MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/m_MTBLS2274_LC-MS_positive_hilic_metabolite_profiling-1_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/a_MTBLS2274_LC-MS_positive_hilic_metabolite_profiling-1.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/s_MTBLS2274.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_035_QC05_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_061_SAM03958_A_lower_48h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_047_QC07_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_042_SAM03942_A_lower_0h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_034_SAM03936_C_upper_24h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_067_Blank_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_052_SAM03951_B_lower_12h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_041_QC06_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_050_SAM03949_D_lower_6h_control.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_029_QC04_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_059_QC09_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_031_SAM03933_D_upper_12h_control.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_025_SAM03928_C_upper_6h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_026_SAM03929_D_upper_6h_control.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_053_QC08_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_062_SAM03959_B_lower_48h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_033_SAM03935_B_upper_24h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_017_QC02_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_037_SAM03938_A_upper_48h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_028_SAM03931_B_upper_12h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_043_SAM03943_B_lower_0h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_016_QC01_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_060_SAM03957_D_lower_24h_control.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_058_SAM03956_C_lower_24h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_065_QC10_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_039_SAM03940_C_upper_48h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_066_QC11_.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_027_SAM03930_A_upper_12h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_048_SAM03947_B_lower_6h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_040_SAM03941_D_upper_48h_control.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_032_SAM03934_A_upper_24h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_044_SAM03944_C_lower_0h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_022_SAM03926_A_upper_6h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_057_SAM03955_B_lower_24h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_036_SAM03937_D_upper_24h_control.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_018_SAM03922_A_upper_0h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_054_SAM03952_C_lower_12h_cells.mzMLftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS2274/191027_S1_LC02MS02_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ChaleckisMetaboLightsPublicLiquid Chromatography MS - positive - hilic<p>Data were acquired on an Agilent 6550 Q-TOF-MS system (ESI source) with a mass range of 40-1200 <em>m/z</em> in the positive all ion fragmentation mode, including 3 sequential experiments at alternating collision energies: 1 full scan at 0 eV, followed by 1 MS/MS scan at 10 eV, and then 1 MS/MS scan at 30 eV. The data acquisition rate was 6 scans/s.</p><p>Metabolite separation was performed on an Agilent 1290 Infinity II system using SeQuant ZIC-HILIC (Merck, Darmstadt, Germany) column. Sample analysis was performed using water with 0.1 % formic acid (solvent A) and acetonitrile with 0.1 % formic acid (solvent B). The elution gradient was as follows: isocratic step at 95 % B for 1.5 min, 95 % B to 40 % B in 12 min and maintained at 40 % B for 2 min. The flow rate was 0.4 mL/min.