This material is based upon work supported by the United States Department of Defense through an interagency agreement (#19-1006-IM) with the Temporary Corneal Repair acquisition program (United States Army Medical Materiel Development Agency).

Before performing this protocol, be aware that there are legal and ethical requirements in place for the use of animals in research and training. If live animals are used for the source of ocular tissue, seek approval by the local ethical or legal authority (IACUC or Ethics committee, etc.) before beginning. If there is any question in obtaining approval for the use of animals, do not proceed. We previously determined and reported that fresh porcine eyes obtained and used within 24 h post-mortem compared closest to in vivo physiology and fared well for these studies (Animal Technologies, Tyler, TX, USA)10,13. No live animals were used throughout this protocol, using a tissue vendor to obtain tissue within 24 h.
NOTE: Prior to tissue arrival, fabricate the organ culture dishes (Supplementary Protocol 1), clamping rings (Supplementary Protocol 1), dish stands (Supplementary Protocol 1), pressure transducer data collection setup (Supplementary Protocol 2), and pneumatic puncture platform (Supplementary Protocol 3). Sterilize the dishes, tools, and supplies and prepare the work areas. It&#39;s useful to have a non-sterile area to perform gross dissection on the eyes, as they usually come with connective, extra orbital tissue attached. Execute these first steps on an open, clean work surface, and then transfer the eyes aseptically into a BSC II cabinet for micro-dissection (cabinet #1). Optimally, the BSC II cabinet utilized for micro-dissection is separated from the dish assembly BSC II cabinet (cabinet #2) to minimize airflow and maximize workspace. Set up the micro-dissection cabinet with a dissecting microscope and a way to visualize the work surface (camera or eyepieces protruding from the cabinet).
1. Sterilization steps, supplies (see Table of Materials for more details), and setup
<ol>
	<li>Prepare&#160;and gas-sterilize the following items (1 kit for each eye): ASOC dish, clamping ring, two fluidic connectors with O-rings, two 18 G needle hubs, four screws, two lengths of PE-100 tubing (length of distance should be long enough to extend from the dish inside the incubator to the syringe pump and pressure transducer data collection setup), two 18 G 90&#176; bent needle hubs, two 3-way valves.</li>
	<li>Prepare and autoclave the following kits.
	<ol>
		<li>Prepare and autoclave the micro-dissection instrument kit, containing one pair of fine forceps, one pair of Vannas scissors, one pair of medium toothed forceps, one pair of large scissors, cotton swabs, and a razor blade or scalpel.</li>
		<li>Prepare and autoclave the assembly instrument kit, containing one pair of medium toothed forceps, one pair of surgical scissors, and one L-key.</li>
		<li>Prepare and autoclave the daily kit (quantity: one per day of culture), containing one L-key to tighten clamping rings to dishes each day as needed.</li>
		<li>Autoclave four 100 mL beakers for disinfecting and storing eyes and anterior segments.</li>
		<li>Autoclave the puncture objects.</li>
	</ol>
	</li>
	<li>Gather the following sterile items: Petri dish (1 dish/eye), gauze (1-2/eye), dish stand, 20 mL syringes (1/eye), 10 mL syringes (1/eye), nylon syringe filters (1/eye).</li>
	<li>Prepare sterile media: DMEM with 4% FBS, 1x Glutamax, 1x Gentamicin, 1x Antibiotic-Antimycotic (AA; approximately 30-40 mL complete media/eye).</li>
	<li>Prepare AA-PBS: PBS with 1x AA (~500 mL).</li>
	<li>Prepare the gross dissection tool pack: clean and dry large surgical scissors and forceps.</li>
	<li>Set up the non-sterile dissection workspace: Gather supplies from gross dissection tool pack, enucleated porcine eyes submerged in PBS and on ice, surgical drape, 100 mL beaker with PBS. Lay out the surgical drape and items required for gross dissection.</li>
	<li>Set up the sterile dissection workspace: Gather supplies from micro-dissection instrument kit, sterile gauze, dissecting microscope, betadine solution, sterile PBS, sterile media, four sterilized 100 mL beakers, sterile Petri dish. Aseptically transfer to BSC II cabinet #1. Set up the cabinet for visualizing eyes on the dissecting microscope.</li>
	<li>Set up the ASOC assembly workspace: Gather gas-sterilized kits (dish kit and lid kit), assembly instrument kit, sterile media, dish stands, sterile Petri dishes, sterile syringes, and syringe filters. Aseptically transfer to BSC II cabinet #2. Set up the cabinet for dish assembly.</li>
