The overall goal of this procedure is to apply minutes scaled intermittent hypoxia to micro scaled pancreatic eyelets to investigate preconditioning effects. This is accomplished by first fabricating a multimodal microfluidic device with a thin gas permeable membrane. The second step is to create a computerized oxygen modulation system.
Next eyelets are carefully loaded into the device, and then liquid and oxygen microfluidics are connected. The final step is to coordinate microscopy with the multimodal microfluidics in order to visualize the eyelet parameters that are changed during the oxygen modulation. Ultimately, the eyelet, intermittent hypoxia technique is used to show that glucose response is clearly impaired in hypoxia.
While temporal modulations of hypoxia can train eyelets to respond with less impairment. The main advantage of this technique over existing methods like dissolved oxygen or using a hypoxic chamber, is that accurate. Oxygen concentration could be delivered to the eyelids in rapid, minuscule time intervals without sheer stress from complicated flows.
It also allows real time monitoring with floor and microscopy. This master can help answer key questions in the Alli transplant field, such as defining hypoxic impairments in transplant sites or preconditioning regimens to improve transplant outcomes. So this method can provide insight into Alli hypoxia.
It can also be applied to other tissues and systems such as the central nervous system, cardiac tissues, the kidney, and other transplant organs. Video demonstration of this method is critical as many of the device fabrication and eyelet handle. The steps are difficult to learn because of the novel interdisciplinary nature of the technique, but inherent the challenging or familiarized Begin by fabricating the cover layer of the microfluidic device.
To accomplish this pour DGAs pre-mixed PDMS to a height of 1.5 millimeters into a blank Petri dish and cure it at 80 degrees Celsius for two hours. Next, fabricate the glucose microfluidic layer with profusion chamber master by spin coating two 350 micron thick layers of SU 8 21 50 to form a single 700 micron layer on a four inch silicone wafer. Then expose it to UV light using the glucose microfluidic layer photo mask to transfer the microchannel pattern onto the SU eight layer.
Rinse away the unexposed area with SU eight developer. Next, pour DGAs pre-mixed PDMS onto the MA up to a height of three millimeters and cure at 80 degrees Celsius for two hours. Once cured, cut the glucose microfluidic layer to shape and use a two millimeter punch to form the inlet and outlet ports, as well as an eight millimeter diameter punch for the main chamber.
Next, fabricate the micro well master for eyelid immobilization by spin coating a 100 micron thick layer of SU 8 2100 onto a clean four inch silicon wafer, and then use UV lithography to transfer the micro well pattern onto this layer. Rinse away the unexposed areas with developer when the microwell master is complete. Add two 100 micron layers of PDMS by spin coating at 900 RPM for 30 seconds, and then curing for 10 minutes at 80 degrees Celsius to produce each layer.
Once finished, punch the two millimeter diameter inlet in outlet ports. Finally fabricate the gas microfluidic master by spinning SU 8 2100 to 100 microns thickness as in the previous layer, and transfer the gas microfluidic patterns again using UV lithography. Rinse away the unexposed area with developer.
Next, pour a 1.5 millimeter thick layer of PDMS onto the gas microfluidic master and cure at 80 degrees Celsius for two hours. Then cut the PDMS to shape with the razor blade. Then punch two millimeter diameter inlet and outlet ports into the cover layer to line up with where they're found on the other layers to bond the multiple layers together.
First, prepare them by cleaning the surfaces with scotch tape, exposing them to a Corona arc, and then aligning the layers by hand in the following order. First, bond the membrane to the bottom gas layer with the micro wells facing up. Then bond the glucose microfluidic layer on top of the micro well membrane.
Finally, bond the inlet and outlet layer on the very top, encapsulating the whole assembly. Assist in forming a tight bond by adding a one kilogram weight on top of the assembly and baking it at 100 degrees Celsius for three hours. Then leak.
Test the device by loading water to the aqueous layer, submerging the device and flowing air through the gas layer with the syringe while looking for leaks. Finally, sterilize the leak-free devices by passing 70%ethanol through the aqueous layer for one to two minutes. Set up the micro dispensers by first connecting the micro dispensers control leads to the included driver units.
Then connect the drivers to the digital IO board at ports corresponding to their lab view controls. Next, connect five volt and 20 volt DC power supplies to their corresponding contacts on the drive units and connect the digital IO to a laptop in order to execute lab view codes for gas control as described in the appendix of the accompanying text protocol. Then connect one micro dispenser to nitrogen and the other to compressed air, both containing 5%carbon dioxide and set both gases to two PS.I hook up the dispenser outputs in a T junction prior to entering the microfluidic device.
Next, mount the device onto the heated stage of an inverted microscope with 0%and 21%oxygen connected to the device. Next place a fiber optic oxygen sensor at the fluid output of the device to measure the dissolved oxygen within the output in real time. Then connect the device to a peristaltic pump and begin to flow water through the device at 250 microliters per minute.
Characterize the transient response of oxygen modulation by cycling the micro dispensers between five and 21%oxygen. Connect a peristaltic pump to the microfluidic device and position the tubing over a 37 degree Celsius hot plate to warm the fluid before it enters the microfluidic device. Also, connect the output port of the glucose microfluidics to a fraction collector to begin dissect C 57 black six mice and isolate pancreatic eyelets by collagenase digestion and F ccal density gradient separation as described in the following videos available from JoVE once isolated.
Incubate the eyelets in RPMI 1640 medium containing 10%FBS, 1%penicillin streptomycin, and 20 millimolar heaps in Petri dishes for 24 to 48 hours. Use the eyelets within two days to ensure consistent results. Next, prepare fresh Krebs ringer bicarbonate buffer as described in the accompanying text protocol with one solution containing two millimolar glucose and another containing 14 millimolar glucose.
Warm the solutions in 50 milliliter conical tubes by placing them in a 37 degree Celsius water bath. Then prepare the stain for the isolated eyelets by adding five micromolar of 4:02 AM and DMSO and 2.5 micromolar of RH 1 23 and 100%ethanol to two milliliters of the Krebs buffer containing two millimolar of glucose. Next, pick up eyelets with a 10 microliter pipette and incubate them in stain for 30 minutes at 37 degrees Celsius.
Once stained load approximately 20 eyelets into the device via the glucose microchannel inlet. Direct the eyelets into the chamber by priming buffer from the outlet back into the inlet. Then perfuse the eyelets and buffer for 10 minutes to wash away excess dyes.
First, draw two millimolar Krebs ringer bicarbonate buffer through the system for five minutes to establish a normal pulse baseline. Then stimulate the chamber with the 14 millimolar glucose buffer for 15 minutes, followed by a 15 minute wash with the two millimolar glucose buffer. During this process, record the 510 nanometer for a two emissions by exciting the diet 340 nanometers and 380 nanometers.
Also, record the 530 emission of RH 1 23 by excit the dye at 480 nanometers. To minimize convective disturbances during oxygen modulation, apply buffer flow during the stimulation and washing steps, and make sure to stop the flow in the other steps during testing. Collect effluence from the glucose outlet at one minute intervals for use in additional analysis using the microfluidic approach shown in this video, it is possible to regulate the percent of oxygen flowing past the thin PDMS membrane rapidly using the computerized micro injectors.
Here's an example of this modulation ranging from one minute to six minutes. It is also possible to provide quantitative sub minute modulations of oxygen between two and 8.5 parts per million by delivering oxygen between five and 21%The blue line shows the percentage of delivered oxygen and green is the resulting dissolved oxygen in parts per million. When this cycling is applied to create intermittent hypoxia at the eyelets, one can observe the benefits of preconditioning eyelets against hypoxia as compared to irregular normoxic pulse by observing the improved for ratio after treatment effects of hypoxia and intermittent hypoxia can be observed in the overshoot and oscillation damping of calcium transients as shown here in blue is the normoxic response.
In red is the hypoxic response, and the green line is the conditioned hypoxic response. The measure of calcium, mitochondrial potential and insulin are shown here. They're the results of real-time fluorescent microscopy and off chipp EISA assay using the microfluidic effluence from the system together, these begin to build a multimodal view of the glucose insulin response under hypoxic transient.
Once mastered, this technique can be completed in two days if performed properly, one day for the fabrication of device and to isolate the eyelid cells and one day for the eyelid experiments themselves. While attempting this procedure, it's important to remember to verify the delivered oxygen concentration by marrying the gas ator of the device After it development. This technique paved the way for researchers in the fetal outlet and other tissue micro physiology to explore how hypoxia and temporary oxygen patterns can affect diseases like diabetes, cardiovascular, and neurological disorders.
After watching this video, you should have a good understanding of how to control oxygen modulation, manipulate micro tissues, and generate repeatable, meaningful measurements of tissue hypoxia using multimodal microfluidic devices.
