We would like to acknowledge the financial support of the Royal British Legion Centre for Blast Injury Studies to HA and funding from the Medical Research Council (M01858X/1) to CAH.

1. Cell Culture and Sample Preparation
<ol>
	<li>Obtain an appropriate cell line (e.g., dermal papilla cells). 
	NOTE: The current protocol has been designed for adherent cells, with rat dermal papilla fibroblasts isolated from vibrissae used as a model.</li>
	<li>Culture the cells in a T-75 flask containing minimum essential medium (MEM) &#945; supplemented with 10% fetal bovine serum and 1% penicillin streptomycin. Keep the cells at standard culture conditions of 37 &#176;C and 5% CO2 in a humidified environment until they reach 80% confluence.</li>
	<li>Aspirate the medium and wash the cells twice in Dulbecco&#39;s phosphate-buffered saline (PBS). Dissociate the cells from the flask by incubating them in 2 mL of trypsin/EDTA at standard culture conditions for 5 min. Briefly check to ensure that cells have successfully dissociated from the flask using a light/phase contrast microscope (with a 10X objective lens).</li>
	<li>Add 4 mL of culture medium to neutralize the trypsinization reaction. Transfer the cell suspension to a 15 mL centrifuge tube.</li>
	<li>Centrifuge at 200 x g for 4 min to pellet the cells.Slowly aspirate the supernatant, taking care not to dislodge the pellet.Re-suspend the pellet in 5 mL of medium.</li>
	<li>Transfer a 20 &#181;L aliquot of the cell suspension to a clean hemocytometer and count the cells using a microscope fitted with a 10X objective lens.</li>
	<li>Prepare the cells for the blast by placing three 35 mm dishes inside a 90 mm dish. Label them appropriately. Add 2 mL of medium to each 35 mm dish and seed a total of 5 x 104 cells per dish, distributing the cells evenly around the dish. Incubate the experimental samples overnight in standard culture conditions to allow for adequate cell attachment before shock wave exposure. 
	NOTE: For fluorescent imaging, cells must be seeded onto sterile glass coverslips.</li>
</ol>
2. Shock Tube Assembly
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55618/55618fig2.jpg" /> 
Figure 2: EVOC Rig and Shock Tube Assembly. (A) Image of the EVOC rig. (B) Schematic of the shock tube apparatus. Shock tube dimensions include an internal diameter of 59 mm and a total length, including the EVOC rig, of 4.13 m. The length of the driven tube and the EVOC rig totals 2.71 m. <a href="http://cloudfront.jove.com/files/ftp_upload/55618/55618fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Wear steel-toe work boots and a laboratory coat during the assembly procedures.</li>
	<li>Insert the two alignment bars through the two bolt holes found in the horizontal plane of the outlet flange. 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 (EVOC) rig. 
	NOTE: The EVOC rig is a custom-designed fixture intended for use with 35 mm Petri dishes (Figure 2A).</li>
	<li>Attach the EVOC rig to the outlet flange of the shock tube by sliding the fixture over the alignment bars, ensuring that it is orientated correctly so that the section that holds the petri dish is located at its base.</li>
	<li>Insert four M24 bolts and washers into the remaining holes. Position the nuts so that they touch the flange.</li>
	<li>Tightly fasten the nuts and bolts sequentially in a diagonally symmetric fashion whilst visually checking that the EVOC rig and shock tube have successfully aligned.</li>
	<li>Attach a pressure transducer to the EVOC rig and connect it to the current source using a sensor cable. Then, using a BNC cable, connect the current source to the oscilloscope.</li>
	<li>Ensure that all release valves and flow controls on the shock tube are closed. Open the built-in external compressed air line and manually turn the pressure regulator clockwise to charge the solenoid to 2.5 bar.</li>
	<li>Open the safety valve on the compressed gas bottle by turning it anticlockwise 180&#176;.</li>
	<li>Slowly turn the pressure regulator, located on top of the gas bottle, clockwise to increase the pressure to approximately 15 bar. 
	NOTE: The setting point of this regulator should be slightly above the highest bursting pressure of the thickest diaphragm that is to be used in the experiment.</li>
	<li>Prepare the diaphragms by cutting a 0.125 mm-thick plastic sheet into 10 cm x 10 cm squares. 
	NOTE: The thickness of the diaphragm will correlate with the bursting pressure, altering the peak pressure of the shock wave (i.e., a thicker diaphragm will result in a shock wave with a larger peak pressure; Table 1).</li>
</ol>
<table fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Mylar Diaphragm dimensions (cm)</td>
			<td>Burst pressure (bar)</td>
			<td>Sensor 3 pressure range (kPa)</td>
		</tr>
		<tr>
			<td>10 x 10 x 0.023</td>
			<td>2 &#177; 0.2</td>
			<td>47 &#177; 7.5</td>
		</tr>
		<tr>
			<td>10 x 10 x 0.050</td>
			<td>4 &#177; 0.2</td>
			<td>72 &#177; 7.5</td>
		</tr>
		<tr>
			<td>10 x 10 x 0.125</td>
			<td>9.5 &#177; 0.5</td>
			<td>127 &#177; 12.5</td>
		</tr>
	</tbody>
</table>
Table 1: Burst Pressure Corresponding to Diaphragm Thickness and Peak Pressure.
<ol start="11">
	<li>Create handles out of tape, fix them to the top and bottom of the diaphragm, and 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.</li>
	<li>Secure this using four M24 bolts and nuts. Tightly fasten the nuts and bolts sequentially in a diagonally symmetric fashion.</li>
</ol>
3. Preparation of Cells Just Prior to Shock Wave Exposure
<ol>
	<li>Prepare a tissue laminar flow hood by performing ultraviolet sterilization. Clean it with a disinfectant solution and 70% ethanol. Gather all items needed for handling cells prior to/post-shock wave exposure and place them within the sterile hood. 