	<li>When eyes are stabilized and ready for puncture (72 h post-setup), aseptically transfer them to a BSC II cabinet. Set up the OG injury induction workspace: Pneumatic powered injury induction device (assembly detailed in Supplemental 3) and Lab Jack and cross-tracking vise to hold the ASOC dish.</li>
</ol>
2. Dissection of tissue
<ol>
	<li>Prepare the porcine tissue using non-sterile dissection workspace.
	<ol>
		<li>Procure enucleated porcine eyes from a local abattoir, animal studies, or vendor. Maintain the eyes on ice submerged in PBS during delivery and use immediately upon receiving.</li>
		<li>Cut away the extraorbital tissue and trim the conjunctiva leaving only the corneoscleral shell and optic nerve. Perform the dissection under non-sterile conditions with large surgical scissors and forceps in a gross dissection tool pack.</li>
		<li>Place the eyes back into fresh PBS on ice until all eyes required for experimental setup have been preliminary/gross dissected.</li>
		<li>Submerge the eyes in 10% betadine solution for 2 min in closed containers and transfer aseptically to BSC II cabinet #1. Perform all the subsequent works under sterile conditions to minimize contaminations during setup.</li>
	</ol>
	</li>
	<li>Sterilely dissect anterior segments.
	<ol>
		<li>After 2 min in betadine solution, transfer the eyes into three serial washes of sterile AA-PBS to remove excess betadine solution from the ocular surface while maintaining sterility of the ocular tissue. After three washes, maintain the tissue in AA-PBS until further use.</li>
		<li>Hemisect the eye using a razor blade/scalpel and curved scissors. Place the eye on an AA-PBS-soaked gauze and create an incision with a sterile razor blade or scalpel near the equator of the eye (60/40 split with 40 on the anterior side). Using curved surgical scissors, hemisect the eye to isolate the anterior eye (corneal half). 
		NOTE: The cut around the anterior segment needs to be continuous to prevent jagged, rough edges in the sclera that will create fluid leaks after setup in the organ culture.</li>
		<li>Use microscissors as a shovel to scoop vitreous humor from the anterior segment. Remove the lens from the anterior segment using microscissors. Leave the anterior segments in AA-PBS until further dissection steps. 
		NOTE: All the eyes that will be dissected can be held at this step and one by one taken through the remainder of the dissection process.</li>
		<li>With a dissecting microscope, cut back iris to iris root gradually, radially until trabecular meshwork (TM) is visible. The TM is a pigmented tissue that comprises fibers circumferentially oriented around the corneoscleral shell. Careful cuts into the iris toward the iris root will expose the depth of the TM under the tissue.</li>
		<li>Cut 360&#176; around the iris at the same depth as the initial cut into the tissue to expose the entire TM region. Clean up any remaining residual iris covering TM as is necessary.</li>
		<li>Trim the ciliary body remnants posterior to the TM, leaving only a thin band of tissue posterior to the TM region (approximately 1 mm).</li>
		<li>Place the dissected anterior segment (AS) in media until further setup in ASOC in BSC II cabinet #2. 
		NOTE: All eyes can be held at this point prior to ASOC setup if a single user is performing dissection and organ culture dish assembly.</li>
	</ol>
	</li>
</ol>
3. Setting up anterior segments in organ culture dishes
<ol>
	<li>Place a single AS in a Petri dish with AS inverted (cup up). Using a cotton swab, wet in the media and gently dab in the center of the cornea to remove any pigment. Using forceps to hold the eye and the same swab, wipe the cotton swab around the sclera to remove extra pigment.</li>
	<li>Invert the AS and place on top of bottom part of the dish over the elevated region, centering the cornea over the elevated region in the dish. Place the clamping ring on top of the newly placed AS.</li>
	<li>Place four screws into the corresponding holes to hold the ring in place with the AS under the ring. Gently hand-tighten the screws with the L-key. 
	NOTE: The tightening step will happen daily throughout the experiment, so the goal of the initial tightening here is to ensure the media does not leak while avoiding breaking the clamping ring.</li>
