The authors wish to thank FORTE Project, funded by Tuscany Region (POR CReO FESR 2007-2013, Bando Unico R&#38;S 2012), the EU FP7 ECHORD++ Experiment LA-ROSES that partially supported the research activities, and the FP7 BiophotonicPlus Project &#8220;LITE&#8221; granted by Tuscany Region.

The study was conducted with the prospectively approval of the hospital&#8217;s Ethics Committee; informed consent was obtained. The study was in adherence to the tenets of the Declaration of Helsinki.
1. Donor Endothelium Preparation
<ol>
	<li>Use a donor cornea as prepared by the local eye bank at room temperature.</li>
	<li>In the surgery room, pull out the donor cornea from its delivery container and set apart the solution for tissue preservation and nutrition that is used for corneal transport.</li>
	<li>Place the donor corneo-scleral rim over an artificial anterior chamber, into which a liquid can flow and create a variable pressure; cover the cornea with the tissue retaining head.</li>
	<li>Connect the artificial anterior chamber (AAC) through a 3-way connector to a syringe filled with the cornea preservation and nutrition liquid.</li>
	<li>Use the compression ring to hold the retainer securely in place.</li>
	<li>Use the syringe connected to the AAC to maintain the intracameral pressure: fill the anterior chamber with the tissue preservation solution until reaching the optimal pressure (see 1.7). Close the connector.</li>
	<li>Test the pressure inside the anterior chamber with a finger. Change the inside pressure until reaching the right internal pressure (in the range 60&#8211;70 mmHg). Close the 3-way connector.</li>
	<li>Measure the thickness of the donor cornea with Optical Coherence Thomography (OCT). Maintain the anterior chamber with that cornea in a fixed position in front of the instrument optics and then take a full thickness-OCT acquisition.</li>
</ol>
2. Femtosecond Laser Preparation of Donor Endothelium
<ol>
	<li>Perform applanation of the donor cornea under the femtosecond laser device. Use the femtosec laser to cut the donor tissue with three subsequent cuts: posterior side cut, full lamellar and anterior side cut. Set the following parameters for the three cuts.
	<ol>
		<li>For the full lamellar cut (raster pattern, start out), set the cut depth corresponding to the thinnest point of the donor cornea (measured with the OCT as described in 1.8), always subtracting 95 &#181;m. Set the pulse energy in the range 0.8-0.9 &#181;J (depending on the working depth). Set the diameter to 8.7 mm. Set the tangential spot separation to 2 &#181;m. Set the radial spot separation to 2 &#181;m.</li>
		<li>For the anterior side cut, set the posterior depth 30 &#181;m deeper than the previous full lamellar cut. Set the pulse energy to 2.10 &#181;J; the diameter to 8.6 mm; the spot separation to 3 &#181;m, while the layer separation to 3 &#181;m.</li>
		<li>For the posterior side cut, locate the anterior depth of the posterior side cut 30 &#181;m anteriorly than full lamellar cut. Set the posterior depth to 900 &#181;m. Set the pulse energy to 2.10 &#181;J; the diameter to 8.3 mm; the spot separation to 2 &#181;m; the layer separation to 2 &#181;m.</li>
	</ol>
	</li>
</ol>
3. Recipient Eye Preparation
<ol>
	<li>Prepare the patient for surgery. Make one 4.00 mm corneal incision at 12 o&#39;clock with a 4.0 mm precalibrated blade. Make one limbal paracentesis with a 1.2 mm precalibrated blade, placed at 2 o&#39;clock. Make another one limbal paracentesis at 6 o&#39;clock with a 30&#176; stab knife.</li>
	<li>Insert an anterior chamber maintainer in the patient&#8217;s anterior chamber, through the 2 o&#39;clock paracentesis.</li>
	<li>Perform one 8.2 mm diameter circular descemetorexis with the dedicated hook.</li>
	<li>Strip the Descemet membrane and endothelium from the posterior stroma and remove the tissues with a Descemet hook.</li>
</ol>
4. Chromophore Preparation
<ol>
	<li>Put 1 mg of Indocyanine Green powder in a 1.5 ml microcentrifuge tube.</li>
	<li>Add 9 mg of sterile water (the ICG water solution is 10% w/w).</li>
	<li>Manually mix the ICG powder and the water with a metal stirrer.</li>
</ol>
5. Staining of Donor Endothelium
<ol>
	<li>Put the donor endothelium onto the wider part of a Busin-injector, with the inner side in contact with the injector surface.</li>
	<li>Stain the inner side of donor endothelium with the chromophore solution, in its peripheral part, using a spatula. The correctly stained tissue has a homogeneous greenish color. Wait 3 min before starting step 5.3.</li>
	<li>Pull the donor endothelium on the anterior part of the Busin-injector, rolled.</li>
</ol>
6. Inserting the Donor Endothelium
<ol>
	<li>Grasp and insert the folded donor endothelium using an atraumatic coaxial forceps for endothelial lenticule.</li>
	<li>Remove the anterior chamber maintainer.</li>
	<li>Suture the corneal incision and the paracentesis (performed in step 3.1) with a Nylon 10.0 single stich.</li>
	<li>Inject an air bubble to unfold the donor tissue and to press it against the recipient cornea. The air bubble must completely fill the anterior chamber space.</li>
</ol>
7. Laser Welding
<ol>
	<li>Place the donor lenticule in the center of the recipient cornea, from inside with a hook or moving the air-bubble from the outside of the cornea with a spatula.</li>
	<li>Use a diode laser emitting at 810 nm, equipped with a 300 &#181;m core diameter sterile fiber optic, with 0.22 as a numerical aperture (NA).</li>
	<li>Keep the fiber tip outside the eyeball, and deliver the laser light towards the stained endothelium, through the transparent corneal tissue. The fiber tip is in non-contact configuration.</li>
	<li>Use the following settings for the laser: single spot emission mode, 70&#8211;80 msec pulse duration, 35&#8211;40 mJ per pulse. He-Ne aiming beam on.</li>
	<li>Deliver single laser spots at the stained periphery of the donor endothelium. Deliver the spots sequentially: the final aspect is a ring of spots in the periphery of the donor lenticule. The distance between two adjacent spots center is the double of a spot diameter.</li>
	<li>Apply a contact lens on the patient eye, along with 0.3% tobramycin and 0.1% dexamethasone ophthalmic suspension.</li>
</ol>