	NOTE: This includes fresh medium, adhesive gas-permeable membranes, sterile scissors, pipettes, and pipette tips.</li>
	<li>Transfer one 90 mm Petri dish containing three experimental samples from the incubator to the sterile hood.</li>
	<li>Cut an adhesive gas-permeable membrane sheet (see the Table of Materials) into six squares, such that each is large enough to cover the top of one 35 mm Petri dish.</li>
	<li>Aspirate the medium from the 35 mm Petri dish, place the lid to one side, and cover with two adhesive gas-permeable membranes sections, ensuring that there is a tight seal between the membrane and the edge of the dish.</li>
	<li>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.</li>
</ol>
4. Shock Tube Operation
<ol>
	<li>Wear ear defenders, eye goggles, safety boots, and a laboratory coat when pressurizing the shock tube system.</li>
	<li>Turn the flow control knob on the regulator clockwise, allowing gas to flow into the flexible hose connecting the compressed air cylinder to the shock tube.</li>
	<li>At the bottom of the control panel, select either the &#34;Double Breech&#34; inlet for a short-duration shock wave or the &#34;Driver Tube&#34; inlet for an increased wave duration.</li>
	<li>Switch on the DC power source and oscilloscope to acquire the waveform data. Set the oscilloscope sampling rate to 50.0 MS/s, with a record length of 1 M points. Set a range of 50 mV for firing at 2 bar and 100 mV for above 4 bar.</li>
	<li>Turn on the safety lights using the switch on the wall opposite the control panel. 
	NOTE: This tells other researchers not to enter the room when the shock tube is charging.</li>
	<li>Turn on the switch on the solenoid control box to close the solenoid. 
	NOTE: This safety feature closes a valve located on the double breech chamber, allowing for the system to be charged.</li>
	<li>On the control panel, slowly open the flow control by turning the knob counterclockwise to gradually increase the pressure within the compression chamber. Monitor the pressure using the control panel display.</li>
	<li>Once the diaphragm burst pressure is reached, the diaphragm will rupture and a &#34;bang&#34; will be heard. Quickly close the flow control by turning the knob. 
	NOTE: The shock wave will travel down the tube over the cell sample and out the end of the tube.</li>
	<li>Open the solenoid by turning off the switch on the solenoid control box and switch off the safety light (using the switch on the wall opposite the control panel).</li>
	<li>Transfer the cell sample to the sterile hood, remove the adhesive gas-permeable membranes, and fill the dish with 2 mL of fresh growth medium. The cells can be used immediately for assessment, as described in step 5, or returned to the incubator for longer-term studies.</li>
</ol>
5. Cell Viability
<ol>
	<li>Assess cell viability following shock wave exposure using the combination of a redox indicator assay and the fluorescent imaging of live and dead cells. 
	NOTE: For fluorescent imaging, cells must be seeded onto sterile glass coverslips during section 1: Cell culture and sample preparation. These can be placed within the 35-mm petri dishes.</li>
	<li>Transfer 200 &#181;L of the redox indictor reagent (see the Table of Materials) to each experimental dish directly after refilling them with 2 mL of medium post-shock wave exposure. Incubate at standard culture conditions for 4 h.</li>
	<li>For each dish, transfer (in a dark sterile hood) 2 x 100 &#181;L aliquots to either a black or clear 96-well plate, depending on whether fluorescence or absorbance will be used to assess viability.</li>
	<li>Transfer the 96-well plate to an appropriate plate reader fitted with the correct filters and read using excitation bands 530ex/590em for fluorescence or 570 nm combined with a 600-nm reference for absorbance. Export and save the data.</li>
	<li>Analyze the data to identify any difference between shock wave-exposed samples and non-exposed controls, as this can indicate a drop in cell viability. 
	NOTE: A negative control should also be included in the experimental design. Furthermore, the interpolation of a standard curve can be used to calculate cell numbers.</li>
	<li>Aspirate the medium containing the redox indicator reagent. Replace it with 2 mL of fresh medium and incubate the cells at standard culture conditions for 24 h.</li>
	<li>Repeat the above steps as necessary to obtain a second viability time point.</li>
	<li>For the fluorescent imaging of live and dead cells, aspirate the medium and wash the cells twice in 1 mL of PBS. Transfer 100 &#181;L of the complete reagent found in the imaging kit (see the Table of Materials) to each sample and incubate at room temperature protected from light for 15 min.</li>
	<li>Aspirate the reagent and wash once using 1 mL of PBS. Using fine point forceps, carefully remove the coverslip from the 35 mm dish and place it face-down onto a glass microscopy slide.</li>
	<li>Acquire images for each sample using a fluorescent microscope (10X objective lens, 0.50 numerical aperture) fitted with the correct excitation and emission filters (ex/em 488 nm/515 nm and 570 nm/602 nm). 
	NOTE: Live cells will emit intense, uniform, green fluorescence. Dead or dying cells with a compromised cellular membrane will uptake the dead kit component, resulting in the emission of red fluorescence, found predominantly in the nuclear region of the cell.</li>
	<li>Use image analysis software to either manually or automatically count the number of live and dead cells from each image.</li>
</ol>

