This model stimulates bio mechanical stimuli experienced by smooth muscle cells in human aorta. Combined with patients derived cells, this system can be used for drug screening and personalized medicine of aortic diseases. This model can mimic and precisely control the bare mechanical parameters of the smooth muscle cells in human aorta, which provides a novel in vitro platform for studying and pathogenesis of aortic diseases.
Demonstrating the procedure will be Mieradilijiang Abudubat, a poster doctor from our laboratory. Begin by washing the right lateral region of the ascending aorta with sterile pbs, once or twice. Remove the intima and a adventitia layers of the tissue with two ophthalmic forceps and retain the media layer to harvest the cells.
Place the media layer onto a 10 centimeter dish and cut it into two to three millimeter pieces. Then, add five milliliters of SMCM containing 10%fbs and 1%penicillin streptomycin to the tissue samples. Move the small tissue into the culture bottle with a sterile dropper, spreading the tissue evenly.
Discard the culture medium as much as possible. Then invert the bottle upside down. Add two milliliters of SMCM into the inverted culture bottle and place it in the incubator with 5%carbon dioxide at 37 degrees Celsius for one to two hours.
Then slowly turn it right side up and add another two milliliters of SMCM. After five to seven days of incubation, exchange the SMCM with four milliliters of fresh SMCM. Slowly discard and add the SMCM when changing the medium.
Generally, sporadic smooth muscle cells climb out in approximately two weeks. Transport the culture bottle gently when moving to a microscope station. Otherwise, the tissues will float and the cells will grow slowly.
When the cells have grown, divide them into two new culture bottles with four milliliters of fresh SMCM. Identify the cells through an immunofluorescence analysis of four specific markers for smooth muscle cells. To polymerize PDMS, add the curing agent or B component to the base or the A component and mix the complex for five minutes.
The volume will depend on the need of the study. Place the prepared PDMS gel in a vacuum extraction tank for 30 to 60 minutes and maintain the pressure below negative 0.8 milli pascals. Using computer aided design software, design the mold.
Custom make the molds of the three layers using a high precision computer numerical control engraving machine. After carving out the frame of the molds and the microchannels using PMMA plates, glue them onto another plate. Pour the prepared PDMS gel onto a designed mold.
Then cross-link at 70 degrees Celsius for one to two hours. Peel off the cross-linked PDMS slabs from the mold and cut the commercialized PDMS membranes into 100 millimeter by 40 millimeter sections. Punch holes on the three PDMs slabs using a one millimeter hole puncher to make the inlet and outlet of air and medium microchannels on the PDMS chip.
Treat the three prepared PDMS slabs and two PDMS membranes with oxygen plasma for five minutes for PDMs surface activation. Set the room air to process gas, pressure to negative 100 kilopascals, current to 180 to 200 milliamp piers, voltage to 200 volts, and the processing time to five minutes. Bond three PDMS slabs and two PDMS membranes together under a stereoscopic microscope, such that the top layer consists of the air channel PDMS slab, then the PDMS membrane.
The medium channel consists of the PDMS slab, then the PDMs membrane, and the bottom layer is the air channel PDMS slab. Place the assembled PDMS chip in a 70 degree Celsius incubator for one hour. Prepare several one millimeter inner diameter and three centimeter length latex hoses.
Insert a one millimeter outer diameter and one centimeter long stainless steel needle into one end of the prepared hose. Then insert a lure into the other end of the hose to create the tube connected to the air and medium microchannels of the PDMS chip. Insert the prepared tubes into the outlets and inlets of the air and medium microchannels on the PDMS chip.
Infuse two milliliters of 80 milligrams per milliliter of mouse collagen into the medium microchannel of the PDMs chip, using a two milliliter syringe, and incubate at room temperature for 30 minutes. After incubation, remove the collagen from the channel with a two milliliter syringe. Place the collagen coded PDMS chips in a 60 degree Celsius incubator overnight.