<ol>
	<li>Tan, D. T., Dart, J. K., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> Lancet. 379 (9827), 1749-1761 (2012).</li><li>El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmol Ther. , (2014).</li><li>Rossi, 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=Laser+tissue+welding+in+ophthalmic+surgery.">Laser tissue welding in ophthalmic surgery.</a> J Biophotonics. 1 (4), 331-342 (2008).</li><li>Pini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+femtosecond+laser+ablation+and+diode+laser+welding+in+lamellar+and+endothelial+corneal+transplants.">Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants.</a> Proc. SPIE Ophthalmic Technologies XVIII. 6 (844), 684411-1-684411-7 (2008).</li><li>Rossi, 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=All-laser'+endothelial+corneal+transplant+in+human+patients.">All-laser' endothelial corneal transplant in human patients.</a> Proc. SPIE Ophthalmic Technologies XXII. 8209, 82091O-1-82091O-3 (2012).</li><li>Rossi, F., Pini, R., Menabuoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+model+analysis+on+the+temperature+dynamics+during+diode+laser+welding+of+the+cornea.">Experimental and model analysis on the temperature dynamics during diode laser welding of the cornea.</a> J Biomed Opt. 12 (1), 014031-1-014031-7 (2007).</li><li>Menabuoni, L., 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=Laser-assisted+corneal+welding+in+cataract+surgery:+retrospective+study.">Laser-assisted corneal welding in cataract surgery: retrospective study.</a> J Cataract Refract Surg. 33 (9), 1608-1612 (2007).</li><li>Buzzonetti, L., 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=Laser+Welding+in+Penetrating+Keratoplasty+and+Cataract+Surgery+in+Pediatric+Patients.+Early+Results.">Laser Welding in Penetrating Keratoplasty and Cataract Surgery in Pediatric Patients. Early Results.</a> J Cataract Refract Surg. 39 (12), 1829-1834 (2013).</li><li>Menabuoni, L., 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+'anvil'+profile+in+femtosecond+laser-assistedpenetrating+keratoplasty.">The 'anvil' profile in femtosecond laser-assistedpenetrating keratoplasty.</a> Acta Ophthalmol. 91 (6), e494-e495 (2013).</li><li>Canovetti, 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=Laser-assisted+penetrating+keratoplasty:+one+year&#8217;s+results+in+patients,+using+a+laser-welded+&#8220;anvil&#8221;-profiled+graft.">Laser-assisted penetrating keratoplasty: one year&#8217;s results in patients, using a laser-welded &#8220;anvil&#8221;-profiled graft.</a> Am J Ophthalmol. 158 (4), 664-670 (2014).</li></ol>