	<li>With a sterile Petri dish set, place the top over the dish and invert the setup. Attach the dish stand. Attach the fluidic connectors with O-rings to threaded ports on the bottom of the dish.</li>
	<li>To one fluidic connector, attach an 18 G 90&#176; bent needle hub, a length of tubing, an 18 G needle hub, a nylon syringe filter, a three-way valve, and a 20 mL syringe filled with media.</li>
	<li>To the second fluidic connector, attach an 18 G 90&#176; degree bent needle hub, length of tubing, an 18 G needle hub, a three-way valve, and the barrel portion of a sterile 10 mL syringe (this will act as a reservoir to catch liquid and bubbles from the ASOC).</li>
	<li>With three-way valves open appropriately to the syringes, gently push media through the system using the fluidics connector port identified in step 3.5 to inflate the AS, fill media in the tubing, and eventually the reservoir. 
	NOTE: If media leaks into the ASOC dish, the AS is not secured tightly enough with the clamping ring.</li>
	<li>Remove bubbles by gently pushing media into the dish and inverting the dish to push out the bubbles and into the reservoir.</li>
	<li>Place the dish and stand upright. Place the bottom portion of a Petri dish underneath the feet of the stand, careful not to ensnarl the tubing.</li>
</ol>
4. Starting anterior segment organ culture
<ol>
	<li>ASOC is now ready for incubation. Place the ASOC dish into the cell culture incubator (37 &#176;C, 5% CO2). Ensure the height of the ASOC dish in the incubator above the pressure transducers is known and accounted for to calculate IOP accurately (Supplementary Protocol 4).</li>
	<li>Direct the tubing lines out through the bottom of the 37 &#176;C, 5% CO2 incubator door so that they do not interfere with the opening and closing of the door. Attach the 20 mL syringe to the syringe pump set at 2.5 &#181;L/min.</li>
	<li>Position the tubing line with the reservoir at the pressure transducer instrument. Connect the side 3-way valve to the pressure transducer setup while flowing PBS through the line to avoid air bubbles entering the tubing lines. 
	NOTE: Empty the PBS from the reservoirs after the system is set up to reduce the likelihood of reservoir contamination with microbial growth for the duration of the organ culture.</li>
	<li>Initiate IOP data collection by first ensuring a microSD card is present for saving data files. Then, turn on the pressure transducer setup to begin data collection. 
	&#8203;NOTE: Details for setting up the pressure transducer data collection device are provided in Supplementary Protocol 2.</li>
</ol>
5. Daily maintenance of ASOC
<ol>
	<li>After the ASOC has had 24 h to equilibrate, remove the dishes from the 37 &#176;C, 5% CO2 incubator and place them into the BSC II cabinet. 
	NOTE: On pressure data acquisition, these time periods look like spikes as the ASOCs are removed from the incubator (height change) and adjusted in the cabinet.</li>
	<li>Check for leaks under each dish on the Petri plate. If present, check for tight fluidic connections under the dish and re-tighten if necessary. Check for leaks in the dish top using a sterile L-key to tighten the screws in the clamping ring. 
	NOTE: The AS sclera tissue will compress and reduce thickness by 24 h, and the clamping ring will need to be tightened.</li>
	<li>Aspirate the media from the dish well. 
	NOTE: The trabecular meshwork is filtering fluid from the media being pumped into the ASOC. Therefore, media will be present in the ASOC dish along the edges.</li>
	<li>Repeat steps 3.7 and 3.8 to remove any trapped air bubbles.</li>
	<li>Refill syringes on the syringe pumps, ensure the syringe pumps are operating, and confirm the alignment of valves for perfusion into the ASOC. Return the ASOC dish to the 37 &#176;C, 5% CO2 incubator. 
	&#8203;NOTE: Optimally, these steps should be performed daily. However, the use of a 20 mL starting volume of media, the ASOC dish well volume, and a pump rate of 2.5 &#181;L/min should be sufficient to let the system run for several days undisturbed.</li>
</ol>
6. OG injury induction with pneumatic-powered puncture device
NOTE: Construction of the pneumatic puncture device is detailed in Supplementary Protocol 3. OG injuries are induced after IOP has stabilized, which normally occurs after 3 days in culture. Acceptable IOP values are 5-20 mmHg based on physiological IOP, which can be determined by evaluating the IOP data files or setting LED indicators in the pressure measurement system as described in Supplementary Protocol 2.
