Name: corneal donor tissue preparation for descemet's membrane endothelial keratoplasty
Uploaded: 2014-09-17T14:00:00.000+00:00
Duration: 10 min 46 s
Description: Watch this Scientific Journal Video about Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty at JoVE.com

1. Setup of Sterile Field
<ol>
	<li>Don proper attire including cap and mask. Wear eye protection when in the processing room.</li>
	<li>Prepare the processing area. When using a processing room, maintain continuous operation of the fan to create positive pressure in the room.</li>
	<li>Gather all supplies. Check expiration dates, package integrity, and sterilization indicator.</li>
	<li>Wash hands and don non-sterile gloves.</li>
	<li>Open the sterile kits to establish a sterile field. When using a processing room, place the kits on the table and aseptically open the wraps. Do not utilize the outer 1&#189; inches of the wrap as part of the sterile field as this segment is considered unsterile.</li>
	<li>Using aseptic technique, open and drop sterile items onto sterile field unless already part of the premade pack.</li>
	<li>Move microscope so that it is above the sterile field, turn on light source of microscope, and adjust oculars as necessary.</li>
	<li>Obtain corneal tissue to be stripped (in preservation media) and place next to the prepared sterile processing area. Loosen cap of viewing chamber and let it rest on top of viewing chamber base with no threads engaged. Discard non-sterile gloves.</li>
	<li>Open one pair of sterile gloves and sterile gown with towel.</li>
	<li>Perform surgical scrub. Put on sterile gown, then sterile gloves.</li>
	<li>Open instrument boxes so that all supplies are accessible. Open moisture impermeable drape to create the work area.</li>
	<li>Attach needle to a 5 cc syringe. Using a hemostat, prepare the 30 G needle by placing a bend 1 mm from the bevel, and another bend 1 mm from the base (to form an &#8220;S&#8221; shape).</li>
	<li>Assemble the glass syringe for the trypan blue stain by placing the plunger into the syringe and finger tightening the plunger. Attach cannula.</li>
	<li>Attach the cannula to the balanced salt solution (BSS) bottle.</li>
	<li>Separate the trephine punch from the vacuum block and set aside. Center the vacuum block within the field of view of the microscope.</li>
	<li>Bring the vacuum block into focus by adjusting the microscope settings.</li>
</ol>
2. Procedure for Donor Tissue Preparation
<ol>
	<li>Lift lid of viewing chamber using foil square as a barrier. With opposite hand and using forceps, gently grasp cornea by sclera rim to remove cornea. Replace lid of viewing chamber.</li>
	<li>Completely depress the suction syringe, then place cornea epithelial side down on the vacuum block and ensure that it is centered. (Figure 1)</li>
	<li>Gently lower seating ring on to the vacuum block using the guide posts. Engage vacuum by slowly releasing piston of the suction syringe. Remove the seating ring and set aside. (Figure 2).</li>
	<li>Perform a partial trephination to score through the Descemet&#8217;s membrane.</li>
	<li>Adjust microscope zoom and focus so that the tissue is well visualized.</li>
	<li>Tilt the vacuum block and use a sterile swab spear placed at the limbus to remove excess medium. (Figure 3)</li>
	<li>Place enough stain at the limbus to stain the edge of the scored Descemet&#8217;s membrane. Allow the stain to remain on the endothelium for 60-90 sec. (Figure 4)</li>
	<li>Remove stain:
	<ol>