<ol>
	<li>Proud, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physical+basis+of+explosion+and+blast+injury+processes.">The physical basis of explosion and blast injury processes.</a> J R Army Med Corps. 159, 4-9 (2013).</li><li>Born, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blast+Trauma:+The+Fourth+Weapon+of+Mass+Destruction.">Blast Trauma: The Fourth Weapon of Mass Destruction.</a> Scand J of Surg. 94 (4), 279-285 (2005).</li><li>Kocsis, J. D., Tessler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathology+of+blast-related+brain+injury.">Pathology of blast-related brain injury.</a> J Rehabil Res Dev. 46 (6), 667-671 (2009).</li><li>Bass, C. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Injuries+from+Blast.">Brain Injuries from Blast.</a> Ann Biomed Eng. 40 (1), 185-202 (2012).</li><li>Cernak, I., Kobeissy, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+Neurotrauma:+Molecular,+Neuropsychological,+and+Rehabilitation+Aspects.">Brain Neurotrauma: Molecular, Neuropsychological, and Rehabilitation Aspects.</a> Frontiers in Neuroengineering. , (2015).</li><li>Alfieri, K. A., Forsberg, J. A., Potter, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blast+injuries+and+heterotopic+ossification.">Blast injuries and heterotopic ossification.</a> Bone Joint Res. 1 (8), 174-179 (2012).</li><li>Potter, B. K., Burns, T. C., Lacap, A. P., Granville, R. R., Gajewski, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+Ossification+Following+Traumatic+and+Combat-Related+Amputations.+Prevalence,+Risk+Factors,+and+Preliminary+Results+of+Excision.">Heterotopic Ossification Following Traumatic and Combat-Related Amputations. Prevalence, Risk Factors, and Preliminary Results of Excision.</a> J Bone Joint Surg Am. 89 (3), 476-486 (2007).</li><li>Sasser, S. M., Sattin, R. W., Hunt, R. C., Krohmer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blast+Lung+Injury.">Blast Lung Injury.</a> Prehosp Emerg Care. 10 (2), 165-172 (2006).</li><li>Cho, S. -. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+Hearing+Loss+after+Blast+Injury+to+the+Ear.">Mechanisms of Hearing Loss after Blast Injury to the Ear.</a> PLoS ONE. 8 (7), 67618 (2013).</li><li>Bull, M. A., Clasper, J., Mahoney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Blast Injury Science and Engineering: A Guide for Clinicians and Researchers. , (2016).</li><li>Arun, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+blast+traumatic+brain+injury+using+in-vitro+model+with+shock+tube.">Studies on blast traumatic brain injury using in-vitro model with shock tube.</a> Neuroreport. 22 (8), 379-384 (2011).</li><li>Ahlers, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+the+effects+of+acute+and+repeated+exposure+to+blast+overpressure+in+rodents:+toward+a+greater+understanding+of+blast+and+the+potential+ramifications+for+injury+in+humans+exposed+to+blast.">Assessment of the effects of acute and repeated exposure to blast overpressure in rodents: toward a greater understanding of blast and the potential ramifications for injury in humans exposed to blast.</a> Front Neurol. 3, 32 (2012).</li><li>Polfer, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+rat+model+to+investigate+the+formation+of+blast-related+post-traumatic+heterotopic+ossification.">The development of a rat model to investigate the formation of blast-related post-traumatic heterotopic ossification.</a> Bone Joint J. 97 (4), 572-576 (2015).</li><li>Miller, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Blast+Overpressure+on+Neurons+and+Glial+Cells+in+Rat+Organotypic+Hippocampal+Slice+Cultures.">Effects of Blast Overpressure on Neurons and Glial Cells in Rat Organotypic Hippocampal Slice Cultures.