The overall goal of this procedure is to create a dynamic microfluidic-based co-culture platform to emulate the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs. By using this micro for this platform, it helps to answer the key questions in the cancer biology field, such as the microenvironment of peritoneal dissemination. The main advantage of this technique is that a 3D co-culture under precise-controlled free-flow better mimics the in vivo communication between ovarian cancer cells and methothedia cells that occurs in a peritoneal cavity.
After designing and drawing the microfluidic channel pattern and having it produced according to the text protocol, spin coat a layer of SU82075 Photoresist onto the silicone wafer with a spinning speed of 1800 RPM. Bake the Photoresist-coated wafer directly onto a hot plate at 65 degrees Celsius for five minutes. And then at 95 degrees Celsius for 25 minutes to remove excess solvent.
Spin coat a second layer of SU82075 Photoresist onto the wafer for a final thickness of 250 micrometers. Place the Photoresist-coated wafer directly on a hot plate at 65 degrees Celsius for seven minutes and then at 95 degrees Celsius for 30 minutes before slowly cooling the wafer to room temperature. Next, align and put the photo mask directly on top of the wafer.
Then expose it to UV light for 20 seconds to cross length the pattern. On a hot plate, bake the coated wafer at 65 degrees Celsius for five minutes and 95 degrees Celsius for 12 minutes to remove the solvent for prelimerization. Remove the noncross-lengthed Photoresist by immersing the wafer in an SU8 developer for 25 minutes.
Then with isopropanal, rinse the wafer and use pressurized air to dry it. Combine the Polydimethylsiloxane or PDMS base and curing agent at a 10-to-one mass ratio and use the centrifugal mixer under the mixing and degas function to mix the solution. Slowly pour the PDMS mixture onto the wafer to a height of approximately five millimeters.
Degas the PDMS in a vacuum desiccator for 40 minutes. Then cure the PDMS in the oven at 65 degrees Celsius for two hours. Carefully peel the PDMS slab off the master and trim the PDMS along the channels until it is the size of a glass slide.
Then with a one-millimeter biopsy punch, punch the PDMS to create the inlets and outlets of the device. Use pressurized air to clean the PDMS surface and then with adhesive tape, remove the dust and PDMS debris. Now place the PDMS slab with the channel side facing upwards into a plasma cleaner.
Then position a clean glass slide into the plasma cleaner alongside the PDMS slab. Treat the PDMS and glass slide under air plasma at a high-radio frequency or RF level for one minute. Then after removing the samples from the plasma cleaner, bind the PDMS to the glass slide to create an irreversible covalent bond.
Place the PDMS chip on a hot plate at 150 degrees Celsius for one hour to enhance the bonding strength and to return the hydrophobicity of the PDMS before use. To coat microfluidic channels with fibronectin, begin by using 30 micro-liters of 75%ethanol to sterilize the channels. Remove the ethanol and use PBS to rinse the channels twice.
Then thoroughly aspirate the PBS from the channels. Prepare 10 micrograms per milliliter of fibronectin solution in serum-free M199MCDB105 medium with 30 microliters of the solution, slowly fill up the channels and use tape to seal them. Then incubate the chip at four degrees Celsius overnight.
To seed primary peritoneal mesothelia cells or HPMCs into the microfluidic channels, after rinsing cultured cells according to the text protocol, use two milliliters of 10%FBS culture medium to re-suspend the HPMCs. Using a hemocytometer, count the cells and adjust the cell concentration to 3.5 times 10 to the sixth cells per milliliter. Warm the fibronectin-coated microfluidic chip in a 37 degrees Celsius incubator for five minutes before use.
Then completely aspirate the fibronectin solution and slowly pipet 30 microliters of HPMC suspension into each channel. Use tape to seal the channels and place the device in the CO2 incubator overnight. To induce ovarian cancer spheroid formation, dissolve 0.5%agarose in sterilized water.
Dispense 50 microliters of the agarose solution into the wells of 96 well plates. After counting previously-cultured human epithelial cancer cells, re-suspend the cells in 5%FBS culture medium at a density of 5, 000 cells per milliliter. Transfer 200 microliters of cell suspension into each agarose-coated well.
And culture the cells at 37 degrees Celsius and five percent CO2 for 48 hours. Transfer the cancer spheroids to a 50-milliliter centrifuge tube prewetted with one milliliter of serum-free medium and spin down the spheroids at 750 times G for five minutes. Following the preparation of 2.5 micrograms per milliliter of CMFDA solution in serum-free M199MCDB105 medium.
Re-suspend the cancer spheroids in one milliliter of the solution and incubate the spheroids at 37 degrees Celsius for 30 minutes. Then count the spheroid number and use serum-free medium to adjust the concentration to 2, 000 spheroids per milliliter. Prior to spheroid loading, slowly inject 100 microliters of serum-free M199MCDB105 medium to wash away the non-adhered mesothelio cells inside the channels.
Load 30 microliters of fluorescent-labeled spheroid suspension into each channel. Then place the microfluidic chip into the CO2 incubator. Assemble a profusion platform by attaching a syringe loaded with fresh serum-free M199MCDB105 medium to a one-meter long tube.
Place the syringe on the syringe pump and secure the plunger and the body of the syringe. Then install the syringe pump in a horizontal position at the same height as the microfluidic chip inside the incubator. Quickly infuse the whole tube with medium by pressing and holding the fast forward button until the medium overflows in the tubing.
Then run the injection at the desired flow rate. Connect the tubing to the inlet of the channel according to the text protocol. Then use a short-output tube to connect the outlet of the channel to a 50-milliliter conical tube to collect the outflow medium.
Finally, use a bright field or fluorescence microscope to observe the HPMCs and cancer spheroids. Using the microfluidic platform described in this video, ovarian cancer spheroids were modeled with mesothelial cells under hydrodynamic conditions. As shown in this figure, mesothelial cells that were cultured in the microdevice for 16 hours successfully formed a monolayer of HPMCs.
In this experiment, multi-cellular spheroids with diameters of approximately 100 micrometers which are similar in size to those found in patient societies were labeled with a green fluorescent dye CMFDA. The spheroids were then transferred to HPMC-coated microfluidic channels and remained in suspension and the cell morphologies were captured. Once mastered, this technique can be done in several hours over three days if it is performed properly.
One in 10 in this procedure, it is important to remember to avoid introducing air bubbles into the microfluidic channels. Following this procedure, other methods, like drug treatment, adhesion assays, mesothelia-clearance assays can be performed to answer additional questions, like the effect of the introperitoneal microenvironment on cancer progression. After its development, this technique paved a way for researchers in the field of cancer biology to explore into peritoneal mestastasis in humans.
After watching this video, you should have a good understanding of how to generate a peritoneal microdevice with fluidic flow to starting ovarian cancer behavior in peritoneal cavity. Don't forget that working with organic sulfurs such as acetone, as a developer and cyline can be extremely hazardous. MP cautions such as wearing gloves and a lab coat should always be taken when you first perform this procedure.
All of the procedures dealing with this office should be performed under extreme cover.