		<li>Tilt the vacuum block to pour the stain off of the cornea and onto gauze pad.</li>
		<li>Use the surgical spears to remove excess stain from the limbal area of the cornea, taking care not to touch the endothelium. (Figure 5)</li>
	</ol>
	</li>
	<li>Use several drops of BSS to rinse the cornea of staining solution, and sterile swab spears to remove excess BSS. (Figure 6)</li>
	<li>Place 1-2 drops of BSS on the sclera rim to prevent tissue from drying. Repeat as necessary throughout the procedure.</li>
	<li>Perform partial dissection of Descemet&#8217;s membrane:
	<ol>
		<li>Beginning where the tubing connects to the vacuum block, using open forceps, place one tip at the edge of the score mark and gently separate Descemet&#8217;s membrane and endothelium away from the stroma. (Figure 7).</li>
		<li>Separate Descemet&#8217;s membrane and endothelium not more than 1 mm from the scored edge and continue dissection 360&#176; around. (Figure 8).</li>
		<li>Note location of any micro tears by placing a dot on the vacuum block with the skin marker. 
		NOTE: If the trephine did not penetrate completely through Descemet&#8217;s membrane, use the beveled edge of the prepared 30 G needle to complete the score through Descemet&#8217;s membrane where necessary, followed by a second application of staining as needed. If Descemet&#8217;s membrane&#8217;s adhesiveness is impeding the partial dissection, remove the endothelium outside the trephinated area as needed.</li>
	</ol>
	</li>
	<li>Use forceps to remove any endothelial tags that overlap the score mark.</li>
	<li>Consider the location of previously marked micro tears and rotate the vacuum block so that the largest is at 6 o&#8217;clock. This will become the hinge of the flap.</li>
	<li>Using the forceps, grasp the Descemet&#8217;s membrane at 12 o&#8217;clock. Gently separate the Descemet&#8217;s membrane from the stroma by peeling towards the hinge. Create the hinge by stopping the separation 2 mm from the score mark. 
	NOTE: Peripheral edges of flap will begin to scroll during separation. (Figure 9).</li>
	<li>Gently lay the flap back in place on the stroma. Use BSS and swab spears to unscroll the endothelium and return it to its original position. (Figure 10A-C)</li>
	<li>Remove residual fluid from between Descemet&#8217;s membrane and stroma with sterile swab spears so the flap stays in position. (Figure 11)</li>
	<li>Use sterile swab spears to soak up any excess moisture from the sclera near the hinge. Use the skin marker to draw an arrowhead on the sclera rim pointing to the center of the hinge, remove any excess ink using a sterile swab spear. (Figure 12)</li>
	<li>Release vacuum by depressing the piston of the syringe connected to the vacuum block. While vacuum is disengaged, use forceps to remove cornea from the vacuum block.</li>
	<li>Lift lid of viewing chamber using foil square as a barrier. Place the cornea in the viewing chamber, endothelial side up, and replace lid of viewing chamber.</li>
	<li>Repeat above procedure steps with mate cornea, if applicable. Use gauze to clean the vacuum block before processing the second tissue.</li>
	<li>Tighten the viewing chamber lid(s) and remove tissue from the processing area.</li>
	<li>Break down the sterile field by disposing of items according to Biohazardous Waste Management Plan. Remove contaminated gloves, and gown. Wash hands and don non-sterile gloves.</li>
	<li>Perform post-cut slit lamp evaluation and specular microscopy of the cornea(s) and record the findings.</li>
</ol>