</a> Front Neurol. 6, 20 (2015).</li><li>Chandra, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+blast+wave+profiles+in+simulated+air+blasts:+experiment+and+computational+modeling.">Evolution of blast wave profiles in simulated air blasts: experiment and computational modeling.</a> Shock Waves. 22 (5), 403-415 (2012).</li><li>Kuriakose, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailoring+the+Blast+Exposure+Conditions+in+the+Shock+Tube+for+Generating+Pure,+Primary+Shock+Waves:+The+End+Plate+Facilitates+Elimination+of+Secondary+Loading+of+the+Specimen.">Tailoring the Blast Exposure Conditions in the Shock Tube for Generating Pure, Primary Shock Waves: The End Plate Facilitates Elimination of Secondary Loading of the Specimen.</a> PLoS ONE. 11 (9), 0161597 (2016).</li><li>Reneer, D. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Multi-Mode+Shock+Tube+for+Investigation+of+Blast-Induced+Traumatic+Brain+Injury.">A Multi-Mode Shock Tube for Investigation of Blast-Induced Traumatic Brain Injury.</a> J Neurotrauma. 28 (1), 95-104 (2011).</li><li>VandeVord, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up-regulation+of+reactivity+and+survival+genes+in+astrocytes+after+exposure+to+short+duration+overpressure.">Up-regulation of reactivity and survival genes in astrocytes after exposure to short duration overpressure.</a> Neurosci Lett. 434 (3), 247-252 (2008).</li><li>Kane, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+gene+expression+in+cultured+microglia+in+response+to+simulated+blast+overpressure:+Possible+role+of+pulse+duration.">Altered gene expression in cultured microglia in response to simulated blast overpressure: Possible role of pulse duration.</a> Neurosci Lett. 522 (1), 47-51 (2012).</li><li>Nienaber, M., Lee, J. S., Feng, R., Lim, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impulsive+pressurization+of+neuronal+cells+for+traumatic+brain+injury+study.">Impulsive pressurization of neuronal cells for traumatic brain injury study.</a> J Vis Exp. (56), (2011).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+Profiling+in+Rat+Hair+Follicles+following+Simulated+Blast+Insult:+A+New+Diagnostic+Tool+for+Traumatic+Brain+Injury.">Transcriptional Profiling in Rat Hair Follicles following Simulated Blast Insult: A New Diagnostic Tool for Traumatic Brain Injury.</a> PLoS ONE. 9 (8), 104518 (2014).</li><li>Bo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+characterization+of+compression-induceddamage+in+live+biological+samples.">Cellular characterization of compression-induceddamage in live biological samples.</a> AIP Conf Proc. 1426 (1), 153-156 (2012).</li><li>Tannous, O., Griffith, C., O'Toole, R. V., Pellegrini, V. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterotopic+ossification+after+extremity+blast+amputation+in+a+Sprague-Dawley+rat+animal+model.">Heterotopic ossification after extremity blast amputation in a Sprague-Dawley rat animal model.</a> J Orthop Trauma. 25 (8), 506-510 (2011).</li><li>Chen, Y. C., Smith, D. H., Meaney, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-Vitro+Approaches+for+Studying+Blast-Induced+Traumatic+Brain+Injury.">In-Vitro Approaches for Studying Blast-Induced Traumatic Brain Injury.</a> J Neurotrauma. 26 (6), 861-876 (2009).</li><li>Nguyen, T. T. N., Wilgeroth, J. M., Proud, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+blast+wave+generation+in+a+shock+tube+for+biological+applications.">Controlling blast wave generation in a shock tube for biological applications.</a> JPCS. 500 (14), 142025 (2014).</li></ol>

