Organ Metabolism Analyzer: Microphysiological Systemreal-time monitoringCross epithelial cell layerresistanceAnd extracellular acidification rate

Skin-on-a-Chip：Transepithelial Electrical Resistance

And Extracellular Acidification Measurements

Through an Automated Air-Liquid Interface

The skin is an important organ in the human body, playing a crucial role in protecting internal organs.Therefore，People have done a lot of work to create artificial skin modelsconductIn vitro skin toxicity test. These organizational models are called reconstructed human epidermiscell model（reconstructed human epidermis，RhE）, used forInPharmaceutical, cosmetics, and environmental fieldsMiddleAssess skin exposure to exogenous substancesMiddleof毒性研究Here, we propose a proposition of nothingmarkofMethod, i.eUtilizedintelligent lab for in vitro diagnostics（IMOLA-IVD）A non-invasive, sensor based platform,ComedetectOn porous membraneRhEcell modelCrossing with adherent cellsepitheliumcelllayerresistance（transepithelial electrical resistance，TEER）Firstly, mouse fibroblasts were cultured on polycarbonate membrane as a test model, using customized biochipsClosed design，And doublemicrochannelStructure, used forContinuous and automatic perfusion of culture materialsDetecting the extracellular acidification rate of L929 cells（Extracellular acidification rate，EAR）andCross epithelial cell layerresistance（transepithelia lelectricalresistance，TEER）.Through this platformMonitoringRhEcell modelThe TEERMore than 48 hours.TEER and EAR tests indicate that,the platformCan be stably cultivated for a long timeOn the chipskinCell models, monitoring metabolic parameters, andOrganizational degradation.

Keywords：TEER；Organ-on-a-Chip；skin models；reconstructed human epidermis；impedance；label-free monitoring

1. INTRODUCTION

As the largest organ in the human body, the skin representsInside the human bodyAnd externallyenvironmentBetween themStructural StudiesBarrier, connecting internal organs with toxins、Isolate the pathogen, andprotectinternal organsProtect from UV rays（UV）Radiation. In addition to important barrier functionsYes, human skinstillPerform several basic functions of the human body, such as thermoregulation, sensation, and excretion. Because skin is humanResist the influence of external environmentThe first protective cover, the new chemicalThe study of matterLike drugs and toxins, they mustAnalysis and evaluationIt isThe ability to regulate skin integrity. Investigating the effects of these compounds, cell biology utilizes cell culture models to gain a deeper understanding of cell behavior regulated by chemicals. in the pastIn ten years,ScientistDevelopment了Various bioengineering modelsUsed for skin toxicity research. The first skin model is based on traditional two-dimensional models（2D）Co culture model, keratinocytesVaccination tocultivateGoodOn fibroblasts. Due to the inability to maintain epithelial cells for a long time when cultured on rigid plastic, andBlocked the cellsstratificationThe field of organizational engineering has developed 3D technologySkin models are used to reproduce similarities within the bodyStructure and design a more physiologically relevant environment by integrating gas-liquid interfaces.

The human skin is composed of the following primary layers, which have appendagesThe function：epidermislayerDermis and subcutaneous tissue.Integrating different skin layers into cell models, current skin models can be divided into those containing keratinReconstruction of human epidermal model with cells, including keratinocytes, andA full layer model of the representative dermal and epidermal compartments of fibroblasts, as well as with additional cell types（Such as melanocytes, stem cells, etc）The full layer modelDue to its simplified structure, it has been redesignedestablishHuman epidermis（RhE）The model has high reproducibility and is therefore widely accepted.RhEcell modelHuman derived keratinCell inoculation inpolycarbonatesemi-transparentmembraneAdd it to the cell culture system and place it in a standard well plate, such ascommercializedTranswell® system（Corning Inc.，Corning，NY，USA）The device places the culture at the gas-liquid interface and exposes the skin surface to the air,formphysiologyenvironmentsimilar toThe cultivation conditions. Currently, there areopenSkin models, as well as commercial models such asEpiDerm™（MatTek in vitro life science laboratories, Bratislava, Slovak Republic），EpiSkin™（EpiSkin, Lyon Cedex, France），SkinEthic™（EpiSkin），EpiCS®（CellSystemsGmbH，Troisdorf，Germany）, andLabcyte.（Japan Tissue Engineering Co., Ltd. [J-TEC], Aichi, Japan）ALEXANDRA Association follows a non-profit approach to production and testingopenThe 3D skin model providesSolution.

