Kinetic Modeling and Numerical Simulation as Tools to Scale Microalgae Cell Membrane Permeabilization by Means of Pulsed Electric Fields (PEF) From Lab to Pilot Plants

Pulsed Electric Fields (PEF) is a promising technology for the gentle and energy efficient disruption of microalgae cells such as Chlorella vulgaris. The technology is based on the exposure of cells to a high voltage electric field, which causes the permeabilization of the cell membrane. Due to the dependency of the effective treatment conditions on the specific design of the treatment chamber, it is difficult to compare data obtained in different chambers or at different scales, e.g., lab or pilot scale. This problem can be overcome by the help of numerical simulation since it enables the accessibility to the local treatment conditions (electric field strength, temperature, flow field) inside a treatment chamber. To date, no kinetic models for the cell membrane permeabilization of microalgae are available what makes it difficult to decide if and in what extent local treatment conditions have an impact on the permeabilization. Therefore, a kinetic model for the perforation of microalgae cells of the species Chlorella vulgaris was developed in the present work. The model describes the fraction of perforated cells as a function of the electric field strength, the temperature and the treatment time by using data which were obtained in a milliliter scale batchwise treatment chamber. Thereafter, the model was implemented in a CFD simulation of a pilot-scale continuous treatment chamber with colinear electrode arrangement. The numerical results were compared to experimental measurements of cell permeabilization in a similar continuous treatment chamber. The predicted values and the experimental data agree reasonably well what demonstrates the validity of the proposed model. Therefore, it can be applied to any possible treatment chamber geometry and can be used as a tool for scaling cell permeabilization of microalgae by means of PEF from lab to pilot scale. The present work provides the first contribution showing the applicability of kinetic modeling and numerical simulation for designing PEF processes for the purpose of biorefining microalgae biomass. This can help to develop new processes and to reduce the costs for the development of new treatment chamber designs.DFG, 414044773, Open Access Publizieren 2019 - 2020 / Technische Universität Berli

MATHEMATICAL MODELING OF MOLECULAR TRANSMEMBRANE TRANSPORT AND CHANGES OF TISSUES´ DIELECTRIC PROPERTIES DUE TO ELECTROPORATION

Visokonapetostni električni pulzi povečajo prepustnost celične membrane (Tsong 1991Weaver 1993Kotnik et al. 2012) skozi pore (Abidor et al. 1979), ki nastanejo na tistih njenih delih, kjer vsiljena transmembranska napetost preseže kritično vrednost (Towhidi et al. 2008Kotnik et al. 2010). Elektroporacija je reverzibilna, če si celica po pulzih opomore, in ireverzibilna, če je škoda preobsežna in celica odmre (Pakhomova et al. 2013bJiang et al. 2015a). Trenutne optične metode por ne morejo zaznati, zato njihov nastanek zaznavamo posredno, bodisi z meritvami vnosa različnih molekul v celice ali z meritvami električnih lastnosti celic (Napotnik in Miklavčič 2017). Uporaba elektroporacije V živilski industriji (Toepfl 2012Toepfl et al. 2014) uporabljamo elektroporacijo oziroma pulzirajoča električna polja (angl. pulsed electric fields), kar je uveljavljen izraz v tej industriji, za uničevanje patogenih organizmov in njihovih produktov (encimov in toksinov). V nasprotju s termično obdelavo hrane električni pulzi ne vplivajo na okus, barvo ali hranilno vrednost. V biotehnologiji uporabljamo elektroporacijo za ekstrakcijo molekul iz mikroorganizmov in rastlin, s čimer se izognemo uporabi kemičnih sredstev in ne uničimo celičnih organelov, torej se izognemo tudi dodatnemu čiščenju končnega produkta (Sack et al. 2010Haberl et al. 2013aMahnič-Kalamiza et al. 2014bKotnik et al. 2015). Primeri: ekstrakcija DNK iz bakterijsladkorja iz sladkorne pese (Haberl et al. 2013b), sokov iz