The authors declare that they have no competing financial interests.

The &#8220;all laser&#8221; endothelial transplant is an original approach to minimally invasive corneal transplantation.
All the procedures described within the protocol were performed in the surgery room, observing the hygienic and sterilization procedure that are common practices during surgeries, such as the use of sterilized gloves, gown, mask and cap. The ICG solution was prepared in the surgery room, soon before its application in staining the donor endothelium. The ICG powder, the wate...

In this work we present an original approach to endothelial keratoplasty, based on the use of a femtosecond laser to prepare donor tissue and a near infrared diode laser to weld it onto the recipient bed. Intraoperative measurement of the donor cornea is necessary to correctly design the donor tissue dimensions. Endothelial keratoplasty has been proposed in the recent years to replace penetrating keratoplasty in treating endothelial disease1,2. The main advantage of this technique is a faster visual recovery, with respect to penetrating keratoplasty, reduced anesthesia during surgery, a decreased risk of graft rejection and the preservation of eye integrity. The main risk factor is postoperative donor lenticule dislocation. The standard technique is performed by inserting the donor endothelium in its final position where it is maintained by the injection of an air bubble: no sutures are used because of the mechanical, biophysical and dimensional characteristics of the endothelium. Moreover, visual acuity recovery can be limited mainly because of a mismatch between donor and recipient tissues due to a thick tissue transplanted.
Here we present a procedure in performing endothelial keratoplasty that can overcome those main problems. The donor endothelium can be assured in its final position by the use of the laser welding technique. This is a controlled and localized photothermal process: it can be induced at the donor/recipient interface. It has been studied in the last ten years and proposed in penetrating keratoplasty and in the transplant of endothelium3-5. The near infrared light (wavelength: 810 nm) emitted by a low power diode laser is delivered towards the biological tissue at the wound site. The cornea is naturally transparent to this wavelength: in order to make this tissue to absorb the laser light, it is necessary to stain it with a chromophore. The proposed dye is a sterile saturated water solution of Indocyanine Green (ICG). We demonstrated that when corneal tissue is properly stained with this ICG preparation, it shows an absorption peak at 810 nm6. Moreover, ICG is widely used in clinical diagnostics and its safety has been already demonstrated in human subjects. The stained cornea absorbs the diode laser light energy and the main resulting effect is a controlled temperature rise at the welding site. No thermal effects are induced in the unstained tissues. The temperature enhancement induces reversible thermal denaturation in the stromal collagen, with an immediate closuring of the wound walls upon cooling. This laser welding effect was firstly demonstrated in cataract surgery7,8 and penetrating keratoplasty9,10. An optimized approach that we are presenting in this paper has been studied for application in endothelial keratoplasty.
In the proposed surgery, single laser spots (lasting tens of msec) are delivered to the tissue, resulting in a photothermal effect localized within the spot dimension (a few hundreds of &#181;m in diameter): the induced effect is a hard laser welding, consisting of a photocoagulation of the collagen confined at the donor/host interface. The result of the collagen denaturation at the welded site is a strong adhesion between the donor and host tissues, thus providing a suturing effect that is impossible to obtain with standard technique (stitches). The tissue regains his natural appearance in a short follow up (1 month) and the adhesion between donor/host tissues is improved by the welding provided in the very early stage of the healing phase.
To avoid the other main risk of the endothelial keratoplasty, i.e. the transplant of a thick donor tissue, intrasurgical optical coherence tomography (OCT) is used: a commercial device measures the thickness of donor cornea, so that a correct cut profile can be designed with the femtosec laser. The proposed &#8220;all laser&#8221; endothelial transplant thus seems to improve the clinical results of this minimally invasive surgery.

