The overall goal of this procedure is to demonstrate the novelty of an an isotropic stretching platform that can be used to investigate single cell responses to complex mechanical deformation and to quantify the mechanical properties of biological tissues. This is accomplished by first fabricating the flexible membrane and mounting this membrane on a set of clamps. The second step is to culture C two C 12 cells on the membrane overnight, and on the following day, mount the clamps on the stretching platform.
Next, in a second set of experiments, thoracic aortic tissues are rejected from a mouse model and cleaned from any connective tissues. Then the aortic tissue is mounted on the stretching platform using a different set of clamps. Ultimately, the stretching platform is used to induce a deformation in cells along two orthogonal directions that can be quantified using an inverted microscope.
Or alternatively, a force transducer is integrated to the stretching device to measure tissue stiffness. The main advantage of this technique over the existing techniques, such as uni actual or biaxial cell stretching, is that it allows us to control independently the deformation in two orthogonal directions in a transparent membrane on which cells are attached, and therefore allowing for live cell imaging. Microscopy also a careful design of a different set of clamps.
With the integration of a four string user on the platform, allows us to perform 10 cell testing on biological tissue for the quantification of their mechanical properties. This video presents the Biaxial Stretching platform, a custom-built apparatus that can be used to investigate the effect of substrate deformation at the cellular level, and perform tensile tests on biological tissues. The design and features of the Biaxial stretching platform is detailed in the accompanying manuscript to investigate the effect of mechanical deformation of single cells, a flexible and transparent substrate on which cells can adhere must first be fabricated.
To begin this procedure, pipette 500 microliters of a fluorescent microsphere solution into a 1.5 milliliter micro centrifuge tube and centrifuge at 16, 200 Gs For 10 minutes, discard the supernat and add 500 microliters of isopropanol, followed by five minutes of vortexing. Put the vial aside overnight in the dark in order to allow any large particle aggregates to sediment on the following morning, carefully transfer the SNAT to a clean micro centrifuge vial. This bead solution can be used to fabricate more than five substrates because the bead solution will continue to sediment for the next three days.
It is important to avoid Resus suspending the pellet. Pour 0.5 grams of the curing agent provided with the PDMS kit into a 1.5 milliliter micro centrifuge vial. Add 15 microliters of fluorescent beads and vortex for one minute.
Repeat this five more times to add a total of 90 microliters of beads Set aside. Weigh 10 grams of PDMS add 0.5 grams of the curing agent, supplemented with fluorescent beads and mix for at least 12 minutes. Next, use the transfer pipette to transfer four milligrams of the PDMS with beads into an SUH 2050 cross shaped mold previously fabricated using standard photolithography techniques.
The mold has a height of 320 micrometers and an area of 13.4 square centimeters and can contain 428 microliters or 440 milligrams of PDMS cure the PDMS for two hours at 80 degrees Celsius. After curing peel off the substrate from the mold, the substrate can be kept in a Petri dish at room temperature for two weeks without showing significant changes and its mechanical properties. Pour droplets of PDMS in a Petri dish with a final size of approximately four millimeters in diameter, cure them upside down for two hours at 80 degrees Celsius, the dish must be maintained upside down to prevent the drops from flattening during the curing process.
These anchoring features can be kept in a Petri dish for weeks. Air plasma treat the substrate and eight anchoring features, bind the features on each end of the substrate at a distance of four millimeters from the square shaped indent present on the substrate to mount the membrane on the clamps. Wrap each end of the substrate around the groove cylindrical part of the clamps, and secure it in place with the two set screws from the top.
Screw the four clamps on the clamp holder. Then pour PDMS at the interface between the substrate and the grooved part of the clamps. Spread the uncured PDMS around the grooved cylindrical part using a 1.5 millimeter hex key.
Pour PDMS in the grooves until completely filled by capillary action and cure the assembly at 80 degrees Celsius for two hours prior to seeding cells on the membrane. Air plasma. Treat the whole assembly to sterilize and functionalize the substrate to allow for collagen coating.
Functionalize the area of the substrate where cells will be seeded with one milliliter of 0.02 molar acetic acid supplemented with 16 micrograms per milliliter of rat tail collagen at room temperature for one hour. The desired final collagen density is five micrograms per square centimeters After one hour, rinse the substrate three times with phosphate buffer and let it dry at room temperature for at least 10 minutes. Add 40 microliters of culture medium, supplemented with 10%fetal bovine serum and 1%penicillin.
Streptomycin containing 2000 cells in the center portion of the substrate to cover an area of one square centimeter. In this example, the cell density is 20 cells per square centimeters. However, cell density can be altered according to experimental requirements.
Put the whole assembly in a standard cell culture incubator with the substrate facing up with the drop of culture medium containing cells on it. The assembly must be kept with the substrate facing up for at least three hours to allow cells to firmly attach to it to prevent evaporation. 30 microliters of warm culture medium is added into the drop on the substrate.
Every 45 minutes after three hours, flip the whole assembly into a Petri dish filled with fresh culture medium to submerge the substrate incubate overnight to allow cells to proliferate on the following day. Mount the setup on an inverted phase contrast or fluorescent microscope. Mount the clamp substrate assembly on the bi axial stretching platform, and motors fill the Petri dish inside the Petri dish heater with a freshly prepared HEPs buffered salt solution.
