This work was supported by the National Natural Science Research Foundation of China Grants-in-Aid (grant nos. 11827803, 31971244, 31570947, 11772036, 61533016, U20A20390 and 32071311), National key research and development program of China (grant nos. 2016YFC1101101 and 2016YFC1102202), the 111 Project (B13003), and the Beijing Natural Science Foundation (4194079).

1. Wafer preparation
NOTE: This protocol is specific for the SU-8 2075 negative photoresist used during this research.
<ol>
	<li>Clean the silicon wafer 3 to 5 times with methanol and isopropanol on a spin coater as follows: first spin for 15 s at 500 rpm, and then spin for 60 s at 3,000 rpm.</li>
	<li>Transfer the silicon wafer to a hotplate, which is preheated to 180 &#176;C and bake the wafer for 10 min.</li>
	<li>Remove the silicon wafer from the hotplate and cool it to room temperature. Clean the wafer again with compressed air before proceeding with the spin coating. Apply 4 mL of the SU-8 2075 photoresist to the center of the wafer.</li>
	<li>Obtain a feature height of 70 &#181;m on the spin coater as follows: first spin for 12 s at 500 rpm, and then spin for 50 s at 2,100 rpm.</li>
	<li>Soft bake the wafer on a hotplate as follows: first bake for 8 min at 65 &#176;C, then bake for 20 min at 95 &#176;C. Next, remove the silicon wafer from the hotplate and cool it to room temperature before lithography.</li>
	<li>Place a photomask onto the photoresist film. Expose the wafer with a lithography equipment to achieve a total exposure of 200 mJ/cm2. 
	NOTE: The photomask contains nine sets of patterns of the chip, so it can fabricate nine microfluidic sprouting chips each time.</li>
	<li>Post expose the wafer on a hotplate as follows: first bake for 5 min at 65 &#176;C, and then bake for 20 min at 95 &#176;C. Next, remove the silicon wafer from the hotplate and cool it to room temperature before developing.</li>
	<li>Transfer the wafer to a glass Petri dish filled with SU-8 developer (PGMEA) to start developing. 
	CAUTION: The developer is irritating to the eyes and respiratory tract. Perform developing in a fume hood. Wear splash goggles, nitrile gloves, and airline mask during the operation.</li>
	<li>Shake the dish gently along the direction of the flow channel and change the developer after 10 min.</li>
	<li>Repeat step 1.9 3-5 times until the patterns can be clearly observed. 
	NOTE: Make sure there is no photoresist residue left on the wafer, otherwise rinse the wafer in the developer again.</li>
	<li>Transfer the wafer to a preheated hotplate set to 120 &#176;C and bake it for 30 min.</li>
	<li>Pipet 35 &#181;L of silane onto a coverslip,&#160;then put the coverslip along with the wafer into a desiccator and pull vacuum. Seal the desiccator and leave the wafer under vacuum for 4 h to silanize the wafer to prevent the adhesion of PDMS during the soft-lithography processes. 
	CAUTION: The silane is toxic. To prevent poisoning, perform silanization in the fume hood and wear nitrile gloves while handling.</li>
	<li>Release the vacuum from the desiccator. Remove the silanized wafer onto a preheated hotplate and bake it at 65 &#176;C for 2 h.</li>
	<li>Store the wafer in a clean Petri dish until required.</li>
</ol>
2. Microfluidic sprouting chip fabrication
<ol>
	<li>Combine 20 g of base agent and 2 g of curing agent (10:1 ratio) in a plastic beaker and mix them thoroughly with a mixing rod.</li>
	<li>Place the beaker into a desiccator and pull vacuum for 1 h to remove air bubbles in the PDMS mixture.</li>
	<li>Pour the PDMS mixture onto the wafer in the Petri dish and place the Petri dish back into the desiccator, degassing for another 30 min. 
	NOTE: It is helpful to use double-sided adhesive tape to glue the wafer onto the bottom of the Petri dish to ensure the wafer is kept horizontal during degassing and curing.</li>
	<li>Remove the Petri dish from the desiccator and place it into an 80 &#176;C dry oven for 3 h to cure.</li>
	<li>Carefully separate the PDMS layer from the wafer and cut the layer to nine chips with a scalpel according to the pattern. 
	NOTE: Keep the feature side up after separation.</li>
	<li>Punch two hydrogel injection ports and four media injection ports out of each chip using a 1 mm and a 3.5 mm biopsy punch, respectively.</li>
	<li>Clean the punched chips with residue-free tape to remove PDMS residue. Place the chips and nine glass coverslips into a plasma cleaner and treat them with oxygen plasma for 30 s to form covalent bonding on the surface.</li>
	<li>Take out the chips and coverslips. Attach the feature side of the chips onto the coverslips.</li>
	<li>Place the attached chips into an 80 &#176;C dry oven for 1 h to intensify the bonding.</li>