</p>Spatiotemporal determination of metabolite activities in the corneal epithelium on a chip. 10.1016/j.exer.2021.108646. PMID:34102209Nagoya City UniversityblankHomo sapiensmass spectrometry<p>Tubes containing 1 mL of dried sample were thawed and 100 μL of water:ACN (1:9, v/v) mixture containing 5 tISs was added (<strong>Table S2</strong> in the paper associated with this study). For the preparation of <strong>QC </strong>samples, 0 h cell culture medium sample was used from our previous study. For each <strong>QC </strong>sample, 1 mL of cell culture medium was evaporated and processed together. After resuspension, the samples were centrifuged at 4 °C for 15 min at 20,000 g. Next, 40 mL of the supernatant was transferred to a 96-well 0.2 mL PCR plate (PCR-96-MJ; BMBio, Tokyo, Japan). The plate was sealed with a pierceable seal (4titude; Wotton, UK) for 3 s at 180 °C by using a plate sealer (BioRad PX-1; CA, USA) and maintained at 4 °C during the LC-MS measurement. The injection volume was 10 μL.</p>https://www.ebi.ac.uk/metabolights/MTBLS2274Craig Wheelock. Gunma University Initiative for Advanced Research (GIAR), Gunma University. Maebashi, Gunma 371-8511, Japan. cewheelock@ki.se.Ken-ichiro Kamei. Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University. Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan. kamei.kenichiro.7r@kyoto-u.ac.jp.Romanas Chaleckis. Gunma University Initiative for Advanced Research (GIAR), Gunma University. Maebashi, Gunma 371-8511, Japan. romanas.chaleckis@gmail.com.Rodi Abdalkader. Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University. Yoshida-Ushinomiya-cho, Sakyo-ku, Kyoto 606-8501, Japan. abdalkader.rodiadeeb.3z@kyoto-u.ac.jp.<p><strong>Raw </strong>data files were were converted to <strong>mzML </strong>format using <strong>MSconvert </strong>(ProteoWizard) and processed in <strong>MS-DIAL version 4.38</strong> (see detailed parameters in <strong>Tables S3</strong> and <strong>S4</strong> in the paper associated with this study).</p>initial time pointChamberLayer<p><strong>Microfluidic device fabrication</strong></p><p>The microfluidic device was fabricated using stereolithographic 3D-printing techniques and solution cast-molding processes<strong>[1][2]</strong>. (Abdalkader and Kamei, 2020; Kamei et al., 2015). In brief, the mold for microfluidic channels was produced using a 3D printer (Keyence Corporation, Osaka, Japan). 2 molds were fabricated: the <strong>upper </strong>and <strong>lower </strong>blocks. Each block contained four chambers (15 mm length, 1.5 mm width, and 0.5 mm height). Before use, the surfaces of the molds were coated with trichloro (1H,1H,2H,2H-perfluorooctyl) silane (Sigma-Aldrich, St. Louis, MO, USA). Sylgard 184 PDMS 2-part elastomer (ratio of pre-polymer to curing agent, 10:1; Dow Corning Corporation, Midland, MI, USA) was mixed, poured into the molds to produce 4-mm and 0.5-mm thick PDMS <strong>upper </strong>and <strong>lower </strong>layers, respectively, and degassed using a vacuum desiccator for 1 h. The PDMS of the lower block was fixed on a glass slide and then cured in an oven at 80 °C for 24 h. After curing, the PDMS was removed from the molds, trimmed, and cleaned. A clear PET membrane (pore size, 0.4 μm; thickness, 10 μm; nominal pore density, 4 x 106 pores cm^2 ) was fixed on each chamber of the lower PDMS block. Both the PDMS blocks were treated with corona plasma (Shinko Denki, Inc., Osaka, Japan) and bonded together by baking in an oven at 80 °C.</p><p><br></p><p><strong>Human corneal epithelial cell culture</strong></p><p><strong>HCE-T cells</strong> were provided by RIKEN Bioresource Research Centre (Ibaraki, Japan). Cells were cultured in DMEM/F12 supplemented with 5% (v/v) fetal bovine serum, 5 μg/mL insulin, 10 ng/mL human epithelial growth factor, and 0.5% dimethyl sulfoxide. The cells were passaged with trypsin-EDTA (0.25–0.02%) solution at a 1:4 subculture ratio.