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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I7 - Development of a Micro-scale Closed-Circuit Peristaltic Pumping System for Long-term Cell Growth Experiments in Microfluidic Channels

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Acute respiratory distress syndrome (ARDS) triggers barrier-function disruption of the lung epithelium allowing plasma proteins to leak into alveoli, thus deactivating lung surfactant. The loss of surfactant leads to airways collapsing and being compromised with fluid, so mechanical ventilation becomes a life-sustaining solution for adequate oxygen exchange in the lungs of ARDS patients. Unfortunately, mechanical ventilation can lead to repetitive recruitment and de-recruitment of sensitive alveoli, leading to ventilator-induced lung injury (VILI). Our goal is to explore in vitro models of VILI and the lung epithelium’s reaction to physiochemical stimuli. To do so, we have developed a micro-size flow circulation system to support the formation of a confluent epithelial cell layer in a non-air permeable microfluidic channel (0.6 mm x 5.0 mm x 50.0 mm). This is critical because a supply of oxygenated growth media is key for stability of cell layer growth in the non-air-permeable micro channel. Commercially available systems, like a conventional syringe pump, are neither cost- nor space- efficient solutions for multi-channel experiments. We have investigated cost and space effective alternatives using 3D printing and micro controller systems. Our goal is to create a micro-size peristaltic pumping system that
recirculates and re-oxygenates growth media for extended duration (up to 6 days) with infrequent interaction. In this presentation, we will demonstrate the new model by establishing precise user-defined flow parameters, exploring the range of flow rates driven by the peristaltic pump, and improving its design to be cost efficient and flexible for any cell culturing conditions.

Materials and

Methods:  This system was fabricated through stereolithographic (SLA) printing (Formlabs Form 3 Printer) using resin (Formlabs Clear Resin V4) that is hardened by UV light. The pump (Figure 1) consists of the resin printed parts, a 5V DC stepper motor, and 6 rotors with attached ball bearings. The pump is driven by an Arduino Uno board (manufactured by Many) with 5V power supply controlled by Arduino IDE software and an A4988 driver with 12V power supply. The pump consists of 6 rotors placed on a rotating shaft within the pump case with tubing (Cole-Parmer Instrument
Company) of ID= 0.0625” and OD = 0.125”. The pump rotates the shaft at an adjustable step rate, squeezing the rotors along the tubing causing a peristaltic flow between the inlet and outlet both attached to the reservoir. The pump circuit is as follows: The pump inlet is located at the bottom-most point of the fluid reservoir and is connected by tubing to the microfluidic channel. The other end of the channel is connected by tubing that passes through the pump casing so that it pulls fluid from the channel and connects at the outlet which feeds back into the reservoir. The outlet’s location slightly above the fluid level within the reservoir exposes media to open air allowing it to be reoxygenated before resupplying the bulk fluid media.

Results, Conclusions, and Discussions: To calibrate the pump flow, we set the pump to various step rates and measured the total volume produced over a set time, creating a relationship between step rate (steps/sec) and flow rate (µL/min). The least squares approximation of the calibration curve in Figure 2 demonstrates a y=0.455x (R2=0.9817) relationship between flow rate and motor step rate. The pump system is thus capable of driving a minimum flow rate of Q=0.455 µL/min (1 step/sec) and maximum flow rate up to Q=250 µL or higher. The new circulation design was able to achieve a wide range of stable flow rates for extended periods of time due to the closed-circuit design paired with re-adjustment of reservoir size. The pump’s reservoir design was also modified to hold up to four different types of fluid media while requiring less total media (Figure 1). The micro-size circulation pump system delivers highly accurate flow rates of the range of 0.455< Q < 250 µL/s and provides an efficient solution for long-term support of cell-culture in a closed environment. In our presentation, we will demonstrate its use for investigating cell seeding and incubation results using electric cell-substrate impedance sensing (ECIS) and immunofluorescent staining investigations of barrier-function responses using in vitro models of alveolar recruitment to elucidate mechanisms of VILI.

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