<ol>
	<li>Fernandez, M., Afshari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+From+DLEK+to+DMEK.">Endothelial Keratoplasty: From DLEK to DMEK.</a> Middle East African Journal of Ophthalmology. 17 (1), 5-8 (2010).</li><li>Kruse, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Stepwise+Approach+to+Donor+Preparation+and+Insertion+Increases+Safety+and+Outcome+of+Descemet+Membrane+Endothelial+Keratoplasty.">A Stepwise Approach to Donor Preparation and Insertion Increases Safety and Outcome of Descemet Membrane Endothelial Keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Dapena, I., Moutsouris, K., Droutsas, K., Ham, L., van Dijk, K., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+'No-Touch'+Technique+for+Descemet+Membrane+Endothelial+Keratoplasty.">Standardized 'No-Touch' Technique for Descemet Membrane Endothelial Keratoplasty.</a> Archives of Ophthalmology. 129, 88-94 (2011).</li><li>Tourtas, T., Laaser, K., Bachmann, B., Cursiefen, C., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+Endothelial+Keratoplasty+versus+Descemet+Stripping+Automated+Endothelial+Keratoplasty.">Descemet Membrane Endothelial Keratoplasty versus Descemet Stripping Automated Endothelial Keratoplasty.</a> American Journal of Ophthalmology. 153 (6), 1082-1090 (2012).</li><li>Guerra, F., Anshu, A., Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+fellow+eyes+comparison+of+Descemet+stripping+automated+endothelial+keratoplasty+and+Descemet+membrane+endothelial+keratoplasty.">Endothelial Keratoplasty: fellow eyes comparison of Descemet stripping automated endothelial keratoplasty and Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (12), 1382-1386 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2012 Eye Banking Statistical Report: Surgical Use and Indications for Corneal Transplant Statistical Report Analysis. , (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+stripping+endothelial+keratoplasty.">Descemet's stripping endothelial keratoplasty.</a> Current Opinion Ophthalmology. 18 (4), 290-294 (2007).</li><li>Woodward, M., Titus, M., Mavin, K., Shtein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Donor+Tissue+Preparation+for+Endothelial+Keratoplasty.">Corneal Donor Tissue Preparation for Endothelial Keratoplasty.</a> Journal of Visualized Experiments. (64), e3847 (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+membrane+endothelial+keratoplasty+surgery:+update+on+the+evidence+and+hurdles+to+acceptance.">Descemet's membrane endothelial keratoplasty surgery: update on the evidence and hurdles to acceptance.</a> Current Opinion Ophthalmology. 24 (4), 329-335 (2013).</li><li>Venzano, D., Pagani, P., Randazzo, N., Cabiddu, F., Traverrso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+air-bubble+separation+in+donor+cornea.">Descemet Membrane air-bubble separation in donor cornea.</a> Journal of Cataract and Refractive Surgery. 36 (12), 2022-2027 (2010).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DSEK: What you need to know about endothelial keratoplasty. , (2009).</li><li>Studeny, P., Sivekova, D., Lieheova, K., Vokrojova, M., Kuchynka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+technique+of+lamellar+keratoplasty+(DMEK-S).">Hybrid technique of lamellar keratoplasty (DMEK-S).</a> Journal of Ophthalmology. 2013, 2013.254383 (2013).</li><li>Muraine, M., Gueudry, J., He, Z., Piselli, S., Lefevre, S., Toubeau, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+technique+for+the+preparation+of+corneal+grafts+for+Descemet+membrane+endothelial+keratoplasty.">Novel technique for the preparation of corneal grafts for Descemet membrane endothelial keratoplasty.</a> American Journal of Ophthalmology. 156 (5), 851-859 (2013).</li><li>Lie, J. T., Birbal, R., Ham, L., van der Wees, J., Melles, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+tissue+preparation+for+Descemet+membrane+endothelial+keratoplasty.">Donor tissue preparation for Descemet membrane endothelial keratoplasty.</a> Journal of Cataract Refractive Surgery. 34 (9), 1578-1583 (2008).</li><li>Kruse, F. E., Laaser, K., Cursiefen, C., Heindl, L. M., Schlotzer-Schrehardt, U., Riss, S., Bachmann, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+approach+to+donor+preparation+and+insertion+increases+safety+and+outcome+of+Descemet+membrane+endothelial+keratoplasty.">A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Rudolph, M., Kaaser, L., Bachmann, B., Cursiefen, C., Epstein, D., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+higher-order+aberrations+after+Descemet&#8217;s+membrane+endothelial+keratoplasty.">Corneal higher-order aberrations after Descemet&#8217;s membrane endothelial keratoplasty.</a> Ophthalmology. 119 (3), 528-535 (2012).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+endothelial+keratoplasty.">Evolution of endothelial keratoplasty.</a> Cornea. 32, 28-32 (2013).</li><li>Dapena, I., Dapena, L., Dirisamer, M., Ham, L., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+and+endothelial+cell+density+following+Descemet+membrane+endothelial+keratoplasty+(DMEK).">Visual acuity and endothelial cell density following Descemet membrane endothelial keratoplasty (DMEK).</a> Arch Soc Esp Oftalmol. 86 (12), 395-401 (2011).</li></ol>

