BaroFuse, a novel pressure-driven, adjustable-throughput perfusion system for tissue maintenance and assessment

Abstract

Objectives: Microfluidic perfusion systems are used for assessing cell and tissue function while assuring cellular viability. Low perfusate flow rates, desired both for conserving reagents and for extending the number of channels and duration of experiments, conventionally depend on peristaltic pumps to maintain flow yet such pumps are unwieldy and scale poorly for high-throughput applications requiring 16 or more channels. The goal of the study was to develop a scalable multichannel microfluidics system capable of maintaining and assessing kinetic responses of small amounts of tissue to drugs or changes in test conditions.

Methods: Here we describe the BaroFuse, a novel, multichannel microfluidics device fabricated using 3D-printing technology that uses gas pressure to drive large numbers of parallel perfusion experiments. The system is versatile with respect to endpoints due to the translucence of the walls of the perifusion chambers, enabling optical methods for interrogating the tissue status. The system was validated by the incorporation of an oxygen detection system that enabled continuous measurement of oxygen consumption rate (OCR).

Results: Stable and low flow rates (1-20 μL/min/channel) were finely controlled by a single pressure regulator (0.5-2 psi). Control of flow in 0.2 μL/min increments was achieved. Low flow rates allowed for changes in OCR in response to glucose to be well resolved with very small numbers of islets (1-10 islets/channel). Effects of acetaminophen on OCR by precision-cut liver slices of were dose dependent and similar to previously published values that used more tissue and peristaltic-pump driven flow.

Conclusions: The very low flow rates and simplicity of design and operation of the BaroFuse device allow for the efficient generation of large number of kinetic profiles in OCR and other endpoints lasting from hours to days. The use of flow enhances the ability to make measurements on primary tissue where some elements of native three-dimensional structure are preserved. We offer the BaroFuse as a powerful tool for physiological studies and for pharmaceutical assessment of drug effects as well as personalized medicine.

Keywords: Bioengineering; Pharmaceutical Chemistry.

Fig. 1

BaroFuse parts and functions. (A) (top) Schematic diagram of the reservoir module topped by the perfusion module that holds the tissue perfusion channels and response measurement. (bottom). (B) An 8-channel BaroFuse prototype, consisting of a perfusion module with 8 vertical perfusion columns, is mounted atop a reservoir module adjoined on a (black) silastic gasket. Ports for independently pressurizing source and/or transfer reservoirs are shown at left, while the transfer conduits are visible as the horizontal passages across the septum. High-resistance, low-resistance and transfer perfusate flow tubes are not visible but one of each of these is associated with a perfusion channel and are contained inside the reservoir module.

Fig. 2

Pressure-flow rate test confirms ability of the BaroFuse to control flow rates. Flow rates were measured for different pressures generated by pressurized gas using 3 different resistance tubes with inner diameters as indicated. Pressure was changed by adjustment of the pressure regulator. Lengths of resistance tubes were all ≈ 100 mm. Due to gravitational forces, the flow continues even when there is no added pressure from the gas tank, and would only become 0 if negative pressure of −0.2 psi would be induced.

Fig. 3

Functional response of pancreatic islets in the BaroFuse. (A) (top) OCR by 10 isolated rat islets was measured in response to glucose simultaneously in 3 of the 8 channels (flow rate was 7 μL/min). (bottom) Outflow fractions were collected in three of the channels and assayed for insulin. (B) OCR by 1 isolated rat islet was measured in response to glucose simultaneously in 3 of the 8 channels (flow rate was 1.5 μL/min). Data for both curves are averages ± standard error of the mean (n = 3).

Fig. 4

Effect of acetaminophen on liver oxygen consumption in the pressure-driven micro-perifusion system. OCR by 1 mg slices of mouse liver was measured in response to acetaminophen. Data are plotted as averages ± standard error of the mean (n = 3).