</p><p><br></p><p><strong>Human corneal epithelial barrier construction in the microfluidic device</strong></p><p>Before use, the microfluidic cell culture devices were placed under ultraviolet light in a biosafety cabinet for 30 min. The microfluidic channels were washed with DMEM/F12. Cells were harvested using trypsin and collected in a 15 mL tube. Following centrifugation, the cells were resuspended in DMEM/F12 medium and introduced into the upper channel of the microfluidic devices via a cell inlet with a cross-sectional area of 0.23 cm^2 at a density of 1 × 106 cells/mL. The lower receiver channel was filled with DMEM/F12 only. The microfluidic devices were then placed in a humidified incubator at 37 °C with 5% CO2 for 7 d. The medium in each chamber was periodically changed every 24 h. </p><p><br></p><p><strong>Extracellular culture medium collection for untargeted metabolomics</strong></p><p>After the <strong>HCE-T </strong>cells were cultured for 7 d, the culture medium was replaced with fresh medium in both the <strong>apical </strong>(<strong>upper</strong>) and <strong>basolateral </strong>(<strong>lower</strong>) channels of the microfluidic device (contains 4 chambers: <strong>A, B, C</strong> (<strong>HCE-T</strong> cells) and <strong>D </strong>(<strong>no cells</strong>). Microfluidic device were then placed at 37 °C with 5% CO2. At <strong>0</strong>, <strong>6</strong>, <strong>12</strong>,<strong> 24</strong> and <strong>48</strong> h time points, a sample consisting of 1 mL of extracellular culturing medium from both the <strong>apical </strong>and <strong>basolateral </strong>channels was collected. The collected samples were dried in a vacuum incubator for 3 h at 25 °C and then preserved at -80 °C.</p><p><br></p><p><strong>Refs:</strong></p><p><strong>[1]</strong> Abdalkader R, Kamei KI. Multi-corneal barrier-on-a-chip to recapitulate eye blinking shear stress forces. Lab Chip. 2020 Apr 21;20(8):1410-1417. doi:10.1039/c9lc01256g. Epub 2020 Mar 23. PMID:32202263.</p><p><strong>[2] </strong>Kamei K, Mashimo Y, Koyama Y, Fockenberg C, Nakashima M, Nakajima M, Li J, Chen Y. 3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients. Biomed Microdevices. 2015 Apr;17(2):36. doi:10.1007/s10544-015-9928-y. PMID:25686903</p>Metabolomicsultra-performance liquid chromatography-mass spectrometrytandem mass spectrometrycell culture mediumuntargeted metabolitesorgan-on-a-chipLongitudinal Studycorneal epitheliumultra-performance liquid chromatography-mass spectrometrytandem mass spectrometrycell culture mediumuntargeted metabolitesorgan-on-a-chipLongitudinal Studycorneal epitheliumblankCorneal Epithelium<p>An <strong>in-house MS2 spectral library</strong> containing experimental MS2 spectra and retention times (RT) for 391 compounds obtained from standards was used for annotation of detected features using 3 criteria: (i) accurate mass (AM) match (tolerance: 0.01 Da), (ii) RT match (tolerance: 1 min), and (iii) MS2 spectrum match (similarity >70%). The MS2 similarity was scored by the simple dot product without any weighting (at least 2 MS2 peaks match with the reference spectra). The MS2 similarities with reference spectra were matched to any of the CorrDec or the MS2Dec deconvoluted MS2 spectra of the 3 collision energies (0, 10, and 30 eV).</p>TaurineAllantoinPhenylacetylglutamineRiboflavinN-Acetyl-D-galactosamineo-Hydroxyhippuric acidN,N-DimethylguanosineFolic acidgamma-Glutamyl-cysteinePantothenolKynurenic acidN1-MethyladenosineProline-HydroxyprolineGlutaryl-carnitineFolinic acidThreoninePalmitoyl-carnitineMethionine4-Aminobutyric acid betaineN-Acetyl-ornithineN-Acetyl-arginineUreaValeryl-carnitineAICA-ribosideMethylhippuric acidcis-Urocanic acidNicotinamide4-HydroxyprolineGlutamic acidPyrantelDecanoyl-carnitinetrans-Urocanic acidPenicillin GGuanineCystine1-MethylnicotinamideQuinaldic acidN1-MethylguanosinePyroglutamic acidKynurenineGlutathioneCreatinineN-Acetylglutamic acidAlaninePhenylalanineN2-Acetyl-lysineN-AcetylputrescinePropionyl-carnitineTryptophanXanthosinePyridoxinePseudouridineXanthineN-Acetyl-glutamine7-MethylguanineValineProline betaineN-Acetyl-leucineCholineAsparagine3-Ureidoisobutyric acidStearoyl-carnitineUracilOctenoyl-carnitineN-Methyl-prolinePantothenic