2. Background

2.1.The latest progress in skin toxicity testing based on reconstructed human epidermis

From a regulatory perspective，To evaluate and predict the harmful effects of toxic compounds and drugs on the skin, 3D modeling systems must go through a wide range of processesTest compoundVerification ofIfThroughEuropean Alternative Methods Validation Center（ECVAM）Confirmation and Organization for Economic Cooperation and Development（OECD）Test Guide（TG）A model with testing techniques is sufficientAs an acceptable toolforSkin toxicity testing. So far, OECD has providedforGuidelines for testing skin corrosion and irritationThe Guide，Based on OECD TG 431 and 439 respectively.

2.2.Organ chip platform and microlifePhysical measurement

Due to the relevance of current models in biologyUp，Provide limited meaning onlySo in the field of toxicologyTesting is moving towards implementing organ chips（Organ-on-Chip，OOC）The development direction of instruments.The organ chip system describes a system that includes perfusionsmallMicrofluidic cell culture device in the room，Cultivate live cells under physiological conditions.However, most OOC platforms still heavily rely on endpoint analysismethod，Lack of different time pointsofTestMethod In addition, chemical labels such as fluorescent labels mayPotential impact on cellular metabolism to alter experimental results. Therefore，Real time reading using unlabeled technologynumberIntegration ofequipment,ForMeasurement for spatial and temporal analysisyesVery meaningful.

２. ３. Skin/Rebuilding the Cross of Human SkinepitheliumCell layerresistance

Due to potential harmful compoundsAffects skin thickness, thickness measurementalsoParameters of early researchone of.Destructive measurements, such as biopsy, or non-destructive measurements, such as confocal Raman spectroscopy and ultrasound imagingAmong them, traditional methods and useful onesApplication of Cell CulturejustIt measures the skin permeability barrier to evaluate epithelial or endothelial tissueofVitality and functionality. herecrossepitheliumCell layerresistance（TEER）Provides a label free and rapid technique for studying skin integrity. TEER valuerepresentsonelayerOr a fewlayercellLayer by layerresistancevalue.

２. ４. CurrentlycrossepitheliumCell layerresistancemeasurementDisadvantages andcrossepitheliumCell layerresistanceAutomationmeasurementThe need

Except for microProcessingMethods, as well as some commercially available external testing systems such as the "volt ohmmeter"（World Precision Instruments, Sarasota, FL, USA）,It can be readily available. Due to the lack of integration with cell culture systems, researchersRequires manualTranslatecell cultureFrom BiologyIncubator placementTo the measurement system. This process lacks reproducibilitySex and standardization,I can't eitherProvide high throughputmeasurementofabilityAlthough the next generation of electrode chambers（asEndohmsroom）Can be directly measured，butculture mediumAnd the reagents must be manually operatedjoin, used forLong term cultivation and drug research casesTherefore, extended researchIt is impossible because old media cannot be usedIt was removed from the cultivation system, but there was no automatic infusionfreshculture mediumIn summary, the current TEER measurement system still heavily relies on manual operationoperationandHandheld systems affect the stability of measurements, thereby impacting the overall performanceexperimentReproducibility of.Organisation for Economic Co operation and Development（OECD）It is believed that,An acceptable testing plan，Reproducibility analysis is a fundamental criterion in validation. Therefore, the TEER field integrates automatic TEER monitoring and acquisition,The system is capable ofculture mediumExecute programming mode under frequent changesmeasurementAnd alsoVery meaningful.

Here, we introduce a new automatic monitoring, testing, and data collection methoddataMicrophysiologymeasurement system（IMOLA-IVD, GermanyCellasys）forTEERMeasurement.Throughonesealdesignofbusinesstransform，Epidermal cells cultured with polycarbonate membraneRhEcell modelApplication of TEER measurement for researchRhECultivate and integrate into automated fluid platforms,canAccurate infusionculture mediumanddosingIn addition, we are able to monitor the extracellular acidification rate（extracellular acidification rate，EAR）The purpose of empirical research is toIntegrated automationIrrigation systemandintegration testingPlatform, implementationReal time dynamicsmeasurementAt the same timeguaranteeMeasurement stability.This study demonstrates a non-invasive approach for assessing skin integrityofVerification and application of measurement methods.

