Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation

Abstract

This paper describes the use of arrays of horizontally-oriented reservoirs to deliver liquids through microchannels at a constant flow rate over extended periods of time (hours to days). The horizontal orientation maintains a constant hydraulic pressure drop across microfluidic channels even as the volumes of liquids within the reservoirs change over time. For a given channel-reservoir system, the magnitude of the flow velocity depends linearly on the height difference between reservoirs. The simple structure and operation mechanism make this pumping system versatile. A one-inlet-one-outlet system was used to continuously deliver media for perfusion cell culture, and an array of inlet reservoirs coupled to an outlet reservoir via microchannels was used to drive flows of multiple laminar streams. The parallel pumping scheme conveniently generated various smooth and step concentration gradients, and allowed evaluation of the effect of colchicine on myoblasts. Since the reservoir arrays are configured to be compatible with commercialized multichannel pipettors designed for 96 well plate handling, this simple pumping scheme is envisioned to be broadly useful for medium to high throughput microfluidic perfusion cell culture assays, cell migration assays, multiple laminar flow drug tests, and any other applications needing multiple microfluidic streams.

A 3D printed microfluidic perfusion device for multicellular spheroid cultures

“…This design configuration is commonly employed to immobilize and culture cells as 3D spheroids or organoids in microfluidic devices [3,25]. In line with the objective of making microfluidic cell culture devices more simple and accessible for biological experimentation, we have also implemented a pump-free perfusion culture system, relying on gravity-driven flow to perform medium perfusion for nutrient and oxygen delivery [28] in a conventional cell culture incubator. Finally, we demonstrated the biological performance of the 3D printed device by performing pump-free perfusion cultures of patientderived oral squamous cell carcinoma (OSCC) tumor and liver cell (HepG2) spheroids with good cell viability and functionality.…”

“…A constant medium flow rate was sustained by a pair of 3 ml media reservoirs, which were secured onto the printed inlets and outlets at a specified height difference and orientated horizontally ( figure 1(C)). This setup allows the maintenance of the liquid meniscus in the inlet and outlet reservoirs at a given height difference to generate a constant hydrostatic pressure to drive medium flow across the microfluidic channel (figure 4(E)) [28,29]. Hence, the entire 3D printed microfluidic perfusion culture device can be placed in a sterile secondary container inside a 37°C, CO 2 incubator and operate as a standalone device independent of pumps and tubing.…”

“…Perfusion culture in the 3D printed device relied on gravitational-induced flow, which was dependent on the height difference between the horizontally-orientated media reservoirs attached to the perfusion inlet and outlet of the device ( figure 4(C)). The horizontal orientation of media reservoirs allowed the liquid meniscus to maintain the fluid height difference between the perfusion inlet and outlet throughout the culture period, generating constant perfusion flow rate [28,29]. We used CFD simulation to estimate the flow velocities ( figure 4(E)) and corresponding shear stresses (figures 4(D), (F)) at the cell immobilization microstructures as a function of height difference between the inlet and outlet media.…”

Polymer-Based Microfluidic Devices for Pharmacy, Biology and Tissue Engineering

“…The system offers possibilities to localize the cells inside the microfluidic chamber and enable cell growth in a homogeneous microenvironment [288]. Many other microfluidic devices for enhanced cell growth, culturing and micro-environmental changes with various perfusion systems have been reported [289][290][291][292][293][294][295].…”

Microfluidics for cell-based high throughput screening platforms—A review

“…Besides commercial syringe pumps which are used in most microfluidic devices for the generation of flows, using gravity driven flow is another possible solution. Zhu et al [45] proposed an improved and simple gravity driven system with horizontally-oriented tubes maintaining a constant hydraulic pressure drop across microfluidic channels. With a similar principle, a pressure-control based microfluidic system has been applied in a three-dimensional cell culture platform for cell-based drug testing in 30 microbioreactors [46,47].…”