sadjapolifenolov iz grozdja za izboljšanje kvalitete vina (Puértolas et al. 2010)vode pri sušenju zelene biomase, ki služi kot vir za biogorivo (Golberg et al. 2016). Elektroporacija je tudi nova metoda pri zamrzovanju celic in tkiv, angl. cryopreservation (Galindo in Dymek 2016Dovgan et al. 2017). Elektroporacijo uporabljamo tudi v medicini (Miklavčič et al. 2010Yarmush et al. 2014), in sicer pri elektrokemoterapiji (Miklavčič et al. 2012Mali et al. 2013Cadossi et al. 2014Miklavčič et al. 2014Campana et al. 2014Serša et al. 2015), netermičnem odstranjevanju tkiva z ireverzibilno elektroporacijo (Davalos et al. 2005Garcia et al. 2010José et al. 2012Cannon et al. 2013Scheffer et al. 2014bJiang et al. 2015aRossmeisl et al. 2015), genski terapiji (Golzio et al. 2002Vasan et al. 2011Gothelf in Gehl 2012Calvet et al. 2014Heller in Heller 2015Trimble et al. 2015) in vnosu učinkovin v kožo in skoznjo (Denet et al. 2004Zorec et al. 2013b). Pri genski terapiji vnesemo v celice plazmide, v katerih je zapisana sinteza določenega proteina, ki lahko spremeni biološko funkcijo celice (Aihara in Miyazaki 1998Heller in Heller 2015). Z elektroporacijo povišamo varnost genske terapije, saj se izognemo uporabi virusov in kemikalij. Mehanizmi genske terapije z elektroporacijo še niso popolnoma pojasnjeni, osnovni koraki so opisani v literaturi (Rosazza et al. 2016). Z elektroporacijo lahko zlivamo različne celice, s čimer pridobivamo celice, ki proizvajajo monoklonska protitelesa ali inzulin (Ramos in Teissié 2000Trontelj et al. 2008Rems et al. 2013). V doktorski disertaciji sem se osredotočila na uporabo elektroporacije v medicini, predvsem pri elektrokemoterapiji, netermičnem odstranjevanju tkiva z ireverzibilno elektroporacijo in pri vnosu učinkovin v kožo in skoznjo je, zato so ti trije posegi podrobneje opisani v naslednjem poglavju. Medicinski posegi z elektroporacijo – elektrokemoterapija, netermično odstranjevanje tkiva z ireverzibilno elektroporacijo in vnos učinkovin v kožo in skoznjo Elektrokemoterapija je kombinacija kemoterapije in električnih pulzov, dovedenih neposredno na tarčno tkivo. Električni pulzi povečajo prepustnost celične membrane za kemoterapevtike, zato povečamo učinkovitost zdravljenja, obenem pa zmanjšamo dovedeno dozo kemoterapevtika in omilimo stranske učinke. Celoten tumor mora biti pokrit z dovolj visokim električnim poljem, da povečamo prepustnost vseh tumorskih celic (Miklavčič et al. 2006a), zagotoviti pa moramo tudi dovolj visoko koncentracijo kemoterapevtika znotraj tumorja (Miklavčič et al. 2014). Okoliško tkivo ne sme biti uničeno, torej mora biti električno polje okoli tumorja pod mejo za ireverzibilno elektroporacijo. Pri elektrokemoterapiji običajno dovajamo osem pulzov dolžine 100 μs s ponavljalno frekvenco 1 Hz. S poskusi določena meja za povišanje prepustnosti tumorskega tkiva je 0,4 kV/cm (Miklavčič et al. 2010). Osem pulzov je bilo določenih kot optimalno število pulzov (Marty et al. 2006Mir et al. 2006), večje število dovedenih pulzov namreč že zmanjšuje preživetje (Dermol in Miklavčič 2015). Za zdravljenje tumorjev z elektrokemoterapijo so bili definirani standardni postopki (angl. standard operating procedures) (Marty et al. 2006Mir et al. 2006), kjer so glede na število tumorjev, njihovo velikost in lokacijo (na koži ali pod kožo) določeni tip elektrod, kemoterapevtik, anestezija in način dovajanja kemoterapevtika. Kemoterapevtik lahko dovedemo lokalno ali sistemsko. V elektrokemoterapiji oz. terapiji z električnimi pulzi sta najbolj razširjena kemoterapevtika cisplatin in bleomicin. Z elektrokemoterapijo je možno zdraviti tudi globlje ležeče tumorje (Miklavčič et al. 2010Pavliha et al. 2013Edhemović et al. 2014Miklavčič in Davalos 2015). V zadnjem času se uveljavlja tudi uničevanje tumorskih celic z visokimi koncentracijami kalcija in električnimi pulzi (Frandsen et al. 2015Frandsen et al. 2016Frandsen et al. 2017). Pri elektrokemoterapiji se pojavijo še dodatni učinki, ki povišajo učinkovitost elektroporacije. Vazokonstrikcija zmanjša spiranje kemoterapevtika iz tumorja in