The authors have no competing financial interests.

Primary injuries obtained from exposure to blast events are not yet fully understood. Identifying and understanding the mechanisms that trigger blast-induced injuries, such as traumatic brain injury3,4 and heterotopic ossification6,7, are important first steps to developing effective methods of prophylaxis. To help achieve this goal, a number of experimental systems have been developed to replicate blast event exposure11,12,13,14,18,19. The technique described here uses shock tube equipment (Figure 2) capable of firing shock waves at a range of pressures at whole-body (i.e., rodent), tissue, or cell samples. The ability to load individual cell types rather than whole tissues gives the ability to parse out distinct cellular responses, as damage can occur concurrently via a range of mechanisms1,2. For example, to model traumatic brain injury, the assessment of individual cell types, such as neurons and astrocytes, can allow for the identification of cell-specific injury. Also, the whole-organ response can be assessed using brain tissue. Both the individual cell types and the tissue specimens have value and can give different information. It is also possible to alter the amount of air that is pressurized to generate the shock by selecting the double-breech or driver-tube inlet. This controls the duration of the shock wave. Another possibility is to change the diaphragm material and thickness to alter the peak pressure25.
Another factor to consider are interference end effects that can be present when the sample housing is located near the exit of the shock tube, such as that found on the EVOC rig described in the present system. Chandra et al. looked at blast wave profiles found on different locations on a compression-driven shock tube and found that the Friedlander waveform was best represented at a location deep within the shock tube15. Kuriakose et al. also studied secondary loading of the sample and found that the placement of an end plate at the end of the shock tube was able to eliminate unwanted reflected waves16. Considering the data found in these publications15,16, future modifications to improve the shock tube system described in this article could involve the placement of the EVOC rig at a deeper location within the driven tube or, alternatively, the inclusion of an end plate on the shock tube. Limitations of the described method could include the relatively low throughput of samples. A single user can operate the shock tube safely at an output of around 6-8 samples per hour. At present, the system is designed around the use of single 35-mm petri dishes. Therefore, larger experiments containing multiple groups and biological replicates can be difficult to achieve.
This methods article shows how the viability of adherent dermal papilla cells was affected by exposure to a single shock wave. A short-duration shock wave (&#60;10 ms) of &#8804;72 kPa did not affect viability when compared to the control (Figure 3 and Figure 4). In contrast, a shock wave at 127 kPa stimulated a significant drop in viability at 24 h post-blast, as shown by both a redox indicator assay (Figure 3) and fluorescent image analysis (Figure 4). Miller et al. reported a similar reduction in cell viability in rat organotypic hippocampal slice cultures when cells were exposed to a either a 147 kPa or 278 kPa shock wave using an open-ended, helium-driven shock tube14. In contrast, VandeVord et al. reported that there was no effect on viability in rat astrocytes exposed to a short-duration overpressure of &#62;200 kPa, although a barochamber was used rather than a shock tube18. It should be noted that the external pressure is reliant on the blast wave, although this creates complex stress waves within the body, therefore making the nature of the loading highly dependent upon the mechanical properties of the tissue or cell. Additional characterization studies of the cellular response to blast events is required. Furthermore, by assessing shock wave exposure at the cellular level, as shown in this technique, biological responses triggered from the injury, such as the perturbation of signaling pathways or epigenetic changes, can be identified and explored further.
In conclusion, this work describes the use of a stainless-steel shock tube and a modified EVOC rig to incorporate primary cell cultures. Shock waves at a range of pressures can be generated and propagated over live cells to replicate the effects that occur from exposure to a blast wave. This protocol demonstrates how to evaluate cell viability, but longer-term changes to individual cell types can also be studied. Going forward, we plan to assess the differential effects that complex shock waves can elicit in different cell types, with the aim of furthering our understanding of blast-induced primary injuries.

