Name: anterior segment organ culture platform for tracking open globe injuries and therapeutic performance
Uploaded: 2021-08-25T13:00:00
Description: Watch this Scientific Journal Video about Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance at JoVE.com

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 i...

<ol>
	<li>Hilber, D., Mitchener, T. A., Stout, J., Hatch, B., Canham-Chervak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+injury+surveillance+in+the+US+Department+of+Defense,+1996-2005.">Eye injury surveillance in the US Department of Defense, 1996-2005.</a> American Journal of Preventive Medicine. 38, 78-85 (2010).</li><li>Linde, A. S., McGinnis, L. J., Thompson, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-Battle+domain-perspective+in+military+medical+simulation+trauma+training.">Multi-Battle domain-perspective in military medical simulation trauma training.</a> Journal of Trauma &amp; Treatment. 06 (04), (2017).</li><li>Riesberg, J., Powell, D., Loos, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+loss+of+the+golden+hour.">The loss of the golden hour.</a> Special Warfare. , 49-51 (2017).</li><li>Townsend, S., Lasher, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The US Army in Multi-Domain Operations 2028. (525-3-1), (2018).</li><li>Blanch, R. J., Bishop, J., Javidi, H., Murray, P. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+time+to+primary+repair+on+final+visual+outcome+after+open+globe+injury.">Effect of time to primary repair on final visual outcome after open globe injury.</a> The British Journal of Ophthalmology. 103 (10), 1491-1494 (2019).</li><li>Lesniak, S. 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=Characteristics+and+outcomes+of+delayed+open+globe+repair.">Characteristics and outcomes of delayed open globe repair.</a> Investigative Ophthalmology &amp; Visual Science. 53 (14), 4954 (2012).</li><li>Loporchio, D., Mukkamala, L., Gorukanti, K., Zarbin, M., Langer, P., Bhagat, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+foreign+bodies:+A+review.">Intraocular foreign bodies: A review.</a> Survey of Ophthalmology. 61 (5), 582-596 (2016).</li><li>Jonas, J. B., Budde, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+versus+late+removal+of+retained+intraocular+foreign+bodies.">Early versus late removal of retained intraocular foreign bodies.</a> Retina. 19 (3), 193-197 (1999).</li><li>Watson, P. G., Jovanovik-Pandova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prolonged+ocular+hypotension:+would+ciliary+tissue+transplantation+help.">Prolonged ocular hypotension: would ciliary tissue transplantation help.</a> Eye. 23 (10), 1916-1925 (2009).</li><li>Snider, E. 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=Development+and+characterization+of+a+benchtop+corneal+puncture+injury+model.">Development and characterization of a benchtop corneal puncture injury model.</a> Scientific Reports. 10 (1), 4218 (2020).</li><li>Snider, E. 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=An+open-globe+porcine+injury+platform+for+assessing+therapeutics+and+characterizing+biological+effects.">An open-globe porcine injury platform for assessing therapeutics and characterizing biological effects.</a> Current Protocols in Toxicology. 86 (1), 98 (2020).</li><li>Snider, E. J., Cornell, L. E., Gross, B., Zamora, D. O., Boice, E. N. <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+commercial+off-the-shelf+tissue+adhesives+for+sealing+military+relevant+corneal+perforation+injuries.">Assessment of commercial off-the-shelf tissue adhesives for sealing military relevant corneal perforation injuries.</a> Military Medicine. , (2021).</li><li>Snider, E. J., Boice, E. N., Butler, J. J., Gross, B., Zamora, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+anterior+segment+organ+culture+model+for+open+globe+injuries.">Characterization of an anterior segment organ culture model for open globe injuries.</a> Scientific Reports. 11 (1), 8546 (2021).</li><li>Erickson-Lamy, K., Rohen, J. W., Grant, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outflow+facility+studies+in+the+perfused+human+ocular+anterior+segment.">Outflow facility studies in the perfused human ocular anterior segment.</a> Experimental Eye Research. 52 (6), 723-731 (1991).</li><li>Johnson, D. H., Tschumper, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+organ+culture+on+human+trabecular+meshwork.">The effect of organ culture on human trabecular meshwork.</a> Experimental Eye Research. 49 (1), 113-127 (1989).</li><li>Johnson, D. H., Tschumper, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+trabecular+meshwork+organ+culture.+A+new+method.">Human trabecular meshwork organ culture. A new method.</a> Investigative Ophthalmology &amp; Visual Science. 28 (6), 945-953 (1987).</li><li>Snider, E. 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=Improving+stem+cell+delivery+to+the+trabecular+meshwork+using+magnetic+nanoparticles.">Improving stem cell delivery to the trabecular meshwork using magnetic nanoparticles.</a> Scientific Reports. 8 (1), 12251 (2018).</li><li>Llobet, A., Gasull, X., Gual, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+trabecular+meshwork+physiology:+a+key+to+the+control+of+intraocular+pressure.">Understanding trabecular meshwork physiology: a key to the control of intraocular pressure.</a> Physiology. 18 (5), 205-209 (2003).</li><li>Goel, M., Picciani, R. G., Lee, R. K., Bhattacharya, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aqueous+humor+dynamics:+A+review.">Aqueous humor dynamics: A review.</a> The Open Ophthalmology Journal. 4, 52-59 (2010).</li><li>Snider, E. 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=Development+of+a+porcine+organ-culture+glaucoma+model+mimicking+trabecular+meshwork+damage.">Development of a porcine organ-culture glaucoma model mimicking trabecular meshwork damage.</a> Investigative Ophthalmology &amp; Visual Science. 62 (3), 18 (2021).</li><li>Ren, H., Wilson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis+in+the+corneal+epithelium.">Apoptosis in the corneal epithelium.</a> Investigative Ophthalmology &amp; Visual Science. 37 (6), 1017-1025 (1996).</li><li>Komuro, A., Hodge, D. O., Gores, G. J., Bourne, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+death+during+corneal+storage+at+4&#176;C.">Cell death during corneal storage at 4&#176;C.</a> Investigative Ophthalmology &amp; Visual Science. 40 (12), 2827-2832 (1999).</li><li>Crespo-Moral, M., Garc&#237;a-Posadas, L., L&#243;pez-Garc&#237;a, A., Diebold, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+immunohistochemical+characterization+of+the+porcine+ocular+surface.">Histological and immunohistochemical characterization of the porcine ocular surface.</a> PLOS One. 15 (1), e0227732 (2020).</li><li>Wilson, S. E., Medeiros, C. S., Santhiago, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+corneal+scarring+in+persistent+epithelial+defects+after+prk+and+other+corneal+injuries.">Pathophysiology of corneal scarring in persistent epithelial defects after prk and other corneal injuries.</a> Journal of Refractive Surgery. 34 (1), 59-64 (2018).</li><li>Auw-Haedrich, 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=Immunohistochemical+expression+of+epithelial+cell+markers+in+corneas+with+congenital+aniridia+and+ocular+cicatrizing+pemphigoid.">Immunohistochemical expression of epithelial cell markers in corneas with congenital aniridia and ocular cicatrizing pemphigoid.</a> Acta Ophthalmologica. 89 (1), 47-53 (2011).</li><li>Lyngholm, 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=Immunohistochemical+markers+for+corneal+stem+cells+in+the+early+developing+human+eye.">Immunohistochemical markers for corneal stem cells in the early developing human eye.</a> Experimental Eye Research. 87 (2), 115-121 (2008).</li><li>Bandamwar, K. 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>

