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PubMed | Fondazione Banca degli Occhi del Veneto and University of Ljubljana
Type: | Journal: Advances in experimental medicine and biology | Year: 2016

Significant advances have been made in the field of ocular regenerative medicine. Promising stem cell-based therapeutic strategies have been translated into the clinical practice over the last few decades. These new stem cell-based therapies offer the possibility of permanently restoring corneal epithelium in patients with severe disabling and blinding ocular surface disease. The European Union has already classified stem cell-based therapies as medicinal products. Therefore, manipulation is strictly regulated according to the defined conditions of good manufacturing practice, with the production of stem cell therapeutics at only accredited production sites authorized by the national regulatory agencies. In this regard, as first medical products are licensed for commercial use in Europe enabling a more widespread access to a stem cell-based therapy, the need for safe, validated and reproducible techniques for ex vivo cultured tissue preservation and distribution are coming to the forefront of research. However, these provide various new challenges for biobanking industry such as the retention of viability, good functionality of stem cells and sterility issues. This chapter provides an overview of the current advances in the field of corneal/limbal epithelial stem cell culture preservation techniques using either hypothermic storage or cryopreservation methods, that were used in different culturing steps (from stem cell isolation to the ex vivo epithelial graft preparation), with the reported impact on the post-thawing product recovery.


Busin M.,Villa Serena Hospital | Busin M.,University of Catanzaro | Scorcia V.,Villa Serena Hospital | Scorcia V.,University of Catanzaro | And 3 more authors.
Ophthalmology | Year: 2010

Purpose: To investigate the feasibility of pneumatic dissection of donor endothelium and the effect of 7 days of storage in tissue culture medium on the endothelial cell count. Design: Experimental study. Participants: We used 20 human donor corneoscleral tissues deemed unsuitable for transplantation. Intervention: Donor corneas were mounted on an artificial anterior chamber and the anterior stroma removed with a 300-micron microkeratome head. Air was injected into the residual donor tissue with a 30-G needle from the endothelial side to detach Descemet's membrane. The "bubble" was expanded as far as possible into the periphery. A silicone weight was attached to the scleral ring and the prepared tissue was stored in tissue culture medium for 7 days. Main Outcome Measures: Complete detachment of Descemet's membrane, size of detachment, pre- and post-storage endothelial cell counts. Results: Complete detachment of Descemet's membrane was achieved in 19 of 20 (95%) cases. In 12 (60%) cases, this was achieved with a single injection; 7 (35%) cases required repeat injections. In 1 case, Descemet's membrane could not be detached despite repeat air injections. The average size of detachment was 8.11±2.0 mm. Endothelial cell loss after 7 days of tissue culture medium storage was 4.44±4.3%. Conclusions: Descemet's membrane and endothelium can be separated from the overlying stroma with a simple technique using air dissection. An adequate size of graft tissue is obtained without the need to manually handle the tissue. The technique allows storage of the tissue in tissue culture medium with low endothelial cell loss. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references. © 2010 American Academy of Ophthalmology.


Busin M.,Villa Serena Hospital | Busin M.,University of Catanzaro | Patel A.K.,Villa Serena Hospital | Scorcia V.,Villa Serena Hospital | And 4 more authors.
Ophthalmology | Year: 2010

Purpose: To evaluate the use of a peripheral stromal support to facilitate and improve the outcomes of Descemet's membrane endothelial keratoplasty (DMEK). Design: Prospective case series. Participants: Ten patients with Fuchs' endothelial dystrophy. Intervention: Pneumatic dissection was used to detach the central part of the Descemet's membrane and endothelium from the deep stroma. Endothelial grafts including a peripheral stromal support were obtained by eccentric punching of donor tissue and used to perform DMEK surgery in 10 patients. Main Outcome Measures: Operative time, graft attachment rate, best spectacle-corrected visual acuity (BSCVA), endothelial cell loss, refraction, and complications. Results: In all cases the surgical time was ≤1 hour. The postoperative course was uneventful in all but 2 cases, which required rebubbling owing to early graft detachment. Final attachment rate was 100%. The average follow-up was 8.4 months (range, 612). Postoperative BSCVA was <20/40 in all cases and no substantial change in refraction was recorded. Postoperative endothelial cell loss averaged 24.1% (range, 8%34.9%). Conclusions: Stromal support facilitates surgery, reduces complications, and appears to maintain the favorable outcomes of DMEK. Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references. © 2010 American Academy of Ophthalmology.