Hassan Tausif and Dr. Navasuja Chandrasekaran have nothing to disclose. Drs. Mian, Shtein, and Woodward are medical directors for the Michigan Eye Bank. Mr. Mavin, Mr. Titus, and Ms. Johnson are employees of Midwest Eye Banks.

The corneal endothelial cell layer plays a critical role in maintaining corneal clarity through dehydration. Diseases of the endothelial cells can result in significant visual impairment. DMEK aims to replace the diseased endothelial cells with a donor graft consisting of Descemet&#8217;s membrane and endothelial cell layer. Donor tissue preparation with an intact DM and adequate endothelial cell count are critical to the survival of the graft tissue. Tearing of DM during donor tissue preparation can result in tissue loss and wastage of donor tissue. Widespread application of DMEK has been limited by the challenges in preparing the graft tissue and mastering the complex surgical technique. With improvements in instrumentation and availability of eye-bank prepared precut tissue, this procedure has the potential to become the standard treatment for corneal endothelial diseases.
Current techniques for donor tissue preparation include use of an air-bubble to separate the DM from the stroma to facilitate the removal of graft tissue from donor cornea10. SCUBA (submerged cornea using backgrounds away) technique of DMEK graft preparation involves harvesting the donor tissue under Optisol or balanced salt solution (BSS), minimizing the surface tension and chances of tearing11. Yet another technique in DMEK graft preparation is leaving a peripheral rim of stroma yielding a graft that is easier to the handle and insert12. The donor tissue may also be prepared with a custom trephine to leave hinges of intact DM while lifting the graft with BSS13.
The overall success rate with our technique for DMEK graft preparation was limited compared to previously published techniques to 76% which may reflect that these were the first 50 tissues being prepared by two eye bank technicians14,15. However, the outcomes did improve in the second half of tissue prepared. In addition, our technique does not require specialized instruments, making DMEK easily accessible as well as more cost effective. The visual recovery can be faster and better with DMEK due to lack of the stromal interface5. Studies have also demonstrated fewer higher order aberrations on the posterior surface of the cornea than DSEK or PKP16. Incidence of graft rejection is significantly lower with DMEK than with other keratoplasty techniques17. The endothelial cell density following DMEK is comparable to Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK)18. DMEK offers a minimally invasive surgery maintaining the structural integrity of the eye and reducing the risk of postoperative astigmatism, wound leaks and postoperative glaucoma linked to prolonged topical steroid use.
In summary, DMEK allows replacement of diseased endothelium without removing recipient corneal stroma or additional tissue. The surgical technique is challenging in part due to the reproducibility of donor tissue preparation. Eye bank preparation for DMEK may allow for improved adaptation of DMEK similar to previous endothelial keratoplasty techniques.

Endothelial Keratoplasty (EK) has rapidly replaced penetrating keratoplasty (PKP) as the procedure of choice for treatment of endothelial diseases accounting for over 50% of all transplants performed in the United States in 20126. PKP is limited by prolonged visual rehabilitation with high astigmatism and refractive instability, loss of globe integrity and suture-related complications. Early generation EK procedures, including Deep Lamellar Endothelial Keratoplasty (DLEK) involved manual dissection of corneal stroma with transplantation of partial thickness donor tissue. These procedures were successful in transplanting donor endothelial tissue while maintaining an intact corneal surface but were technically challenging and resulted in limited best corrected visual acuity due to an irregular corneal lamellar interface1. The current EK procedure of choice is Descemet&#8217;s Stripping Endothelial Keratoplasty (DSEK) which offers corneal clarity with faster recovery of vision, minimizes postoperative refractive error and astigmatism, and decreases risk of traumatic wound dehiscence while maintaining the corneal surface7. This procedure involves removal of the diseased Descemet&#39;s membrane and endothelium and replacement with partial thickness donor corneal tissue comprising a new endothelial cell layer. The donor cornea is dissected with a microkeratome to prepare the posterior tissue being transplanted8. Preparation of the donor tissue by the eye-banks has been instrumental in the rapid growth and, arguably, the success of this procedure. The near 16-fold increase in DSEK procedures in the United States between 2005 and 2012 corresponds with almost half of the tissue currently being prepared by the eye-banks6.
Although DSEK can improve visual acuity to 20/40 or better, most patients do not achieve 20/20 vision due to stromal interface haze and increased light scatter from the addition of donor stromal tissue7. The EK surgical technique has progressed to Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) which involves transplantation of only donor Descemet&#39;s membrane (DM) and endothelium. This procedure has a higher proportion of patients who achieve 20/20 vision, faster visual recovery, and a lower risk of endothelial rejection9. However, preparation of donor tissue is more challenging due to the thinner graft dimensions.