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10-32 Polycarbonate straight plug, male threaded pipe connector</td>
			<td>McMaster-Carr</td>
			<td>51525K431</td>
			<td></td>
		</tr>
		<tr>
			<td>10-32 Socket cap screw, &#189;&#34;</td>
			<td>McMaster-Carr</td>
			<td>92196A269</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringe</td>
			<td>BD</td>
			<td>302995</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td>BD</td>
			<td>302830</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Anti</td>
			<td>Gibco</td>
			<td>15240-096</td>
			<td></td>
		</tr>
		<tr>
			<td>Ball-End L key</td>
			<td>McMaster-Carr</td>
			<td>5020A25</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Fisher Scientific</td>
			<td>NC1696484</td>
			<td></td>
		</tr>
		<tr>
			<td>BD&#160;Intramedic&#160;PE 160 Tubing</td>
			<td>Fisher Scientific</td>
			<td>14-170-12E</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Puritan</td>
			<td>25-8061WC</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM media</td>
			<td>ATCC</td>
			<td>30-2002</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>ATCC</td>
			<td>30-2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>World Precision Instruments</td>
			<td>15914</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Covidien</td>
			<td>8044</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>High temperature silicone O-ring, 2 mm wide, 4 mm ID</td>
			<td>McMaster-Carr</td>
			<td>5233T47</td>
			<td></td>
		</tr>
		<tr>
			<td>Large forceps</td>
			<td>World Precision Instruments</td>
			<td>500365</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>World Precision Instruments</td>
			<td>503261</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium toothed forceps</td>
			<td>World Precision Instruments</td>
			<td>501217</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail (puncture object)</td>
			<td>McMaster-Carr</td>
			<td>97808A503</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon syringe filters</td>
			<td>Fisher</td>
			<td>09-719C</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (100 mm)</td>
			<td>Fisher</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate, three-way, stopcock with male&#160;luer&#160;lock</td>
			<td>Fisher</td>
			<td>NC9593742</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel 18 G&#160;90 degree&#160;angle dispensing needle</td>
			<td>McMaster-Carr</td>
			<td>75165A81</td>
			<td></td>
		</tr>
		<tr>
			<td>Stainless steel 18 G straight &#189;&#39;&#8217; dispensing needle</td>
			<td>McMaster-Carr</td>
			<td>75165A675</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 100 mL beakers with lids</td>
			<td>VWR</td>
			<td>15704-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas&#160;scissors</td>
			<td>World Precision Instruments</td>
			<td>WP5070</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