<ol>
	<li>Prepare the BSC II cabinet for OG injury induction as detailed in step 1.10. Connect the puncture platform to a compressed air line. Attach the sterile puncture object to the chuck. 
	NOTE: An air compressor can be used to power the device, but tank compressed air or built-in laboratory lines can suffice if pressure is greater than 50 psi.</li>
	<li>Set the pressure regulator on the puncture platform to 50 psi for adequate puncture force on objects up to 4.5 mm in diameter. Position the cross-tracking vise on the lab jack in front of the puncture platform to hold the ASOC dish during injury induction.</li>
	<li>Remove ASOC setup from 37 &#176;C, 5% CO2 incubator, and place in the cross-tracking vise perpendicular to the puncture platform (Figure 2) after removing the lid and setting it aside. Keep the anterior segment perfusing but close off the 3-way valve port to the pressure transducer to prevent over-pressurization damage to the transducer.</li>
	<li>Extend the piston arm to its maximal distance and position the corneal apex within 1 mm of the puncture object. Retract the piston arm and bring the anterior segment 1 cm closer to the puncture object. 
	NOTE: This distance has been optimized for high-efficiency injury induction without hitting the ASOC dish.</li>
	<li>Fire the puncture device by turning it on and opening the solenoid valve with the second switch on the device. To retract the device, press the second switch again to remove the puncture device from the eye. Verify proper injury induction by visual inspection and media leaking from the injury site.</li>
	<li>Remove ASOC dish from the vise; place the lid back onto the dish assembly and open the fluidic line to the pressure transducer. Place the ASOC back into the 37 &#176;C, 5% CO2 incubator. 
	&#8203;NOTE: At this point, therapeutic can be applied to the AS to assess its efficacy for sealing OG injuries.</li>
</ol>
7. Removing ASOC from culture
NOTE: Depending on endpoint analysis (see Representative Results for possible endpoint methods), the AS needs to remain in the ASOC dish inflated while other methods require AS tissue isolated from the culture chamber. The below methodology describes how to take AS out of the organ culture dishes and to remove the rest of the setup.
<ol>
	<li>Remove the ASOC dishes from the 37 &#176;C, 5% CO2 incubator. Close the 3-way valve to the syringe and reservoir and disconnect the tubing from the system. Discard the syringe, reservoir, and filter. Place the 3-way valves, tubing, and needle hubs in a separate container for washing and sterilizing.</li>
	<li>Disconnect the needle hubs from the fluidic connections on the bottom of the dish. Unthread the fluidic connectors and o-rings. Place all items in a container for washing and sterilizing.</li>
	<li>Remove the four screws from the clamping ring using the L-key. Carefully remove the clamping ring.</li>
	<li>Using forceps, remove the AS from the dish and, depending on endpoint analysis and image, place in fixative or appropriate biohazard waste.</li>
</ol>
8. IOP data analysis
<ol>
	<li>Connect the microSD card to a computer to remove the .txt file containing data from the most recent experimental run. 
	NOTE: The file is named in the code controlling the microcontroller and should be updated for each experiment (see Supplementary Protocol 2).</li>
	<li>Import the data into a spreadsheet.</li>
	<li>Organize the data into 12 columns: Time (in min) and mV signal for each of 11 pressure transducer channels. The first ten channels correspond to ten ASOC experimental setups. The final transducer signal is for a sensor open to air as a control channel to confirm mV signal did not alter due to changes in the input signal. Plot control channel versus time to confirm mV signal was consistent throughout.</li>
	<li>Convert the mV signals for ten channels into mmHg using slope-intercept equations generated from the initial calibration of each sensor (see Supplementary Protocol 4).</li>
	<li>Plot time (convert to days to simplify data interpretation) versus each of the ten channels to determine how the IOP fluctuated across the experimental time course.</li>
	<li>Determine average IOP values at key points in the data to more easily compare values between each and how they are altered before and after OG injury induction. Average 2-3 h of data for each 24 h interval to determine IOP at each day of ASOC. 
	NOTE: Representative IOP results are shown in Figure 4.</li>
</ol>

