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Genska elektrotransfekcija na poroznih membranah
ID Vindiš, Tina (Author), ID Rems, Lea (Mentor) More about this mentor... This link opens in a new window

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Abstract
Genska elektrotransfekcija je ena izmed glavnih nevirusnih metod za dostavo plazmidne DNK v notranjost bioloških celic. Ta metoda temelji na pojavu elektroporacije, pri katerem se ob izpostavitvi celic pulzirajočemu električnemu polju začasno poveča prepustnost celičnih membran, kar pa omogoča vnos terapevtskih molekul v celice. Uporaba naprav z nanostrukturiranimi geometrijami, kot so nanoslamice, nanokanali in nanopore, omogoča lokalizacijo električnega polja na majhnih delih celične membrane, s čimer je mogoče znatno povečati učinkovitost genske elektrotransfekcije in izboljšati preživetje celic. Slabosti takšnih nanostrukturiranih naprav so, da niso splošno dostopne in zahtevajo strokovno znanje postopkov nanofabrikacije ter dostop do čistih prostorov. Nedavno pa je bilo dokazano, da je mogoče doseči zelo učinkovito elektrotransfekcijo plazmidov tudi z uporabo komercialnih polikarbonatnih membran z nanoporami polmera 0,05 μm. Zato v tej raziskovalni nalogi preučujem in želim oblikovati pristop elektrotransfekcije, ki temelji na komercialno dostopnih vstavkih s poroznimi membranami iz polietilentereftalata (PET), namenjenim za kulturo in preučevanje biologije celic. Najprej z numeričnim modeliranjem na ravni ene celice določim parametre električnega pulza, velikost por porozne membrane in druge dejavnike, za katere se pričakuje, da bodo povzročili znotrajcelično dostavo plazmidov. Na podlagi numeričnih rezultatov oblikujem preprosto napravo, ki vsebuje vstavek, porozno membrano s porami polmera 0,2 m, 0,5 m ali 1,5 m, večjamično ploščo in par žičnih elektrod. Napravo nato tudi numerično modeliram, da načrtam ustrezno postavitev in konfiguracijo elektrod, ki omogoči homogeno porazdelitev električne napetosti vzdolž porozne membrane. Napravo nato preizkusim na treh celičnih linijah sesalcev in ovrednotim učinkovitost genske transfekcije ter preživetje celic. Pridobljeni rezultati so primerljivi z rezultati klasične genske elektrotransfekcije, pri kateri suspenzijo celic ali celice, pritrjene na podlago, postavimo med par vzporednih elektrod in jih izpostavimo homogenemu električnemu polju. Vendar uporaba poroznih vstavkov omogoča dovajanje nizkonapetostnih pulzov, ki ne zahtevajo uporabe dragih visokonapetostnih elektroporatorjev in je možno pulzni generator izdelati tudi doma. Na koncu diskutiram še o možnostih nadaljnjih raziskav, vezanih na vpliv lastnosti porozne membrane, kot so debelina, poroznost in polmer por, na učinkovitost transfekcije.

Language:Slovenian
Keywords:Genska elektrotransfekcija, elektroporacija, transfekcija, porozna membrana, lokalizirana elektroporacija, numerično modeliranje, ovarijske celice kitajskega hrčka (CHO), mioblasti, celična linija C2C12, celična linija H9C2
Work type:Master's thesis/paper
Organization:FE - Faculty of Electrical Engineering
Year:2022
PID:20.500.12556/RUL-139811 This link opens in a new window
COBISS.SI-ID:120770051 This link opens in a new window
Publication date in RUL:07.09.2022
Views:646
Downloads:101
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Secondary language

Language:English
Title:Gene electrotransfer using porous membranes
Abstract:
Gene electrotransfer is one of the main non-viral methods for intracellular delivery of plasmid DNA, wherein exposure of biological cells to pulsed electric field induces electroporation, that is a transient increase in cell membrane permebility to exogenous molecules. By localizing the electric field on small parts of the cell membrane using nanostructured geometries such as nanostraws, nanochannels, and nanopores, the efficiency of gene electrotransfer and cell viability can be significantly increased. Disadvantages of such nanofabricated devices are that they are not widely available and require nanofabrication expertise and access to cleanroom. It has recently been shown that highly efficient electrotransfer of plasmids can also be achieved using commercial polycarbonate membranes with nanopores of 0.05 μm in radius. Therefore, in this thesis I study and design an electrotransfer approach based on commercially available cell culture inserts with porous membrane from polyethylene terephthalate (PET). First, I use numerical modeling at the single cell level to determine electric pulse parameters, size of pores in the porous membrane, and other factors expected to result in successful gene electrotransfer. Based on the numerical results, I design a simple device that contains an insert with porous membrane containing pores with radius of 0,2 µm, 0,5 µm or 1,5 µm, a multiwell plate, and a pair of wire electrodes. I numerically model the device to improve the placement and configuration of the electrodes, in order to achieve a homogenous voltage along the entire porous membrane. I then test the device using three mammalian cell lines and assess the transfection efficiency and cell viability. The results are comparable to those obtained in previous studies using classical gene electrotransfer, where cells in suspension or adhered to surface are placed between a pair of parallel electrodes and exposed to homogenous electric field. However, the porous membranes enable application of low-voltage pulses, which do not require the use of expensive high-voltage electroporators. Finally, I discuss further research directions to assess the influence of porous membrane properties, such as thickness, porosity, and pore radius on transfection efficiency and cell viability.

Keywords:Gene electrotransfer, electroporation, transfection, cell culture insert, localized electroporation, numerical modeling, Chinese hamster ovary cells (CHO), myoblasts, C2C12 cell line, H9C2 cell line

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