Patel A.K.,Villa Serena Hospital | Luccarelli S.,Villa Serena Hospital | Ponzin D.,Fondazione Banca degli Occhi del Veneto | Busin M.,Villa Serena Hospital
American Journal of Ophthalmology | Year: 2011

• Purpose: To describe a technique that uses a transcorneal suture for safe delivery and fixation of donor tissue during Descemet stripping automated endothelial keratoplasty in patients that are at risk of graft dislocation into the vitreous cavity as a result of minimal or absent iris-lens diaphragm. • Design: Interventional case series. • Methods: Thirteen eyes with endothelial decompensation and inadequate iris-lens diaphragm underwent modified Descemet stripping automated endothelial keratoplasty surgery. A 10-0 Prolene suture (Ethicon Inc) was passed through the endothelial graft and used to pull the graft into the eye and anchor it onto the recipient cornea. Best-corrected visual acuity, refraction, and complications were recorded. • Results: Preoperative best-corrected visual acuity was less than 20/200 in all cases. Eleven patients had a pre-existing comorbidity (glaucoma, n = 8; previous retinal detachment, n = 2; epiretinal membrane n = 1). Average follow-up was 11.3 months (range, 3 to 36 months). No graft dislocation occurred during surgery. After surgery, graft detachment was noted in 2 cases and rebubbling succeeded in achieving reattachment. All patients had successful attachment of the endothelial graft. Postoperative best-corrected visual acuity improved in 11 of 13 patients and remained unchanged in 2 patients. • Conclusions: In patients with insufficient iris-lens diaphragm, this technique allowed safe graft delivery, prevented intraoperative and postoperative graft dislocation, and facilitated successful rebubbling in case of postoperative graft detachment. © 2011 Elsevier Inc.


Busin M.,University of Catanzaro | Busin M.,Villa Igea Hospital | Scorcia V.,University of Catanzaro | Zambianchi L.,Villa Igea Hospital | Ponzin D.,Fondazione Banca degli Occhi del Veneto
Archives of Ophthalmology | Year: 2012

To improve visual and refractive outcomes, microkeratome-assisted lamellar keratoplasty for the treatment of keratoconus (exchange of a 9.0-mm anterior recipient lamella with a 9.0-mm donor lamella, using a 200-μm head for the former and a 300-μm head for the latter) was modified by adding a 6.5-mm incomplete full-thickness incision in the recipient bed before suturing the donor graft in place. After complete suture removal, 1 year postoperatively, best spectacle-corrected visual acuity was 20/40 or better in 92 of 97 eyes and 20/25 or better in 67 of 97 eyes; regular astigmatism was 4.5 diopters or worse in 86 of 97 eyes; endothelial cell loss averaged 20.4%. The disruption of the recipient's architecture induced by the full-thickness circular incision makes the final corneal shape closely resemble the physiologic curvature of the donor cornea, thus optimizing postoperative refractive error and spectacle-corrected visual acuity.


Ruzza A.,Fondazione Banca Degli Occhi Del Veneto | Salvalaio G.,Fondazione Banca Degli Occhi Del Veneto | Bruni A.,Fondazione Banca Degli Occhi Del Veneto | Frigo A.C.,University of Padua | And 2 more authors.
Cornea | Year: 2013

PURPOSE: The demands for precut lamellar grafts for Descemet stripping automated endothelial keratoplasty rose in our eye bank from 74 in 2007 to 408 in 2010. To meet this expanding requirement, we explored the possibility to preserve these preparations in organ culture. METHODS: Organ cultured corneas, stored in a medium containing 6% dextran, were mounted on a Moria artificial anterior chamber, deprived of the epithelium and then cut with a microkeratome. The posterior lamella was protected by positioning the anterior stromal cap, trephined at a diameter of 8.5 mm and stored at 31°C in the medium containing dextran. The endothelium was examined with trypan blue and alizarin staining and tested for its glycolytic activity (conversion of glucose into lactate). RESULTS: Incubation for a period of 1 week caused a small increase in the cell loss observed after trephination (from 6.2% to 10.6%). After 2 weeks, the decrease in endothelial cell density was 19.9% but the endothelial organization remained intact. The rate of glycolysis remained unchanged during the 2 weeks of preservation, with the majority of glucose uptake accounted for by lactate production. The thickness of the lenticules remained constant, ranging from 170 to 180 μm during the preservation. CONCLUSIONS: The lamellar grafts for Descemet stripping automated endothelial keratoplasty may be stored in organ culture for 2 weeks without damaging the endothelium or increasing the overall thickness. Copyright © 2012 by Lippincott Williams & Wilkins.


