title: An in vitro hepatic zonation model with a continuous oxygen gradient in a microdevice.
abstract: In a hepatic lobule, different sets of metabolic enzymes are expressed in the periportal (PP) and pericentral (PC) regions, forming a functional zonation, and the oxygen gradient is considered a determinant of zone formation. It is desirable to reproduce lobular microenvironment in vitro, but incubation of primary hepatocytes in conventional culture dishes has been limited at fixed oxygen concentrations due to technical difficulties. We designed a cell culture microdevice with an oxygen gradient to reproduce the hepatic microenvironment in vitro. The oxygen gradient during cell culture was monitored using a laser-assisted phosphorescence quenching method, and the cellular oxygen consumption rate could be estimated from changes in the gradient. Culture medium was continuously exchanged through microchannels installed in the device to maintain the oxygen gradient for a long term without transient hyper-oxygenation. The oxygen consumption rates of hepatocytes at 70.0mmHg and 31.4mmHg of partial oxygen pressure, which correspond to PP and PC regions in the microdevice, were 3.67×10(-10) and 3.15×10(-10)mol/s/10(6) cells, respectively. Antimycin A changed the oxygen gradient profile, indicating that cellular respiration can be estimated during cell culture. RT-PCR analysis of hepatocytes cultured under the oxygen gradient showed that mRNA expression of PEPCK and GK significantly increased in culture areas corresponding to PP and PC regions, respectively. These results indicate that the developed microdevice can reproduce the hepatic lobular microenvironment. The oxygen gradient in the microdevice can be closely controlled by changing the sizes of gas channels and the ambient oxygen concentration around the device; therefore, it could be expected to mimic the oxygen gradient of various organs, and it may be applicable to other pathological models.
authors.affiliation: Graduate School of Fundamental Science and Technology, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Sato
authors.affiliation: Department of Applied Physics and Physico-Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Kanae
authors.initials: K
authors.lastname: Kadokura
authors.affiliation: Graduate School of Fundamental Science and Technology, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama, Kanagawa 223-8522, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Uchida
authors.affiliation: Graduate School of Fundamental Science and Technology, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama, Kanagawa 223-8522, Japan; Department of Applied Physics and Physico-Informatics, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kouhoku-ku, Yokohama, Kanagawa 223-8522, Japan. Electronic address: ktsukada@appi.keio.ac.jp.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Tsukada
mesh_terms: D000818:Animals; D061249:Batch Cell Culture Techniques / Q000295:instrumentation*; D019149:Bioreactors; D002478:Cells, Cultured; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation*; D022781:Hepatocytes / Q000145:classification / Q000166:cytology* / Q000378:metabolism*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008904:Miniaturization; D009924:Organ Culture Techniques / Q000295:instrumentation; D010100:Oxygen / Q000378:metabolism*; D010101:Oxygen Consumption / Q000502:physiology
chemical_list: D010100:Oxygen
keywords: Hypoxia; Liver; Metabolic zonation; Microdevice; Oxygen gradient