With the recent trend towards the use of improvised explosive devices in modern warfare and terrorist actions on civilians, understanding the effects of explosive events on the human body is of great importance. Injuries obtained through exposure to blast events can be deadly and lethal, with the physical processes of injury being divided into four categories. Primary injuries result from direct exposure to the blast wave, which interacts locally with the body in a compressive and subsequently expansive manner, causing the disruption of membranes and soft tissues1. Secondary injuries include blunt trauma or penetrative wounds caused by impact with low-mass objects propelled at high velocity by the blast wave. Tertiary injuries occur when the blast wave has sufficient energy to throw objects of high mass or individuals against objects. Lastly, quaternary blast injuries are defined by other miscellaneous injuries that do not fit the other categories, such as flash burns2. Following exposure to such blast events, primary injuries include traumatic brain injury3,4,5, heterotopic ossification6,7, blast lung injury8, loss of hearing9, and others10.
A commonly observed waveform from blast events is the Friedlander wave, representing a free-field, as opposed to an enclosed-space, explosion. The waveform consists of a blast front that can be defined as a sharp and rapid rise in positive pressure. This is immediately followed by a blast wind of air moving at high speed and a release wave that reduces the pressure to below atmospheric levels. A partial vacuum is left at the region of the initial explosion, which results in the slow backflow of air. The positive and negative phases of the wave (Figure 1A) result in the push-pull movement of the blast wave1. To help elucidate the mechanisms behind primary blast injuries, experimental models have been created to produce waveforms, such as the Friedlander wave, which cells and tissues will face when exposed to a genuine blast event. Current systems listed in the literature include shock tubes11,12,13,14,15,16,17, barochambers18,19, the Kolsky bar20, advanced blast simulators21, the Split Hopkinson pressure bar22, and the recreation of alternative blast events in a controlled environment using pentaerythritol tetranitrate23. Despite the wide range of models available, many variables influence the injury obtained from blast waves, including the pre-stress applied to, and the mechanical properties of, the individual cell types or tissues under evaluation24. While the study of tissue or organs can shed light on tissue deformation and gross morphological changes sustained as a result of blast events, analysis at the cellular level can uncover transcriptional and epigenetic changes influenced by the shock wave.
This methods article describes a technique to propagate shock waves at a range of pressures over live cells in a monolayer. This allows for the immediate characterization of cell viability, elucidating potential damage thresholds from shock waves. Furthermore, viable cells can be returned to standard culture conditions, and long-term biological effects from the blast event can be assessed. The protocol below describes two cell viability techniques that can be used on cells in culture.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55618/55618fig1.jpg" /> 
Figure 1: Approximation of a Friedlander Wave. (A) An approximation of a Friedlander wave observed at sensor 3 on the shock tube. (B) Representative data showing the different pressure profiles observed at sensors 1, 2, and 3 on the shock tube. <a href="http://cloudflare.jove.com/files/ftp_upload/55618/55618fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="1124">
	<tbody>
		<tr height="21">
			<td>MEM &#945;, nucleosides</td>
			<td>ThermoFisher</td>
			<td>22571020</td>
		</tr>
		<tr height="21">
