96-Well Oxygen Control Using a 3D-Printed Device

Abstract

Oxygen concentration varies tremendously within the body and has proven to be a critical variable in cell differentiation, proliferation, and drug metabolism among many other physiological processes. Currently, researchers study the gas's role in biology using low-throughput gas control incubators or hypoxia chambers in which all cells in a vessel are exposed to a single oxygen concentration. Here, we introduce a device that can simultaneously deliver 12 unique oxygen concentrations to cells in a 96-well plate and seamlessly integrate into biomedical research workflows. The device inserts into 96-well plates and delivers gas to the headspace, thus avoiding undesirable contact with media. This simple approach isolates each well using gas-tight pressure-resistant gaskets effectively creating 96 "mini-incubators". Each of the 12 columns of the plate is supplied by a distinct oxygen concentration from a gas-mixing gradient generator supplied by two feed gases. The wells within each column are then supplied by an equal flow-splitting distribution network. Using equal feed flow rates, concentrations ranging from 0.6 to 20.5% were generated within a single plate. A549 lung carcinoma cells were then used to show that O 2 levels below 9% caused a stepwise increase in cell death for cells treated with the hypoxia-activated anticancer drug tirapirizamine (TPZ). Additionally, the 96-well plate was further leveraged to simultaneously test multiple TPZ concentrations over an oxygen gradient and generate a three-dimensional (3D) dose−response landscape. The results presented here show how microfluidic technologies can be integrated into, rather than replace, ubiquitous biomedical labware allowing for increased throughput oxygen studies.

Emulating clinical pressure waveforms in cell culture using an Arduino-controlled millifluidic 3D-printed platform for 96-well plates

“…In recent years, methods for cell culture within multiwell plates have grown increasingly complex and more accurate at mimicking in vivo conditions. By incorporating micro and millifluidic elements, insert devices and custom-made plates now can incorporate a growing number of physiological variables such as gas tension, 13,[23][24][25] perfusion, 26 matrixstiffness, 27 mechanical confinement, 28,29 and cell-cell interaction. 30 Multiwell plates are a powerful tool to investigate many conditions simultaneously and are the workhorse for high-throughput screening.…”

“…The X-profile O-ring design and PTFE backup rings used in this device allow for easier insertion and removal as well as a 30 psi improvement in burst pressure compared to our previous work. 13 Next, to validate that our platform was compatible with cell growth, we grew HUVECs under a full physiologic spectrum of both static and dynamic pressure profiles. To our surprise, cells under all conditions grew at a comparable or even slightly faster rate than controls.…”

“…8,10 which do not resemble those experienced by cells in vivo. However, recent advances in opensource microfluidic pressure control 12 and methods developed by our laboratory to control gaseous environments within the headspace of 96-well plates 13 have set the stage for precise control of complex waveforms within cell culture. Building on the strengths of the gas pressure method and overcoming its weakness, we introduce a high-throughput, workflowcompatible, precise-waveform device for in vitro hydrostatic pressure control in 96-well plates.…”

Intolerance of profligacy: an aptamer concentration gradient-tailored unicellular array for high-throughput biologics-mediated phenotyping

“…In addition, the examination of bulk sums of cells may conceal specific information regarding intratumoral cell heterogeneity. [10][11][12] Hence, the microfluidics-based integration of a gradient generator and single-cell trap array has been developed for assessing the results of cellular stress reactions, aimed at reagent optimization. Additionally, microfluidics techniques appear to use only hundredths of the amounts of precious reagents with respect to traditional operations.…”

Influence of the Hypercapnic Tumor Microenvironment on the Viability of Hela Cells Screened by a CO2-Gradient-Generating Device

“…Eddington and co-workers recently developed a 3D-printed gas delivery device that adapts to the standard 96-well plate . In their design, a linear oxygen (O 2 ) profile was produced by mixing two gases through distribution networks, and O 2 of 12 quantized concentrations was equally delivered to the wells of each column.…”