	<li>Autoclave the chips before use and keep them sterile for the rest of the procedure.</li>
</ol>
3. Surface modification and hydrogel injection
<ol>
	<li>For each chip, pipet 40 &#181;L of 1 mg/mL poly-D-lysine (PDL) and inject it into channels in the chip from hydrogel injection port. 
	NOTE: Make sure the PDL fills channels, especially in narrow channels near the ports.</li>
	<li>Incubate the chips at 37 &#176;C for 4 h to modify the surface of the PDMS .</li>
	<li>Pipet 200 &#181;L of sterile water and inject it into channels in the chip from hydrogel injection port to wash out PDL.</li>
	<li>Remove the chips into a 120 &#176;C dry oven for 3 h to restore hydrophobicity.</li>
	<li>Place on ice a sterile tube and calculate the volume of Type I collagen to be used as the following equation. 
	Final volume (50 &#181;L) x Final collagen concentration (3 mg/mL in this research) / Concentration in bottle = volume collagen to be added</li>
	<li>Calculate the volume of NaOH to be used as the following equation. 
	(volume collagen to be added) x 0.023 = volume 1 N NaOH</li>
	<li>Prepare 50 &#181;L of hydrogel in the tube as the following protocol: add 5 &#181;L of 10x PBS, 0.5 &#181;L of phenol red, calculated volume of 1 N NaOH, 4 &#181;L of 1 mg/mL Fibronectin, calculated volume of Type I collagen in turn. Add the proper amount of dH2O so that the total volume reaches 50 &#181;L. Mix all the contents in the tube thoroughly. 
	NOTE: Perform all the operations on ice. Use phenol red as an acid-based indicator to assist in visual determination of the pH of the hydrogel. The final hydrogel ends up orange when the pH is about 7.4 and the stiffness is about 15 kPa49.</li>
	<li>Pipet 2-3 &#181;L of hydrogel for each chip and slowly inject it into the central hydrogel channel from hydrogel injection port.</li>
	<li>Incubate the chips at 37 &#176;C for 30 min to allow gelation. 
	&#8203;NOTE: Seal the chips into a sealed box with 1 mL of sterile water if they are not used immediately. Sealed chips can be stored at 37 &#176;C for at most 24 h.</li>
</ol>
4. Cell seeding
<ol>
	<li>Pipet 20 &#181;L of 125 &#181;g/mL Fibronectin into one media injection port of the cell culture channel.</li>
	<li>Cut a pipette tip to fit the port of the cell culture channel with scissors.</li>
	<li>Insert the pipette tip into the other media injection port of the cell culture channel. Then, pipet out air from the cell culture channel to fill it with Fibronectin.</li>
	<li>Incubate the chips at 37 &#176;C for 1 h.</li>
	<li>Before cell seeding, pipet 20 &#181;L of ECM media into each media injection port and incubate the chips at 37 &#176;C for 30 min.</li>
	<li>Then, pipet out all the media in all media injection ports.</li>
	<li>Next, pipet 5 &#181;L 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. 
	NOTE: Prepare the cell suspension by trypsinizing human umbilical vein endothelial cells (HUVECs) from the culture flask and centrifuging them at 400 x g. Then, resuspend the cells to 107 cells/mL in a tube.</li>
	<li>Add about 4-6 &#181;L of ECM media to the other port to adjust the hydrostatic pressure and stop cell moving.</li>
	<li>Remove the chips to the cell incubator. Then, turn over the chips every 30 min until endothelial cells coat around the internal surface of the cell culture channel 2 h later (Figure 1). 
	NOTE: To make the chip upside down, pipet a little water on the back of the coverslip. Then the chip can attach to the cover of the Petri dish.</li>
	<li>Use the pipette tip to remove the attached cells in the injection ports very carefully.</li>
	<li>Then, insert four barbed female Luer adaptors into the media injection ports and fill with ECM media. 
	&#8203;NOTE: The adaptors can function as fluid reservoirs to provide nutrients for the cells in the channel.</li>
	<li>Remove the chips to cell incubator. Change ECM media in Luer adaptors every 12 h.</li>
</ol>
5. Measurement of FITC-dextran diffusional permeability
NOTE: To assess barrier function of the micro-vessel, diffusional permeability of the EC culture channel with or without cell lining is assessed.
<ol>
	<li>Take out a microfluidic sprouting chip with hydrogel injected.</li>
	<li>Repeat steps 4.1-4.6.</li>
	<li>Remove all the Luer adaptors and pipet out all the media in four media injection ports.</li>
	<li>Place the chip onto the confocal laser scanning microscope.</li>
	<li>Pipet 5 &#181;L of culture media containing 500 &#181;g/mL 40 kDa FITC-dextran to one port of cell culture channel. 
	NOTE: 40 kDa FITC-dextran has similar molecular size to VEGF-165 (39-45 kDa).</li>
	<li>Capture images every 3 s for 30 s. Thus, the diffusional permeability without cell lining is measured.</li>