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L., Papas, E. B., Garrett, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+staining+and+physiological+state+of+corneal+epithelial+cells.">Fluorescein staining and physiological state of corneal epithelial cells.</a> Contact Lens &amp; Anterior Eye: The Journal of the British Contact Lens Association. 37 (3), 213-223 (2014).</li><li>Bandamwar, K. L., Garrett, Q., Papas, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+fluorescein+staining+of+the+corneal+epithelium:+What+does+it+mean+at+a+cellular+level.">Sodium fluorescein staining of the corneal epithelium: What does it mean at a cellular level.</a> Investigative Ophthalmology &amp; Visual Science. 52 (14), 6496 (2011).</li><li>Sherwood, J. M., Reina-Torres, E., Bertrand, J. A., Rowe, B., Overby, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+outflow+facility+using+iPerfusion.">Measurement of outflow facility using iPerfusion.</a> PLoS One. 11 (3), (2016).</li><li>Weichel, E. D., Colyer, M. H., Ludlow, S. E., Bower, K. S., Eiseman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combat+ocular+trauma+visual+outcomes+during+operations+iraqi+and+enduring+freedom.">Combat ocular trauma visual outcomes during operations iraqi and enduring freedom.</a> Ophthalmology. 115 (12), 2235-2245 (2008).</li><li>Colyer, M. H., 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=Delayed+intraocular+foreign+body+removal+without+endophthalmitis+during+Operations+Iraqi+Freedom+and+Enduring+Freedom.">Delayed intraocular foreign body removal without endophthalmitis during Operations Iraqi Freedom and Enduring Freedom.</a> Ophthalmology. 114 (8), 1439-1447 (2007).</li><li>Geggel, H. S., Maza, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+stromal+puncture+with+the+Nd:YAG+laser.">Anterior stromal puncture with the Nd:YAG laser.</a> Investigative Ophthalmology &amp; Visual Science. 31 (8), 1555-1559 (1990).</li><li>Matthews, A., 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=Indentation+and+needle+insertion+properties+of+the+human+eye.">Indentation and needle insertion properties of the human eye.</a> Eye. 28 (7), 880-887 (2014).</li><li>Rau, A., 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+mechanics+of+corneal+deformation+and+rupture+for+penetrating+injury+in+the+human+eye.">The mechanics of corneal deformation and rupture for penetrating injury in the human eye.</a> Injury. 49 (2), 230-235 (2018).</li><li>Agrawal, R., Ho, S. W., Teoh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-operative+variables+affecting+final+vision+outcome+with+a+critical+review+of+ocular+trauma+classification+for+posterior+open+globe+(zone+III)+injury.">Pre-operative variables affecting final vision outcome with a critical review of ocular trauma classification for posterior open globe (zone III) injury.</a> Indian Journal of Ophthalmology. 61 (10), 541 (2013).</li><li>Knyazer, B., 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=Prognostic+factors+in+posterior+open+globe+injuries+(zone-III+injuries).">Prognostic factors in posterior open globe injuries (zone-III injuries).</a> Clinical &amp; Experimental Ophthalmology. 36 (9), 836-841 (2008).</li><li>Tan, 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=C3+Transferase-Expressing+scAAV2+Transduces+Ocular+Anterior+Segment+Tissues+and+Lowers+Intraocular+Pressure+in+Mouse+and+Monkey.">C3 Transferase-Expressing scAAV2 Transduces Ocular Anterior Segment Tissues and Lowers Intraocular Pressure in Mouse and Monkey.</a> Molecular Therapy - Methods &amp; Clinical Development. 17, 143-155 (2020).</li><li>Bhattacharya, S. K., Gabelt, B. T., Ruiz, J., Picciani, R., Kaufman, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cochlin+Expression+in+Anterior+Segment+Organ+Culture+Models+after+TGF&#946;2+Treatment.">Cochlin Expression in Anterior Segment Organ Culture Models after TGF&#946;2 Treatment.</a> Investigative Ophthalmology &amp; Visual Science. 50 (2), 551-559 (2009).</li><li>Zhu, W., Godwin, C. R., Cheng, L., Scheetz, T. E., Kuehn, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+iPSC-TM+stimulates+division+of+trabecular+meshwork+cells+in+human+eyes.">Transplantation of iPSC-TM stimulates division of trabecular meshwork cells in human eyes.</a> Scientific Reports. 10 (1), 2905 (2020).</li></ol>

