Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy Georg Linz,Suzana Djeljadini,Lea Steinbeck,Gurbet Köse,Fabian Kiessling,Matthias Wessling Highlights Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy Georg Linz,Suzana Djeljadini,Lea Steinbeck,Gurbet Köse,Fabian Kiessling,Matthias Wessling •Four-electrode electrical impedance spectroscopy in transwell inlets •Subsequent electrical equivalent circuit ﬁtting for barrier characterization •Resistance and capacitance give insight into cellular barrier behavior Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy Georg Linza,b, Suzana Djeljadinia,b, Lea Steinbecka,b, Gurbet Kösec, Fabian Kiesslingcand Matthias Wesslinga,b,< aDWI - Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52056 Aachen, Germany bRWTH Aachen University, Aachener Verfahrenstechnik-Chemical Process Engineering, Forckenbeckstrasse 51, 52074, Aachen, Germany cInstitute for Experimental Molecular Imaging, Faculty of Medicine, RWTH Aachen University, 52074 Aachen, Germany ARTICLE INFO Keywords : Caco-2 membrane capacitance electricalimpedancespectroscopy(EIS) sonoporation transepithelialelectricalresistance(TEER) transwell insertABSTRACT Wedescribeanimpedance-basedmethodforcellbarrierintegritytesting. Afour-electrodeelectrical impedancespectroscopy(EIS)setupcanberealizedbysimplyconnectingacommercialchopstick-like electrode (STX-1) to a potentiostat allowing monitoring cell barriers cultivated in transwell inserts. Subsequentelectriccircuitmodelingoftheelectricalimpedanceresultsthecapacitivepropertiesofthe barriernexttothewell-knowntransepithelialelectricalresistance(TEER).Theversatilityofthenew methodwasanalyzedbytheEISanalysisofaCaco-2monolayerinresponseto(a)diﬀerentmembrane coating materials, (b) two diﬀerent permeability enhancers ethylene glycol-bis(2-aminoethylether)- N,N,N’,N’-tetraacetic acid (EGTA) and saponin, and (c) sonoporation. For the diﬀerent membrane coating materials, the TEERs of the standard and new protocol coincide and increase during cul- tivation, while the capacitance shows a distinct maximum for three diﬀerent surface materials (no coating, Matrigel®, and collagen I). The permeability enhancers cause a decline in the TEER value, butonlysaponinaltersthecapacitanceofthecelllayerbytwoordersofmagnitude. Hence,celllayer capacitance and TEER represent two independent properties characterizing the monolayer. The use of commercial chopstick-like electrodes to access the impedance of a barrier cultivated in transwell inserts enables remarkable insight into the behavior of the cellular barrier with no extra work for the researcher. This simple method could evolve into a standard protocol used in cell barrier research. 1. Introduction Cellbarriersforminterfacesbetweentissuecompartments and control the exchange of substances. Tight cell contacts limit the unhindered diﬀusion between compartments and bring single cells to act as a barrier. Cell-cell connections are formed by diﬀerent protein complexes, with the most prominent representative called tight junctions. They also regulate the paracellular transport and limit the diﬀusion of moleculesandionsthroughintercellularspaces(Tsukitaetal., 2001). These protein complexes enable the polarization of cellsbydiﬀerentcompositionsoflipidsandtransmembrane proteins, leading to the formation of the apical and basolat- eral sides (Ebnet, 2015). Passing a cell barrier is crucial for manybiologicalprocessesandamajorchallengeindrugde- livery (Benson et al., 2013). Research has so far focused on developing the in-vitro modelsofvariouscellbarriers. Amongothers,barriermod- els for lungs (Hermanns et al., 2004), intestinal (Hidalgo et al., 1989), and blood-brain (Czupalla et al., 2014) have beendescribed. Acommonmethodfor in-vitroexperiments withbarrier-formingcellsistocultivatethecellsonaremov- ableporousmembranesupportforastandardwellplate. The removable insert allows access to the apical and basolateral sides (Benson et al., 2013). Monitoring the development and integrity of cell barri- ersduringmaturationandexperimentsiscrucialforallstud- <Corresponding author, Phone: +49-241-8095470, Fax: +49-241- 8092252, Manuscripts.cvt@avt.rwth-aachen.de, Forckenbeckstrasse 51, 52074 Aachen, Germanyiesperformedonbarriers. Methodsforaccessingthebarrier permeabilityarebasedonthetransportoftracersubstances, e.g.,mannoseorﬂuorescentdyes,immunoﬂuorescentstain- ingofproteinsrelatedtothetightjunctioncomplex,ormea- surement of the electrical resistance (Yeste et al., 2018). In this article, we focus on the latter considering that electric resistance is noninvasive, comparably easy to perform, and the most widely used method (Srinivasan et al., 2015). In particular, we focus on an important extension of the current methodology: by using the complex impedance of aTEERmeasurementmoredetailedinformationcanbeob- tained about the various contributions to the barrier devel- opment. 