The hypoxic insert device has six pillars that nest into the wells of a six. Well plate gas flows across the microfluidic network at the base of the pillar and can diffuse across the gas permeable PDMS membrane, separating the gas network and culture media. This device minimizes the diffusion path length between the oxygen source, the microfluidic network at the base of the pillar, and the cells permitting more rapid oxygen concentration.Equilibration.
The device also permits far greater control over the spatial pattern of oxygen exposed to cells, which is determined by the microfluidic gas network design made by standard photolithography using photo masks. Hi, I'm Sean Ard from the Dr.David Edington lab in the Department of Bioengineering at the University of Illinois at Chicago. Hi, I'm Ellie also from the Edington Lab.
Today we'll show you how to fabricate, validate, and utilize a custom device to expose in vitro cell cultures in a six-fold plate to different oxygen concentrations. We've use this procedure in our laboratory to study the effect of oxygen on wound healing, reactive oxygen species generation and migration. So let's get started.
To begin fabrication of the oxygenating insert, prepare Polymethyl Sloane or PDMS in a 10 to one ratio between pre polymer and curing agent. According to manufacturer's instructions, the PDMS mixture is added to a previously machined Dell ran mold patterned to mimic the shape of a standard six well culture plate. Once cured at 75 degrees Celsius overnight, the resultant replica molded pillar array will form the first of three components that comprise the oxygenating insert in order to fabricate The second key component of the oxygenating insert.
An SU eight master is prepared by first spinning SU 8 21 50. On a silicon wafer, a photo mask is applied to the wafer and the SU eight is exposed to UV through the photo mask to create a master mold with a microchannel thickness of 300 micrometers. Pour PDMS on top of the SU eight Master and cure for two hours at 75 degrees Celsius to yield a positive mold which will ultimately form the microchannels at the bottom of each pillar.
The third component of the device is fabricated by spinning PDMS on a silicon wafer at 500 RPMs for 10 seconds, followed by 900 RPMs for 30 seconds to achieve a 100 micrometer thick membrane brain. As PDMs is gas permeable, oxygen can be delivered into the micro channels directly through this membrane. Once all components are prepared, each bonding surface is subjected to oxygen plasma treatment for one minute.
Using a handheld plasma device first bond the six microchannel components to the pillar array, then puncture two holes in each pillar to serve as the gas inlets and outlets. Finally bond the membrane to the bottom of the microchannel to facilitate bonding and expel any bubbles. The plasma treated surfaces are pressed together for about a minute using the blend, end of a pair of tweezers, wait one hour between each bonding to ensure the device is completely bonded and can be calibrated.
Once a complete device has been fabricated, the level of oxygen received by cells when using the oxygenating insert in actual experiments needs to be calibrated. For calibration use a fluorescent oxygen sensor or foxy slide, which contains a fluorescent ruthenium dye coating that is quenched by oxygen. Foxy slides are precut to fit into the bottom of a six well plate.
The foxy slide should be cut to cover as much as possible of the well surface. Since the foxy slide has a thickness of about one millimeter, modify the device to maintain the desired distance for adequate membrane. Slide diffusion.
This distance depends on the experiment, but for most cases should be below one millimeter using the handheld plasma device bond four cut one millimeter thick glass microscope. Slide posts of roughly six millimeters in length and four millimeters in width to the bottom of the device. The device will rest on posts atop the slide cut.
Glass cover slips can also be used for a diffusion gap of 0.17 millimeters to validate oxygen usage. At three milliliters of deionized water at room temperature into the wells of the six well plate where the oxygen will be measured. Place the device in the plate and using metamorph software, select the number and location of the desired positions on the foxy sensor.
Slide to assay oxygen concentrations. Connect tai gun tubing between the oxygen gas tank and the hypoxic insert device. A precision flow regulator set to a flow rate of 50 to 100 milliliters per minute is used for fine control of the delivered gas.
Making sure the precision flow regulator is closed. Open the tank top regulator first, followed by the precision regulator. Reduce the flow rate to between 10 to 20 per minute after 15 minutes.