The authors declare no competing interests. The views expressed in this article are those of the author(s) and do not reflect the official policy or position of the US Army Medical Department, Department of the Army, Department of Defense, or the US Government.

There are critical steps with the ASOC OG injury platform that should be highlighted to improve the likelihood of success when using the methodology. First, during the anterior segment dissection, preserving the trabecular meshwork is essential but challenging to do correctly. If the TM is disrupted, the eye will not maintain physiological pressure and will not meet eligibility criteria for experimental use. It is recommended to practice the dissection process under normal conditions first rather than introducing the add...

Open globe (OG) injuries can result in permanent loss of vision when not treated or at least stabilized following injury1. Delays, however, are prevalent in remote areas where access to ophthalmic intervention is not readily available, such as in rural areas or on the battlefield in military scenarios. When treatment is not readily available, the current standard of care is to protect the eye with a rigid shield until medical intervention is possible. In military medicine, this delay is currently up to 24 h, but it is anticipated to increase up to 72 h in future combat operations in urban environments where air evacuation is not possible2,3,4. These delays can be even longer in rural, remote civilian applications where access to ophthalmic intervention is limited5,6. An untreated OG injury is highly susceptible to infection and loss of intraocular pressure (IOP) due to the watertight seal of the eye being compromised7,8. Loss of IOP can impact tissue viability, making any medical intervention unlikely to restore vision if the delay between injury and therapeutic is too long9.
To enable the development of easy-to-apply therapeutics for sealing OG injuries until an ophthalmic specialist can be reached, a benchtop OG injury model was previously developed10,11. With this model, high-speed injuries were created in whole porcine eyes while IOP was captured by pressure transducers. Therapeutics can then be applied to assess their ability to seal the OG injury site12. However, as this model uses whole porcine eyes, it can only assess immediate therapeutic performance with no way of tracking longer-term performance across the possible 72 h window in which the therapeutic must stabilize the injury site until the patient reaches specialty care. As a result, an anterior segment organ culture (ASOC) OG injury model was developed and detailed in this protocol as a platform for tracking long-term therapeutic performance13.
ASOC is a widely used technique for maintaining avascular tissue of the anterior segment, such as the cornea, for multiple weeks post-enucleation14,15,16,17. The anterior segment is maintained under physiological IOP by perfusing fluid at physiological flow rates and preserving the trabecular meshwork outflow region, the tissue responsible for regulating IOP, during ASOC setup18,19. The ASOC platform can maintain tissue physiologically, induce an OG injury using a pneumatic-powered device, apply a therapeutic, and track injury stabilization for at least 72 h post-injury13.
Here, the protocol provides a step-by-step methodology for using the ASOC platform. First it details how to set up and fabricate the ASOC platform. Next, the protocol details how to aseptically dissect the anterior segment and maintain the trabecular meshwork, followed by setting up anterior segment tissue in custom-built organ culture dishes. Then, it details how to create open globe injuries and apply therapeutic immediately following injury. Lastly, the protocol provides an overview on characterization parameters that are possible for use with this method that assesses functional, mechanical, and biological properties of the eye and how well the injury was stabilized. Overall, this model provides a much-needed platform to accelerate product development for stabilizing and treating open globe injuries and improve the poor vision prognosis following injury.