2. Background 2.1. TEER Electrical resistance rises from the high transport resis- tance of ions through a cell barrier and can, therefore, be usedasameasureoftheintegrityofabarrier. TheTEERis often measured with a chopstick-like electrode (Yeste et al., 2018). The standard method for measuring TEER is to connect the chopstick-like electrode with an Epithelial voltohmme- ter. The voltohmmeter imposes a square-wave current of 10Awith12.5polarityreversalseverysecondbetweenthe current-injecting electrodes. The two voltage pick-up elec- trodes on the inside of the sticks measure the potential be- tween the two sides. Resistance is determined by applying Ohm’s law. This direct current method measures the resis- Linz et al.: Preprint submitted to Elsevier Page 1 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy Figure 1: Graphic B displays the experimental setup of a chopstick-like electrode inserted in a transwell insert (B). When using the chopstick-like electrode with an endothelial voltohmmeter (standard method), the cell layer resistance is gained by applying a constant current and recording the resulting potential (A). The resistance can be calculated with Ohm’s law and is the sum of the media resistance RMediaand the cell layer resistance RTEER(D). In the proposed method, the input signal is changed to an alternating current signal with varying frequencies (C). By equivalent electric circuit ﬁtting cell layer resistances ( R1;R2) and capacitances ( C1;C2) are extracted from the obtained impedance data. The ﬁtted impedance data leads to direct determination between media and cell layer resistance and additional cell layer capacitance data (E). tances of the medium, the ﬁlter, and the cell layer in sum. The course of the input and output signal is described in 1 A)andanequivalentelectricalcircuitisdepictedinD).Cell layerresistancecanbeobtainedbysubtractingtheresistance of a sample without cells from that of a sample with cells. The result is highly susceptible to wrong positioning of the electrode. Therefore,carefulpositioningoftheelectrodesis required (Srinivasan et al., 2015). 2.2. Impedance By changing the input signal from direct to alternating currents with varying frequencies, many limitations of di- rect current resistance methods can be overcome. Ohm’s lawdeﬁnesthequotientofvoltageandcurrentasresistance. Analogous to Ohm’s law for direct current, impedance Z is deﬁned as Z=Ut_Itas the time-dependent quotient of voltageUtand currentItfor alternating current. Therefore, thefrequency-dependentelectricalresistanceofcellbarriers becomesaccessible. Currentandvoltagecharacteristicscan be described by sinusoidal functions: Ut=U0sin.!t+/ It=I0sin.!t/:(1) WhereU0andI0are the amplitudes, !is the radial fre- quency, andis the phase shift by which the current lags the voltage.The impedance has a magnitude of Z0=U0_I0and by Euler’s relationship, the impedance can be expressed as a complex number. Z=U.t/ I.t/=Z0ei=Z0.cos+jsin/=Z¨+jZ¨¨(2) Hence, the impedance can be expressed as a complex num- ber wherej=ù.*1/,Z¨is the real part, and Z¨¨is the imaginarypartoftheimpedance. Therealpartoftheimpe- dance represents the resistance to current ﬂow and the im- aginary part of the capability of a system to store electrical energy. Impedance data measured at a range of frequencies can be used to derive the physical and structural properties of a cell barrier by representing them with equivalent circuit models. Combining several diﬀerent equivalent circuit ele- mentsinparallelorseriesformssuchamodel. Twostandard circuit elements are predominantly used for modeling. The ﬁrst one is the frequency-independent resistance ( ZR=R). The second equivalent circuit element is an ideal capacitor, whose impedance is frequency-dependent and can be ex- pressed asZC=1_j!C, whereCis the capacitance. Thebasicelementscanbecombinedtoﬁttheimpedance responseofanelectrochemicalsystem. AccordingtoKirch- hoﬀ’s law, the total impedance of two impedances that are Linz et al.: Preprint submitted to Elsevier Page 2 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy connectedinseriescanbecalculatedbythesumoftheindi- vidual impedances ( Z=Z1+Z2). In case two impedance elementsaresetinparallel,theinverseofthetotalimpedance is the sum of the inverse of the individual impedances (1_Z=1_Z1+1_Z2) (Orazem and Tribollet, 2017). 