s tem ohranja visoko koncentracijo kemoterapevtika v tumorju, obenem se zmanjša pretok krvi skozi tumor, kar povzroči hipoksijo in pomanjkanje hranilnih snovi (Mir 2006Serša et al. 2008). Elektrokemoterapija sproži tudi odziv imunskega sistema, ki nato odstrani preostale tumorske celice (Serša et al. 2015). Z ireverzibilno elektroporacijo netermično odstranjujemo tumorje brez uporabe kemoterapevtika (Jiang et al. 2015a). Tako se popolnoma izognemo stranskim učinkom kemoterapevtikov, vendar na račun več dovedene energije in posledično Joulovega gretja. Pri ireverzibilni elektroporaciji dovajamo več (okoli 90) električnih pulzov, dolgih od 50 μs do 100 μs, s ponavljalno frekvenco 1 Hz. Dovedeno električno polje je v rangu nekaj kV/cm, kar je dosti več kot pri elektrokemoterapiji. Pri ireverzibilni elektroporaciji lahko z visoko natančnostjo odstranimo želeno tkivo – območje med uničenim in nepoškodovanim tkivom je široko le nekaj premerov celic (Rubinsky et al. 2007). Za odstranjevanje tumorjev tradicionalno uporabljamo termične metode (Hall et al. 2014) – radiofrekvenčno odstranjevanje in odstranjevanje s tekočim dušikom, kjer tkivo uničujemo z visoko oz. z nizko temperaturo. Prednost ireverzibilne elektroporacije pred uveljavljenimi termičnimi metodami je krajši čas zdravljenja, izognemo se učinkom hlajenja oz. gretja tkiva zaradi bližine žil (Golberg et al. 2015), pri čemer ostanejo okoliške pomembne strukture (žile, živci) nedotaknjene (Jiang et al. 2015a). Tudi pri ireverzibilni elektroporaciji je v dokončno odstranitev tumorskih celic vpleten imunski sistem (Neal et al. 2013). Pri elektrokemoterapiji in ireverzibilni elektroporaciji se zaradi daljših pulzov in ponavljalne frekvence 1 Hz pojavljajo težave zaradi krčenja mišic (Miklavčič et al. 2005), bolečine med dovajanjem pulzov, heterogenosti električnih lastnosti tkiv v tem frekvenčnem področju ter zaradi možnosti srčnih aritmij (Ball et al. 2010). Bolečini in krčenju mišic se lahko izognemo, če pulze dovajamo z višjo frekvenco, npr. 5 kHz (Županič et al. 2007Serša et al. 2010). Srčnim aritmijam se izognemo tako, da s sinhroniziramo dovedene električne pulze z električno aktivnostjo srčne mišice (Mali et al. 2008Deodhar et al. 2011aMali et al. 2015). Bolečini, krčenju mišic in heterogenosti električnih lastnosti tkiv se lahko izognemo z dovajanjem 1 μs bipolarnih pulzov (Arena et al. 2011Arena in Davalos 2012Sano et al. 2015). V zadnjem času so se pojavile tudi metode, s katerimi so vnos barvil v celico dosegli brezkontaktno s t. i. magnetoporacijo (Chen et al. 2010Towhidi et al. 2012Kardos in Rabussay 2012Novickij et al. 2015Kranjc et al. 2016Novickij et al. 2017bNovickij et al. 2017a). Elektroporacijo lahko uporabljamo ne le za zdravljenje tumorjev, temveč tudi za vnos učinkovin v kožo in skoznjo. Vnos učinkovin skozi kožo je neinvaziven, poleg tega pa se izognemo degradaciji učinkovin pri prehodu skozi prebavni trakt. Skozi kožo lahko preide le malo molekul, zato uporabljamo različne metode za povečanje prehoda učinkovin – iontoforezo, radiofrekvenčno mikroablacijo, laser, mikroigle, ultrazvok in elektroporacijo (Zorec et al. 2013b). Proces elektroporacije kože je slabo razumljen. Predpostavljamo, da pri dovajanju visokonapetostnih električnih pulzov v roženi plasti nastanejo lokalna transportna območja, kjer sta povišani električna prevodnost in prepustnost (Pliquett et al. 1996Pliquett et al. 1998Pliquett et al. 1998Pavšelj in Miklavčič 2008a). Skozi lokalna transportna območja lahko nato učinkovine še nekaj ur po dovedenih pulzih vstopajo skozi kožo v krvni obtok (Zorec et al. 2013a). Gostota teh območij je odvisna od električnega polja v koži – višje električno polje jih povzroči več. Velikost lokalnih transportnih območij je odvisna od trajanja pulza. Med samim pulzom se zaradi Joulovega gretja topijo lipidi v roženi plasti, kar povzroči njihovo širjenje (Pliquett et al. 1996Prausnitz et al. 1996Pliquett et al. 1998Weaver et al. 1999Vanbever et al. 1999Gowrishankar et al. 1999b). Načrtovanje posegov elektrokemoterapije in netermičnega