acidGuanosineCreatineBetaineButyryl-carnitineCytosineCysteine-S-sulfateMethionine sulfoneTyrosine7,8-Dihydrobiopterin3-Ureidopropionic acidCytidineCyanocobalaminCarnitineHypoxanthine2-PhenylglycinePipecolic acid betaine2-Aminoadipic acidHippuric acidMethionine sulfoxideSerine5-FluorocytosineAllantoic acidProlineN-Acetyl-serineLeucineCHESN6-SuccinyladenosineAspartic acidIsoleucineAdenosineThymineGlycochenodeoxycholic acidOleoyl-carnitine1-Methyluric acidUric acidPIPESDeoxycytidineAcetylcarnitine2-O-MethylinosineInosineDimethylglycineGlutamineThe corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of metabolism and determine the transportation of endogenous and exogenous molecules across the corneal epithelium barrier. This study introduces multiple corneal epitheliums on a chip namely, Corneal Epithelium on a Chip (CEpOC), which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography-mass spectrometry metabolomics method, and 104 metabolites were annotated. We observed the spatiotemporal secretion of biologically relevant metabolites (i.e., antioxidant, glutathione and uric acid) as well as the depletion of essential nutrients such as amino acids and vitamins mimicking the in vivo molecules trafficking across the human corneal epithelium. Through the shifts of extracellular metabolites and quantitative analysis of mRNA associated with transporters, we were able to investigate the secretion and transportation activities across the polarized barrier in a correlation with the expression of corneal transporters. Thus, CEpOC can provide a non-invasive, simple, yet effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as advanced experimental model of the human corneal epithelium.Spatiotemporal determination of metabolite activities in the corneal epithelium on a chip.Abdalkader Rodi R, Chaleckis Romanas R, Wheelock Craig E CE, Kamei Ken-Ichiro KIbiochemical pathways, Safeties, 9-dihydro-, Metabolic Process, Biological Markers, Viral Marker, Surrogate Endpoints, epithelium posterius corneae, determination, Laboratory, Commuting, Aminosaeure, tissue distribution, Metabolic Concepts, Metabonomic, Biochemical, Amino acid, Monohydrate, Endpoint, Metabonomics, Serum, Monosodium, eye surface, cornea epithelium, Techniques, Laboratory Markers, Method, Biological, vitamin, gamma-L-Glutamyl-L-cysteinyl-glycine, eye, Concepts, 2, globe, Analysis, 6, Metabolism Concept, 7, Phenomenon, Nutrient, 2310040B03Rik, Kinetics, Mass Spectrum Analysis, amino acids, enzymes, Analyses, catabolism, Chip, ChIP, Glycine, eyes, Reduced, membrane region, visual system, 5-L-Glutamyl-L-cysteinylglycine, Drug Kinetics, CHIP, Bodily, metabolic process resulting in cell growth, Urate, eye surface region, behavioral response to stimulus, procedures, Acid Urate, inhibition of homeostatic process, Glutathione, Reduced glutathione, epithelial tissue of cornea, chip, Secretion, Immune, Markers, Methodological Studies, medicine, Secretions, Viral Markers, AW046544, homeostasis, biotransformation, associated, SIMPLE, Catabolism, epithelium anterius (cornea), single-organism behavior, eye globe, l(2)k04405, Viral, epithelium anterius corneae, Surrogate Endpoint, Process, external secretion, Aminokarbonsaeure, metabolism resulting in cell growth, LADMER, Liquid Chromatography, integral to membrane, gamma-L-Glu-L-Cys-Gly, vertebrate eye, Biochemical Markers, membranous organ component, Procedure, corneal endothelium, Biologic Marker, Spectrum Analysis, alpha-amino carboxylic acids, Acceptance Processes, Liberation, Spectroscopy, exocrine gland fluid/secretion, Eyes, endothelium camerae anterioris bulbi, Bodily Secretion, Acceptance Process, cornea epithelial tissue, epithelial, positive regulation of homeostatic process, Marker, PIG7, precocious, secretion, eyeball, Trioxopurine, SDCCAG7, Uric, Acid, Amino Acid, antioxidants, membrane of organ, Vitamin, dLdb, protein_coding_transcript, Amino acids, End Points, vitamines, Spectrometry, Methodological, Immunologic, Laboratory Marker, Methodological Study, GSH, human, early, experimental procedures, Ammonium Acid, uric acids, ADME, orbital part of