3．Materials and Methods

3.1.Overview of Intelligent Mobile Laboratory for In Vitro Diagnosis

IMOLA-CVD is a laboratory on chip technologymeasurementSystem for automatic monitoring of L929 mouse fibroblasts andEpiDerm™rebuildHuman epidermiscellTEER and EAR of the model. The basic operation of IMOLA-IVD has been described in previous work，Simply put, each IMOLA-IVD includes a power supply, analog and digital modules, as well as a bio chip integrated with sensors that passively monitor the microenvironment of the cell model. The standard IMOLA-CVD experiment is conducted by loading data acquisition and linking applicationsDALiA 2.0softwareImplementing automation on personal computers to control oneA peristaltic pump andconnectionSix IMOLA-IVD systemsmicrofluidicspathwayThe pump circulates between ON and OFF states. InOFFPump shutdown cycle, cells can metabolize nutrients in the culture medium；InONpumpopenperiod，nutritiousThe culture medium is perfusedTo cellsMiddle.

3.2improvedBioChip-DKOMURO

biochipmoduleIncludes a sensor chip connected to the circuit board and connected to aA cylindrical sealed chamber,in order toProtect the connection of the chip andConnecting cells andCulture medium. oneFluid headdirectlyConnect the sealed chamber, forming a liquid seal、Static micro volume（~ 6ML）The reaction chamber,InIntegrationofSensor surfacemeasurementExtracellular acidification rate（EAR）andOxygenation for use （Figure 1a）However, measurement is requiredRhEIn this case,Cultivation on porous membraneThe cellsNot suitable for standard biochips or standard IMOLA-IVD forked electrode sensors（IDES）measurementresistanceTo overcome these limitations, the newSealed chamberDesign of fluid head to maintain air-water compatibility using Pro/Engineer Wildfire 4.0 （PTC Inc., Needham, MA, USA）.sealuseUltimaker 2 （UltimakerBV，Geldermalsen，Netherlands）Using polylactic acid（PLA）And silicone rubberMedical Adhesives（Corning Inc., New York, NY, USA）Connect to the BiochipBioChipRedesignedBioChipSmall rooms are suitable forExpand the training roomand othersCultivation on porous membraneThe cells（Figure 1b）ofRhE.

3.3.L929 cells andrebuildHuman epidermiscellPreparation and cultivation of

Record mouse fibroblasts（L929）andrebuildHuman epidermisCell（RhEofcrossepithelial cellslayerresistanceValue. L929 cells added with 10%Fetal Bovine Serum（FBS） and10 Mg/ml gentamycin（THermo Fisher,Waltham, MA, USA）. CellsAccording toStandardofLaboratory Regulations（GLP）Cultivate and store in 37℃After cultivation in a 5% incubator, at 95%Degree of integrationWhen cells are passaged, 100000 cellsplantAt 12mmTranswell® Up，Aperture 3MM'sPorous membrane（Corning Inc.）.transwellSubsequently placed in 6ConfuciusOn the board，And incubate in 1ml DMEM for another 24 hours, then transfer tomodifiedBioChipIn the middle. L929 cells are the standard cell line used in our laboratory for validationexperiment.

TranslateEpiDerm™RhEcell modelTransfer to a 6-hole plate and place at 5.5mm/5mLMatTek serum-free long-term culture medium（#EPI-100-NMM-250） （MatTek in vitro life science laboratories）Exchange the culture medium every 48 hours until it is transferred toBioChipsMiddleTEERExperiment 24 hours before the start of the experiment, reduce the culture medium to 0.9 mL and place it at 12orifice plateInside.Modified BioChip-DAfter sterilization at room temperature in 70% ethanol for 20 minutes, rinse with sterile deionized water. biochipperfusionNon buffered DMEM treatment was performed for 24 hours to stabilize the temperature, and then the membrane culture was placed on the chip.

3.4.AutomationIntroduction to Irrigation System

Designed an automatic systemperfusionThe system is hereAutomationMonitoringCultivate on a microchip for 48 hoursaboveofRhEcell modelThe TEER. The system consists of two fluid modules（FM）form,ThroughandTygon® E-3603（ProLiquidGmbH,Transfers, Germany）PipelineconnectionbecomeindependentofPipelineThese independent networks are connected throughIMOLA-IVD 泵StandardSettingsON/OFFSwitchProgram,Provide nutrient rich cell culture medium to basal cells，And regularly submit toTranswell® membraneoftopRegion,Pump in phosphate buffered salinesolution（PBS）As shown in Figure 2.Flow of culture mediumModule in non buffered DMEMAnd 0.2%SDS'sculture mediumSwitching between，provide toBottom of membraneThe cellsThe TEER measurement module is connected to PBS for TEER measurement or testing, which can be used for vaccinationRhEsuperficialWater soluble test substance.