2.3. Cell barrier impedance A cell barrier posseses a capacitive and resistive char- acter. The capacitive eﬀects rise from the impermeability of charged species through the phospholipid membrane of cells. The conductivity of the cell membrane is 107times lower compared with the extracellular medium (Kotnik and Miklav/uni010Di/uni010D,2000). Whensinglecellsstarttoconnectthrough tight junctions, they control the ionic ﬂux across the inter- cellular space, and form a barrier. The capacitance of a cell membrane is approximately 1 F/cm2(Cole, 1968) but can appear much larger when the cell membrane is folded (Lo et al., 1995). In addition, the composition (Wegener et al., 2004)andthecelllayerthickness(VanDriesscheetal.,1999) inﬂuence the capacity. The biological meaning of the cell layercapacitancecan,therefore,berelatedtomorphological characteristics such as microvilli formation or other mem- branealterations(Czupallaetal.,2014). Athighfrequencies (>10kHz),theimpedanceofthecelllayerreacheszero,be- cause of the capacitively coupled pathway through the cell barrier. Theimpedanceathighfrequenciesonlyresultsfrom the media and ﬁlter resistance. Highelectricalresistancerepresentsastrong,intactbar- rierthatcontrolstheﬂuxofsubstances(Groeberetal.,2015). Ifstronglypolarizedcellsaremeasured,thesurfaceareaand protein concentration may diﬀer from the apical to basolat- eral sides, which leads to diﬀerent capacitances for the api- cal and basolateral cell membrane. The resistances and ca- pacitancescanbeusedtodrawconclusionsaboutbiological processes in and across the barrier. For accessing the electrical impedance, a small electri- calpotentialorcurrentacrossthecelllayermustbecreated. Therefore, at least two electrically conductive electrodes on eachsideofthecellbarrierareneeded. Inafrequencyrange between 10 Hz and 1 MHz, the impedance of the media and cell support is frequency independent. However, the electrode-electrolyte interface forms a frequency dependent electrodepolarizationimpedance,whichdisturbsacellbar- riermeasurement. Therefore,theelectrode-electrolyteimpe- dancecontributionmustbeconsideredintheequivalentelec- triccircuitmodeling,orthecurrent-carryingelectrodemust be separated from the potential sensing electrode. The lat- tererasestheimpedancecontributionofthecurrent-carrying electrodesandthereforesimplifytheequivalentelectriccir- cuit(Schwan,1966). Thecommerciallyavailablesystemfor measuring EIS in transwell inserts CellZscope® (nanoAnalytics,Münster,Germany)usestwoﬁxedsteelelec- trodes and considers the contribution of the electrode sur- face. Other groups integrated the four-electrode EIS setup viaﬁxedwires(Onnelaetal.,2012)orasrecentlypresented by Nikulin et al. an approach where a chopstick-like elec- trodeisconnectedtoanimpedancegenerator(Nikulinetal.,2019). In this work we demonstrate the powerful combina- tionofachopstick-likeelectrodeandEISinthreesetsofex- periments. In all experiments the colon carcinoma cell line (Caco-2) was used, which reassembles the small intestine barrier (Hidalgo et al., 1989): 1. Wecomparedthestandardepithelialvoltohmmethod forTEERmeasurementwiththeEISmethodbymon- itoring the development of a cell layer. The electri- cal impedance of a Caco-2 barrier, which was culti- vated in diﬀerent coated transwell inserts, was ana- lyzed over twelve consecutive days and subsequently the capacitance and resistance extracted by electrical circuit modeling. 2. Theresponseofafullydevelopedcellbarriertodiﬀer- entchemicalswasmonitoredwiththeEIS,andthecell layer resistance and capacitance were used to charac- terizetheeﬀectsofthesubstances. Thecomplexbio- chemicalprocessesoftheCaco-2layeraftertheaddi- tion of forskolin to the media were monitored. Fors- kolin is known to shorten the brush border microvilli (Rousset et al., 1985) and increase the apical HCO* 3secretion (Laohapitakworn et al., 2011) among other complex biochemical reactions related to a high con- centration of the second messenger cyclic adenosine monophosphate (cAMP). 