odstranjevanja tkiva z ireverzibilno elektroporacijo Pri zdravljenju tumorjev z elektroporacijo lahko uporabimo standardne oblike in postavitve elektrod z že določenimi parametri električnih pulzov (Marty et al. 2006Mir et al. 2006Campana et al. 2014). Če zdravimo velike tumorje ali tumorje nepravilnih oblik, ki pogosto ležijo globlje, s standardno postavitvijo elektrod ne moremo zagotoviti ustrezne pokritosti tumorja z dovolj visokim električnim poljem. V tem primeru lahko elektrode med samim posegom večkrat premaknemo ali pa prilagodimo njihovo število in postavitev. Pri tem moramo prej pripraviti načrt posega (Kos et al. 2010Miklavčič et al. 2010Pavliha et al. 2012Linnert et al. 2012Edhemović et al. 2014). V njem zagotovimo, da bo cel tumor izpostavljen dovolj visokemu električnemu polju (Miklavčič et al. 2006a), obenem pa škoda na okoliškem tkivu minimalna. Načrtovanje posega poteka v več korakih: 1. zajem medicinskih slik (računalniška tomografija, magnetna resonanca) tumorja in okoliškega tkiva2. obdelava slik3. razgradnja slik in določitev geometrije tkiva4. vzpostavitev tridimenzionalnega modela5. optimizacija postavitve elektrod glede na obliko in velikost tumorja6. izdelava modela elektroporacije (izračun električnega polja in spremembe električne prevodnosti tkiva)7. optimizacija napetosti med elektrodami in položaja elektrod (Pavliha et al. 2012). Na sliki 1 lahko vidimo izračunano električno polje v tumorju in okoliškem tkivu pri eni izmed možnih postavitev elektrod.Electroporation is a phenomenon, which occurs when short high voltage pulses are applied to cells and tissues resulting in a transient increase in membrane permeability or cell death, presumably due to pore formation. If cells recover after pulse application, this is reversible electroporation. If cells die, this is irreversible electroporation. Electroporation is used in biotechnology for biocompound extraction and cryopreservation, in food processing for sterilization and pasteurization of liquid food and in medicine for treating tumors by electrochemotherapy or irreversible electroporation as an ablation technique, for gene electrotransfer, transdermal drug delivery, DNA vaccination, and cell fusion. In electroporation-based medical treatments, we can treat tumors with predefined electrode geometry and parameters of electric pulses. When we treat larger tumors of irregular shape treatment plan of the position of the electrodes and parameters of the electric pulses has to be calculated before each treatment to assure coverage of the tumor with a sufficient electric field. In treatment plans, currently, 1) we assume that above an experimentally determined critical electric field all cells are affected and below not, although, in reality, the transition between non-electroporated and electroporated state is continuous. 2) We do not take into account the excitability of some tissues. 3) The increase in tissues’ conductivity is described phenomenologically and does not include mechanisms of electroporation. 4) Transport of chemotherapeutics into the tumor cells in electrochemotherapy treatments is not included in the treatment plan although it is vital for a successful treatment. We focused on the mathematical and numerical models of electroporation with the aim of including them in the treatment planning of electroporation-based medical treatments. We aimed to model processes happening during electroporation of tissues, relevant in the clinical procedures, by taking into account processes happening at the single cell level. First, we used mathematical models of cell membrane permeability and cell death which are phenomenological descriptions of experimental data. The models were chosen on the basis of the best fit with the experimental data. However, they did not include mechanisms of electroporation, and their transferability to tissues was questionable. We modeled time dynamics of dye uptake due to increased cell membrane permeability in several electroporation buffers with regard to the electrosensitization, i.e., delayed