			<td>Fetal Bovine Serum, certified, US origin</td>
			<td>ThermoFisher</td>
			<td>16000044</td>
			<td>Supplement to create complete growth media.</td>
		</tr>
		<tr height="21">
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Penicillin-Streptomycin&#160;</td>
			<td>ThermoFisher</td>
			<td>15070-063</td>
			<td>Supplement to create complete growth media.</td>
		</tr>
		<tr height="21">
			<td>Trypsin-EDTA (0.5%), no phenol red</td>
			<td>ThermoFisher</td>
			<td>15400-054</td>
			<td>Dilute 1 in 10 before use.</td>
		</tr>
		<tr height="21">
			<td>CytoOne T-75 Flask, TC-Treated, vented</td>
			<td>Starlab</td>
			<td>CC7682-4875</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>TC Dish (PS) 35mm, 8.5 cm2</td>
			<td>Triple Red</td>
			<td>TCD010035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Petri dish (PS) 90x14.2mm no vent</td>
			<td>VWR UK</td>
			<td>391-0453&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Gas Permeable Adhesive Plate Seals</td>
			<td>ThermoFisher</td>
			<td>AB-0718</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>LIVE/DEAD Cell Imaging Kit (488/570)</td>
			<td>ThermoFisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Alamarblue cell viability reagent</td>
			<td>Fisher Scientific</td>
			<td>13494309</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Virkon tablets</td>
			<td>VWR UK</td>
			<td>115-0020</td>
			<td>Use to create 2% solution as viability control reagent.</td>
		</tr>
		<tr height="21">
			<td>Dumont forceps</td>
			<td>SurgicalTools</td>
			<td>11295-10</td>
			<td>Use to remove coverslips from petri dish.</td>
		</tr>
		<tr height="21">
			<td>Cover glass, square</td>
			<td>VWR UK</td>
			<td>631-0125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>Microscope slides</td>
			<td>VWR UK</td>
			<td>631-1553</td>
			<td></td>
		</tr>
		<tr height="21">
			<td>96 Well plate, solid black</td>
			<td>AppletonWoods</td>
			<td>CC760</td>
			<td>Plate to be used for fluorescence measurements.</td>
		</tr>
		<tr height="21">
			<td>96 Well plate, clear, (PS)</td>
			<td>VWR UK</td>
			<td>734-1799</td>
			<td>Plate to be used for absorbance measurments.</td>
		</tr>
		<tr height="21">
			<td>Leica DMi1 Camera stand outfit</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Optical microscope used for cell culture.</td>
		</tr>
		<tr height="21">
			<td>Zeiss PALM MicroBeam Laser Capture Microdisseciton</td>
			<td>Zeiss</td>
			<td></td>
			<td>Fluorescence microsope used for LIVE/DEAD imaging.</td>
		</tr>
		<tr height="21">
			<td>EnVision Multilabel Reader</td>
			<td>PerkinElmer</td>
			<td>2104-0010A</td>
			<td>Plate reader to be used for fluorescence/absorbance readings.</td>
		</tr>
		<tr height="42">
			<td>Mylar Electrical &#38; Chemical Insulating Film, 304mm x 200mm x 0.023mm&#160;</td>
			<td>RS Components</td>
			<td>785-0782</td>
			<td>Use to create shock tube diaphragm.</td>
		</tr>
		<tr height="42">
			<td>Mylar Electrical &#38; Chemical Insulating Film, 304mm x 200mm x 0.05mm&#160;</td>
			<td>RS Components</td>
			<td>785-0786</td>
			<td>Use to creatw shock tube diaphragm.</td>
		</tr>
		<tr height="42">
			<td>Mylar Electrical &#38; Chemical Insulating Film, 304mm x 200mm x 0.125mm&#160;</td>
			<td>RS Components</td>
			<td>785-0798</td>
			<td>Use to create shock tube diaphragm.</td>
		</tr>
		<tr height="21">
			<td>Current source power unit</td>
			<td>Dytran Intruments Inc.</td>
			<td>4103C</td>
			<td>Power source for 2300V1 sensor.</td>
		</tr>
		<tr height="21">
			<td>IEPE Pressure Sensor</td>
			<td>Dytran Intruments Inc.</td>
			<td>2300V1</td>
			<td>Pressure sensor located on shock tube.</td>
		</tr>
		<tr height="21">
			<td>Digital Phosphor Oscilloscope</td>
			<td>Tektronix</td>
			<td>DPO 4104B</td>
			<td>Use to gather and save sensor 2300V1 data.</td>
		</tr>
	</tbody>
</table>