3. TheeﬀectofsonoporationwasmonitoredbyEIS.Dur- ingsonoporation,ultrasoundisusedtooscillateorde- stroy gas-ﬁlled microbubbles, which temporarily in- crease cell membrane permeability, and thus is a pro- misingtooltotransportsubstancesacrossbarriers(Sni- pstadetal.,2018). Thepenetrationofcellbarrierscan beinvestigatedviatheimpedance-deriveddataTEER and capacitance (Kooiman et al., 2009; Lelu et al., 2016; Stewart et al., 2018). EIS can be performed during the sonoporation procedure, which gives on- line information of the cell barrier disintegration. In long term experiments the recovery of the sonopora- tionofthecellbarriercanbemeasuredbyEISaswell. 3. Materials and methods 3.1. Cultivation Caco-2cellline,mediumcomponents,collagenI,Matri- gel®, and TEER enhancers were all purchased from Sigma Aldrich. Eagle’s minimum essential medium (EMEM) was prepared according to the manufacturer, and supplemented with 2 mM glutamine, 1% non-essential amino acids, 1% penicillin/streptomycin,and10%fetalbovineserum. Forthe reported experiments, cell passage 71 was used. The cells wereseededwithacelldensityof 105cells/cm2in24-trans- well ﬁlter (Polyester, 0.4 m pore size, Corning Inc., Corn- ing, NY, USA). The day before seeding, the inserts were coated with collagen I or Matrigel®for cell adhesion and growth. Finalconcentrationsof50 g/mLforcollagenIand 250g/mL for Matrigel®were prepared with sterile dem- ineralized water. A total of 50 L of the corresponding di- luted solution was applied on each insert and incubated at Linz et al.: Preprint submitted to Elsevier Page 3 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy 37ýC for at least 4 h. Afterward, the coating solution was discarded, and the inserts were kept in EMEM medium at 37ýC until cell seeding. The cells were seeded and cul- tivated in 0.2 ml of cell culture medium with 1 ml of the samemediuminthebasolateralcompartmentandincubated at 37ýC and 5% CO2. The medium was exchanged every day. 3.2. Chopstick-like electrode preparation RemovingtheRJ-11plugandconnectingthefourwires fromthechopstick-likeelectrodeSTX-1(Merck,Darmstadt, Germany) to a Versastat 3F potentiostat galvanostat (AME- TEK, Berwyn, PA, USA) upgrades the chopstick-like elec- trode to a four-electrode electrical impedance spectroscopy setup. On the inside of each tip, a silver/silver chloride pel- let measures the voltage and is connected to the working sense and reference electrode of the potentiostat. The outer electrodes were connected to the working and counter elec- trodes. If not in use, the chopstick-like electrode was stored in phosphor buﬀered saline (PBS) with the two reference electrodesinshortcircuittoallowtheelectrodepairtoequi- librate and limit the potential diﬀerence. The working pro- cedure regarding cell culture experiments was equal to the standardTEERmeasurementwithchopstick-likeelectrodes in transwell inserts and was described by the manufacturer of chopstick-like electrodes. In short, the electrodes were sterilized with 70% ethanol and dipped afterward in fresh cell culture media to remove the excess ethanol. The cells grown in transwell inserts were taken out of the incubator and placed in a sterile workbench. The chopstick-like elec- trodes were immersed in the transwell with the short tip on the inside and the long tip outside of the insert. The EIS measurements were performed in galvanostatic mode with anamplitudeof10 Aandatafrequencyrangeof10Hzto 100kHzwithtenprobesperdecade. RepeatedEISmeasure- mentsshowednoinﬂuenceontheimpedanceresponseofthe celllayer,whichledtotheconclusionthatthedesignedEIS measurementisanoninvasivemethodandthatthecellswere notharmedbythemeasurement. Moreover,nodiﬀerencein theimpedanceresponsewasfoundbychangingbetweenpo- tentiostatic and galvanostatic modes. The impedance spec- trawereanalyzedwiththeﬁttingsoftwareZview2(Version 3.5b,ScribnerAssociatesInc.,SP,USA).Thedatawereﬁt- ted with an equivalent electric circuit, as shown in Figure 1 E), using a nonlinear least-squares ﬁtting algorithm. For datacomparisonbetweentheTEERvaluesgeneratedbyEIS measurement and standard voltohm meters (e.g. EVOM, Millicell),similarsettingswereused. Accordingtotheman- ufacturer of the EVOM device, a direct current of 10 A with 12.5 polarity reversal per second is the measurement signalofthemostfrequentlyuseddevicesforTEERcharac- terization. We adapted the settings by chronopotentiometry measurements with a direct current of 10 A or -10A al- ternating for 80 mS. The measurement was canceled after ten positive and negative currents. At the end of each cycle, the potential wasrecorded,andtheresistancewascalculated. Themeansof all twenty resistances were the cell layer resistances af- ter subtracting the resistances of an empty transwell. The formationoftheepithelialcelllayerwasmonitoreddailyby EIS and the above described voltohm method. 