<table><tbody><tr><td>VisionBlue</td><td>D.O.R.C., Zuiland, The Netherlands</td><td>NA</td><td></td></tr><tr><td>Barron vacuum punch</td><td>Katena, Denville, New Jersey</td><td>K20-2112</td><td></td></tr></tbody></table>

corneal donor tissue preparation for descemet's membrane endothelial keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Proper separation of DM and endothelium from stroma is essential in preparing precut tissue for DMEK. Fifty donor corneas with a mean age of 65 &#177; 6 years (range 55-75), death to processing time of 5.9 &#177; 2.3 days (range 1-10) and endothelial cell density (ECD) of 2,616 &#177; 321 cells/mm2 (range 2,049-3,247) were used to assess feasibility and reproducibility of the surgical preparation technique by two eye bank technicians. The success rate was 72% in the first 25 cases and increased to 80% in the second half with an overall success rate of 76%. Point biserial correlation (Rpb) was used to determine correlation with preoperative tissue factors. The donor age (Rpb = 0.18), death to processing time (Rpb = 0.07) and ECD (Rpb = -0.11) did not correlate with successful preparation of tissue. The post-preparation ECD (n = 37) of 2,676 &#177; 284 (range 2,041-3,205) was not significantly different from the pre-preparation ECD (p = 0.72). Unsuccessful outcomes resulted from DM tears (10%), and severe cell loss (14%). These cases were also associated with difficulty remounting tissue and incomplete staining of DM edge.
Eye-bank assessment of patient outcomes provides essential data for clinical success and adverse events associated with DMEK which may help improve the adaptation of the surgical technique in practice and utilization of eye-bank prepared tissue. Nine pre-cut DMEK tissue were provided to five surgeons for 9 patients. One case had a dislocation at 1 day and 1 week without need for rebubbling. Visual acuity at 1 day ranged from 20/400 to light perception and by 1 week improved to 20/103 (range 20/30-20/200).
<img alt="Figure 1" src="/files/ftp_upload/51919/51919fig1highres.jpg" /> 
Figure 1. Tissue mounting. The corneal tissue is mounted and centered on the trephination block.
<img alt="Figure 2" src="/files/ftp_upload/51919/51919fig2highres.jpg" /> 
Figure 2. Tissue trephination. The vacuum ring is seated and the corneal tissue is partially trephined including Descemet&#8217;s membrane.
<img alt="Figure 3" src="/files/ftp_upload/51919/51919fig3highres.jpg" /> 
Figure 3. Storage media removal. Excess storage media is removed prior to stain application.
<img alt="Figure 4" src="/files/ftp_upload/51919/51919fig4highres.jpg" /> 
Figure 4. Trypan blue application. Apply trypan blue stain on Descemet&#8217;s membrane surface with filling of the well for 60-90 sec.
<img alt="Figure 5" src="/files/ftp_upload/51919/51919fig5highres.jpg" /> 
Figure 5. Stain removal. Rinse trypan blue with BSS.
<img alt="Figure 6" src="/files/ftp_upload/51919/51919fig6highres.jpg" /> 
Figure 6. Stain removal. Complete removal of stain.
<img alt="Figure 7" src="/files/ftp_upload/51919/51919fig7highres.jpg" /> 
Figure 7. Descemet&#8217;s membrane dissection. Tying forceps are used to perform 360&#176; partial dissection.
<img alt="Figure 8" src="/files/ftp_upload/51919/51919fig8highres.jpg" /> 
Figure 8. Descemet&#8217;s membrane stripping. Begin to strip Descemet&#8217;s membrane.
<img alt="Figure 9" src="/files/ftp_upload/51919/51919fig9highres.jpg" /> 
Figure 9. Descemet&#8217;s membrane stripping. Continue to strip Descemet&#8217;s membrane.
<img alt="Figure 10" src="/files/ftp_upload/51919/51919fig10highres.jpg" /> 
Figure 10. Reposition Descemet&#8217;s membrane. (A) Return Descemet&#8217;s membrane to the stromal bed, (B) BSS application to aid in Descemet&#8217;s membrane unfurling, and (C) complete unfurling of Descemet&#8217;s membrane to original position.
<img alt="Figure 11" src="/files/ftp_upload/51919/51919fig11highres.jpg" /> 
Figure 11. Balanced salt solution removal. Removal of extra BSS from beneath the flap.
<img alt="Figure 12" src="/files/ftp_upload/51919/51919fig12highres.jpg" /> 
Figure 12. Hinge mark. Identification of hinge with skin marker.