evaluating primary blast effects in vitro

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such blast-induced injuries is of great importance considering the recent trend towards the use of explosives in modern warfare and terrorist-related incidents. To fully understand blast-induced injury, we must first be able to replicate such blast events in a controlled environment using a reproducible method. In this technique using shock tube equipment, shock waves at a range of pressures can be propagated over live cells grown in 2D, and markers of cell viability can be immediately analyzed using a redox indicator assay and the fluorescent imaging of live and dead cells. This method demonstrated that increasing the peak blast overpressure to 127 kPa can stimulate a significant drop in cell viability when compared to untreated controls. Test samples are not limited to adherent cells, but can include cell suspensions,&#160;whole-body and tissue samples, through minor modifications to the shock tube setup. Replicating the exact conditions that tissues and cells experience when exposed to a genuine blast event is difficult. Techniques such as the one presented in this article can help to define damage thresholds and identify the transcriptional and epigenetic changes within cells that arise from shock wave exposure.

Using the method described above, cells grown in a monolayer were exposed in triplicate to shock waves generated using a shock tube (Figure 2B). Markers of cell viability were assessed. Using a redox indicator assay, it was found that the application of a 127 kPa shock wave was able to significantly reduce the viability of dermal papilla cells compared to controls after 24 h in culture (Figure 3). The application of a shock wave &#8804;72 kPa did not reduce viability. To support these observations, a fluorescent image assay capable of fluorescently labelling live or dead cells with green or red fluorophores, respectively, was used. The quantification of live and dead cells from 13 fluorescent images per biological replicate demonstrated a reduction in cell viability in those exposed to the 127 kPa shock wave when compared to the control (Figure 4). Statistical analysis was carried out using a one-way ANOVA followed by Tukey&#39;s multiple comparison test, with p &#60;0.05 deemed statistically significant. The sample size per group totaled 9 for the redox indicator assay and 39 for the quantitative fluorescence imaging analysis.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55618/55618fig3.jpg" /> 
Figure 3: Redox Indicator Assay Data Showing Time Course of Cell Viability After Shock Wave Exposure. Significantly fewer cells were observed after 24 h in the 127-kPa shock wave-exposed group when compared to the untreated control. The negative control that consisted of a 30-s treatment with 2% disinfectant (see the Table of Materials) showed significantly fewer cells at both T0 and 24 h compared to all other groups. Each bar represents the mean &#177; 1 standard deviation (SD); biological replicates, N = 3; technical replicates, n = 3. **** = p &#60;0.0001. * = p &#60;0.05. <a href="http://cloudflare.jove.com/files/ftp_upload/55618/55618fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55618/55618fig4.jpg" /> 
Figure 4: Fluorescence Imaging. (A) Quantitative data gathered from fluorescent images of live and dead cells captured using a fluorescence microscope at 24 h post-shock wave exposure. Significantly fewer viable cells were observed in the 127 kPa (mean = 93.42) shock wave-exposed sample compared to the 47 kPa (mean = 98.18), 72 kPa (mean = 98.87), and control groups (mean = 99.10). No viable cells were observed on the negative control group after 24 h (mean = 0). (B) Representative fluorescence images. Green shows live cells, whilst red shows dead cells. Each box plot shows the upper and lower quartiles with Tukey whiskers; biological replicates, N = 3; technical replicates, n = 13. **** = p &#60;0.0001. Scale bar = 300 &#181;m. <a href="http://cloudflare.jove.com/files/ftp_upload/55618/55618fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluating Primary Blast Effects In Vitro at JoVE.com

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.

Evaluating Primary Blast Effects In Vitro

Exposure to blast events can cause severe trauma to vital organs such as the lungs, ears, and brain. Understanding the mechanisms behind such ...

evaluating-primary-blast-effects-in-vitro

Research

JoVE Journal

Bioengineering

7.9K Views.  Imperial College London. 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.

Video: Evaluating Primary Blast Effects In Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles

Biodegradable nanoparticles have emerged as a versatile platform for the design and implementation of new intranasal vaccines against respiratory infectious diseases. Specifically, polyanhydride nanoparticles composed of the aliphatic sebacic acid (SA), the aromatic 1,6-bis(p-carboxyphenoxy)hexane (CPH), or the amphiphilic 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) display unique bulk and surface erosion kinetics1,2 and can be exploited to slowly release functional biomolecules (e.g., protein antigens, immunoglobulins, etc.) in vivo3,4,5. These nanoparticles also possess intrinsic adjuvant activity, making them an excellent choice for a vaccine delivery platform6,7,8.
	In order to elucidate the mechanisms governing the activation of innate immunity following intranasal mucosal vaccination, one must evaluate the molecular and cellular responses of the antigen presenting cells (APCs) responsible for initiating immune responses. Dendritic cells are the principal APCs found in conducting airways, while alveolar macrophages (AM&#632;) predominate in the lung parenchyma9,10,11. AM&#632; are highly efficient in clearing the lungs of microbial pathogens and cell debris12,13. In addition, this cell type plays a valuable role in the transport of microbial antigens to the draining lymph nodes, which is an important first step in the initiation of an adaptive immune response9. AM&#632; also express elevated levels of innate pattern recognition and scavenger receptors, secrete pro-inflammatory mediators, and prime na&iuml;ve T cells12,14. A relatively pure population of AM&#632; (e.g., greater than 80%) can easily be obtained via lung lavage for study in the laboratory. Resident AM&#632; harvested from immune competent animals provide a representative phenotype of the macrophages that will encounter the particle-based vaccine in vivo. Herein, we describe the protocols used to harvest and culture AM&#632; from mice and examine the activation phenotype of the macrophages following treatment with polyanhydride nanoparticles in vitro.

Herein, we describe protocols for harvesting murine alveolar macrophages, which are resident innate immune cells in the lung, and examining their activation in response to co-culture with polyanhydride nanoparticles.

Biodegradable nanoparticles have emerged as a  versatile platform for the design and implementation of new intranasal vaccines  against respiratory ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...