Pressure-Driven Medium Exchange in Physoxia

Abstract

Nowadays Organ-on-Chip research is looking for replacements for In vivo studies by enhancing cell-based models in vitro. Human cell metabolism and drug studies have been done on a dish in regular incubators for decades, but result does not reliably present physiologically relevant situation. Therefore, increasing biomimicry of Organ-on-Chip enables to achieve more physiological relevant research. Physiological oxygen is one of the most crucial parameters to control in Organ-on-Chip models. Most of the cells experience physiological oxygen concentration between 1-10 % and therefore low-concentration oxygen control is needed. Cell culture medium must be changed regularly in long-term In vitro studies to remove metabolic waste and maintain enough amount of nutrients. Goal of this thesis was to study how the different medium exchange methods affect 1-well’s oxygen concentration and develop perfusion-based medium exchange system which could maintain stable oxygen conditions. Thus, thesis project is application for long-term cell studies under stable physoxia. Physoxia is physiologically relevant oxygen concentration in tissues while the hypoxia is unnormal low-oxygen concentration in tissues. The atmosphere in the 1-well structures was controlled by supplying premixed gas around the structure which was sealed with a custom-made cover. Four methods were tested to change cell culture medium in the 1-well structures, while measuring oxygen concentration. The following four types of medium exchange methods were tested: 1) conventional pipetting, 2) syringe injection through the 1-well structure, 3) non-conditioned continuous perfusion and 4) conditioned continuous perfusion. The measurements were done with the cell cultivation 1-well with a volume of 1 ml and in each exchange method the whole volume of the 1-well were exchange. For first two methods, the medium exchange was simulated by changing 1 ml dH2O for the same volume of pre-conditioned dH2O. For the case 3) continuous perfusion, we used unconditioned dH2O to simulate how the slowly perfused unconditioned medium affects the oxygen concentration in the cell culture. The exchanging methods were executed on top of a non-invasive optical oxygen sensor with integrated temperature control. In this study, physoxic and hypoxic oxygen atmospheres (5 % O2, 1 % O2 and 0 % O2 and all with 5 % CO2) were used. As expected, the conventional pipetting method showed the largest peaks (in average 11.1 % O2) in oxygen data in physoxia (5 % O2) and the stabilization back to 5 % O2 took over one hour. Syringe injection through a closed silicone-made chamber was more promising producing only 7.8 % O2 peaks and recovering time was 6.5 minutes on average. Non-conditioned continuous perfusion was tested with large variety of different perfusion flow rates between 1 ml/hour to 1 ml/day. With the flow rate of 1 ml/day, the oxygen concentration stays inside 4.9 % - 5.5 % limits under 5 % oxygen atmosphere but with a significantly faster perfusion speed 1ml/hour, the oxygen concentration varies between 5 – 12 % with 1 ml/hour, which indicates that the chamber could not condition fast flows steadily. Therefore, this study also investigates pre-conditioned continuous perfusion flow, which was tested with 0 %, 1 % and 5 % oxygen concentrations. The results show that with the highest oxygen concentration (5 % O2) the oxygen levels in the chamber stay between 4.9 %-5.3 %.With 1 % oxygen supply, the oxygen level in the cell culture compartment varies between 0.7 % -1.6 % O2 and with 0 % oxygen supply the variation is between 0.1 % and 1.7 % and it is not really achieving the 0 % oxygen level. For a conclusion, results show that the conditioned continuous perfusion is the most promising choice to make cell medium exchanges in physoxia. At least, the pre-conditioning unit should be revised to enhance cell medium pre-conditioning. On the other hand, the syringe-based method could be a viable method to fast, small volume injections. Injecting does not affect the oxygen concentration significantly. A slow perfusion unit could be an enhancement for long-term cell studies under stable and controlled physoxia and hypoxia.