This physiological solution is used to maintain the cells on the microscope stage by mimicking the normal tissue blood environment. The same bi axial stretching platform can also be used to quantify the stiffness of small tissue samples. Isolated mouse aortas will be used in this demonstration.
Remove the diaphragm, the thoracic cage, and the lung lobes from the euthanized mouse to minimize the risk of damaging the tissue. Keep the heart attached to the aorta and avoid touching the vessel directly but manipulated. Using the heart.
Remove the heart, the aortic root and the thoracic aorta by gently cutting between the vessel and the spine to keep the inner structure of the tissue intact. Be careful not to induce any elongation in the vessel during the incision. Immediately immerse and keep the heart and aorta in Krebs solution, which is prepared daily and kept at 37 degrees Celsius during the experiments.
Cut and carefully wash the aorta in Krebs solution to remove any blood clots working under a surgical dissecting microscope. Remove connective tissue using micro scissors and tweezers. It is important to keep all the vessel length and use the aortic root to determine the vessel orientation.
The unloaded vessel dimensions are required to determine the stiffness of the vessel. Cut an aortic ring of about two millimeters in length and precisely measure its length using a calibrated microscope setting. Put this segment aside in Kreb's solution.
Next, cut two small aortic rings from the sections of aorta left after the removal of the two millimeter segment. One ring from the section of aorta before the two millimeter segment, and another ring from the section of aorta. After the two millimeter segment, put both aortic rings on a microscope glass slide with the lumen facing up and measure the wall thickness using a calibrated microscope setting.
Aortic wall thickness is measured and averaged using these two rings. Fill the Petri dish on the biaxial stretching platform with Krebs solution and insert the two millimeter aorta ring segment on the pulling pins. In this protocol, the Biaxial stretching platform was used to investigate the mechanical response of the nucleus in single mouse myoblast cells exposed to a substrate deformation of 25%displacement deformation.
Relationships from performing standard and pure uni axial stretching are shown in these representative results. Fluorescent beads are manually selected on the first frame of the video and tracked from one frame to the other until maximum stretch is reached along the horizontal and vertical directions. A MATLAB script automatically computes the components of the green strain tensor for each frame, and produces a calibration curve of the strain in respect to motor displacement.
In these graphs, the blue and red lines correspond to the component of the green strained heer along the horizontal and vertical directions respectively. These results illustrate the capabilities of the Biaxial stretching platform for inducing a deformation in the substrate along two orthogonal directions. In addition to standard uni axial of EQU Biaxial strain fields, the biaxial stretching platform can induce complex strain fields in the substrate.
Panel G shows that stretching the same substrate by four millimeters along the x axis and slightly stretching along the Y axis by 1.5 millimeters highlighted in red, produces a pure uni axial field with no compressive deformation in the substrate. Panel, H shows that stretching the substrate along the x axis by 3.5 millimeters produces a deformation of 25%and a compressive deformation of 7%when the ends of the substrate along the vertical axis are fixed, highlighted in red With the ability to perform live cell microscopy imaging during stretching nuclei was stained with a live cell fluorescent dye that binds the DNA and then stretched by 25%along each orthogonal stretching axis as illustrated here between each stretching cycle phase, contrast images and epi fluorescent images of the unformed and deformed nucleus were acquired. The lengths of the major and minor nuclear axis along the unformed state and when the nucleus was sequentially stretched along its major and minor axis were determined using an image J plugin and used to calculate the relative change in length.
Along each axis in this bar graph, the X or major axis is shown in blue, and the y or minor axis is shown in red. The deform ability of the nucleus exhibits a mechanical anisotropy as it displays a significantly higher deform ability along its minor axis in, in comparison to its major axis, the role of the actin and microtubules cytoskeleton in regulating nuclear deform ability was examined by depolymerizing, the actin filaments or microtubules selectively using cyto D and neol respectively depolymerizing. The actin filaments or the microtubules was found to induce a loss of the anisotropic de formability of the nucleus.
These results support the findings that cytoskeletal components transmit forces to the nucleus from the substrate and are also essential for preserving the natural mechanical behavior of the nucleus. During deformation, the bi axial stretching platform was also used to assess the effect of chronic overexpression of heat shock protein 27 or HP 27 on vessel stiffness HSP over expression. In an atherosclerosis prone mouse model, A POE homozygous negative modifies the histological composition of atherosclerosis lesions by increasing intimal smooth muscle cells and collagen content.
To determine the impact of this histological remodeling on vessel mechanical properties, the stretching platform was used to measure aortic stiffness. This graph shows a typical stress strain curve obtained from stretching an aortic vessel while the stiffness is computed at 30%of strain. The stiffness is the slope of the tangent indicated by the red line to the curve.
It was found that the stiffness of aortic segments of HSP 27 over expressing mice was significantly increased by 41%compared to the control mouse model. Taken together the mechanical assessment combined with the vessel and plaque histology demonstrated that the overexpression of HSP 27 is characterized by increased vessel stiffness and collagen smooth muscle cell content. These results suggest that HSP 27 could potentially increase stability of atherosclerotic lesions and therefore decrease the risk of plaque rupture After its development.
This technique pays a new way for researcher in the field of bioengineering to explore cell response to complex and inotropic mechanical deformation for applications in mechanical biology, but also to monitor biological tissue mechanics in disease.
