Neovascularization, the generating of new blood vessels is a highly regulated process in both normal and pathological events. To better understand the neovascularization, it's important to reconstruct the process in natural cellular microenvironment in vitro. We've developed a microfluidic chip and automatic control.
Highly efficient circulations is done to form perfusion micro-tubes and simulate to use the protein to recapitulate to the initial event of new vascularization. The microfluidic sprouting chip was consisted of three channels. An endothelial cell culture channel and a liquid channel were separated by a wide central hydrogel channel.
The microfluidic control system was consisted of a microsyringe pump and electromagnetic pinch valve. A bubble trap chip, a microfluidic sprouting chip, a micro-peristaltic pump, and a culture medium reservoir. With a microfluidic system, endothelial cells could be stimulated by high luminal shear stress, physiological level of transendothelial flow and various vascular endothelial growth factor distributions simultaneously.
Pipette 20 microliter of 125 microgram per milliliter of fibronectin into one media injection port of cell culture channel. Cut a pipette tip to fit the port of cell culture channel with scissors. Insert the pipette tip into the other media injection port of cell culture channel.
Then, pipette out air from cell culture channel to fill it with fibronectin. Incubate the chips at 37 degrees Celsius for one hour. Before cell seeding, pipette 20 microliter of endothelial cell medium into each media injection port and incubate the chips at 37 degrees Celsius for 30 minutes.
Then, pipette out all the media in all media injection ports. Next, pipette five microliter of cell suspension into one media injection port of cell culture channel. Then, endothelial cells quickly spread over the entire channel under differential hydrostatic pressure.
Add about four to six microliter of ECM media to the other port to adjust the hydrostatic pressure and stop cell moving. Remote the chips to cell incubator. Then, turn over the chips every 30 minutes until endothelial cells coat around the internal surface of the cell culture channel, two hours later.
Use pipette tip to remove attached cells in the injection ports very carefully. Then, insert four barbed female Luer adapters into the media injection ports and fill with ECM media. Remove the chips to cell incubator, change ECM media and Luer adapters every 12 hours.
To assemble the microfluidic control system, prepare two polytetrafluoroethylene tubes, two short silicone tubes, three long silicone tubes, one barbed female Luer adaptor, one Y type connector, and then three L type connectors. Fill the syringe with 10 milliliter of preheated ECM medium. Connect a polytetrafluoroethylene tube to the syringe by a barbed female Luer adaptor.
Then, connect the other end of polytetrafluoroethylene tube to a Y type connector. Next, connect two long silicone tubes to the other two ends of Y type connector. We use one tube to connect to reservoir and the other tube to connect to bubble trap chip.
Connect another long silicone tube to reservoir. Next, use two short silicone tubes to connect all inlet and outlet holes on the top layer of bubble trap chip. Connect a polytetrafluoroethylene tube to the backend of the chip.
Next, fix a syringe onto microsyringe pump. Clip two long silicone tubes into the electromagnetic pinch valve. Next, switch the electromagnetic pinch valve to open the pipeline between syringe and the reservoir.
Inject media to the reservoir using microsyringe pump to exhaust air in the tube. Then, switch the valve again to open the pipeline between syringe and the bubble trap chip. Inject media to fill the liquid chamber and backend tube of bubble trap chip.
Take out endothelial sprouting chips from cell incubator, then remove Luer adaptors on the cell culture side. Insert two pipe plugs into the hydrogel injection ports of microfluidic sprouting chip. Connect the back-end tube of bubble trap chip to one port of cell culture channel.
Insert a T type connector to the other port and connect it to long silicon tube connected with reservoir. Clip the long silicone tube into the micro peristaltic pump. Then, insert an air filter to the reservoir.
Next, assemble the microfluidic sprouting chip to the stage top incubator. Next, connect the vacuum pump to the holes in the bottom layer of bubble trap chip by a TPU tube. Set opposite circulation volume and a flow rate in the custom program which controls the microsyringe pump and electromagnetic pinch valve simultaneously.
Next, set up the flow rate of micro peristaltic pump. Turn on the microsyringe pump. Then, the circulation control system is established.
We test the barrier function of some micro-vessels in our model by measuring the diffusional permeability coefficient of 40 kDa F-I-T-C dextrin. As it is shown in the figure, the diffusional permeability coefficient of static culture chip with cell lining is 0.1 plus or minus 0.3 micrometer per second. And the diffusional permeability coefficient of empty channel without cell lining is 5.4 plus or minus 0.7 micrometer per second.
Endothelial sprouting assay showed that endothelial cells sprouted into the central hydrogel largely after 24 hours of static culturing, while the degree of sprouting significantly decreased with the increase of luminal shear stress. Quantitatively, luminal shear stress decreased endothelial sprouting significantly in terms of area of sprouting, average sprouting length, and the longest sprout length. With our system, shear stress on endothelial cells could be optionally changed at any time during the experiment while transendothelial flow stayed at physiological level.
This in vitro 3D biomimetic model can be directly applied to the study of neovasularization mechanism, and holds the potential promise as a low cost platform for drug screening and toxicology applications.
