Evaluating Primary Blast Effects In Vitro

Understanding how cells are modulated by exposure to shock waves can help identify the mechanisms behind injuries triggered from blast events. This protocol uses custom-built shock tube equipment to apply shock waves at a range of pressures to cell monolayers and to identify the subsequent effects on cell viability.

Transcript

The overall goal of this method is to generate representative shock waves that can occur during blast events and evaluate the impact of this mechanical shock on the viability of primary cells in culture. This method can help us answer key questions relating to the response of individual cell types to high energy events such as exposure to a shock wave. The main advantage of this method is it enables us to evaluate distinct cell responses to a single shock wave as oppose to tissue or organ level responses.

The implications of this technique extends towards identifying the cellular mechanisms altered as a result of primary blast injuries, which are caused by exposure to a shock wave. Though this method is being demonstrated with a specific cell type of interest, any cells can be cultured and placed within the test setup to assess cell-specific responses to a shock wave. To begin, culture the cell line of interest at standard culture until they reach 80%confluence.

Then, aspirate the medium. Wash the cells twice with PBS, and add two milliliters of Trypsin-EDTA. Incubate them at standard culture conditions for five minutes.

Briefly check to ensure the cells have successfully dissociated from the flask. And then add four milliliters of FBS containing culture medium to the flask in order to neutralize the Trypsin and transfer the cells to a 15-milliliter conical tube. Centrifuge the cells at 200 times g for four minutes to pellet the cells.

Then, slowly aspirate the supernatant taking care not to dislodge the pellet. And resuspend the pellet in five milliliters of medium. Place three labeled 35-milliliter dishes inside a 90-milliliter dish.

Then, add two milliliters of medium to each dish and seed them each with 70 thousand cells. Incubate the cells overnight to allow for adequate cell attachment before exposing them to the shock wave. Prior to beginning shock tube assembly, put on the proper personal protective equipment, including steel-toe work boots and a laboratory coat.

Start by inserting two alignment bars through the bolt holes found in the horizontal plane of the outlet flange. Then, place a nitrile rubber gasket sheet over the end of the alignment bars so that it sits between the outlet flange and the ex vivo organ culture rig. Attach the EVOC rig to the outlet flange of the shock tube by sliding the fixture over the alignment bars.

Ensure that it is oriented correctly so that the section that holds the Petri dish is located at its base. Next, insert four M24 bolts and washers into the remaining holes and tightly fasten the nuts and bolts sequentially in a diagonally symmetric fashion, while visually checking that the EVOC rig and shock tube have successfully aligned. Then, attach a pressure transducer to the EVOC rig and connect it to the current source using a sensor cable.

In addition, use a BNC cable to connect the current source to the oscilloscope. Ensure that all release valves and flow controls on the shock tube are closed. Then, open the built-in external compressed airline and manually turn the pressure regulator clockwise to charge the solenoid to 2.5 bar.

Open the compressed air cylinder, then slowly turn the pressure regulator clockwise to increase the pressure to approximately 15 bar. Next, prepare diaphragms by cutting a 0.125-milliliter thick plastic sheet into 10 centimeter by 10 centimeter squares. Create handles out of tape, and fix them to the top and bottom of the diaphragm.

Use them to carefully position the diaphragm next to the front small flange by the breech chamber. Ensure that the diaphragm is straight and correctly positioned before closing the breech. And then secure it in place using four M24 bolts and nuts.

Inside the sterile hood, prepare adhesive gas permeable membranes so that each is large enough to cover the top of one 35-milliliter Petri dish. Then, transfer a 90-milliliter Petri dish containing three experimental samples into the hood. Aspirate the medium from one of the dishes, and cover it with two membrane sections, ensuring that there is a tight seal between the membrane and the edge of the dish.

Transfer the sample to the EVOC rig and secure it to the base of the fixture so that the top of the dish is aligned with the inner base of the shock tube. At this point, place on ear defenders, eye goggles, safety boots, and a laboratory coat. Pressurize the shock tube system by turning the flow control knob on the regulator clockwise.

At the bottom of the control panel, select either the double breech inlet for a short duration shock wave, or the driver tube inlet for an increased wave duration. Then, switch on the DC power source and the oscilloscope to require the waveform data. Next, turn on safety lights using the switch on the wall opposite the control panel in order to notify other researchers not to enter the room when the shock tube is charging.

On the solenoid control box, press the switch to close the solenoid. Then, on the control panel, slowly open the flow control by turning the knob counter clockwise to gradually increase the pressure within the compression chamber. When the diaphragm burst pressure is reached, the diaphragm will rupture and a bang will be heard.

Once this noise is heard, quickly close the flow control knob. Then, open the solenoid by turning off the switch on the solenoid control box, and switch off the safety light. Transfer the cell sample to the sterile hood.

There, remove the adhesive gas-permeable membrane, and fill the dish with two milliliters of fresh growth medium. Add 200 microliters of redox indicator reagent to each experimental dish directly after refilling them with two milliliters of fresh growth medium. Then, transfer the dish to a standard incubator for four hours.

Following incubation, transfer two 100-microliter aliquots from each dish to an appropriate 96-well plate. Read the plate on a fluorescence plate reader and then export and save the data. For fluorescent imaging of live and dead cells, cells need to be initially plated on a glass cover slip inside the 35-milliliter culture plates.

Following shock wave treatment, aspirate the medium, and wash the cells twice in one milliliter of PBS. Then, transfer 100 microliters of the live dead reagent from the imaging kit to each sample. And incubate the cells and reagent at room temperature, protect it from light for 15 minutes.

After incubation, remove the reagent, and wash the cells once using one milliliter of PBS. Using fine point forceps, carefully remove the cover slip from the 35-milliliter dish and place it facedown onto a glass microscopy slide. Acquire images for each sample using a fluorescent microscope equipped with a 5X objective lens having a numerical aperture of 0.25.

Then, use image analysis software to either manually or automatically count the number of live green cells and red dead cells from each image. Using the method described here, cells grown in a monolayer were exposed to shock waves generated with a shock tube. Sensors in the shock tube display the pressure profiles observed at different locations along the tube.

Using a redox indication assay, it was found that the application of a 127-kilopascal shock wave was able to significantly reduce the viability of dermal papilla cells compared to controls after 24 hours in culture. The application of a shock wave's 72 kilopascals or less did not reduce viability. To support these observations, a live dead assay also demonstrated a reduction of cell viability in those exposed to the 127-kilopascal shock wave when compared to the control.

Once mastered, this technique can be done variably quickly at a rate of 60 samples per hour. The shock tube can be used to generate shock waves to explore a range of primary blast injuries. These can range from blast lung through to heterotopic ossification, which is the inappropriate formation of bone within the soft tissues of the body.

While attempting this procedure, it's important to remember the risks involved when working with compressed gases and high energy shock waves, and to always follow the correct safety guidelines. Minor modifications in a shock tube can enable us to alter the shock wave. We can then asses the cellular response to different pressure profiles and intensities, which represent different blast scenarios.

Following this procedure, next-generation sequencing can be performed to answer additional research questions, like, How does exposure to a shock wave alter our natural inscription within cells?

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Journal (Bioengineering)

Journal (Bioengineering)

Evaluating Primary Blast Effects In Vitro

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