Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.
1. Device function
2. Device fabrication
3. Device setup
4. Device Validation
5. Applications
6. Representative Results
Figure 1. Schematic and diagrams illustrating device features. The oxygen insert device is fabricated by conventional photolithography (microfluidic network), replica molding (microfluidic network and insert scaffold), and defined spinning of PDMS (gas-permeable membrane). A) The oxygen device nested into a 6-well plate. B) Examples of 24 and 96-well pillar arrays. C) A cross-sectional schematic of a pillar. Oxygen flows into the device through the inlet and travels across a microfluidic network at the bottom of the pillar. Oxygen can freely diffuse across the gas-permeable PDMS membrane at the bottom of the pillar and dissolve into the culture media. D) A microscope image showing the various features of a single-channel pillar from above, with bonded glass posts for the equilibration studies.
Figure 2. Validation of the device with oxygen sensors. Oxygen tension within each well was characterized using a planar ruthenium oxygen sensor. All oxygen mixtures contained balanced nitrogen and 5% CO2 for media buffering. A) Plot illustrating the effect of post height, and thus oxygen diffusion distance between the membrane and cells, on the equilibration time and effectiveness. Heights were established by cut-glass posts bound to the bottom of the device. All three post sizes yield equilibration times much improved over the hypoxic chamber. Note that time is on a log scale. B) Plot depicting the rapid oxygen equilibration response time of the 0.2 mm gap device. C) Multi-position linescans were also taken across the well under the microchannel to ensure homogeneity of the oxygen concentration introduced by the device. Graph depicts the oxygen concentration measured after infusing 0%, 10%, and 21% oxygen for 10 min. D) Device effectively maintains 10% oxygen over 5 days.
Figure 3. Experimentation with more complex oxygen microchannel designs. A) Dual-condition microchannel setup yields a stable 0% and 21% oxygen profile over 14 days. B) An interdigitated and winding pattern of 500 μm width microchannels extending across the pillar results in a cyclic oxygen profile. Note that the data only depicts one representative trial as microchannel alignment was difficult.
Figure 4. Timelapse images of wound closure 0, 7, and 17 hours after initial scratch. Cells were delivered 21% oxygen throughout duration of experiment.
Figure 5. Effect of oxygen concentration on wound healing rate in a scratch assay.
The device is fabricated by standard SU-8 photolithography, replica molding, and defined spinning and made entirely of polydimethylsiloxane. Gas is introduced into the device to establish a concentration gradient between the pillar microchannel and the culture media, driving the system towards a desired equilibrium oxygen concentration. The device has been shown to effectively modulate the temporal and spatial oxygenation inside a well, as well as modulate cellular behavior appropriately. The spatial patterning of oxygenation is defined by the microchannel at the base of the pillar, so a variety of designs could be implemented in crafting the photomask. Additionally, infusion of the desired gas into the gas-phase of the well is expected to improve equilibration time and extent of hypoxia. A microfluidic mixing network could be adapted to the device to provide a means to produce novel gas mixtures from only a few stock gas tanks. Finally, a mechanism for media exchange would eliminate the need for removal of the device from the multiwell plate, of which the cells may respond.
The device has applications in any in vitro or ex vivo experiment requiring control over oxygen concentration. As oxygen is an important physiological variable affecting a vast majority of signaling pathways, the areas of research that would benefit is limited by the creativity of the researcher. Some fields that would benefit from the enhanced temporal control of oxygen concentration include cancer metastasis, sleep apnea, and cardiac ischemia reperfusion injury, among many others. For example, intermittent hypoxia has been correlated with more invasive cancers, upregulating a number of metastastis-associated genes relative to continuous hypoxia and normoxia. Spatial control is also important, as oxygen gradients are critical in development, liver zonation, drug toxicity, and the stem cell niche. The device presented in this article will benefit a number of areas of research by providing a system with a smaller lab footprint, relatively simple operational requirements, and far greater control over oxygen exposure to cells.
This project was funded by the Illinois Department of Public Health and the National Science Foundation (DBI-0852416).
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
PDMS-Sylgard 184 | Dow Corning | |||
Planar FOXY sensor | Ocean Optics | FOXY-SGS-M | Coated microscope slide | |
Gas regulator | Omega | FL-1472-G | ||
Gas | Airgas | Custom mixes | All have 5% CO2 | |
SU-8 2150 | Microchem | |||
MDCK Growth Medium w/ L-Glutamine | SAFC Biosciences | M3803 | ||
Fetal Bovine Serum | ATCC | 30-2020 | ||
Trypsin-EDTA | Sigma | T4049 | ||
L-Glutamine solution | Sigma | G7513 |