	<li>Take out the microfluidic sprouting chip after HUVECs are confluent in the cell culture channel.</li>
	<li>Repeat steps 5.3-5.5.</li>
	<li>Capture images every 3 s for 30 s. Thus, the diffusional permeability with cell lining is measured.</li>
	<li>Calculate the diffusional permeability by quantifying changes of fluorescent intensity over time using the following modified equation50. 
	Pd= (I2- I1) / ((I1- Ib)&#183;&#916;t) &#183;S/d 
	where, Pd is the diffusional permeability coefficient, I1 is the average intensity at an initial time point, I2 is the average intensity after delta time (&#916;t), Ib is the background intensity, S is the area of the channel in fluorescence images, and d is the total interval of micro-posts in fluorescence images. In the present work, &#916;t is set as 9 s. 
	&#8203;NOTE: The present equation is slightly different from the original50, due to the diffusion direction of the fluorescence in the chip is only from the EC channel to hydrogel channel, which is not like the circular tube diffusing in all the radial direction.</li>
</ol>
6. Microfluidic control system setup
NOTE: The microfluidic control system in the present study is consisted of a micro-syringe pump, an electromagnetic pinch valve, a bubble trap chip, a microfluidic chip, a micro-peristaltic pump, and a reservoir. Each part of the system can be replaced by alternatives able to perform the same function.
<ol>
	<li>Bubble trap chip fabrication 
	NOTE: The bubble trap chip is used to remove air bubbles in circulation. The chip consists of three PDMS layers. The top layer is constructed using soft lithography to form grid structure as the liquid chamber. Each channel of the grid structure is 100 &#181;m wide. The bottom layer is a PDMS chunk with a hole. Between the two layers, a 100 &#181;m thin PDMS film is laid.
	<ol>
		<li>Repeat step 1 to prepare the wafer for bubble trap chip.</li>
		<li>Repeat steps 2.1-2.5 to fabricate the top layer and bottom layer of the bubble trap chip. 
		NOTE: The photomask of the top layer contains three sets of patterns, so cut the PDMS layer to three chips according to the pattern.</li>
		<li>Punch six holes on the end of inlet and outlet channels of the top layer using a 3.5 mm biopsy punch.</li>
		<li>Punch two holes on the corresponding position of bottom layer using a 6 mm biopsy punch according to the pattern on the top layer.</li>
		<li>Repeat steps 2.1-2.2 to prepare 10 g of PDMS mixture and pour it on the center of a cleaned silicon wafer.</li>
		<li>Fabricate a 100 &#181;m PDMS film by applying the following spin protocol: spin for 15 s at 500 rpm, increase the spin speed to 1,300 rpm and hold here for 45 s.</li>
		<li>Transfer the wafer to a preheated hotplate set to 180 &#176;C and bake it for 30 min.</li>
		<li>Clean the punched layers with residue-free tape to remove PDMS residue. Place the top layers and wafer into a plasma cleaner and treat them with oxygen plasma for 30 s to form covalent bonding on the surface.</li>
		<li>Take out top layers and wafer. Attach the feature side of top layers onto the PDMS film.</li>
		<li>Cut the film carefully along the edge of the top layers with a needle.</li>
		<li>Slowly separate the film from the wafer and turn over the chips to make the film side up after separation.</li>
		<li>Place the bottom layers and attached chips into a plasma cleaner and treat them with oxygen plasma for 30 s again.</li>
		<li>Take out bottom layers and attached chips. Attach the bottom layers onto the PDMS film and the bubble trap chips are done. 
		NOTE: Align the holes on the bottom layer with the patterns on the top layer when attaching.</li>
		<li>Place the chips into an 80 &#176;C dry oven for 1 h to intensify the bonding.</li>
	</ol>
	</li>
	<li>Assemble the microfluidic control system 
	NOTE: All the parts of the system, such as bubble trap chip, reservoir, tubes, and connectors are used after autoclave sterilization, except electronic equipment. Assemble the microfluidic control system on a clean bench.
	<ol>
		<li>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 three L type connectors. 
		NOTE: The advantage of polytetrafluoroethylene tube is low elasticity, therefore, less media is needed in pipeline. Silicone tube, in contrast, has high elasticity, therefore, it is suitable for the pinch valve and peristaltic pump.</li>
		<li>Fill the syringe with 10 mL of preheated (37 &#176;C) ECM medium. 
		NOTE: Preheating helps the medium release dissolved gas.</li>
		<li>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.</li>
		<li>Next, connect two long silicone tubes to the other two ends of Y type connector with one tube connecting to the reservoir and the other tube connecting to the bubble trap chip.</li>