<table><tbody><tr><td>Indocyanine Green</td><td>Pulsion Medical Systems, Germany</td><td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td><td>Alternative product: IC-GREEN, Akorn&amp;nbsp; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td></tr><tr><td>Femtosecond Laser&amp;nbsp;</td><td>Abbott Medical Optics,&amp;nbsp; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td><td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td><td></td></tr><tr><td>Optical Coherence Tomography (OCT)</td><td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td><td>Visante</td><td></td></tr><tr><td>Diode Laser</td><td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td><td>Mod. WELD 800</td><td></td></tr><tr><td>Artificial Anterior Chamber</td><td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td><td>Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_&lt;br /&gt;anterior_chamber.html)</td><td></td></tr><tr><td>Solution for tissue preservation and nutrition&amp;nbsp;&amp;nbsp;</td><td>AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&amp;ograve; - PD&lt;br/&gt; &amp;nbsp;ITALY</td><td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&amp;deg;-c-eb/carry-c-eb)</td><td></td></tr></tbody></table>

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.

The &#8220;all laser&#8221; surgical procedure is proposed to perform minimally invasive corneal transplantation. The procedure is easy to perform (see Figure 1): with respect to a standard endothelial transplant only the steps of measuring the corneal thickness, staining the donor tissue and delivering the laser light are added. The achieved advantages largely compensate an increased surgical time of a few min. The use of intraoperative OCT to measure the donor cornea thickness and the femtosecond laser...

Watch this Scientific Journal Video about An Ã¢â‚¬Å“All-laserÃ¢â‚¬Â Endothelial Transplant at JoVE.com

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

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 ...

Research

JoVE Journal

Medicine

9.5K Views.  Italian National Research Council. 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.

Video: An “All-laser” Endothelial Transplant

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk of thromboemboli formation1,2,3. We have developed a method to line the inside surface of Ti tubes with autologous blood-derived human or porcine endothelial progenitor cells (EPCs)4. By implanting Ti tubes containing a confluent layer of porcine EPCs in the inferior vena cava (IVC) of pigs, we tested the improved biocompatibility of the cell-seeded surface in the prothrombotic environment of a large animal model and compared it to unmodified bare metal surfaces5,6,7 (Figure 1). This method can be used to endothelialize devices within minutes of implantation and test their antithrombotic function in vivo. 
Peripheral blood was obtained from 50 kg Yorkshire swine and its mononuclear cell fraction cultured to isolate EPCs4,8. Ti tubes (9.4 mm ID) were pre-cut into three 4.5 cm longitudinal sections and reassembled with heat-shrink tubing. A seeding device was built, which allows for slow rotation of the Ti tubes. 
We performed a laparotomy on the pigs and externalized the intestine and urinary bladder. Sharp and blunt dissection was used to skeletonize the IVC from its bifurcation distal to the right renal artery proximal. The Ti tubes were then filled with fluorescently-labeled autologous EPC suspension and rotated at 10 RPH x 30 min to achieve uniform cell-coating9. After administration of 100 USP/ kg heparin, both ends of the IVC and a lumbar vein were clamped. A 4 cm veinotomy was performed and the device inserted and filled with phosphate-buffered saline. As the veinotomy was closed with a 4-0 Prolene running suture, one clamp was removed to de-air the IVC. At the end of the procedure, the fascia was approximated with 0-PDS (polydioxanone suture), the subcutaneous space closed with 2-0 Vicryl and the skin stapled closed. 
After 3 - 21 days, pigs were euthanized, the device explanted en-block and fixed. The Ti tubes were disassembled and the inner surfaces imaged with a fluorescent microscope.
We found that the bare metal Ti tubes fully occluded whereas the EPC-seeded tubes remained patent. Further, we were able to demonstrate a confluent layer of EPCs on the inside blood-contacting surface.
Concluding, our technology can be used to endothelialize Ti tubes within minutes of implantation with autologous EPCs to prevent thrombosis of the device. Our surgical method allows for testing the improved biocompatibility of such modified devices with minimal blood loss and EPC-seeded surface disruption.