3.3. EIS response to TEER enhancer TheimpedanceresponseofafullydevelopedCaco-2cell barrier to three diﬀerent TEER enhancers, namely, EGTA, saponin and forskolin was recorded. The stock solutions of EGTAandsaponinwerepreparedinculturemedium. Theﬁ- nalconcentrationswerechosenaccordingtoliterature,hence 0.05%w/v(Chaoetal.,1998)forsaponinand10mM(Sun et al., 2010) for EGTA. 10 mg forskolin was ﬁrst dissolved in 1 ml ethanol (=24 mmol/l), and 18.5 L of the forskolin solution in ethanol was added to 45 ml medium leading to a 10M solution (Rousset et al., 1985). The chopstick-like electrode was placed in a transwell insert. Prior to the ex- periment,afullEISspectrumwasrecordedtodeterminethe starting value for TEER and cell layer capacitance. All the inserts showed at least a starting TEER of 1500 cm2and a total membrane capacitance of 3 F/cm2. After an EIS spectrum, the media on the apical side were replaced with medium spiked with a TEER enhancer and EIS was per- formed in a loop to monitor the immediate response of the Caco-2 cell layer to the TEER enhancer. The measurement was terminated when the alteration between the loops was negligible. The values for TEER and cell layer capacitance were extracted by equivalent electric circuit ﬁtting. 3.4. Sonoporation For analyzing the inﬂuence of sonoporation to a Caco-2 celllayer,theimpedanceresponsewasmeasuredduringthe application. Fortheexperiments,microbubbleswithapoly- meric n-butyl cyanoacrylate shell were used. The used mi- crobubblesaredescribedelsewhere(Appoldetal.,2017). A 1MHztransducer(Olympus,Waltham,MA)wasplacedina waterbathandthetranswellplatewasmountedcloselyunder thewatersurface. Ultrasoundwasappliedwithapressureof 600kPaforthreemin. Themicrobubbleswerefreshlymixed before each sonoporation experiment to a concentration of 1.8 Mio bubbles per ml medium and injected to the lower compartmentofthewellplateasthemicrobubblesﬂoat. The Caco-2cellswerecultivatedonthelowersideofaninsertfor direct contact between the cells and microbubbles. 3.5. Fluorescence microscopy ThetranswellinsertswererinsedwithcooledPBSbuﬀer andﬁxatedby4%paraformaldehydeinPBSfor15min. Fol- lowedbythreewashingstepswithPBScontaining5%bovine serumalbumin(BSA)and5%fetalcalfserumfor30minat room temperature. The cells were then washed three times for 15 min with PBS containing 1% BSA, and the inserts wereincubatedovernightat4ýCwiththeﬁrstantibodyrab- bitanti-ZO-1(ThermoFisherScientiﬁcInc.,Waltham,MA, USA) which was diluted in PBS with 1% BSA to a con- centration of 2.5 g/mL. After decanting the solution and washing three times for 15 min in PBS with 1 % BSA the second antibody goat anti-rabbit with Alexa-488 (Thermo Linz et al.: Preprint submitted to Elsevier Page 4 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy Fisher Scientiﬁc Inc., Waltham, MA, USA) with a concen- tration of 5 g/mL was applied to the cells for 60 min at roomtemperatureinthedark. Lastly,afteronewashingstep inPBSwith1%BSAandtwowashingstepsinPBSwithout BSA for 15 min, the cells were incubated with 100 g/mL DAPI (4’,6-diamidino-2-phenylindole) diluted in PBS for 5 min in the dark and at the end washed with PBS. The im- ages were taken with the Leica SP8 and ZEISS ApoTome confocal laser scanning microscopes. 4. Results and discussions The ﬁrst part of this section explains the principles fol- lowed for the development of an electrical equivalent cir- cuitmodelwhichiscapableofdescribingtheimpedancere- sponse of a cell layer. Subsequently, the results of diﬀerent experiments show the versatility of the model. 