Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

This protocol is significant as it allows for elucidation of the role cellular forces play in many physiological and pathological processes, such as wound healing and cancer metastases, for example. The main advantage of this mechanics-based technique is its ability to simultaneously disrupt cell-cell junctions and measure its impact on cell-generated mechanical forces, mainly tractions and intercellular stressors. Our protocol is applicable to a wide range of pathophysiological processes, such as cancer metastases, atheroscleroses, and hypertension, for example.

Our methods can be integrated to perform both biochemical and biomechanical analysis with organ-on-chip models, bio-engineered tissue constructs, in vitro drug delivery systems, and tissue co-culture systems. Our protocol is fairly straightforward to perform. However, we advise those doing this protocol for the first time to have patience and not rush through the steps of this protocol.

Our protocol has unique steps that may not be clear to first-time users. Therefore, we encourage users to follow our written step-by-step procedures and use visual demonstrations as guidance. To begin, prepare Blind-silane solution by mixing 200 milliliters of ultra pure water with 80 microliters of acetic acid and 50 microliters of silane methacrylate.

Stir the Bind-silane solution on a stir plate for at least one hour. Treat the center well of a Petri dish with the Bind-silane solution for 45 minutes. Remove the Bind-silane solution and rinse the Petri dish with ultra pure water two to three times.

Dry the Petri dish surface and store at room temperature for future use. To prepare the hydrogel solution, mix 12.49 milliliters of ultra pure water to 2.062 milliliters of 40%acrylamide and 375 microliters of 2%bisacrylamide in a 15-milliliter centrifuge tube. Add 80 microliters of fluorescent beads to the hydrogel solution.

Gently shake the tube to mix the beads with the gel solution. Keep the cap loose on the centrifuge tube and place it in a vacuum chamber. De-gas the gel solution for at least 45 minutes in the vacuum chamber.

To polymerize the hydrogel, first add 75 microliters of 10%ammonium persulphate and then add eight microliters of TEMED to the hydrogel solution. Place 24 microliters of the hydrogel solution at the center of the Petri dish. Press onto the hydrogel with an 18 millimeter cover slip to flatten the hydrogel and make a height of approximately 100 micrometers.

Wait at least 30 to 40 minutes for gel polymerization. Submerge the polymerized hydrogel in ultra pure water to prevent gel dehydration. Cover the Petri dish with aluminum foil to prevent photobleaching of fluorescent beads and store at four degrees Celsius.

First, mix the PDMS silicone base with the silicone curing agent at a ratio of 20 to one in a 50-milliliter centrifuge tube. Invert the tube upside down and shake vigorously multiple times to ensure proper mixing of the PDMS solution. Remove the bubbles in the PDMS solution by centrifuging for one minute at 190 times G.Pour five to six milliliters of the PDMS solution in the center of a 100-millimeter Petri dish and agitate the dish until the PDMS solution covers the entire Petri dish surface.

Cure the PDMS solution overnight at 50 to 60 degrees Celsius. Next, remove a circular 16-millimeter diameter PDMS stencil with a hole puncher. Use a 1.25 millimeter diameter biopsy punch to create small holes in the PDMS stencil.

Sterilize PDMS stencils by submerging them in 70%ethanol for two to three minutes, aspirating off excess ethanol and then placing under a UV light for five minutes. Aspirate any excess liquid and remove the cover slip from the hydrogel. Place the PDMS stencil on the hydrogel surface and use tweezers to apply light pressure to the PDMS stencil to ensure a water-tight seal between the PDMS stencil and the hydrogel surface.

Cover the hydrogel's surface with Sulfo-SAMPA dissolved in 0.1 molar HEPES buffer solution at a concentration of one to 1, 000 and place the hydrogel under a UV lamp with a power of 36 watts for eight minutes. Aspirate the excess Sulfo-SAMPA and HEPES solution and rinse the hydrogel twice with 0.1 molar HEPES followed by an additional two rinses with ultra pure water. Aspirate excess ultra pure water and use a 200-microliter pipette to coat the hydrogel with 0.1 milligrams per milliliter of Collagen-1.

Cover the dish to protect the fluorescent beads from photobleaching and store the hydrogel at four degrees Celsius overnight. Now, add three milliliters of 1X Trypsin into the flask and place it in the incubator at 37 degrees Celsius for three to five minutes to detach the cells from the tissue culture flask. After trypsinization, add three milliliters of cell culture media into the flask, swirl to mix, and transfer the mixture into a 15-milliliter centrifuge tube.

Centrifuge the cell solution for three minutes at 1, 710 times G.Aspirate the supernatant and re-suspend the cells in media to a concentration of 50 times 10 to the four cells per milliliter. Take out the hydrogel dish from the refrigerator. Remove the Collagen-1 from the hydrogel and rinse one time with PBS.

Add 75 times 10 to the third cells to the top of the PDMS stencil and allow the cells to attach to the hydrogel surface for at least one hour in the incubator at 37 degrees Celsius and 5%carbon dioxide. Remove the PDMS stencil and submerge the PDMS stencil in 10X Trypsin for 10 to 15 seconds to remove any attached cells. Add at least two milliliters of media to the Petri dish and sterilize the stencil by spraying with 70%ethanol, and then placing under the UV light for five minutes.

Place the Petri dish in the incubator for at least 36 hours, or until a confluent monolayer is observed. Phase contrast images of 30 minutes before chalcone treatment and two hours after chalcone treatment were compared. Cell-induced bead displacements were observed to decrease in both low-dose chalcone and high-dose chalcone conditions when compared to control HUVEC monolayers.

Prior to chalcone treatment, RMS tractions were around 51 Pascals for all conditions. After chalcone treatment, there was a small increase in RMS tractions to 59 Pascals in low-dose chalcone treated monolayers and an almost two-fold decrease in RMS tractions to 18 Pascals in high-dose chalcone treated monolayers compared to the control. Chalcone treatment increased the average normal intercellular stress magnitude with low-dose, but significantly decreased the average normal intercellular stress magnitude with high dose when compared to control.

The chalcone treatment also decreased the maximum sheer intercellular stresses at both low and high chalcone concentration when compared to the control. The impact of chalcone treatment is statistically significant based on both T-tests and single factor ANOVA test. While placing PDMS stencil on top of the gel, make sure the gel is dry enough to ensure a water-tight seal between the PDMS stencil and the gel.

This protocol can also be applied to probe the biomechanical contribution of a variety of cell-cell junctions, cytoskeletal proteins, cell matrix adhesion molecules, or extracellular molecules with live imaging. With this in vitro protocol, researchers can now successfully investigate a host of biomechanical, as well as biochemical properties during tissue repair, wound healing, or cancer metastases without animal model. We advise our viewers to be very careful while handling chalcone and DMSO as inhalation of these drugs is reported to be harmful.