Watch this Scientific Journal Video about Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty at JoVE.com

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

15.2K Views.  University of Michigan. Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet's membranes from donor corneoscleral buttons for transplantation2.

Video: Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Developmental Biology

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the retrieval of ocular tissues, especially cornea, by cutting it separate from the ocular globe. Evisceration is the process of removing the internal organs referred here as retina. The ocular globe consists of the cornea, the sclera, the vitreous body, the lens, the iris, the retina, the choroid, muscles etc (Suppl. Figure 1). When a patient is suffering from corneal damage, the cornea needs to be removed and a healthy one must be transplanted by keratoplastic surgeries. Genetic disorders or defects in retinal function can compromise vision. Human ocular globes can be used for various surgical procedures such as eye banking, transplantation of human cornea or sclera and research on ocular tissues. However, there is little information available on human corneal and retinal excision, probably due to the limited accessibility to human tissues. Most of the studies describing similar procedures are performed on animal models. Research scientists rely on the availability of properly dissected and well-conserved ocular tissues in order to extend the knowledge on human eye development, homeostasis and function. As we receive high amount of ocular globes out of which approximately 40% (Table 1) of them are used for research purposes, we are able to perform huge amount of experiments on these tissues, defining techniques to excise and preserve them regularly.
The cornea is an avascular tissue which enables the transmission of light onto the retina and for this purpose should always maintain a good degree of transparency. Within the cornea, the limbus region, which is a reservoir of the stem cells, helps the reconstruction of epithelial cells and restricts the overgrowth of the conjunctiva maintaining corneal transparency and clarity. The size and thickness of the cornea are critical for clear vision, as changes in either of them could lead to distracted, unclear vision. The cornea comprises of 5 layers; a) epithelium, b) Bowman's layer, c) stroma, d) Descemet's membrane and e) endothelium. All layers should function properly to ensure clear vision4,5,6. The choroid is the intermediate tunic between the sclera and retina, bounded on the interior by the Bruch's membrane and is responsible for blood flow in the eye. The choroid also helps to regulate the temperature and supplies nourishment to the outer layers of the retina5,6. The retina is a layer of nervous tissue that covers the back of the ocular globe (Suppl. Figure 1) and consists of two parts: a photoreceptive part and a non-receptive part. The retina helps to receive the light from the cornea and lens and converts it into the chemical energy eventually transmitted to the brain with help of the optic nerve5,6.
The aim of this paper is to provide a protocol for the dissection of corneal and retinal tissues from human ocular globes. Avoiding cross-contamination with adjacent tissues and preserving RNA integrity is of fundamental importance as such tissues are indispensable for research purposes aimed at (i) characterizing the transcriptome of the ocular tissues, (ii) isolating stem cells for regenerative medicine projects, and (iii) evaluating histological differences between tissues from normal/affected subjects. In this paper we describe the technique we currently use to remove the cornea, the choroid and retinal tissues from an ocular globe. Here we provide a detailed protocol for the dissection of the human ocular globe and the excision of corneal and retinal tissues. The accompanying video will help researchers to learn an appropriate technique for the retrieval of precious human tissues which are difficult to find regularly.

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

The paper describes a simplified technique to excise corneal and to eviscerate retinal tissues from the ocular globe of human cadaveric donors. The technique described here will help to excise good quality tissues to be used for transplantation, surgical or research purposes without damaging other tissues of the ocular globe.

Enucleation is the process of retrieving the ocular globe from a cadaveric donor leaving the rest of the globe undisturbed. Excision refers to the ...

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

Immunology and Infection

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

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Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

A Simplified Technique for In situ Excision of Cornea and Evisceration of Retinal Tissue from Human Ocular Globe

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

An “All-laser” Endothelial Transplant

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss