The overall goal of this protocol is to describe the fabrication of organ chipped microfluidic devices for recapitulating organ level functionality in vitro. This protocol describes a way to fabricate organ chipped devices recapitulating organ level function in vitro. The func, the devices, such as these, are actually fabricated using 3D printed molds out of a soft silicon rubber.
This rubber enables us to actually imbue these devices with mechanical cues that enables us to stretch the tissue as you would get in let's say a lung or a gut. We also add profusion, which mimics blood flow and the flow of other bodily fluids within organ systems. Now taken together, these devices enable us to actually recreate and try to understand the complex physiology that happens in vivo.
But do this in vitro. So essentially we're experimenting on humans, but not on people. And so it's a very effective way of bridging the divide between animal studies, that are always done in pre-clinical development of therapeutics, and then before human studies, that are called clinical trials, where there is a much greater risk to the human, to human safety.
And so these devices help bridge the divide and actually enable us to develop new therapeutics and understand the basic biology that actually happens in complex systems such as human. Top channel preparation. Wipe down the glossy side of each polyurethane piece with ethanol and clean room wipes.
Place glossy side of the polyurethane over the open side of the mold in place mold. Place the mold and polyurethane assemblies into the jig, textured side against the end of the jig. Continue to do this until all the molds have been placed within the jig.
Tighten the jig by turning its handle using a wrench, until the jig spacing is 25 millimeters in width. Make a boat of aluminum foil surrounding the mold in place jig. Pour PDMS into each mold's well until full.
Once the entire jig is filled, place the jig into the vacuum desiccator. After one hour, remove from desiccator. And place into 60 degree Centigrade oven for at least four hours.
Disassemble the jig using a wrench. Remove the polyurethane strips from each mold. And discard.
Carefully demold PDMS parts from their molds and lay them feature side up. Line up the blade of the tile scraper at the end tab notch. And cut away each end to cingulate the top components.
Store finished parts in square Petri dishes. Store parts in positive pressure cabinets at room temperature. Bottom channel preparation.
Pour 10.5 grams of PDMS into the mold. The air gun can be used very gently to move the PDMS over the space. Place molds into vacuum desiccator.
After one hour, move the molds to a level 60 degree Centigrade oven. Place mold on table in a laminar flow hood. Loosen the PDMS from edge of the mold.
Grip one corner and gently peel back the PDMS from the mold's surface. When fully removed, invert and lay on work surface so that channel features are face up. Cut along outside edges with tile cutter.
With flat paddle forceps, lay parts feature side up on tape to remove any debris. Remove part from packing tape. Drag the loose end of the part across the slide.
The loose end will laminate with the glass. Cover features with Scotch tape. Store parts in positive pressure cabinets at room temperature.
PDMS membrane preparation. Check that the wafers are free of PDMS on the back. Place each membrane wafer in the designated slots.
Use the one mL syringe to place 09 mL of PDMS onto the center of each membrane wafer post array. Let the PDMS sit for a minimum of five minutes. Allow PDMS to spread throughout the post of the membrane wafer.
Do not proceed to the next step until at least 75%of the post array is covered in PDMS. Plasma treat the polycarbonate strip using the conditions described in the protocol. Remove polycarbonate sheet from the plasma machine and use scissors to cut the polycarbonate sheets into 45 millimeter by 45 millimeter squares.
Lay the plasma treated side of the polycarbonate squares onto the PDMS, centered on the membrane wafer. Place PDMS spacer on the center of the polycarbonate square. Then place precut textured polycarbonate.
Insert trays so that tray three is in the back, tray two is in the middle, and tray one is in the front. Tray one has a notch for alignment. Open the output pressure valve, and very slowly open the input pressure valve.
This is so that the four kilograms of force is gradually applied to each membrane wafer, as opposed to instantly which may break the wafers. Flip the AMF switch to on to begin the curing cycle. Then close the input pressure valve and open the output pressure valves to release the pressure from the air cylinders.
Remove the trays and bring them to the laminar flow hood. Carefully peel off the textured polycarbonate. Carefully remove the PDMS spacer.
Inspect the PDMS membrane for areas with through holes. Use a marker to trace the outline of the through hole area. Also mark any holes or defects on the membranes.
This is an example of an unmarked and marked membrane. Using wafer handling tweezers, loosen wafers from the tray. Remove each membrane from the wafer and place on Petri dish.
The PDMS membrane will demold from the wafer and will be bonded to the polycarbonate backing. Store parts in positive pressure cabinets at room temperature. Top assembly and preparation.
Using matte finished Scotch tape, clean the PDMS membranes as well as the insides of the Petri dish to remove debris. Thoroughly tape the feature side of each tall channel top to remove debris. Place tops in Petri dish with PDMS membrane.
Be aware that some membranes may take one or two top parts. The main channels of each top part should fit within the marked area within the membrane. Load the Petri dishes into the plasma.
Plasma treat membrane and top according to the written protocol. Once the bonding cycle has finished, remove the dishes and lay the activated top parts onto the membrane. Place in 60 degree Centigrade oven for at least two hours.
Using a scalpel, trace around the perimeter of the bonded top to separate it from the polycarbonate carrier. Once the part is traced, peel the top from the polycarbonate. The PDMS membrane that has bonded to the top should peel from the carrier as well.
Using tweezers remove the membrane from the ports that access the bottom channel. Do not leave any parts of the membrane covering the access port. Additionally remove any debris or dust with tweezers.
Chip assembly. Feature side up, plasma treat top assemblies with bottoms. Under an inverted microscope, align the top assembly with the bottom half.
Place in 60 degree Centigrade oven for at least two hours.Results. The protocol presented here describes the scalable fabrication of PDMS organ chips. These devices enable culture of two distinct profuse tissue types on an elastic porous membrane.
The PDMS channels are cast using 3D printed molds, which accelerates prototyping of new designs. Top channels are cast in molds under compression against a compliant polyurethane gasket to produce components with molded ports. While bottom channel components are cast in trays and handled on microscope slide backing.
This fabrication approach combines multi scale patterning of the parts into a single step, which saves time, improves reproducibility and traceability, and reduces debris generated by port punching and multiple cutting steps. The porous membranes are critical to the function of the organ chip, and the fabrication approach based on casting against patterned silicon wafers results in membranes of consistent thickness and surface finish. Handling via polycarbonate carriers allows for larger batch production and storage.
The assembled organ chip consists of two profusion channels in an optically transparent package. In the overlapping region, a porous PDMS membrane enables tissue-tissue interaction of metabolites, proteins, therapeutics, pathogens, and cells to recapitulate organ chip function while two parallel channels on either side are used to provide mechanical strain, using cyclic vacuum actuation. The porosity of the PDMS membrane bio-medically supports the flux of metabolites, growth factors, and even cells between the vasculature and organ parenchyma.
The apparent permeability of the membrane was determined using the di-concentration in the outlet channels with and without Caco-2 gut cells. The gut chip cell layers provide a significantly increased barrier to permeability. The organ chip can be actuated using the parallel vacuum channels to quantitatively and reproducibly apply cyclic strain loading to the membrane, and therefore the cultured tissues.
This cyclic strain combined with media profusion supports cellular differentiation to better mimic in vivo organ physiology, such as formation of villi in the gut chip. Using the protocol described here, you should now be able to fabricate a stretchable PDMS organ chip.
