Sato Asako, Kadokura Kanae, Uchida Hideyuki, Tsukada Kosuke
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.
Biochem Biophys Res Commun. 2014 Oct 31;453(4):767-71. doi: 10.1016/j.bbrc.2014.10.017. Epub 2014 Oct 14.
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.
在肝小叶中，不同组的代谢酶在门静脉周围（PP）和中央静脉周围（PC）区域表达，形成功能分区，而氧梯度被认为是分区形成的决定因素。在体外重现小叶微环境是很有必要的，但由于技术困难，在传统培养皿中培养原代肝细胞时，氧气浓度固定不变。我们设计了一种具有氧梯度的细胞培养微装置，以在体外重现肝脏微环境。使用激光辅助磷光猝灭法监测细胞培养过程中的氧梯度，并且可以根据梯度变化估算细胞耗氧率。通过安装在装置中的微通道连续更换培养基，以长期维持氧梯度，而不会出现短暂的高氧状态。在微装置中分别对应于PP和PC区域的70.0mmHg和31.4mmHg的部分氧分压下，肝细胞的耗氧率分别为3.67×10（-10）和3.15×10（-10）mol/s/10（6）个细胞。抗霉素A改变了氧梯度分布，表明在细胞培养过程中可以估算细胞呼吸。对在氧梯度下培养的肝细胞进行的RT-PCR分析表明，磷酸烯醇丙酮酸羧激酶（PEPCK）和葡萄糖激酶（GK）的mRNA表达分别在对应于PP和PC区域的培养区域中显著增加。这些结果表明，所开发的微装置可以重现肝小叶微环境。通过改变气体通道的尺寸和装置周围的环境氧浓度，可以紧密控制微装置中的氧梯度；因此，可以预期它能模拟各种器官的氧梯度，并且可能适用于其他病理模型。
