Chinese hamster ovary (CHO) cells were grown and directly electroporated in nanofibrous blend electrospun fiber mats. An electric pulse was applied in the presence of propidium iodide and CellTracker Green to determine viable permeabilized cell counts using fluorescence microscopy. Cell recovery was evaluated using metabolic MTS assay 24 h post-electroporation. Electric field intensity and distribution within a 3D reconstructed fiber mat was simulated and visualized using COMSOL software. The results demonstrate that with developed setup it is feasible to electroporate around 80% of attached cells with 80% viability after electroporation when electric field strength was ≥1.7 kV/cm. COMSOL simulations showed local increases of electric field at intersection points of numerous fibers which may in part contribute to the observed drop in cell viability post-electroporation. Future studies anticipate implementation of the developed approach in effective biopreservation of stem cells on electrospun fiber mats as a model of tissue-engineered constructs.
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References
Elliott, G.D., Wang, S., Fuller, B.J.: Cryoprotectants: a review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology 76, 74–91 (2017). https://doi.org/10.1016/j.cryobiol.2017.04.004
Awan, M., Buriak, I., Fleck, R., Fuller, B., Goltsev, A., Kerby, J., Lowdell, M., Mericka, P., Petrenko, A., Petrenko, Y., Rogulska, O., Stolzing, A., Stacey, G.N.: Dimethyl sulfoxide: a central player since the dawn of cryobiology, is efficacy balanced by toxicity? Regen. Med. 15, 1463–1491 (2020). https://doi.org/10.2217/rme-2019-0145
Chatterjee, A., Saha, D., Glasmacher, B., Hofmann, N.: Chilling without regrets: deciphering the effects of cryopreservation on the epigenetic properties of frozen cells will benefit the applications of cryo-technology. EMBO Rep. 17, 292–295 (2016). https://doi.org/10.15252/embr.201642069
Lauterboeck, L., Saha, D., Chatterjee, A., Hofmann, N., Glasmacher, B.: Xeno-free cryopreservation of bone marrow derived multipotent stromal cells from Callithrix jacchus. Biopreserv. Biobank 4(6), 530–538 (2016). https://doi.org/10.1089/bio.2016.0038
Weng, L., Beauchesne, P.R.: Dimethyl sulfoxide-free cryopreservation for cell therapy: a review. Cryobiology 94, 9–17 (2020). https://doi.org/10.1016/j.cryobiol.2020.03.012
Stewart, S., He, X.: Intracellular delivery of trehalose for cell banking. Langmuir 35, 7414–7422 (2019). https://doi.org/10.1021/acs.langmuir.8b02015
Kotnik, T., Rems, L., Tarek, M., Miklavčič, D.: Membrane electroporation and electropermeabilization: mechanisms and models. Annu. Rev. Biophys. 48, 63–91 (2019). https://doi.org/10.1146/annurev-biophys-052118-115451
Katkov, I.: Electroporation of cells in applications to cryobiology: summary of 20-year experience. Probl. Cryobiol. 2, 3–8 (2002)
Dovgan, B., Barlič, A., Knežević, M., Miklavčič, D.: Cryopreservation of human adipose-derived stem cells in combination with trehalose and reversible electroporation. J. Membrane Biol. 250(1), 1–9 (2016). https://doi.org/10.1007/s00232-016-9916-z
Mutsenko, V., Barlič, A., Pezić, T., Dermol-Černe, J., Dovgan, B., Sydykov, B., Wolkers, W.F., Katkov, I.I., Glasmacher, B., Miklavčič, D., Gryshkov, O.: Me2SO- and serum-free cryopreservation of human umbilical cord mesenchymal stem cells using electroporation-assisted delivery of sugars. Cryobiology 91, 104–114 (2019). https://doi.org/10.1016/j.cryobiol.2019.10.002
Brun, P., Dettin, M., Campana, L.G., Dughiero, F., Sgarbossa, P., Bernardello, C., Tosi, A.L., Zamuner, A., Sieni, E.: Cell-seeded 3D scaffolds as in vitro models for electroporation. Bioelectrochemistry 125, 15–24 (2019). https://doi.org/10.1016/j.bioelechem.2018.08.006
Pavšelj, N., Miklavčič, D.: Numerical modeling in electroporation-based biomedical applications. RadiolOncol 42, 159–168 (2008). https://doi.org/10.2478/v10019-008-0008-2
Polajžer, T., Miklavčič, D.: Development of adaptive resistance to electric pulsed field treatment in CHO cell line in vitro. Sci. Rep. 10, 9988 (2020). https://doi.org/10.1038/s41598-020-66879-w
Dermol, J., Miklavčič, D.: Mathematical models describing Chinese Hamster ovary cell death due to electroporation in vitro. J. Membrane Biol. 248(5), 865–881 (2015). https://doi.org/10.1007/s00232-015-9825-6
Brees, C., Fransen, M.: A cost-effective approach to microporate mammalian cells with the Neon Transfection System. Anal. Biochem. 466, 49–50 (2014). https://doi.org/10.1016/j.ab.2014.08.017
Sherba, J.J., Hogquist, S., Lin, H., Shan, J.W., Shreiber, D.I., Zahn, J.D.: The effects of electroporation buffer composition on cell viability and electro-transfection efficiency. Sci. Rep. 10, 3053 (2020). https://doi.org/10.1038/s41598-020-59790-x
Acknowledgements
This work was in part performed in the framework of student exchange by Tobias Pfister whose stay at the University of Ljubljana was sponsored by the Dr. Jürgen and Irmgard Ulderup Foundation. The authors acknowledge technical assistance of the student. Experimental work associated with electropopration and cell culture as well as simulation of electric field distribution was performed in the Infrastructural Center Cellular Electrical Engineering funded by Slovenian Research Agency. Development of the main concept of the current work, design and production of the electrode system, production and characterization of the fiber mats were conducted at the Institute for Multiphase Processes (Leibniz University Hannover).
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Gryshkov, O., Mutsenko, V., Dermol-Černe, J., Miklavčič, D., Glasmacher, B. (2021). Electroporation of Cell-Seeded Electrospun Fiber Mats for Cryopreservation. In: Jarm, T., Cvetkoska, A., Mahnič-Kalamiza, S., Miklavcic, D. (eds) 8th European Medical and Biological Engineering Conference. EMBEC 2020. IFMBE Proceedings, vol 80. Springer, Cham. https://doi.org/10.1007/978-3-030-64610-3_55
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