4.1. Development of the equivalent electrical circuit Theequivalentelectricalcircuitshouldfulﬁllthefollow- ing criteria: •The impedance response of a cell layer between 10 Hz and 100 kHz should be described at all stages of maturationandadditionallydetectcomplexbiological processes. •The amount of ﬁtting parameters must be kept to a minimum in order to achieve a proper physical repre- sentation of single elements. •The cell layer and the measurement path must be re- ﬂected. •A direct comparison with the TEER measured with a chopstick-like electrode connected to a voltohm de- vice(DC-measurement)shouldbepossible,sincethis isthemostwidespreadmethodtocharacterizebarrier- forming cells. Usingafour-electrodeEISsetuphasthebeneﬁtofelimi- natingelectrodepolarizationresistancefromtheimpedance spectra, leading to a frequency-independent measurement setup. Therefore, without cells, the impedance response is purelyresistiveandarisesfromthecellculturemedia,mem- brane support and wires. These resistances are summarized in a single resistor element Rmedia. Anequivalentelectricalcircuitthatdescribesacelllayer needs at least a resistive and a capacitive element in paral- lel. Resistance TEER describes the ﬂux of ions through the barrier. The capacitance represents the capacitive behavior of the phospholipid membrane of the cells. If the cell layer ispolarized,whichmeansthatthecellmembraneoftheapi- cal and basolateral sides diﬀers in surface area and protein composition, two capacitances must be considered. The polarized Caco-2 cell layer was ﬁtted with a resis- tanceandacapacitanceinparallelfortheapicalandanother resistanceandcapacitanceforthebasolateralcellmembrane. Figure 2: Nyquist plots of the EIS measurements of a Caco-2 layer between 10 Hz and 30 kHz. The data were ﬁtted with the equivalent circuit presented in Figure 1 E): A) before the additionofforskolintothecellculturemediaandthreeandfour days after addition respectively. Three days after the forskolin addition two semicircles appear. B) The development of the Caco-2 barrier at three diﬀerent days showing good agreement between measurement and ﬁtting. Figure 1 E) presents the complete equivalent electrical cir- cuit. This circuit allows for ﬁtting complex biological pro- cesses as demonstrated by the addition of forskolin, as well asleakyandtightepithelia,asshowninFigure2. Forskolin alterstheelectrochemicalbehaviorofthemembranesurface by shortening the brush border microvilli (Rousset et al., 1985) and increasing the apical HCO* 3secretion (Laohapi- takworn et al., 2011) among other complex biochemical re- actions related to a high concentration second messenger concentration cAMP. Figure 2 A) shows ﬁrst a section of the nyquist plot for the impedance measurement before the addition of forskolin. After day 3 of forskolin addition, two semicircles appear in the Nyquist plot. The two semicircles appearwhenthetimeconstant( =R*C)ofthetwoRCele- mentsdiﬀersuﬃciently. Figure2B)showsthreeexemplary nyquist plots during the development of the barrier and the corresponding ﬁt. The equation for the used equivalent electrical circuit is depicted here: Z=Rmedia+R1 1+j!R1C1+R2 1+j!R2C2(3) Anadditionalresistorisoftenusedparalleltotheapicaland basolateral resistor and capacitance to diﬀerentiate between paracellularandtranscellulartransports. Theincreasednum- ber of undeﬁned parameters must be solved by further sim- plifyingassumptionsorspecializedexperiments(Lewisand De Moura, 1984; Krug et al., 2009). Summing up, for mea- surements with polarized cells, two resistance and capac- itance elements are necessary. However, considering api- cal and basolateral resistances and capacitances separately is often impractical. Therefore, the derived resistances and capacitances may be summarized according to Eq. (4) in TEER and total membrane capacitance C as reported in lit- erature (Erlij et al., 1994; Bertrand et al., 1998). Linz et al.: Preprint submitted to Elsevier Page 5 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy Figure 3: The eﬀect of membrane coating on the development of a Caco-2 cell barrier was studied with respect to resistance and capacitance measurements over a time period of 14 days. The inserts were coated with Matrigel®and collagen I and compared with inserts without coating. The standard voltohm settings (10 A direct current with 12.5 polarity reversal per second) were used to verify the resistance measured with the proposed chopstick-like electrode impedance method. A) shows the development of the TEER measured with both methods and B) shows the corresponding overall cell layer capacitance. The three lower graphs show the development of capacitances C1andC2separately. The measurements were done at least in triplicate and capacitances higher 25F_cm2were neglected. TEER=R1+R21 C=1 C1+1 C2(4) Theseassumptionsenablethecomparisonwithexperiments performedwiththecommercialEISdevicefortranswellin- serts CellZscope®or TEER data from chopstick-like elec- trodes connected to an EVOM or Millicell voltohmmeter. 