hypersensitivity to electric pulses caused by pretreating cells with electric pulses. We also modeled the strength-duration depolarization curve and cell membrane permeability curve of excitable and non-excitable cell lines which could be used to optimize pulse parameters to achieve maximal drug uptake at minimal tissue excitation. Second, we modeled change in dielectric properties of tissues during electroporation. Model of change in dielectric properties of tissues was built for skin and validated with current-voltage measurements. Dielectric properties of separate layers of skin before electroporation were determined by taking into account geometric and dielectric properties of single cells, i.e., keratinocytes, corneocytes. Dielectric properties of separate layers during electroporation were obtained from cell-level models of pore formation on single cells of lower skin layers (keratinocytes in epidermis and lipid spheres in papillary dermis) and local transport region formation in the stratum corneum. Current-voltage measurements of long low-voltage pulses were accurately described taking into account local transport region formation, pore formation in the cells of lower layers and electrode polarization. Voltage measurements of short high-voltage pulses were also accurately described in a similar way as with long low-voltage pulseshowever, the model underestimated the current, probably due to electrochemical reactions taking place at the electrode-electrolyte interface. Third, we modeled the transport of chemotherapeutics during electrochemotherapy in vivo. In electrochemotherapy treatments, transport of chemotherapeutics in sufficient amounts into the cell is vital for a successful treatment. We performed experiments in vitro and measured the intracellular platinum mass as a function of pulse number and electric field by inductively coupled plasma – mass spectrometry. Using the dualporosity model, we calculated the in vitro permeability coefficient as a function of electric field and number of applied pulses. The in vitro determined permeability coefficient was then used in the numerical model of mouse melanoma tumor to describe the transport of cisplatin to the tumor cells. We took into account the differences in the transport of cisplatin in vitro and in vivo caused by the decreased mobility of molecules and decreased membrane area available for the uptake in vivo due to the high volume fraction of cells, the presence of cell matrix and close cell connections. Our model accurately described the experimental results obtained in electrochemotherapy of tumors and could be used to predict the efficiency of electrochemotherapy in vitro thus reducing the number of needed animal experiments. In the thesis, we connected the models at the cell level to the models at the tissue level with respect to cell membrane permeability and depolarization, cell death, change in dielectric properties and transport. Our models offer a step forward in modeling and understanding electroporation at the tissue level. In future, our models could be used to improve treatment planning of electroporation-based medical treatments

An Electrically Active Microneedle Electroporation Array for Intracellular Delivery of Biomolecules

The objective of this research is the development of an electrically active microneedle array that can deliver biomolecules such as DNA and drugs to epidermal cells by means of electroporation. Properly metallized microneedles could serve as microelectrodes essential for electroporation. Furthermore, the close needle-to-needle spacing of microneedle electrodes provides the advantage of utilizing reduced voltage, which is essential for safety as well as portable applications, while maintaining the large electric fields required for electroporation. Therefore, microneedle arrays can potentially be used as part of a minimally invasive, highly-localized electroporation system for cells in the epidermis layer of the skin. This research consists of three parts: development of the 3-D microfabrication technology to