L929 cellsCultivate inOn the porous membrane chamber,RhECell model culture in Modified BioChip-On D，ThroughUnbuffered DMEM andperfusionpumpconduct36 hoursperiodofcultivateFor L929To monitor cellular acidification,pumpONtimeFor 5 minutes, while the pump is turned offtimeFor 5 minutes, 10 minutes, 30 minutes, and 55 minutes. ForRhEcell model, pumpperiodFor 5-minute ON, 10 minute OFF, 5-minute ON, 25 minute OFF, 5-minute ON, 10 minute OFF. At 25Minute pumpOFF stage, by pumping PBS into the membraneTop chamber, on the chipresistanceEstablish a circuit between the sensor and the wire electrode of the fluid headConnect and record TEER measurements. Transfer 750 μ L at a rate of 60 μ L/minThe PBSPump onto the membrane on the chip and remove at a rate of 120 μ L/min. TEER measurement was performed during a 25 minute pump shutdown period. After the predetermined stable measurement cycle（L929 cells andRhEcell model47 and 36 hours respectively）Pump 0.2% SDS buffer free DMEM into the chip as a positive control. Infuse SDS medium for at least 12 hours to confirm the effect of the control group on the measured TEER.

4. Results and Discussion

4.1.Redesigned epitheliumresistancesealfunction

TranslateporousInsert the membrane into the culture chamber and determine a volume of~170 μ L.Matching prototypeInject into the cavity through the inlet hole formed under the membrane，The pores at the bottom of the membrane allow for the passive diffusion of fresh nutrients to the basal layer of cells and cellular wasteSpread out cellsDuring the pump ON cycle,After nutrient consumptionThe culture medium is pumped out of the chamber（Figure 2）.RhEThe top surface of the culture is exposed to ambient air, and after a long period of cultivation, it is stimulated to differentiate into aALI'slayered. To measure the TEER of these cultures, the redesigned fluid head includes a bidirectional probeofThe inlet/outlet valve periodically distributes a certain amount of PBS solution. This kind ofmethodConnecting porous membranesLead electrode of the top cavity（Refer to Figure 1b）The circuit between the planar IDES electrodes on the sensor chip. The overflow valve submerges the TEER electrode in a stable stateculture mediumPrevent overflow within the volume. continuous5 minutesRecord the TEER of the membrane and then measure itAfterwardsRemove PBS to maintain ALI.

4.2.Mouse fibroblast cellsofDetermination of extracellular acidification rate

Use automatic measurementReal time measurement of programsEAR， Take L929Cells grow inPolycarbonate film. Use unbuffered DMEM at 50MPerfuse L929 cells at a rate of L/min. Figure 3 shows thatpumpPH value measured in alternating stages（In mV units）The red bar represents the pumpOFFstage, perfuse fresh culture medium, cellsactiveGround nutrient substancesgenerationThankFor acidic waste, metal oxide sensors on biochips will detect thesemetaboliteThe green bar indicates the pumpONStage. InthisstageMiddle, membranebottomInfuse fresh culture medium,Provide nutrition toCells cultured on the membraneFrom the graph, it can be seen that whenculture mediumDuring acidification, the pump is turned offstage55Within minutesThe voltage will increase by~5 mV for a shorter period of timeInnerThere will be no significant voltage changes.

4.3.Metabolic response of mouse fibroblasts to dodecyl * medium

Once the pumpONAt the end of the cycle, the measured voltage rapidly drops until it reaches the baseline（steady state）Value. There is a brief period before the declineDelay, which may be due to the presence of fresh culture medium and chipsThe acidificationculture mediumMixing between themlead toof，Because the location of the export is higher than that of the import. At 58 hours, the measured signal decreased. This may be due to membrane rupture and release of intracellular contents into the culture medium. At 56 hours, the cells had already dissolved, so no waste was produced to acidify the surrounding medium, resulting in a pump in 55 minutesOFFThe stage signal becomes flat. Figure 4 depicts the OFF phase during the experimental duration of 55 minutessustainMeasure EAR.It can be seen thatjoinAfter SDS, EAR increased sharply and then rapidly dropped below zero, indicating cell lysis. Then it gradually approaches zero until the end of the experiment.