4.2. Caco-2 cell layer development The most common devices for TEER measurement in transwell inserts are voltohmmeters. These devices apply a 10Adirectcurrentwithpolarizationreversalevery80ms. WeadoptedthevoltohmsettingstocomparetheTEERdata derivedfromvoltohmwiththeTEERdataderivedfromEIS. The voltohm and EIS settings were implemented as a se- quencetomeasureconsecutivelywithoutchangingthechop- stick-likeelectrodeposition. ThedevelopmentoftheTEER on diﬀerent coated inserts was monitored over two weeksandisshowninFigure3A).TheTEERvaluesderivedfrom voltohmorEISsettingsareingoodagreementduringallthe stagesofbarriermaturation. ThecellsgrownonMatrigel®- coatedinsertsdevelopedthefastestandreachedroughlythe same end value as the inserts without coating. The cells grownoncollagen-coatedinsertsdevelopedthesameTEER asthecellsgrownwithoutpreviouscoatingsuntilday9and stayed lower after day 9 to around half of the TEER of Ma- trigel®or without coating. The development of cell layer capacitance shows a dif- ferent course over time. The corresponding total membrane capacitance of the cell layer can be seen in Figure 3 B) and is only accessible through the proposed EIS method. While the total membrane capacitance shows a similar trend, the twocapacitancesrevealdiﬀerencesinthetemporaldevelop- ment. The total membrane capacitance is dominated by ca- pacitanceC1,whichisreportedtocorrelatetotheapicalcell membrane. Thebasolateralsideisrepresentedby C2(Schif- Linz et al.: Preprint submitted to Elsevier Page 6 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy ferdecker and Frömter, 1978; Bertrand et al., 1998). The membraneareaofthebasolateralsideislargerthantheapi- calsidebecausetheintercellularspaceisincluded(Clausen etal.,1979). Theapicalandbasolateralcapacitancearesep- arately presented in Figure 3 C) - E). Thecoatinghadastrongeﬀectonthecapacitanceofthe cell layer. Allthe cell layerspeaked between days8 and 10. The lowest peak total membrane capacitance was shown by thecellsgrownonMatrigel®withapproximately3 F_cm2 compared with almost 6 F_cm2for the uncoated inserts. Thedatainterpretationforthecapacitancedevelopmentover the 14 days of cultivation is challenging. Leﬀers et al. re- portedcomparableTEERandcapacitancevalueswith1500 cm2and4F_cm2,respectively,forcollagen-coatedtran- swell inserts (Leﬀers et al., 2013). The capacitance corre- lates with the cell membrane area and protein composition. Furtherresearchisnecessarytocorrelatethecapacityofthe cell layer with biological processes, such as the degree of polarization. 4.3. Impedance response to TEER enhancers and forskolin TheresponseofaCaco-2barriertoEGTA,saponin,and forskolin was assessed by measuring the impedance spec- tra with a chopstick-like electrode. The TEER and capac- itance were extracted by ﬁtting the impedance data to the equivalent circuit depicted in Figure 1 E). The resistances andcapacitancesweresummarizedaccordingtoEq.(4)and normalized to the initial values and presented in Figure 4. While all three substances decreased the TEER value sub- stantially (4 A), only saponin altered the capacitance (4 B) signiﬁcantly, due to the hemolytic eﬀect of this non-ionic surfactant (Narai et al., 1997). The tremendous increase in capacitanceiscorrelatedtotheexistenceofapoptoticorde- tached cells (Lohren et al., 2016). The calcium-chelating agent EGTA reduced the TEER within the ﬁrst 20 min by approximately90%,butthecapacitanceremainedconstant, thereby leading to the assumption that only the tight junc- tionsareaﬀectedbyEGTA.Forskolinwithitscomplexbio- logicaleﬀectsresultedinaninstantdropintheresistanceof nearly 95 % within the ﬁrst 5 min. Analogous to the exper- iments with EGTA, the capacitance stayed almost constant with respect to the initial value, which leads to the assump- tion that forskolin has an immediate eﬀect on the cell–cell connection but no eﬀect on the morphology as this would alter the capacitance. 