Open globe injuries can lead to permanent loss of vision if left untreated. This anterior segment organ culture model allows assessment of these injuries within the need for animal testing. This protocol allows tracking open globe injuries, and therapeutics, to stabilize the injury for three or more days with an organ culture platform.Demonstrating the procedure will be Trent Pearson, a research technician from Dr.Boice laboratory. To begin, lay the surgical drape and the instruments required for the gross dissection. Set up the bio safety cabinet for visualizing the eyes on the dissecting microscope.Set up the bio safety cabinet for dish assembly. Set up the open globe injury induction workspace inside the bio safety cabinet. Position the cross tracking vice on the lab jack, in front of the puncture platform.After removing the extra orbital tissues and washing the eyes with antibiotic antimycotic PBS, to hemisect the eye place the eye on the antibiotic antimycotic PBS soaked gauze. Use a sterile razorblade or scalpel to create an incision near the equator of the eye. Then hemisect the eye using curved surgical scissors to isolate the anterior eye.Use micro scissors as a shovel to scoop the vitreous humor from the anterior segment and remove the lens from the anterior segment with micro scissors. Gradually cut the iris to the iris root radially until the trabecular meshwork is visible. Extend the cut 360 degrees around the iris, to expose the entire trabecular meshwork region and clean up any remaining residual iris covering the trabecular meshwork if necessary.Trim the ciliary body remnants posterior to the trabecular meshwork region leaving only a one millimeter thin band of the tissue. After placing the anterior segment in the Petri dish. Wet a cotton swab in the media and gently dab in the center of the cornea to remove any pigment.Hold the eyes with forceps and remove extra pigment around the sclera by wiping. Place the inverted anterior segment over the elevated region of the bottom dish, ensuring that the cornea remains in the center of the elevated region. Position the clamping ring on top of the newly placed anterior segment, and gently tighten the screws with the L key.Attach both the fluidic connectors with O-rings to threaded ports on the bottom of the dish. To the first fluidic connector attach 18 gauge 90 degree bent needle hub, a length of tubing, an 18 gauge needle hub, a nylon syringe filter, a three-way valve and a 20 millimeter syringe filled with media. To the second fluidic connector attach 18 gauge 90 degree bent needle hub, a length of tubing, an 18 gauge needle hub, a three-way valve as demonstrated for the first fluid connector and then attach the barrel portion of a sterile 10 milliliter syringe, which will act as a reservoir to catch the liquid and bubbles.Keeping the three-way valves open appropriately to the syringes, gently push the media through the system, using the first fluidics connector port. To inflate the anterior segment, fill the tubing with media and eventually fill the reservoir. Remove the bubbles by gently pushing the media into the dish and invert the dish to push out the bubbles.Place the anterior segment organ culture dish into the cell culture incubator. Direct the tubing lines out through the bottom of the incubator door to prevent interference with the door opening and closing. Position the tubing lines with the reservoir at the pressure transducer instrument.Connect the side three way valve to the pressure transducer set up while PBS flows through the line to avoid the air bubbles from entering the tubing lines. Inside the prepared bio safety cabinet for the open injury induction set the pressure regulator on the puncture platform to 50 pound force per square inch for generating adequate puncture force on the objects of up to 4.5 millimeters in diameter. Position the cross tracking vice on the lab jack in front of the puncture platform.Extend the piston arm to its maximal distance and position the corneal apex within the one millimeter region of the puncturing object, retract the piston arm and bring the anterior segment one centimeter closer to the puncturing object. To fire the puncture device, turn on the device and open the solenoid valve with the second switch on the device. To retract the device from the eye, press the second switch, verify proper injury induction by visual inspection and media leaking from the injury site.After the surgery, the corneal epithelium integrity was assessed via imaging using optical coherence tomography. The injured anterior segment tissue was imaged immediately after injury and 72 hours post-injury. The controlled eyes show no noticeable disruption in the cornea while clear injuries were located through the cornea after the injury.From the top-down maximum intensity projection, it was evident that injuries were irregular in shape and size, but the injury size was decreased over 72 hours. Interocular pressure was observed for the first three days before the surgery to determine pressure stability in the acceptable range. In the representative results three out of five eyes fall within the acceptable interocular pressure range.The interocular pressure changes due to the open globe injury and therapeutic intervention. After the open globe injury induction, the pressure significantly decreased and remained low until the experiment was complete. Treatment of the injured tissues with dermabond adhesive resulted in an increased interocular pressure compared to untreated tissue, which indicates that this method can be used to measure the efficacy of the therapeutics for restoring the intraocular pressure.The initial setup of the segment in organ culture is critical for the long-term stability of the tissue. Over tightening the ring can cause stress on the tissue and reduce compliance.

Research

JoVE Journal

Bioengineering

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 ...

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  ...

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 ...

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 ...

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

Fabrication of Inverted Colloidal Crystal Polyethylene 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 ...

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 ...

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 ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

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 ...

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 ...

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 ...