Place the PDMS chips in a UV sterilizer for more than one hour. Place the sterilized PDMS chips on an ultra clean bench in preparation for the cell experiment. Culture four times 10 to the five primary human aortic smooth muscle cells from patients using SMCM containing 2%FBS and 1%penicillin streptomycin in a 5%carbon dioxide incubator, set at 37 degrees celsius.
When the smooth muscle cell density reaches 80%discard the SMCM, and wash the cells with two milliliters of PPS. Digest the cells using one milliliter of 0.25%tripsin for two minutes and centrifuge at 100 RCF for five minutes. Remove the supernatant and resuspend the cell pellet in one milliliter of fresh SMCM.
Use eight milliliters of SMCM to culture the cells on a 10 centimeter culture dish. Using a cytometer, calculate the cell number and proceed with the final concentration of two times 10 to the five cells per milliliter. Slowly pour two milliliters of PBS into the collagen coded and sterilized PDMS chip medium microchannel and later discard using a two milliliter syringe.
Slowly pour two milliliters of two times 10 to the five cells per milliliters smooth muscle cell suspension into the media microchannel of the PDMS chip. Then, close the lure at the entrance and exit of the PDMS chip. Place the PDMS chip in an incubator, set at 37 degrees celsius with 5%carbon dioxide for one day.
After incubation, when the cells are attached to the PDMS membrane in the PDMS chip, connect the outlet of the air microchannel on the PDMS chip to the vacuum controller system. An elongated normal cellular form will be seen under the microscope when the cell is attached. Contrasting with the suspended round cells.
Turn on the solenoid valve and the vacuum pump. Open the vacuum regulator and adjust the pressure level depending on the strains. Then, place the PDMS chips and an incubator set at 37 degrees celsius with 5%carbon dioxide for 24 hours.
Prepare several solenoid valves, vacuum filters, vacuum regulators, a vacuum pump, a peristaltic pump, and a PLC controlling the solenoid valve. Program the PLC controller and set the on off time interval to one hertz. Connect the solenoid valves to the programmed controller.
Connect the inlet of the vacuum pump to the vacuum filters, and then connect the outlet of the vacuum filters to the vacuum regulators. Connect the outlet of the vacuum regulators to solenoid valves. And finally, connect the outlet of the solenoid valves to the outlets of the air microchannels of the PDMS chips.
Connect the outlet of the peristaltic pump to the inlet of the medium microchannel of the PDMS chip and the inlet of the peristaltic pump to the outlet of the medium microchannel PDMS chip for culture medium replacement and drug handling. Adjust the amplitude of the strain by the regulator and the strain frequency by the micro controller. Viability of the human aortic smooth muscle cell line, CRL 1990, in the PDMS chip, was estimated using a live and dead assay on the third and fifth day of cell culture.
The cell viability was found higher than 90%on day three and day five. Cytoskeleton F-actin staining of CRL 1990, in the PDMS chip, showed normal cell morphology and cells aligned perpendicularly to the strain after stretching for 24 hours. Immunofluorescence of the contractile markers SM22 and CNN1, was higher under strain conditions compared to the static conditions.
Also, the SM22 and CNN1 genes were up-regulated in strain conditions of smooth muscle cells. The F-actin staining of BAV and TAV, thoracic aortic aneurysm and dissection of patient-derived primary human aortic smooth muscle cells, showed normal cell morphology and cells aligned perpendicularly to the strain direction after stretching for 24 hours. The SM22 and CNN1 fluorescence in BAVTAAD and TAV thoracic aortic aneurysm and dissection were higher under strain conditions than under static conditions.
Similar to the gene expression levels of SM22 and CNN1, which were also up-regulated in strain conditions compared to static conditions. Attention should be paid to the principle of sterility when isolating primary HASMC. After the PDMS labs and the membranes are treated with plasma, the surfaces should not be soiled.
Based on this protocol, endothelial cells can be co-cultured with smooth muscle cells and the sheer stress can be given to the cells with cyclic strain to further simulate more mechanical forces.