4.4. Fluorescence microscopy The staining of ZO-1 protein, which is part of the tight- junctioncomplex,andthenucleusrevealedthecellmorphol- ogy in Figure 5. A conﬂuent cell layer with distinct tight junctions was visualized by ﬂuorescence microscopy, and the Caco-2 cells were grown in monolayer according to the confocal microscopy images. Unfortunately, no diﬀerence between cells grown on diﬀerent coated membranes can be observed. Figure 4: The graphs show the immediate TEER (A) and ca- pacitance(B)responseofCaco-2celllayerstoEGTA,forskolin, and saponin. The values were normalized to the beginning TEER and capacitance value. All three chemicals sharply de- crease the TEER, but only saponin alters the cell layer capac- itance signiﬁcantly. Data shown represent the mean of three cell monolayers. Linz et al.: Preprint submitted to Elsevier Page 7 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy Figure 5: A) Fluorescence microscopy revealed a dense and homogenous Caco-2 epithelial cell layer. In addition, z-scans showed that the cells were grown in monolayer B). Blue: DAPI staining of the cell nucleus, green: ZO-1 antibody staining. Cells grown on collagen I or Matrigel®coated membranes showed no diﬀerences in ﬂuorescence microscopy. 4.5. Sonoporation Figure 6 shows the course of the TEER and total mem- brane capacitance during a sonoporation of a Caco-2 cell layer. Byapplyingultrasoundincombinationwithmicrobub- blesincontactwiththecelllayertheTEERinstantlydropped to less than 10% of the initial value. During the sonopora- tion the TEER stayed constant. 45 min after turning oﬀ the ultrasound, the TEER increased to 54%. After nearly 6.5 h, the initial TEER was almost reached with a percentage of 99%. Thecapacitancestayedalmostconstantandﬂuctuated in a range of 4% around the initial value. No change in the impedancesignalwasobservedwhenusingmicrobubblesor ultrasound alone. Figure 6: Normalized TEER and total membrane capacitance during and after sonoporation. The gray area shows the time when the ultrasound (US) was applied. The resistance was re- duced directly with the start of the sonoporation and increased to the initial value again within 6 hours. The total membrane capacitance remained almost constant. 5. Conclusions The use of a chopstick-like electrode is the most conve- nient way to measure TEER from cells grown in transwell Linz et al.: Preprint submitted to Elsevier Page 8 of 10 Improved Cell Barrier Characterization in Transwell Inserts by Electrical Impedance Spectroscopy inserts. The TEER represents the transport of ionic species throughthebarrierandcanbeusedtomeasurethecelllayer conﬂuenceandmonitortheexperimentsrelatedtoionictrans- port. By changing the input signal from direct to alternating currents with varying frequencies, the frequency-dependent resistance(impedance)canbecalculated. Byﬁttingthedata to an equivalent electric circuit, the media resistance, cell layerresistance,andcelllayercapacitancecanbeextracted. The cell layer capacitance correlates to the surface area and compositionofthecellmembrane. Inaﬁrststep,weshowed thattheTEERsderivedfromthestandardandtheintroduced method are comparable during all the stages of barrier for- mation. The additional cell layer capacitance can be used to further monitor the morphological behavior of the cells, e.g.,thedevelopmentofapolarizedcelllayer. Theproposed methodwasthenusedtomonitorthebehavioroffullydevel- oped Caco-2 barriers to the permeability enhancers EGTA and saponin. Both decreased the TEER substantially, but only saponin altered the capacitance. Without the proposed impedance method the diﬀerentiation between the two sub- stances would not be possible. A crucial step in impedance spectroscopy is to use an appropriate equivalent electrical circuit. Theusedcircuitallowstheﬁttingoftheexperimen- tal data during all the stages of barrier formation and dur- ing complex biological events, such as after the addition of forskolin. The number of ﬁtting parameters is summarized to gain two easy-to-handle values, TEER, and total mem- brane capacitance. In the last set of experiments, sonopo- ration was monitored. The TEER drops immediately while thecapacitanceremainsconstantduringultrasoundandmi- crobubbles exposure. Our introduced method can help barrier researchers to monitorandevaluatetheirexperimentsbytheadditionalca- pacitance data without changing the established workﬂows. Conﬂict of interest statement The authors declare that there is no conﬂict of interest. Acknowledgements M.W. acknowledges the support through the Gottfried Wilhelm Leibniz award. This project has received funding fromtheEuropeanResearchCouncil(ERC)underthegrant agreement no. 694946. Part of the work was performed at theCenterforChemicalPolymerTechnologyCPT,whichis supported by the EU and the federal state of North Rhine- Westphalia (grant no. EFRE 30 00 883 02).