		<li>Connect another long silicone tube to the reservoir.</li>
		<li>Next, use two short silicone tubes to connect all inlet and outlet holes on the top layer of the bubble trap chip. Connect a polytetrafluoroethylene tube to the backend of the chip.</li>
		<li>Next, fix the syringe onto the micro-syringe pump.</li>
		<li>Clip two long silicone tubes into the electromagnetic pinch valve.</li>
		<li>Next, switch the electromagnetic pinch valve to open the pipeline between the syringe and the reservoir. Inject the media to the reservoir using a micro-syringe pump to exhaust air in the tube.</li>
		<li>Then, switch the valve again to open the pipeline between the syringe and the bubble trap chip. Inject the media to fill the liquid chamber and the backend tube of the bubble trap chip.</li>
	</ol>
	</li>
</ol>
7. Endothelial sprouting assay
NOTE: A stage top incubator assembled with phase contrast microscope is used in the present study to observe the process of sprouting in real time. The stage top incubator can maintain the temperature, humidity, and CO2 control on microscope stages, being good for live cell imaging. But the equipment is not necessary for the assay. The protocols provided here can also be worked in a basic cell incubator.
<ol>
	<li>Take out endothelial sprouting chips from the cell incubator.</li>
	<li>Then, remove Luer adaptors on the cell culture side. Insert two pipe plugs into the hydrogel injection ports of the microfluidic sprouting chip. 
	NOTE: The plugs can transform from needles and their main function is to prevent media from spilling.</li>
	<li>Connect the backend tube of bubble trap chip to one port of cell culture channel. 
	NOTE: Make sure there are no bubbles in the tube before connection.</li>
	<li>Insert a T type connector to the other port and connect it to long silicone tube connected with the reservoir.</li>
	<li>Clip the long silicone tube into the micro-peristaltic pump.</li>
	<li>Then, insert an air filter to the reservoir.</li>
	<li>Next, assemble the microfluidic sprouting chip to the stage top incubator.</li>
	<li>Next, connect the vacuum pump to the holes in the bottom layer of bubble trap chip with a TPU tube.</li>
	<li>Set up the circulation volume and flow rate in the custom program, which controls the micro-syringe pump and electromagnetic pinch valve simultaneously (Figure 2). 
	NOTE: The flow rate across the endothelial cell culture channel is calculated according to the classic equation. 
	&#964;&#160;= 6&#181;Q / Wh2 
	where, &#964; is shear stress (dyn/cm2), &#181; is viscosity of the medium (8.8 x 10-4 Pa&#8226;s), Q is the flow rate across the endothelial cell culture channel (ml/s), h is channel height (70 &#181;m), and W is channel width (1,000 &#181;m). The viscosity of the medium is measured using a coaxial cylinder type rotational viscometer. The height and width of the channel are predetermined and manually confirm using a phase contrast microscope at 4x magnification. The circulation volume is 5 mL and flow rate is 85 &#181;L/min (average 0.2 m/s in cell culture channel) for 15 dyn/cm2 shear stress according to calculation.</li>
	<li>Next, set up the flow rate of micro-peristaltic pump. 
	NOTE: The flow rate of micro-peristaltic pump is slightly higher than micro-syringe pump, in order to prevent the media from spilling.</li>
	<li>Turn on the micro-syringe pump. Then, the circulation control system is established.</li>
</ol>
8. Data analysis
NOTE: To quantify the sprouts, the normalized area of sprouting, average sprout length, and longest sprout length were calculated. Results represent mean &#177; SEM obtained from three independent studies. Statistical significance (P &#60; 0.05) is assessed by Student&#39;s t-test.
<ol>
	<li>Fix cells and sprouts in the microfluidic sprouting chip after experiments with 4% paraformaldehyde in PBS for 15 min. Stain nuclei with 4&#8242;,6-diamidino-2-phenylindole dihydrochloride (DAPI; 1: 1000; Sigma-Aldrich) for 10 min and then stain cytoskeleton with TRITC phalloidin (P5285, 1: 100; Sigma-Aldrich) for 1 h. Wash cells with PBS three times at 5 min intervals between each step.</li>
	<li>Take confocal images of the chips in a tiling mode and stitch them using image editing software.</li>
	<li>Count the number of fluorescent pixels of Z-projection images using a custom code in programming software to quantify normalized area of sprouting (See Supplemental File).</li>
	<li>Manually identify and label each tip of sprouts in Z-projection images and calculate distances between sprouts tips to ECs basement membrane using another custom code to quantify average sprout length and longest sprout length (See Supplemental File).</li>
</ol>

The authors have nothing to disclose.

For a long time, real-time observation of neovascularization has been a problem. Several approaches have been developed recently to create perfused vessels lining with ECs and adjacent to extracellular matrix for sprouting22,32,40,46,54, but the mechanical microenvironment is still hard to maintain constantly. It remains a difficult subject to mimic the initial biomechanical microenvironment of ECs, which are subjected to high luminal shear stress and low velocity of transendothelial flow. Here, we presented a MIEN model that firstly simulates the initial event of neovascularization in mimic physiological microenvironment. With the MIEN model, automatic, efficient and bubble-free long-term perfusion is achieved. Luminal shear stress on ECs could be optionally changed at any time during experiments with transendothelial flow staying at physiological level.
The key of the MIEN model is decoupling the effect of luminal flow on ECs from transendothelial flow. To this end, a T-type connector is creatively inserted to the outlet port of the EC culture channel. One end of the connector is connected to the reservoir through the micro-peristaltic pump. The other end of the connector is exposed to the air of the incubator. It keeps pressure inside the endothelial cell culture channel the same as the atmospheric pressure, since there is neither surplus media gathering to increase pressure nor excess media being drawn away to form negative pressure in the channel. In this way, the flow resistance after microfluidic sprouting chip is very small so that the transendothelial flow can be maintained at physiological range even under high luminal shear stress.
One critical step within the protocol is the successful injection of hydrogel in central hydrogel channel. Huang et al. proposed that successful filling of the gels depends on balancing the capillary forces and surface tension within the microfluidic gel channel55. They found three different variables to control the balance: the spacing between micro-posts, the surface properties of the device, and the viscosity of the hydrogel precursor solutions. In the present model, the design of the microfluidic sprouting chip is optimized from previous works22,23,37,40,51,52,53. The geometry and spacing of micro-posts to separate three channels are determined based on previous calculations and numerous experiments. Further, PDL coating and high temperature baking are performed to modify the PDMS surface; hence, adjust the balance between hydrophilicity and hydrophobicity before hydrogel injection.
The other critical step of the protocol is the removal of bubbles. A large amount of air will dissolve into circulation medium during the experiment due to the T-type connector and micro-peristaltic pump, resulting in bubbles in circulation and greatly affecting the function of endothelial cells. To remove bubbles, a bubble trap chip is introduced before microfluidic sprouting chip. It works based on the high gas permeability of the PDMS membrane. When bubbles get into the trap, they are dispersed by the small and dense grid structure and trapped due to the negative pressure generated by the vacuum pump so that they fail to move forward into the microfluidic sprouting chip. Further, the bubbles transport through the PDMS membrane into the negative pressure hole connected to the vacuum pump and disappear eventually. Even so, great attention should be paid to the formation of bubbles during the experiment. As soon as bubbles are found before microfluidic sprouting chip in the pipeline, stop the micro-syringe pump immediately. Gently flinch the pipeline with the finger to let the bubbles move to bubble trap chip and be removed.
Although the initial microenvironment of neovascularization is partially recapitulated, there are some limitations to this MIEN model. The mechanical microenvironments of endothelial cells such as blood induced shear stress and extracellular matrix stiffness are changing during the neovascularization processes. Before neovascularization, the endothelial basement membrane is still intact with complete barrier function, resulting in high luminal shear stress with low velocity of transendothelial flow and interstitial flow8,9. At the onset of angiogenesis and arteriogenesis, a hallmark event is matrix metalloproteases (MMPs) mediation of basement degradation, which will increase ECs permeability and remodel matrix, leading to changes in mechanical microenvironments such as blood induced shear stress and extracellular matrix stiffness. In the present work, we focus on the initiation of neovascularization so the mechanical environments within the microfluidic sprouting chip are designed to keep stable to simulate the physiological conditions. However, changes of mechanical microenvironments will occur with the ECs sprouting, just like in vivo. Besides, similar to many previous studies34,53,56, the present model takes collagen hydrogel as an extracellular matrix. As we focus on the effect of flow induced shear stress, only one stiffness hydrogel is used. However, considering the important effect of matrix stiffness on cell behavior and neovascularization, different stiffnesses of collagen should be studied and can be achieved by regulating the pH of collagen since the mechanical properties of collagen is depending on its pH49. But it is important to note that the hydrogel needs to be thoroughly rinsed with PBS to remove residual acids or bases before cell seeding to prevent damage to the cells. Further, to mimic the complex components of ECM in vivo, various hydrogels such as Matrigel, hyaluronic acid (HA), and fibrinogen etc. should be used in the future to improve the present model. Blood pressure induced cyclic strain is another important hemodynamic force that modulates the morphology and functions of vascular cells57. Previous studies have shown that tensile strain enhanced expression of angiogenic factors in human mesenchymal stem cells58 and induced angiogenesis via degradation of type IV collagen in the vascular endothelial basement membrane59. These results indicated that blood pressure induced strain may affect neovascularization too. The present model focuses on the effect of flow induced shear stress so it isn&#39;t applicable to introduce strain as the hydrogel doesn&#39;t stick tightly enough to the channel and will fall off when stretched. We will pay attention to overcome this difficulty in future works.

Neovascularization happens in many normal and pathological processes1,2,3,4, which include two major processes in adults, angiogenesis and arteriogenesis5. Besides the best-known growth factors, such as vascular endothelial growth factor (VEGF)6, mechanical stimulations, in particular the blood flow induced shear stress, is important in the regulation of neovascularization7. As we know, the magnitude and forms of shear stress vary dramatically and dynamically in different parts of the vasculature, resulting in important effects on vascular cells8,9,10,11,12. Previous studies have shown that shear stress may affect various aspects of ECs, including cell phenotypic changes, signal transduction, gene expression, and the communication with mural cells13,14,15,16,17,18,19,20; hence, regulate neovascularization21,22,23,24.
Therefore, to better understand neovascularization, it is important to reconstruct the process in natural cellular microenvironment in vitro. Recently, many models have been established to create micro-vessels and provide precise control of microenvironment25,26,27, taking advantage of advances in microfabrication and microfluidic technology. In these models, micro-vessels can be generated by hydrogel28,29, polydimethylsiloxane (PDMS) microfluidic chips30,31,32 or 3D bioprinting33,34. Some aspects of the microenvironment, such as luminal shear stress22,23,35,36, transendothelial flow37,38,39,40, biochemical gradient of angiogenic factors41,42, strain/stretch43,44,45, and co-cultured with other types of cells32,46 have been mimicked and controlled. Usually, a large reservoir or syringe pump was used to provide perfused medium. Transendothelial flow in these models was created by pressure drop between the reservoir and micro-tube22,23,38,40. However, the mechanical microenvironment was hard to maintain constantly in this way. Transendothelial flow would increase and then exceed the physiological level if a high flow rate with high shear stress was used for perfusion. Previous study showed that at the initial period of neovascularization, the velocity of transendothelial flow is very low due to the intact ECs and basement membrane, usually under 0.05 &#181;m/s8. Meanwhile, though luminal shear stress in vascular system varies greatly, it is relatively high with mean values of 5-20 dyn/cm2,11,47. For now, the velocity of transendothelial flow in previous works have been generally kept between 0.5-15 &#181;m/s22,38,39,40, and the luminal shear stress was usually under 10 dyn/cm2&#160;23. It remains a difficult subject to constantly expose ECs to high luminal shear stress and physiological level of transendothelial flow simultaneously.
In the present study, we describe an in vitro 3D model to mimic the initial event of neovascularization (MIEN). We developed a microfluidic chip and an automatic control, highly efficient circulation system to form perfusion micro-tubes and simulate the process of sprouting48. With the MIEN model, the microenvironment of ECs stimulated at the initial period of neovascularization are firstly recapitulated. ECs can be stimulated by high luminal shear stress, physiological level of transendothelial flow and various VEGF distribution simultaneously. We describe the steps of establishing the MIEN model in detail and the key points to be paid attention to, hoping to provide a reference for other researchers.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>Genview</td><td>GP3108</td><td></td></tr><tr><td>Collagen I, rat tail</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D9542</td><td></td></tr><tr><td>Electromagnetic pinch valve</td><td>Wokun Technology</td><td>WK02-308-1/3</td><td></td></tr><tr><td>Endothelial cell medium (ECM)</td><td>Sciencell</td><td>1001</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Every Green</td><td>NA</td><td></td></tr><tr><td>Fibronectin</td><td>Corning</td><td>354008</td><td></td></tr><tr><td>FITC-dextran</td><td>Miragen</td><td>60842-46-8</td><td></td></tr><tr><td>Graphical programming environment</td><td>Lab VIEW</td><td>NA</td><td></td></tr><tr><td>Image editing software</td><td>PhotoShop</td><td>NA</td><td></td></tr><tr><td>Image processing program</td><td>ImageJ</td><td>NA</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>91237</td><td></td></tr><tr><td>Lithography equipment</td><td>Institute of optics and electronics, Chinese academy of sciences</td><td>URE-2000/35</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>82762</td><td></td></tr><tr><td>Micro-peristaltic pump</td><td>Lead Fluid</td><td>BT101L</td><td></td></tr><tr><td>Micro-syringe pump</td><td>Lead Fluid</td><td>TYD01</td><td></td></tr><tr><td>Oxygen plasma</td><td>MING HENG</td><td>PDC-MG</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>PBS (10x)</td><td>Beyotime</td><td>ST448</td><td></td></tr><tr><td>Permanent epoxy negative photoresist</td><td>Microchem</td><td>SU-8 2075</td><td></td></tr><tr><td>Phenol Red sodium salt</td><td>Sigma-Aldrich</td><td>P5530</td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>Sylgard 184</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide (PDL)</td><td>Sigma-Aldrich</td><td>P7886</td><td></td></tr><tr><td>Polytetrafluoroethylene</td><td>Teflon</td><td>NA</td><td></td></tr><tr><td>Program software</td><td>MATLAB</td><td>NA</td><td></td></tr><tr><td>Recombinant Human VEGF-165</td><td>StemImmune LLC</td><td>HVG-VF5</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>1.06498</td><td></td></tr><tr><td>Stage top incubator</td><td>Tokai Hit</td><td>NA</td><td></td></tr><tr><td>SU-8 developer</td><td>Microchem</td><td>NA</td><td></td></tr><tr><td>Trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td><td>Sigma-Aldrich</td><td>448931</td><td></td></tr><tr><td>TRITC Phalloidin</td><td>Sigma-Aldrich</td><td>P5285</td><td></td></tr></tbody></table>

microfluidic model to mimic initial event of neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

The in vitro 3D model to mimic the initial event of neovascularization (MIEN) presented here consisted of a microfluidic sprouting chip and a microfluidic control system. The microfluidic sprouting chip was optimized from previous publications22,23,37,40,51,52,53. Briefly, it contained three channels and six ports: an endothelial cell culture channel and a liquid channel with four media injection ports, and a central hydrogel channel with two hydrogel injection ports (Figure 3). The microfluidic control system consisted of a micro-syringe pump, an electromagnetic pinch valve, a bubble trap chip, a micro-peristaltic pump, and a culture medium reservoir (Figure 4). A custom program was used to control the micro-syringe pump and electromagnetic pinch valve simultaneously, with which the flow rate and circulation volume can be set up. A compensation volume was introduced to correct the slight change of volume after multiple cycles due to systemic error. To minimize the medium used for perfusion, an electromagnetic pinch valve was introduced for medium recycle. The electromagnetic pinch valve could switch between two states to make the microfluidic system in two phases in a cycle. In the injection phase, culture medium was slowly injected from the micro-syringe pump to the microfluidic sprouting chip. While in the recycle phase, culture medium was very rapidly extracted from the reservoir back to the micro-syringe pump.
The barrier function of the micro-vessel in our MIEN model was assessed by measuring the diffusional permeability coefficient (Pd) of 40 kDa FITC-dextran. As it is shown in Figure 5, the Pd of static cultured chip with cell lining is 0.1 &#177; 0.3 &#181;m/s, and the Pd of empty channel without cell lining is 5.4 &#177; 0.7 &#181;m/s. Then, endothelial sprouting assay under static and perfusion was performed in the MIEN model. In static conditions, endothelial sprouting occurred about 4 h after seeding and the sprouts would migrate across the central hydrogel channel to the other side in about 48 h. The sprouts degraded gradually after 48 h. While after 24 h of exposure to 5 or 15 dyn/cm2 shear stress (average 0.07 or 0.2 m/s in cell culture channel), ECs aligned in the flow direction changing from polygonal, cobblestone shape into fusiform, and the degree of sprouting decreased with the increase of shear stress (Figure 6). However, the sprouts under shear conditions were more stable than under static culture conditions. They could maintain over 48 h under perfusion. Quantitative statistics showed that endothelial sprouting decreased significantly in terms of area of sprouting, average sprout length, and longest sprout length (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62003/62003fig01.jpg" /> 
Figure 1: Endothelial cells coat around the internal surface of the cell culture channel. HUVECs are evenly dispersed in the cell culture channel 2 h after cell seeding. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/62003fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62003/62003fig02.jpg" /> 
Figure 2: The custom program used to control the micro-syringe pump and electromagnetic pinch valve simultaneously. Main page (up) and subpage (down) of the custom program with which the flow rate and circulation volume can be set up. A compensation volume was introduced to correct the slight change of volume after multiple cycles due to systemic error. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/62003fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62003/62003fig03.jpg" /> 
Figure 3: The schematic of the microfluidic sprouting chip. The chip contains three channels and six ports: an endothelial cell culture channel and a liquid channel with four media injection ports, and a central hydrogel channel with two hydrogel injection ports. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/62003fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62003/62003fig04.jpg" /> 
Figure 4: The schematic of the microfluidic control system. The microfluidic control system consists of a micro-syringe pump, an electromagnetic pinch valve, a bubble trap chip, a micro-peristaltic pump, and a culture medium reservoir. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/62003fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62003/62003fig05.jpg" /> 
Figure 5: Diffusional permeability (Pd) of 40 kDa FITC-dextran. The Pd of static cultured chip with cell lining is 0.1 &#177; 0.3 &#181;m/s, and the Pd of empty channel without cell lining is 5.4 &#177; 0.7 &#181;m/s. **, p &#60; 0.01. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/62003fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62003/62003fig06.jpg" /> 
Figure 6: Representative images of endothelial sprouting under different shear conditions. After 24 h of static culturing, HUVECs invade from the cell culture channel into the adjacent hydrogel channel through micro-posts and a large number of sprouts in the hydrogel. While after 24 h of exposure to 5 or 15 dyn/cm2 shear stress, the degree of sprouting decrease obviously. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/62003fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62003/62003fig07.jpg" /> 
Figure 7:&#160;Quantified area of sprouting, average sprout length, and longest sprout length. After 24 h of static culture (S0) or exposure to 5 (S5) or 15 (S15) dyn/cm2 shear stress, the degree of sprouting decrease with the increase of shear stress. *, p &#60; 0.05; **, p &#60; 0.01. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/62003fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/62003/Supplemental coding files.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Microfluidic Model to Mimic Initial Event of Neovascularization at JoVE.com

Microfluidic Model to Mimic Initial Event of Neovascularization

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...

microfluidic-model-to-mimic-initial-event-of-neovascularization

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

4.6K Views.  Beihang University. Neovascularisatie, het genereren van nieuwe bloedvaten is een sterk gereguleerd proces in zowel normale als pathologische gebeurtenissen. Om de neovascularisatie beter te begrijpen, is het belangrijk om het proces in natuurlijke cellulaire micro-omgeving in vitro te reconstrueren. We hebben een microfluïdische chip en automatische besturing ontwikkeld.Zeer efficiënte circulaties worden gedaan om perfusiemicrobuizen te vormen en te simuleren om het eiwit te gebruiken om het eerste gev...

Video: Microfluidic Model to Mimic Initial Event of Neovascularization

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Neuroscience

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Preparing a Microfluidics-Based Vascularized Human Micro-Tumor Model

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

Author Spotlight: Creating Human Vascularized Micro-Tumors as Models for Translational Cancer Research 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

Cancer Research

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Samenvatting

Meer video's verkennen