create the microneedle array, fabrication and characterization of the microneedle array, and the electroporation studies performed with the microneedle array. A 3-D fabrication process was developed to produce a microneedle array using an inclined UV exposure technique combined with micromolding technology, potentially enabling low cost mass-manufacture. The developed technology is also capable of fabricating 3-D microstructures of various heights using a single mask. The fabricated microneedle array was then tested to demonstrate its feasibility for through-skin electrical and mechanical functionality using a skin insertion test. It was found that the microneedles were able to penetrate skin without breakage. To study the electrical properties of the array, a finite element simulation was performed to examine the electric field distribution. From these simulation results, a predictive model was constructed to estimate the effective volume for electroporation. Finally, studies to determine hemoglobin release from bovine red blood cells (RBC) and the delivery of molecules such as calcein and bovine serum albumin (BSA) into human prostate cancer cells were used to verify the electrical functionality of this device. This work established that this device can be used to lyse RBC and to deliver molecules, e.g. calcein, into cells, thus supporting our contention that this metallized microneedle array can be used to perform electroporation at reduced voltage. Further studies to show efficacy in skin should now be performed.Ph.D.Committee Chair: Mark G. Allen; Committee Member: Mark R. Prausnitz; Committee Member: Oliver Brand; Committee Member: Pamela Bhatti; Committee Member: Shyh-Chiang She

Electromanipulation of Ellipsoidal Cells in Fluidic Micro-Electrode Systems

Recently, electromanipulation technologies for handling and characterizing individual cells or particles have been applied to lab-on-chip devices. These devices play a role in pharmacological and clinical applications as well as environmental and nanotechnologies. Electromanipulation of ellipsoidal cells in fluidic micro-electrode systems has been studied by numerical simulations, theoretical analysis and experiment. The field distributions in electrorotation chip chambers were analyzed using numerical field simulations in combination with analytical post-processing. The optimal design for two-dimensional electrorotation chips features electrodes with pyramidal rounded tips. Moreover, the three-dimensional electric field distributions in the electroporation and electrorotation chambers were analyzed. The advantage of electroporation chip chambers is to avoid strongly increasing temperatures after pulse application. New chips may be developed for nanoscale applications in the future. New simplified analytical equations have been developed for the transmembrane potential (delta_phi) induced in cells resembling ellipsoids of rotation, i.e. spheroids, by homogeneous DC or AC fields. The new equations avoid the complicated description by the depolarizing factors. Also the dielectrophoretic force expression for spheroidal objects has been simplified. Furthermore, the effects of cell orientation and electric field frequency on the delta_phi induced in ellipsoidal cells were studied. Simplified equations were derived. They show that the membrane surface points for the maximum of delta_phi depend on cell shape, cell orientation, electric cell parameters and field frequency. The theoretical results were compared to electropermeabilization experiments with chicken red blood cells. Experiments confirmed that equations for the transmembrane potential were advantageous for describing the transmembrane potential induced in arbitrarily oriented ellipsoidal cells.In letzter Zeit sind Elektromanipulations-Technologien für die Manipulation und die Charakterisierung von einzelnen Zellen oder Partikeln in Lab-on-Chip Systeme integriert worden. Die neuen Systeme spielen eine Rolle in pharmakologischen und klinischen Anwendungen sowie in Umwelt- und Nanotechnologien. Die