4.4.Mouse fibroblast cellsThe TEER（TransepithelialCell layerresistance）Monitoring

In addition to monitoring the metabolic rate of L929 cells, pump at 25 minutesOFFduringstillcanMonitor TEER values. During the TEER measurement process, as PBS is pumped into the measurementKOMUROWithConnect the circuitMeasurements will be taken, and the values will change. TEER beginscountlessvalueBecauseThere is a gap between the electrode pairsopen circuit. Embedded inFluid headThe wire electrode is immersed in PBS and connected to the membrane belowOn the chipconduct electricityElectrode togetherConnection formationCircuit. PBS stays on cells for 5 minutes, pumpOFFTo conduct stable monitoring. Then PBS is pumped out for measurementSmall chamber, until the next measurement cycle.

Figure 4 shows the EAR and TEER measured from 25 hours onwards. In the pumpOFFIn the stage, EAR is calculated by linear regression. Table 1 summarizes SDSjoinfront and rearjoinThe subsequent result. Experiment started 24After hours, the measured real numbersresistanceStable around 190 ohms, imaginary partresistanceIt is -40 ohms. Because it is known that L929Mouse fibroblasts maintained in cultureSingle layer, expectedresistanceStabilize at a low level. After being exposed to 0.2% SDS medium for 8 hours（timeFor the th56 hours），real numberresistanceA decrease of 14.8%,Corresponding to joiningSDSlead toCell death.imaginary partresistanceDecreased by 6.11%. These results indicate that the developedsealDesign and PumpmoduleCapable of automationresistancemeasurement.

4.4.RhEcell modelofTEER（TransepithelialCell layerresistance）Monitoring

Using the same settings as above重建人表皮Cell model（reconstructed human epidermis，RhE）Perform continuous monitoring of TEER for approximately 50 hours. In the first 12 hours, the TEER value remained relatively stable, as shown in Figure 5. After 36 hours, cells were treated with 0.2% SDS for 2 hours, with an average TEERThe value suddenly drops.（b）SDS culture mediumjoinPreviously, the TEER value wasLinear increaseYes, using PBSjoinAfter the top cavity, quickly stabilize and maintain until the 5-minute measurement cycle ends. When fromUpper chamberMiddleRemove the culture mediumAfterDue toshort circuit， TEER againBecome valueless.

InchangeAfter 2 hours in SDS medium, the changes in TEER curve can be detected. At first, TEER values were related tojoinSame before SDS culture mediumButThe TEER value steadily decreases during the measurement period until it stabilizes at a lower value. This leads to a decrease in the measured average TEER, which is related tojoinCell lysis induced by SDSmatchIn addition, with SDSAddition of culture mediumWith the increase of time,resistanceThe initial peak amplitude decreases. Despite exposure to SDS, the persistence of the initial peak seems to indicate cell survival. It is worth noting that the previous IMOLA-IVDThe study was conducted on simple monolayer cells,Relatively low concentration of 0.2% SDScanKill cells in a shorter period of timeButmoreseverecellcleavageProcessRelatively slowPossible attribution to cells cultured in more complex 3D skin modelslifeEnhancement of power.

5. Conclusions

Here, we propose an innovative method for non-invasive monitoring of cell culture in two-dimensional and three-dimensional porous membranes in various forms. Our BiochipSealed design has been provenSupport for monolayers of mouse fibroblasts and more complex ones重建人表皮Cell model（reconstructed human epidermis，RhE）In addition, a two-part automated fluid system was designed to handle the transportation of PBS solution andjointotopPerform TEER measurement on the chamber. A standard procedure was designed using L929 cellsmethodBy regularlymeasurementTEER is used to monitor metabolic rate and barrier integrity. SDS culture mediumjoinPreviously maintainedcell viability，As a positive control.

After conducting preliminary experiments using L929 cells重建人表皮cell modelMonitor with TEER. TEERThe value shows that the skin model can be culturedAt least 24 hours,joinSDS culture mediumThe subsequent cell lysis alsocanDelay. Higher levels can be used in future experimentsSDSConcentration is used to improve the response time of the control. Overall, this work is a proof of conceptexperiment, demonstrating the ability of IMOLA-IVD for complex 3D tissue models, asNon invasiveTools for automated cell analysis.