Millifluidic Devices for Multiwell Environmental Control

The multiwell plate is an essential tool for biologists. It consists of an array of "test tubes'', or wells, in which cells can be grown and tested for susceptibility to drugs or other factors. Its power lies in the ability to test several conditions at once, with each well serving as a unique test environment. While excellent for this purpose, they lack important environmental factors found in-vivo. To create more realistic cell culture conditions, we introduce three 3D-printed millifluidic devices for environmental control within multiwell plates. Cells are most often grown inside a standard cell culture incubator under ambient room oxygen levels. However, oxygen concentrations within the body vary from tissue to tissue and are most often considerably lower. To address this, we introduce a 3D-printed oxygen-control device for 96-well plates. This device allows users to grow cells under twelve unique oxygen concentrations which can be tuned by changing the relative flow rates of gas entering the device. By growing lung carcinoma epithelial cells under different oxygen environments and exposing them to varying concentrations of the hypoxia-activated drug, TPZ, we were able to visualize cell viability as a function of both oxygen concentration and drug concentration, thus creating a three-dimensional dose-response landscape. Hydrostatic pressure (HP) is also a key physiological variable missing from standard cell culture. Pressure waveforms in the body vary depending on the distance from the heart and each organ's unique biomechanical environment. To mimic these conditions in-vitro, we designed a platform capable of generating HP waveforms within 96-well plates. A 3D-printed device creates a gas-tight seal within each well, and a PID feedback control system opens and closes valves to dynamically pressurize the headspace above each well. Twelve unique waveforms can be delivered simultaneously, one for each column of the plate. To validate the device, human umbilical vein endothelial cells were shown to proliferate under 12 unique static or dynamic arterial pressure waveform conditions ranging from 10-400 mm Hg over 3 days. Additionally, any custom waveform can be generated by the device. To demonstrate, we created a small pressure waveform library from publicly available clinical waveform data. Cells within the body are constantly perfused by a supply of fresh oxygen and nutrients carried by the bloodstream. Certain large 3D cell culture models, such as brain organoids, require perfused or agitated culture conditions for adequate diffusion of oxygen and nutrients. We introduce a 3D-printed millifluidic insert for continuously stirred brain organoid culture in 12-well plates. Angled nozzles direct flow around the well and lift organoids allowing for stirred suspension culture. These systems allow for more accurate representation of in-vivo conditions within standard cell cultureware. It is our hope that this may help researchers understand the role of these physiological variables in health and disease as well as incorporate them into drug discovery workflows.