Elektromanipulation von ellipsoiden Zellen in fluidischen Mikro-Elektrodensystemen wurde mit Hilfe numerischer Simulation, theoretischer Analyse sowie Experimenten beschrieben. Die Feldverteilung in Elektrorotationskammern wurde mit numerischen Simulationen analysiert und optimert. Als geeignetes Elektrodendesign in zweidimensionalen Elektrorotationskammern erwiesen sich pyramidale, abgerundete Elektrodenspitzen. Zusätzlich wurden die drei-dimensionalen Feldverteilungen in den Elektroporations- und Elektrorotationskammern analysiert, um starke Temperaturerhöhungen durch den elektrischen Puls zu vermeiden. Mit diesen Ergebnissen könnten neue Chips für Anwendungen im Nanometerbereich entwickelt werden. Neue und vereinfachte analytische Gleichungen für das Transmembranpotential (delta_phi), welches in einem homogenen Gleich- oder Wechselfeld in Zellen ähnlich Rotationsellipsoiden, d.h. Spheroide, induziert wird, wurden unter Vermeidung der Depolarisierungsfaktoren hergeleitet. Ebenso wurde die Gleichung für die dielektrophoretische Kraft auf spheroide Objekte vereinfacht, sowie die Effekte von Zellorientierung und Frequenz des Wechselfeldes auf das delta_phi von ellipsoiden Zellen untersucht und vereinfachte Gleichungen abgeleitet. Sie zeigen, dass die Membranpunkte mit maximalem delta_phi abhängig sind von der Zellform, der Zellorientierung, den elektrischen Eigenschaften der Zelle und der Frequenz des Wechselfeldes. Die theoretischen Ergebnisse wurden mit Experimenten zur Elektropermeabilität von Hühnererythrozyten verglichen, die bestätigten, dass die vereinfachten Gleichungen das in beliebig orientierten elliptischen Zellen induzierte Transmembranpotential richtig beschreiben

Different sensitivity of normal and tumor cells to pulsed radiofrequency exposure

The effect of nanosecond radiofrequency pulses (nsRF) on tumor and normal cells has been studied. To determine the viability of cells, an MTT test was used, as well as a real time system for analyzing cell cultures-iCELLigence. It has been shown that ns RF pulses under certain combinations of operating conditions reduce cell proliferation of both tumor and normal cells. Double exposure to 1000 pulses leads to the most effective inhibition of tumor cell proliferation and was 40% after 5 days. Inhibition of the proliferative activity of normal cells was 10% and was maximum after 3 days, then cell growth resumed. The results obtained allow to consider ns RF pulses with different parameters as a promising effective factor for controlling cellular processes for biomedical purposes

Discrimination of different cell monolayers before and after exposure to nanosecond pulsed electric fields based on Cole-Cole and multivariate analysis

Normal and cancer cells, which were grown in monolayers, were investigated and discriminated by electrical bioimpedance spectroscopy (EBIS) before and after exposures to nanosecond pulsed electric fields (nsPEFs). Bioimpedance data were analysed with a Cole-Cole model and the principal component analysis (PCA). Normal and cancer cells could be clearly distinguished from each other either from Cole parameters (R 0, a, t) or from two dominant principal components. The trend of changes for Cole parameters indicated distinctively different post-nsPEF-effects between normal and cancer cells. PCA was also able to distinguish characteristic impedance spectra 30 min after exposures. The first principal component suggested that post-nsPEF-effects for normal cells were revealed especially at lower frequencies. The results indicated further that the extracellular resistance, which is dominated by cell-cell connections, might be an important factor with respect to selective nsPEF-effects on cancer cells that are organized in a monolayer or a tissue, respectively. Accordingly, the results support the application of EBIS as an early, non-invasive, label-free, and time-saving approach for the classification of cells to provide in particular predictive information on the success of cancer treatments with nsPEFs. © 2019 IOP Publishing Ltd