To avoid creating bubbles in the water, watch the flow rate value closely over the next hour and make adjustments as needed since the pressure drop will change while the system equilibrates altering the flow rate to capture images of the plate and foxy sensors. A fluorescence equipped Olympus IX 71 microscope a charge couple device camera from Q Imaging Tiga, SRV and Metamorpho image acquisition software are used. The microscope is equipped with an Olympus 3 1 0 2 0, a foxy compatible fluorescent filter with an excitation wavelength of 475 nanometers and emission wavelength of 600 nanometers.
To test for equilibration time and extent of oxygenation for the device, choose three points on the foxy slide in which to measure the oxygen concentration. Capture an image of the slide immediately before starting the flow of gas. Capture images and intervals of 10 seconds over 30 minutes as the gas flows through the device to test for heterogeneity and oxygen delivery.
Establish multiple points at one millimeter intervals spanning the width of the channel and measure the oxygen concentration at the well surface using metamorph. Export the mean image intensity for each position and plot oxygenation concentration as dependent on fluorescence intensity. Generate a calibration curve by fitting linear curves to the zero to 10%line and 10 to 21%line relating fluorescence intensity to oxygen concentration For each position measured raw intensity data can be exported from metamorph and analyzed further with Microsoft Excel or other statistical software.
Once the device has been calibrated, experiments such as wound healing assays can commence autoclave the calibrated insert at 121 degrees Celsius for 15 minutes within 24 hours prior to the experiment and work with it in a laminar flow culture hood to prevent subsequent contamination. Soak the sterilized PDMS insert in serum free medium for 24 hours to inhibit gas bubbles when inserting into the plate wells culture, MDCK cells or other adherent cells to 100%co fluency in a six well plate create straight scratches in the mono layers with a P 200 pipette tip. The scratches simulate wounds in the adherent monolayer.
Aspirate the cell media without disturbing the monolayer and rinse with five milliliters of media. Aspirate again and refill the wells with three milliliters of serum free. Medium gently insert the device into the plate, making sure to avoid bubbles.
Angling the device during insertion helps to expel bubbles out one side. Be careful to avoid putting excess pressure on the device, which could deform the PDMS enough to crush the underlying cells. Place the plate and the device onto a 37 degree Celsius heated microscope stage and connect tubing from the source gas tank to the inlet and outlet ports of the device.
The culture incubator should have a hole to permit tubing entry and the tubing should be connected After placing the device into the incubator, follow the same procedure for starting the flow of gas as described previously to assess wound healing capture time-lapse images of the cells every 20 minutes for 15 to 18 hours using equipment and imaging software as described previously, T scratch a wound measuring algorithm can be used to analyze the unhealed surface area after a desired experimental duration. Stop gas flow, remove the plate and proceed to further cell processing if desired. The hypoxic insert device requires less than two minutes to stabilize to 0.5%oxygen as shown here.
The device membrane well bottom gap size was a critical factor in determining equilibration efficiency with larger gap sizes requiring more time to reach steady state oxygen concentration values. The device also permits a great deal of control over the spatial oxygenation in a single well, allowing the formation of multiple conditions and even generating a cyclic oxygen profile across the surface of the well. MDCK cell monolayers were exposed to 10%or 21%oxygen and the surface area of the wound was analyzed over time using MATLAB Ts scratch wound tailing algorithm.
After 11 hours outta 17 total hours, 6.87%of the original wound remained when exposed to 10%oxygen while 64.4%of the wound remained under 21%As shown here, oxygen concentrations are negatively correlated with wound open area for both oxygen concentrations. For the duration of the experiment, We've just shown you how to fabricate, validate, and utilize the hypoxic insert device for a scratch wound healing assay. The device offers a number of advantages over other oxygenation systems such as a smaller lab footprint and far greater control over the spatial and temporal oxygenation at the cell surface.
The device can be utilized by virtually any lab that studies the effect of oxygen or other gases like carbon dioxide on in vitro cell cultures. When doing this procedure, it's important to soak the sterilized device and culture media overnights, minimize the bubble formation during the experiment. In addition, allow the system to equate before starting the experiment.
This is because the flow rate change as the pressure in the system equates. So that's it. Thanks for watching and good luck with your experiments.
