(G-256) Development of a Closed-Circuit Cell Culturing System for Long-Term Cell Incubation in Microchannels

Introduction:: Acute respiratory distress syndrome (ARDS) causes flooding and eventual collapsing of lung airways due to the deactivation of lung surfactant. The pressure gradient surrounding penetrating fingers of air during airway reopening imposes damage on the respiratory zone of the lung leading to a high mortality rate as a result of ventilator-induced lung injury (VILI). To investigate the VILI mechanism in vitro and culture of lung epithelial cells in micro-channels in order to interrogate cellular responses to physicochemical stimuli utilizing electric cell-substrate impedance sensing (ECIS) measurements. The channel flow apparatus must be made of non-air permeable hard materials to perform stress-induced damage tests under dynamic multiphase flow environments. Our previous study suggests that it requires cell culturing with continuous feeding flow about 10-100 times faster than the flow rate necessary to supply oxygen and nutrition for 3-4 days. (We found that shear stress from the flow impacts a uniformity of H441 cell layer formation.) The requirement for an excess amount of growth media also raised concerns about contamination during cell incubation as well. Our goal is to make a closed-circuit growth media circulation system that can generate variable feeding flow rates in a manner suitable for long incubation periods. The system will not require us to open the incubation chamber during cell incubation. We also aim for a low production cost and customization flexibility in accordance with the needs for cell culturing in various sterilization environments combined with different types of micro-scale pumping systems.




Materials and Methods:: To test the closed system, Stereolithography (SLA) 3D-printing technology is utilized to manufacture the system. The peristaltic pump is composed of a 12V Nema 17 DC stepper motor, with the shaft attached to a top and bottom casing made with the resin. This pump is controlled with an Arduino Uno, powered using a 12V power supply. The peristaltic pump has a 3D-printed rotor with 6 rollers composed of two bearings and a shaft snap fitted onto the rotor. The rotor is placed inside the case, along with flexible tubing of inner diameter 0.0625” and outer diameter 0.125” which is placed along the edges to minimize backward flow at low flow rates. This pump assembly is connected the motor using a shaft coupler. The rotor pushes the bearing against the tube when rotating, which causes the movement of liquid. A more detailed cross-sectioned view of the pump is shown in Figure 2. This tube is then connected from a reservoir to the microchannel outflow, specifically the ECIS chip, which connects back into the reservoir. The reservoir is sealed with a semi-permeable membrane to prevent contaminants from entering the media, as well as simultaneously letting air come in contact with the media.




Results, Conclusions, and Discussions:: This design is capable of pushing circulated media at a constant speed from the reservoir through the channel. We are able to culture the cells in the non-air permeable channel without opening the incubator for 3-4 days with this system. The new system reduces the risk of contamination during the incubation, as well as the waste of growth media. We will demonstrate cell layer formation under various flow conditions and channel geometries using ECIS. We will present a working prototype of the system, along with ECIS and immunofluorescent results of the cell culture under constant flow, and in vitro stress-induced damage experiments.



Acknowledgements (Optional): : This research is supported by NIH R01 HL142702



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