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>
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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.

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

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.

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

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 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 ...