The overall goal of this procedure is to develop a multi depth circular cross-sectional endothelial lives micro channels on a chip, which mimics the 3D geometry of in vivo, microvessels, and runs under controlled continuous profusion flow. This is accomplished by first photo lithographically fabricating a master mold with a semi-circular cross-sectional microchannel network using positive reflowable photo resist. The second step is to use the master mold to replicate two poly dimethyl soane microchannels, align them and bond them to create a cylindrical microchannel network.
Next, the primary human umbilical vein endothelial cells are seated inside the microchannel network before culturing the cells under controlled continuous medium perfusion conditions for a time period between four days and two weeks. Ultimately, a confluence cell monolayer indicated by membrane standing and nuclei standing under the microscopes is developed along the inner surface of the microchannel network Calculation of functional micro vessels in which could provide a platform for the study of complex vascular phenomenon. However, conventional individual micro vessel assays such as anso cell migration assays, andSo two formation assays, and the right and mos tic ring assays are limited to recreate individual micro vessels with respect to three dimensional geometry and continuous flow control.
The main advantage of our techniques, our existing microfabrication method, is that it can conventionally fabricate a multi depth microfluidic channel network that mimics the complex 3D geometries of in vivo micro vessels that have rounded cross sections. It also allows to design microvascular biomagnetic systems, which approximately obey more slow and maintain the fluid flow at a required level so that overall channel resistant is low and the flow we lost is a more uniform throughout the network demonstrating the procedure will be a graduate student from my laboratory. This procedure begins with the fabrication of a photoresist master mold consisting of micro channels with diameters between 30 microns and 60 microns as detailed in the text protocol, briefly transfer the reflow photo resist from the refrigerator at four degrees Celsius to the clean room 24 hours prior to use, and allow it to warm to room temperature.
Begin by spin coating the positive reflow photo resist layer onto a pre-clean silicon substrate following the procedure in the text protocol. Then expose the photoresist to UV light through a patterned mask before developing the patterned micro channels. Finally, after reflow, create a semi-circular cross-sectional microchannel network.
Once the master mold is ready, prepare polymethyl soane or PDMS solution at the weight ratio of 10 to one base to curing agent and mix it thoroughly using a planetary centrifugal mixer. Cast the PDMS solution onto the reflowed photo resist master mold. Place the casted PDMS in a desiccate for 15 minutes to Degas.
Use nitrogen gas to remove any remaining bubbles if necessary. Bake the PDMS in an oven at a temperature of 60 degrees Celsius for three hours to allow it to cure. Then remove the cured PDMS layer from the master mold.
Use a sharpened puncher to create inlet and outlet holes by punching holes in the channel network. Clean the surface of the PDMS using nitrogen gas. Treat two PDMS layers with oxygen plasma for 30 seconds inside a plasma cleaner at an operating pressure of 45 milato and an oxygen flow rate of 3.5 cubic feet per minute.
Then align the surfaces of the PDMS manually under an optical microscope. Use a drop of water if necessary for better control of the alignment. Finally, bake the device in an oven at 60 degrees Celsius for 30 minutes to achieve permanent bonding culture.
Primary human umbilical vein endothelial cells or VE in culture medium with L-glutamine supplemented with 10%fetal bovine serum. Treat the device with oxygen plasma for five minutes with an operating pressure of 45 milit and an oxygen flow rate of 3.5 cubic feet per minute. Then load the device with deionized water and treat with UV light for eight hours in a laminar biosafety hood for sterilization.
One day before the cells are ready, wash the device with one x phosphate buffered saline or PBS and then coat with fibronectin. Incubate in the refrigerator at four degrees Celsius overnight. Once the cells are confluent, harvest them by first rinsing the cells with heaps buffered saline solution, and then treating the cells with trypsin EDTA following addition of trypsin.
Incubate the cells for two to six minutes at 37 degrees Celsius. After the trypsin, isation is complete, neutralize the tripsin EDTA with tripsin neutralizing solution. Count the cells, then centrifuge them before resus.
Suspending in culture medium with 8%dextrin dextrin is used to increase the medium viscosity to aid in better cell seating and attachment following the FI enc ENC coating. Wash the device with one XPBS then load the device with culture medium before incubating at a temperature of 37 degrees Celsius for 15 minutes. Next cells in 8%Dextrin culture medium at a concentration of three to 4 million cells per milliliter are loaded into the device.
Place a 20 microliter droplet of cells at one inlet of the device and tilt it to introduce the cells into the microfluidic channel. After 15 to 20 minutes, the cells will begin to attach to the sidewalls of the channels. Rotate the device every 15 minutes to create a more uniform distribution of cells.
If necessary, additional loading can be performed. After five to six hours of static culture, the attached cells will start to fully spread out. Set up long-term perfusion using a remote controlled syringe pump system with a steady flow of 10 microliters per hour, perfusion can be adjusted for a higher flow rate and can last for a time period between four days and two weeks.
When the cells reach confluence inside the device, first, wash the device with one XPBS to thoroughly remove the medium. Then load the device with diluted red dye following incubation of the device in the dark for five minutes at room temperature. Wash it with culture medium to stop the staining.
Long incubation of the dye can cause cellular toxicity and dis adhesion. Then load the device with blue dye diluted with one XPBS incubate in the dark for five minutes at room temperature before thoroughly washing the device with one XPBS. Examine the cell staining under an inverted optical microscope.
If the staining is good, load the microchannels with fixing medium, then submerge the device in fixing medium and completely cover it with aluminum foil. Store the device in the refrigerator at a temperature of four degrees Celsius to prevent the device from drying out and bleaching the fixed device is now ready for confocal imaging, which can be done by a laser scanning confocal microscope, scanning electron microscopy and optical microscopy. Were used to characterize the reflow photoresist and the replicated PDMS channel before and after reflow progress.
The geometric features of the PDMS Microchannel network were characterized and shown here. Circular cross sections of PDMS molds show channel dimensions at each branching level. The results show that the photo resist reflow technique can create multi depth branching channel networks in a more convenient approach by photoresist reflow techniques and allow designing of the microvascular biomimetic systems, which approximately obey Murray's law shown here are microscopy images using fluorescent cell membrane dye in red and cell nuclear dye in blue.
The circular cross-sectional views revealed that the VE lined the inner surface of a cylindrical microchannel network at different branching regions. Because of the complex geometries of in vivo micro vasculature, realtime monitoring of these small vessels is difficult. The developed P DM S based chip offers good optical properties and allows for high quality and realtime imaging of the endothelial micro channels as shown in this confocal movie of the cell lining along the circular channel network.
After watching this video, you should have a good understanding of how to fabricate the reflow for this mass mode. Create circular cross-section PDMS micro channel network, load the endo cell cells into the devices, and set up long-term media profusion. In summary, the developed endo serialized microchannels on a chip provides a rapid and reproducible approach to create circular cross-section multi depths microchannel networks, which mimics the geometry of InVivo micro vessels.
This procedure illustrates the use of unique capabilities in advanced micro manufacturing and micro foodic technologies to create a microvascular model with a long term continuous perfusion control, as well as high quality and the real time imaging capability with the increasing utility of micro foodic channels for cell biology, tissue engineering, and bioengineering applications. The endo serialized micro channels on a chip is a potential essay for microvascular research.