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			<td>McMaster-Carr</td>
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anterior segment organ culture platform for tracking open globe injuries and therapeutic performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Images captured via Optical Coherence Tomography (OCT) are shown for OG injured eyes to illustrate how a successful injury induction looks. Figure 3 shows images for control and OG injured AS tissue immediately after injury and 72 h later. Two views are shown: cross-sectional images through the injury site and top-down maximum intensity projection (MIPs) to visualize the surface area of the image. Control eyes show no noticeable disruption in the cornea, while clear injuries can be located t...

Watch this Scientific Journal Video about Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance at JoVE.com

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

anterior-segment-organ-culture-platform-for-tracking-open-globe

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Bioengineering

1.8K Views.  Fort Sam Houston. Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

Video: Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

The intervertebral disc (IVD) is the joint of the spine connecting vertebra to vertebra. It functions to transmit loading of the spine and give flexibility to the spine. It composes of three compartments: the innermost nucleus pulposus (NP) encompassing by the annulus fibrosus (AF), and two cartilaginous endplates connecting the NP and AF to the vertebral body on both sides. Discogenic pain possibly caused by degenerative intervertebral disc disease (DDD) and disc herniations has been identified as a major problem in our modern society. To study possible mechanisms of IVD degeneration, in vitro organ culture systems with live disc cells are highly appealing. The in vitro culture of intact bovine coccygeal IVDs has advanced to a relevant model system, which allows the study of mechano-biological aspects in a well-controlled physiological and mechanical environment. Bovine tail IVDs can be obtained relatively easy in higher numbers and are very similar to the human lumbar IVDs with respect to cell density, cell population and dimensions. However, previous bovine caudal IVD harvesting techniques retaining cartilaginous endplates and bony endplates failed after 1-2 days of culture since the nutrition pathways were obviously blocked by clotted blood. IVDs are the biggest avascular organs, thus, the nutrients to the cells in the NP are solely dependent on diffusion via the capillary buds from the adjacent vertebral body. Presence of bone debris and clotted blood on the endplate surfaces can hinder nutrient diffusion into the center of the disc and compromise cell viability. Our group established a relatively quick protocol to "crack"-out the IVDs from the tail with a low risk for contamination. We are able to permeabilize the freshly-cut bony endplate surfaces by using a surgical jet lavage system, which removes the blood clots and cutting debris and very efficiently reopens the nutrition diffusion pathway to the center of the IVD. The presence of growth plates on both sides of the vertebral bone has to be avoided and to be removed prior to culture. In this video, we outline the crucial steps during preparation and demonstrate the key to a successful organ culture maintaining high cell viability for 14 days under free swelling culture. The culture time could be extended when appropriate mechanical environment can be maintained by using mechanical loading bioreactor. The technique demonstrated here can be extended to other animal species such as porcine, ovine and leporine caudal and lumbar IVD isolation.

This protocol illustrates a harvesting technique for coccygeal bovine intervertebral discs for organ culture for in vitro organ culture.

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

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 ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

A Performance-testing Platform for a Conduction Micropump with an FR-4 Copper-clad Electrode Plate

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is fabricated. It is used to investigate the influence of chamber dimensions on the performance of a conduction micropump and to determine the reliability of the conduction pump when acetone is used as the working fluid. A testing platform is set up to evaluate conduction micropump performance under different conditions. When the chamber height is 0.2 mm, the pump pressure reaches its peak value.

This paper presents a protocol for the fabrication of a conduction micropump using symmetric planar electrodes on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) to test the influence of chamber dimensions on the performance of a conduction micropump.

Here, a conduction micropump with symmetric planar electrode pairs prepared on flame-retardant glass-reinforced epoxy (FR-4) copper-clad laminate (CCL) is ...