A method for seeding titanium blood-contacting biomaterials with autologous cells and testing biocompatibility is described. This method uses endothelial progenitor cells and titanium tubes, seeded within minutes of surgical implantation into porcine venae cavae. This technique is adaptable to many other implantable biomedical devices.

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

Bioengineering

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 ...

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This pathology causes symptoms such as intermittent claudication (pain with walking), painful ischemic ulcerations, or even limb-threatening gangrene. It is generally believed that the vascular endothelium, a monolayer of endothelial cells that invests the luminal surface of all blood and lymphatic vessels, plays a dominant role in vascular homeostasis and vascular regeneration. As a result, stem cell-based regeneration of the endothelium may be a promising approach for treating PAD.In this video, we demonstrate the transplantation of embryonic stem cell (ESC)-derived endothelial cells for treatment of unilateral hindimb ischemia as a model of PAD, followed by non-invasive tracking of cell homing and survival by bioluminescence imaging. The specific materials and procedures for cell delivery and imaging will be described. This protocol follows another publication in describing the induction of hindlimb ischemia by Niiyama et al.1

The surgical procedure for delivery of embryonic stem cell-derived endothelial cells to the ischemic hindlimb is demonstrated, with non-invasive tracking by bioluminescence imaging.

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

Biology

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

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.

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, ...

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. As an alternative, an increasing number of these patients are choosing to cryopreserve ovarian tissue for autograft following recovery and remission. Despite improvements in outcomes among patients undergoing auto-transplantation of cryopreserved ovarian tissue, efficient revascularization of grafted tissue remains a major obstacle. To mitigate ischemia and thus improve outcomes in patients undergoing auto-transplantation, we developed a vascular cell-based strategy for accelerating perfusion of ovarian tissue. We describe a method for co-transplantation of exogenous endothelial cells (ExECs) with cryopreserved ovarian tissue in a mouse xenograft model. We extend this approach to employ ExECs that have been engineered to constitutively express Anti-Mullerian hormone (AMH), thus enabling sustained paracrine signaling input to ovarian grafts. Co-transplantation with ExECs increased follicular volume and improved antral follicle development, and AMH-expressing ExECs promoted retention of quiescent primordial follicles. This combined strategy may be a useful tool for mitigating ischemia and modulating follicular activation in the context of fertility preservation and/or infertility at large.

For some patients, the only option for fertility preservation is cryopreservation of ovarian tissue. Unfortunately, delayed revascularization undermines follicular viability. Here, we present a protocol to co-transplant human ovarian tissue with endothelial cells for utilization as a cell-based strategy combining accelerated perfusion with a direct paracrine delivery of bioactive molecules.

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

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 ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

In Vivo Heart Transplantation to Study Endothelial-to-Mesenchymal Transition

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

Author Spotlight: A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...

An “All-laser” Endothelial Transplant

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