2.5D Model for Ex Vivo Mechanical Characterization of Sprouting Angiogenesis in Living Tissue

Quantifying the cell mechanics that drives sprouting Angiogenesis in 3D living tissues is still difficult, and yet is necessary to continue making progress in the field of regenerative medicine. This protocol makes the task more approachable by combining the accessibility of 2D quantitative methods with the representativity of 3D tissues. Existing methods for the quantification of cellular forces during angiogenic sprouting in living tissue are limited to complex and computationally expensive 3D force inference.

Our protocol bridges in vitro control with in vivo relevance by adapting traction force microscopy for X-vivo systems offering an easy to use yet physiologically relevant method for spatial temporal mechanical force characterization. 2 Nav-D model preserves a physiological context while allowing for quantitative analysis of cellular mechanics, offering a unique approach to study the mechanics driving sprouting angiogenesis. To begin, pipette 120 microliters of the bind silane solution into each well of a glass bottom, 12-well plate.

Incubate the plate at room temperature for one hour. After incubation wash the wells three times with absolute ethanol using a spray bottle, dry the plate using nitrogen gas. Pipette 11.5 microliters of the freshly prepared polyacrylamide or PAA gel mixture onto the glass bottom of each well.

Gently place a 13 millimeter cover slip on top of each droplet. After polymerization, add PBS to the well. Using a bent needle, gently loosen the cover slip from the PAA gel.

Then use tweezers to remove the cover slip from the well. Add 75 microliters of one milligram per milliliter of sulfa sampa, dissolved in ultrapure water onto the PAA gels. Place the plate under 365 nanometer ultraviolet light for five minutes.

Quickly rinse the sulfa sampa on the gels with PBS. Then wash the functionalized PAA gels two times with PBS for 10 minutes each. Pipette 50 microliters of collagen 4 solution onto the functionalized PAA gels and incubate overnight at four degrees Celsius.

The next day, wash the gels two times with PBS and allow them to dry for five minutes. Add 50 microliters of endothelial cell growth or ECG medium on top of the gels. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for one hour.

To begin, using sterile surgical tweezers, transfer the porcine carotid artery from the transport bottle containing modified CREB solution into a large Petri dish filled with sterile PBS. Remove excessive tissue surrounding the carotid artery using surgical tweezers and a scalpel. Cut off two to three centimeters from both ends of the artery using a scalpel to eliminate areas near artery bifurcations.

With the help of fine round-tip tweezers and a scalpel, remove the arterial fascia surrounding the carotid artery. Using tweezers, transfer the cleaned carotid artery to a new large Petri dish filled with PBS. Cut the artery into approximately two millimeters wide rings.

Then transfer the rings to a prewarmed small Petri dish containing ECG medium. Place the rings at 37 degrees Celsius. To begin, using round-tip tweezers, transfer the porcine carotid artery ring from the ECG medium to a medium sized Petri dish filled with PBS.

With the help of round-tip tweezers and a scalpel, cut the ring in half, then dissect half a ring into sheets of approximately two millimeters width to create arterial sheets with dimensions of two by two millimeters. Using round-tip tweezers, hold the arterial sheet at the back and position the sheet at the edge of the PAA gel with the endothelial inner lining facing the PAA substrate. Next, with the help of tweezers, move the arterial sheet gently to the center of the PAA hydrogel without touching the gel.

Add 50 microliters of ECG medium on top of the arterial sheet while ensuring the droplet remains on the gel. Using the round-tipped tweezers, place a dry 13 millimeter cover slip on top of the arterial sheet on the PAA substrate in the medium. Use the inner rim of the glass bottom to gently lower the cover slip until the medium droplet spreads underneath.

Allow the tissue to attach at 37 degrees Celsius and 5%carbon dioxide for five hours. Afterward, add one milliliter of ECG medium to each well. Incubate the arterial sheet on the PAA substrate at 37 degrees Celsius and 5%carbon dioxide for 24 hours.

The next day, using a bent needle, carefully lift the cover slip and remove it from the arterial sheet with tweezers. Remove the medium from the well and surrounding tissue using a vacuum suction without touching the tissue, add a 10 microliter droplet of collagen type 1 gel mixture onto each arterial sheet. Allow the gel to polymerize for one hour at 37 degrees Celsius and 5%carbon dioxide.

After incubation, gently add one milliliter of prewarmed ECG medium to each well. Transfer the plate to an incubator at 37 degrees Celsius and 5%carbon dioxide for 24 hours. Increased attachment efficiency of arterial sheets was observed when a 13 millimeter untreated cover slip was used, minimizing sheer forces during its removal.

After 24 hours of porcine carotid artery culture in the 2.5D system, check for sprouting angiogenesis. If sprouting angiogenesis is observed, refresh the ECG medium and place the 12 well plate in the stage holder within the 37 degree Celsius prewarmed incubation box of the microscope. Select the microscope objective based on the imaging needs.

Set the exposure time and adjust the focus plane using phase contrast imaging. Then add the fluorescent channel for imaging of the fluorescent beads and set exposure time again. Select multiple regions of interest in the sample and adjust the focus plane for each position.

Define the time-lapse of interest by selecting a time interval of 5 to 20 minutes and a duration of 4 to 24 hours. Turn on the focus system to maintain a stable focus throughout the time-lapse imaging. Next for traction force microscopy, add 5%SDS in ultrapure water to the well and wait for a few minutes for the outgrowing cells to detach.

Acquire a Z stack of fluorescent markers in the PAA substrate for each selected position to obtain a relaxed state of the fluorescent markers as the reference image. Using customized MATLAB codes, select time-lapse and reference images for image processing and define parameters for analysis. Align and crop the time-lapse images relative to the best reference image For precise analysis.

Measure the displacement of fluorescent markers in the PAA hydrogel by performing particle image vel asymmetry between the time-lapse image and the reference image. Configure the image vel asymmetry analysis to divide the images into interrogation windows of 32 by 32 pixels with a 0.5 overlap. Finally, calculate the cellular traction forces based on the mechanical properties of the PAA gel.

Formation of cellular sprouts was observed within the 2.5D and 3D models of the porcine arterial sheet, resembling angiogenic patterns, while the 2D model showed no sprouting angiogenesis. Soft substrate stiffness of the PAA hydrogel promoted early arterial sprouting angiogenesis compared to a stiffer PAA substrate, demonstrating matrix stiffness effects. Traction force microscopy revealed that cellular sprouts exhibited pulling forces at protrusions and pushing forces along the sprout axis, driven by leader and follower cells.