Live Cell Imaging during Mechanical Stretch

The overall goal of the following experiment is to observe the role of mechanical strain on cell mechanobiology in near real time. This is achieved by first constructing a system with flexible membranes on which cells are grown and stretched. As a second step stretch the membrane to calibrate motor counts with membrane strain.

Next, mouse lung epithelial cells are stretched and nuclei are imaged. In order to show that the membrane strain is transmitted to cells, the stretchers then incorporated into the A FM for nano indentation in order to measure the stiffness of the individual cells before and after stretch. The results show direct damage on the cells due to applied stretch.

Also based on fluorescence imaging, reactive oxygen species production is shown to increase with stretch. Well, this method can answer key questions in the lung mechanical biology field, such as whether or not the epithelial cells can be injured with mechanical ventilation. This method can provide insight into lung cell, mechanical biology, and can also be applied to other soft tissue such as liver or growth plate slices.

Generally, individuals new to this method will struggle because of the unconventional combination of tools used in these experiments such as the atomic force microscope. To begin, prepare a collagen coated PDMS membrane modified for use in cell culture as described in the accompanying text protocol. Next, seed mouse lung epithelial cells at 2.5 million cells per well onto a sterile, flexible collagen coated membrane.

Incubate the cells at 37 degrees Celsius and 5%CO2 for two days until the cells reach con fluency. Then use a 1.5 millimeter biopsy punch to punch two holes in each of the six clamp tabs, 20.5 millimeters from the center of the construct if no pretension is desired. And remove the cell culture media.

Make sure the mechanical actuator is in a zero strain position, and position the membrane on the stretcher so that the punch holes line up with the pins and the clamps. Place top clamps in place, and then tighten the screws one at a time, alternating different sides. Add one milliliter cell culture media before placing the device on the microscope stage.

Center the device on the light path. Next, fix the device to the stage. Focus on the membrane by adjusting the in plane and vertical positions of the mechanical device with a stage controller of the microscope in the destination input block.

Enter a motor count value, which corresponds to the desired strain level to be applied. Next, set the value in the velocity input block to 10 RPM or less to observe the same field before and after stretch as the field may shift with respect to the microscope objective, and it may be necessary to adjust the microscope stage during stretch. Once everything is set, click on go.

Begin by making adjustments to the atomic force microscope. Increase the height of the a FM head to its maximum position in the Z direction. Then put extenders on the legs to lift the plane at which a FM cantilever contacts the sample.

Next, remove the A FM scanner plate and the desired objective from the microscope. Add a spacer to the objective and then mount it back on the microscope. Then place the scanner plate back on the A FM.The height of the spacer will depend on the objective and specific a FM setup, but it is necessary to use the spacer if optical imaging is desired since the observation plan will be shifted in the Z direction.

Next, start the A FM software, the optical microscope software, and all necessary light sources, including the light source for fluorescence measurements. Mount a chip with a cantilever beam that has a stiffness of 200 pico newtons per nanometers or less for measuring the elastic modulus of live cells on the A FM.Then align the laser next mount a glass cover slip on the device and use it to calibrate the cantilever stiffness according to the manufacturer's suggestions. Immediately before mounting the membrane to the stretching device, cut the walls to about one millimeter in height.

This reduces the interference experienced between the membrane walls and the load cell. Then remove the cell culture media and mount the membrane on the mechanical device. Place the mechanical device with the adapter on the scanner using the software.

Stretch the membrane to the desired tensile strain level prior to nano indentation. Add 0.5 milliliters or less of media onto the cells to keep them hydrated, but also take care to avoid a spill which may damage the a FM scanner or microscope. Then engage the cantilever beam with the membrane.

Follow the protocol of the particular A FM device to scan areas of interest. When a 20%strain was applied to cells seeded on the membrane, the cells displaced 20%as indicated here, the impact of cells on the production of reactive oxygen species was measured in bronchial epithelial cells using a cumulative mitochondrial superoxide sensor as described in the accompanying text protocol. In the absence of mechanical stretch, ROS production did not significantly increase over 60 minutes when a single 17%stretch was applied and maintained.

There was an increase in mitochondrial ROS that persisted for another 60 minutes with the incorporation of the mechanical stretcher into the atomic force microscope. Elastic modulus, maps of mouse lung epithelial cells before and after 10%tensile strain were obtained from the regions outlined here. 300 individual forced deflection curves were recorded over a 40 by 40 micrometer area showing a change in the localization of high modulus regions with the application of strain.

Once mastered, cell stretching can be done in 15 minutes. While a FM nano indentation may consume two hours of perform properly While attempting this procedure, it is important to remember to handle the cells carefully yet quickly as the results may be adversely affected by agitation and prolonged room air exposure before the data is reported. Don't forget that working with a FM can be extremely challenging.

Precautions such as keeping all the liquids away from the scanner must be taken while performing this procedure.