face, Biochemical Marker, Biocatalyst, Monosodium Urate Monohydrate, Acids, epithelium of cornea, exocrine gland secretion, vitaminum, time, HSPABP2, Ammonium, advanced, vitamine, human being, Procedures, exocrine gland fluid, regio orbitalis, experimental, exocrine gland fluid or secretion, Clinical Markers, Biocatalysts, Processes, ADMET, Clinical Marker, antoxidant, 8(3H)-trione, 2210017D18Rik, dCHIP, Aminocarbonsaeure, number, antioxydant, gamma-L-Glutamyl-L-Cysteinylglycine, ocular surface, SCAR16, Spectrum Analyses, alpha-amino acid, Metabolic Processes, epithelium corneæ anterior layer, vitamina, presence, 1H-Purine-2, Surrogate End Points, period, N-(N-L-gamma-glutamyl-L-cysteinyl)-, Surrogate Markers, method, DmelCG5203, Elimination, camera-type eye plus associated structures, integral component of membrane, Metabolism, method used in an experiment, Studies, Mass, Distribution, Metabolomic, Metabolism Phenomena, Technique, Mass Spectroscopy, light-detecting organ, study, Biomarker, methods, NY-CO-7, Clinical, cornea epithelium., experimental section, Potassium Urate, nutrients, Biological Marker, endothelium corneale, Absorption, Metabolic Concept, vitaminas, messenger RNA, N-(N-gamma-L-Glutamyl-L-cysteinyl)glycine, external epithelium of cornea, man, gamma L Glutamyl L Cysteinylglycine, Study, secreted substance, template RNA, Enzyme, drugs, Immunologic Markers, 10^[-6], bodily secretion, DmelCG3924, gamma L Glu L Cys Gly, region of membrane, negative regulation of homeostatic process, Sodium Acid, vitamins, 8-Trihydroxypurine, UBOX1, Behaviors, 0610033N24Rik, L-gamma-glutamyl-L-cysteinylglycine, e. anterius corneae, behaviour, Sodium Urate Monohydrate, Immunologic Marker, Potassium, Macronutrient, Biologic, Sodium Acid Urate Monohydrate, TP53I7, Autoregulation, Ammonium Acid Urate, membrane, nutrient, and Toxicology, and Elimination, degradation, anterior endothelium of cornea, drug, Serum Markers, CG5203, End Point, bulbus oculi, enzyme activity, alpha-amino acids, l(2)04405, visual apparatus, Immune Marker, Concept, Metabolic Phenomena, Macronutrients, Metabolism Concepts, count in organism, toxicokinetics, Surrogate End Point, chemical analysis, Phenomena, whole membrane, INSDC_feature:mRNA, Sodium, techniques, regulation of homeostatic process, metabolism, Reduced Glutathione, Mass Spectrum Analyses, epitheliocyte, Metabolic Phenomenon, Biologic Markers, Acceptance, Mass Spectrum, multicellular organism metabolic process, transmembrane, Serum Marker, biodegradation, Metabolic, mRNA, Ldb, LDB, Urate Monohydrate, Surrogate, anterior corneal epithelium, Endpoints, Monosodium Urate, CG3924, Sodium Acid Urate, Amino, Sodium Urate, Surrogate Marker, plan specification, Drug, extracellular, single-organism metabolic process, activation of homeostatic process, behavioural response to stimulus, dLDB/Chip, orbital region, endothelium anterius corneae, and Response, ADME-Tox, Glutathione-SH, assay, quantitative, methodology, Anabolism, presence or absence in organism, Immune Markersbiochemical pathways, Safeties, 9-dihydro-, Metabolic Process, Biological Markers, Viral Marker, Surrogate Endpoints, epithelium posterius corneae, determination, Laboratory, Commuting, Aminosaeure, tissue distribution, Metabolic Concepts, Metabonomic, Biochemical, Amino acid, Monohydrate, Endpoint, Metabonomics, Serum, Monosodium, eye surface, cornea epithelium, Techniques, Laboratory Markers, Method, Biological, vitamin, gamma-L-Glutamyl-L-cysteinyl-glycine, eye, Concepts, 2, globe, Analysis, 6, Metabolism Concept, 7, Phenomenon, Nutrient, 2310040B03Rik, Kinetics, Mass Spectrum Analysis, amino acids, enzymes, Analyses, catabolism, Chip, ChIP, Glycine, eyes, Reduced, membrane region, visual system, 5-L-Glutamyl-L-cysteinylglycine, Drug Kinetics, CHIP, Bodily, metabolic process resulting in cell growth, Urate, eye surface region, behavioral response to stimulus, procedures, Acid Urate, inhibition of homeostatic process, Glutathione, Reduced glutathione, epithelial tissue of cornea, chip, Secretion, Immune, Markers, Methodological Studies, medicine, Secretions, Viral Markers, AW046544, homeostasis, biotransformation, associated, SIMPLE, Catabolism, epithelium anterius (cornea), single-organism behavior, eye globe, l(2)k04405, Viral, epithelium anterius corneae, Surrogate Endpoint, Process, external secretion, Aminokarbonsaeure, metabolism resulting in cell growth, LADMER, Liquid Chromatography, integral to membrane, gamma-L-Glu-L-Cys-Gly, vertebrate eye, Biochemical Markers, membranous organ component, Procedure, corneal endothelium, Biologic Marker, Spectrum Analysis, alpha-amino carboxylic acids, Acceptance Processes, Liberation, Spectroscopy, exocrine gland fluid/secretion, Eyes, endothelium camerae anterioris bulbi, Bodily Secretion, Acceptance Process, cornea epithelial tissue, epithelial, positive regulation of homeostatic process, Marker, PIG7, precocious, secretion, eyeball, Trioxopurine, SDCCAG7, Uric, Acid, Amino Acid, antioxidants, membrane of organ, Vitamin, dLdb, protein_coding_transcript, Amino acids, End Points, vitamines, Spectrometry, Methodological, Immunologic, Laboratory Marker, Methodological Study, GSH, human, early, experimental procedures, Ammonium Acid, uric acids, ADME, orbital part of face, Biochemical Marker, Biocatalyst, Monosodium Urate Monohydrate, Acids, epithelium of cornea, exocrine gland secretion, vitaminum, time, HSPABP2, Ammonium, advanced, vitamine, human being, Procedures, exocrine gland fluid, regio orbitalis, experimental, exocrine gland fluid or secretion, Clinical Markers, Biocatalysts, Processes, ADMET, Clinical Marker, antoxidant, 8(3H)-trione, 2210017D18Rik, dCHIP, Aminocarbonsaeure, number, antioxydant, gamma-L-Glutamyl-L-Cysteinylglycine, ocular surface, SCAR16, Spectrum Analyses, alpha-amino acid, Metabolic Processes, epithelium corneæ anterior layer, vitamina, presence, 1H-Purine-2, Surrogate End Points, period, N-(N-L-gamma-glutamyl-L-cysteinyl)-, Surrogate Markers, method, DmelCG5203, Elimination, camera-type eye plus associated structures, integral component of membrane, Metabolism, method used in an experiment, Studies, Mass, Distribution, Metabolomic, Metabolism Phenomena, Technique, Mass Spectroscopy, light-detecting organ, study, Biomarker, methods, NY-CO-7, Clinical, cornea epithelium., experimental section, Potassium Urate, nutrients, Biological Marker, endothelium corneale, Absorption, Metabolic Concept, vitaminas, messenger RNA, N-(N-gamma-L-Glutamyl-L-cysteinyl)glycine, external epithelium of cornea, man, gamma L Glutamyl L Cysteinylglycine, Study, secreted substance, template RNA, Enzyme, drugs, Immunologic Markers, 10^[-6], bodily secretion, DmelCG3924, gamma L Glu L Cys Gly, region of membrane, negative regulation of homeostatic process, Sodium Acid, vitamins, 8-Trihydroxypurine, UBOX1, Behaviors, 0610033N24Rik, L-gamma-glutamyl-L-cysteinylglycine, e. anterius corneae, behaviour, Sodium Urate Monohydrate, Immunologic Marker, Potassium, Macronutrient, Biologic, Sodium Acid Urate Monohydrate, TP53I7, Autoregulation, Ammonium Acid Urate, membrane, nutrient, and Toxicology, and Elimination, degradation, anterior endothelium of cornea, drug, Serum Markers, CG5203, End Point, bulbus oculi, enzyme activity, alpha-amino acids, l(2)04405, visual apparatus, Immune Marker, Concept, Metabolic Phenomena, Macronutrients, Metabolism Concepts, count in organism, toxicokinetics, Surrogate End Point, chemical analysis, Phenomena, whole membrane, INSDC_feature:mRNA, Sodium, techniques, regulation of homeostatic process, metabolism, Reduced Glutathione, Mass Spectrum Analyses, epitheliocyte, Metabolic Phenomenon, Biologic Markers, Acceptance, Mass Spectrum, multicellular organism metabolic process, transmembrane, Serum Marker, biodegradation, Metabolic, mRNA, Ldb, LDB, Urate Monohydrate, Surrogate, anterior corneal epithelium, Endpoints, Monosodium Urate, CG3924, Sodium Acid Urate, Amino, Sodium Urate, Surrogate Marker, plan specification, Drug, extracellular, single-organism metabolic process, activation of homeostatic process, behavioural response to stimulus, dLDB/Chip, orbital region, endothelium anterius corneae, and Response, ADME-Tox, Glutathione-SH, assay, quantitative, methodology, Anabolism, presence or absence in organism, Immune MarkersHSPABP2, l(2)k04405, NY-CO-7, dLdb, epithelium anterius corneae, epithelium posterius corneae, anterior endothelium of cornea, Ldb, LDB, Chip, ChIP, anterior corneal epithelium, 2210017D18Rik, dCHIP, endothelium corneale, CG5203, secondary metabolites, metabolite, CHIP, external epithelium of cornea, SCAR16, epithelium corneæ anterior layer, CG3924, corneal endothelium, l(2)04405, cornea epithelium, epithelial tissue of cornea, chip, SDCCAG7., endothelium camerae anterioris bulbi, cornea epithelial tissue, primary metabolites, dLDB/Chip, DmelCG5203, endothelium anterius corneae, metabolites, DmelCG3924, AW046544, epithelium of cornea, UBOX1, 0610033N24Rik, e. anterius corneae, epithelium anterius (cornea), 2310040B03RikHSPABP2, l(2)k04405, NY-CO-7, dLdb, epithelium anterius corneae, epithelium posterius corneae, anterior endothelium of cornea, Ldb, LDB, Chip, ChIP, anterior corneal epithelium, 2210017D18Rik, dCHIP, endothelium corneale, CG5203, secondary metabolites, metabolite, CHIP, external epithelium of cornea, SCAR16, epithelium corneæ anterior layer, CG3924, corneal endothelium, l(2)04405, cornea epithelium, epithelial tissue of cornea, chip, SDCCAG7., endothelium camerae anterioris bulbi, cornea epithelial tissue, primary metabolites, dLDB/Chip, DmelCG5203, endothelium anterius corneae, metabolites, DmelCG3924, AW046544, epithelium of cornea, UBOX1, 0610033N24Rik, e. anterius corneae, epithelium anterius (cornea), 2310040B03Rik0falseSpatiotemporal determination of metabolite activities in the corneal epithelium on a chipThe corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of metabolism and determine the transportation of endogenous and exogenous molecules across the corneal epithelium barrier. This study introduces multiple corneal epitheliums on a chip namely, Corneal Epithelium on a Chip (CEpOC), which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography-mass spectrometry metabolomics method, and 104 metabolites were annotated. We observed the spatiotemporal secretion of biologically relevant metabolites (i.e., antioxidant, glutathione and uric acid) as well as the depletion of essential nutrients such as amino acids and vitamins mimicking the in vivo molecules trafficking across the human corneal epithelium. Through the shifts of extracellular metabolites and quantitative analysis of mRNA associated with transporters, we were able to investigate the secretion and transportation activities across the polarized barrier in a correlation with the expression of corneal transporters. Thus, CEpOC can provide a non-invasive, simple, yet effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as advanced experimental model of the human corneal epithelium.2021-07-132020-11-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:71012CHEBI:27891CHEBI:16919CHEBI:57589CHEBI:74767CHEBI:27373CHEBI:125449CHEBI:17724CHEBI:5100CHEBI:16643CHEBI:15676CHEBI:44302CHEBI:16244CHEBI:18386CHEBI:17295CHEBI:17053CHEBI:35280CHEBI:18261CHEBI:82952CHEBI:30837CHEBI:17439CHEBI:17568CHEBI:16235CHEBI:17203CHEBI:16199CHEBI:68467CHEBI:17562CHEBI:16750CHEBI:18095CHEBI:48517CHEBI:19062CHEBI:27470CHEBI:16977CHEBI:16856CHEBI:16737CHEBI:16857CHEBI:40521CHEBI:46905CHEBI:36274CHEBI:9008CHEBI:16347CHEBI:15891CHEBI:15375CHEBI:18089CHEBI:17154CHEBI:28037CHEBI:85095CHEBI:17821CHEBI:16335CHEBI:17786CHEBI:15640CHEBI:19289CHEBI:28664CHEBI:84644CHEBI:68441CHEBI:45441CHEBI:17768CHEBI:17802CHEBI:18107CHEBI:37023CHEBI:8654CHEBI:17533CHEBI:17775CHEBI:68830CHEBI:17015CHEBI:17895CHEBI:15354CHEBI:18344CHEBI:132188CHEBI:16283CHEBI:16040CHEBI:17490CHEBI:18183CHEBI:16709CHEBI:16828CHEBI:84651CHEBI:18208CHEBI:16946CHEBI:17515CHEBI:70869CHEBI:7676CHEBI:16797CHEBI:17368CHEBI:85465CHEBI:17884CHEBI:55484CHEBI:18050CHEBI:1670CHEBI:15603CHEBI:16543CHEBI:90344CHEBI:17115CHEBI:15698CHEBI:17596CHEBI:17750CHEBI:17196CHEBI:16020CHEBI:17191CHEBI:49033CHEBI:71169CHEBI:30818CHEBI:30817CHEBI:35704CHEBI:44933CHEBI:16015CHEBI:16414CHEBI:5757CHEBI:21553