96-Well High-Throughput Hydrostatic Pressure Waveform Control

We introduce a 3D-printed insert device for controlling hydrostatic pressure waveforms within 96-well plates. Hydrostatic pressure (HP) is an important and often overlooked physiological variable which is known to effect cell proliferation, signaling, differentiation, and apoptosis. Abnormally elevated arterial HP, also known as hypertension, is a leading risk factor for heart disease—the number one cause of death within the United States. Despite this, HP is rarely studied or considered in cell culture experiments. Cells are normally grown in an incubator under static, atmospheric, pressure conditions. Current methods to control HP in cell culture allow for only a single condition to be studied at a time. Most commonly, bulky stainless steel pressure chambers are used, however, smaller microfluidic methods have also been developed which use a column of media or a syringe pump to apply pressure. Despite a smaller footprint, these devices have failed to reach mainstream use due to specialized fabrication process and low throughput. Our device aims to overcome these problems by integrating with a piece of ubiquitous cell culture labware, the 96-well plate, and pressurizing the headspace of each well to deliver 12 dynamic, physiologic (or pathophysiologic), pressure conditions. The 3D-printed insert creates a gas-tight environment for each well using a radial double X-ring seal and connects the 8 wells of each column (12 total) to a unique pressure inlet so that each column receives its own unique pressure condition. The waveforms are controlled by proportional solenoid valves and piezoresistive pressure sensors. Using Simulink software and Arduino microcontrollers, pressure is maintained at a setpoint (either static or dynamic) by a PID control algorithm. Using this method, we have generated 12 unique physiological and pathophysiological arterial pressure waveforms within a 96-well plate ranging from 120/80 mm Hg (normotension) to 20/10 mm Hg (extreme hypotension) and 300/200 mm Hg (extreme hypertension). Pressures from 0-55 psi were demonstrated by swapping pressure sensors for higher range measurement making it possible to study high pressure environments within the body such as the joints and cartilage. A compact control box was constructed to house valves, sensors, and the associated electronics and to allow for easy connection of pneumatic components to the plate insert. Preliminary results with Human Umbilical Venous Endothelial Cells (HUVECs) show that pressure (up to 200 mm Hg) increases proliferation after 36 hours of exposure. By bonding a thin sheet of elastomeric Polydimethyl siloxane (PDMS) to bottomless 96-well plates, we can use hydrostatic pressure to grow cells on a dynamically expanding and contracting substrate. It is our goal to use this method to model barotrauma seen in the lungs after mechanical ventilation. By simultaneously controlling 12 unique waveforms, researchers can more effectively study HP as a variable in cell culture.