Nahum Y.,Tel Aviv University | Canton V.,University of Milan | Ponzin D.,Fondazione Banca degli Occhi del Veneto | Busin M.,Villa Igea Hospital
The British journal of ophthalmology | Year: 2015

AIM: To evaluate the prevalence of guttae in donor grafts following corneal transplantation and to examine the possible effect of guttae on postoperative results.METHODS: Retrospective cohort study. We reviewed the medical records of all keratoplasties performed at the Villa Serena-Villa Igea private hospitals (Forlì, Italy) between January 2005 and July 2014. Endothelial specular microscopy images were examined to identify the presence of guttae. Donor's age, patient's age, indication for surgery, surgical procedure, postoperative visual acuity, and endothelial cell density were also noted.RESULTS: A total of 11 068 postoperative specular microscopy pictures were available for 1116 of 2332 eyes (47.9%) that underwent keratoplasty at our institution. Guttae were identified in 42 of 946 eyes (4.44%) following penetrating or endothelial keratoplasty, and in 3 of 170 eyes (1.76%) following anterior lamellar keratoplasty. Twenty-seven of these photos demonstrated a few isolated scattered guttae, nine showed widespread guttae with small patches, and nine demonstrated large patches of guttae. Last documented best spectacle-corrected visual acuity did not differ between patients with or without guttae (logarithm of the minimum angle of resolution (logMAR) 0.22±0.24 (approximately 20/32) vs 0.29±0.45 (approximately 20/40), p=0.25) nor did the groups differ in their 24-month postoperative endothelial cell density (1633±427 vs 1555±454 cells/mm(2), p=0.56). No graft with postoperative guttae failed during the follow-up period of this study.CONCLUSIONS: Guttae can be found in approximately 4% of post-keratoplasty grafts. At least for the initial two postoperative years, they do not negatively affect vision, endothelial cell density or graft survival. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/


Patel A.K.,Villa Serena Hospital | Scorcia V.,Villa Serena Hospital | Scorcia V.,University of Catanzaro | Kadyan A.,Birmingham and Midland Eye Center | And 4 more authors.
Cornea | Year: 2012

Methods: All patients with post-penetrating keratoplasty anterior stromal opacities treated with microkeratome-assisted superficial anterior lamellar keratoplasty between July 2005 and June 2007 were reviewed. A 130-μm superficial keratectomy was performed, followed by the placement of an appropriately sized donor graft, which was secured with overlay sutures. Refraction, corneal topography, and uncorrected and best-corrected visual acuities (UCVA, BCVA, respectively) were noted at each examination.Results: Nine eyes of 8 consecutive patients were identified. Causes of anterior stromal opacities included dystrophy recurrence (n = 3), post-photorefractive keratectomy haze (n = 2), and scarring after stromal melt (n = 4). BCVA improved in all 9 eyes at final follow-up, and 7 of 9 eyes achieved ≥20/40 within the first month. Average follow-up period was 28 ± 3.9 months. Refractive astigmatism also improved by an average of 0.7 diopters.Conclusions: Superficial anterior lamellar keratoplasty is a viable and effective alternative to repeat PK in treating anterior stromal scars. It avoids open-globe surgery and exposure to endothelial rejection associated with repeat PK, and visual rehabilitation is considerably quicker.Purpose: To describe the surgical technique and report the outcomes of patients treated with microkeratome-assisted superficial anterior lamellar keratoplasty for anterior stromal corneal opacities developing after penetrating keratoplasty (PK). Copyright © 2011 by Lippincott Williams & Wilkins.


PubMed | University of Padua, Fondazione Banca degli Occhi del Veneto, University of Ferrara and Center for Retinitis Pigmentosa of Veneto Region
Type: | Journal: Scientific reports | Year: 2016

The aim of this study was to describe a new pathogenic variant in the mutational hot spot exon ORF15 of retinitis pigmentosa GTPase regulator (RPGR) gene within an Italian family with X-linked retinitis pigmentosa (RP), detailing its distinctive genotype-phenotype correlation with pathologic myopia (PM). All members of this RP-PM family underwent a complete ophthalmic examination. The entire open reading frames of RPGR and retinitis pigmentosa 2 genes were analyzed by Sanger sequencing. A novel frame-shift mutation in exon ORF15 of RPGR gene (c.2091_2092insA; p.A697fs) was identified as hemizygous variant in the male proband with RP, and as heterozygous variant in the females of this pedigree who invariably exhibited symmetrical PM in both eyes. The c.2091_2092insA mutation coherently co-segregated with the observed phenotypes. These findings expand the spectrum of X-linked RP variants. Interestingly, focusing on Caucasian ethnicity, just three RPGR mutations are hitherto reported in RP-PM families: one of these is located in exon ORF15, but none appears to be characterized by a high penetrance of PM trait as observed in the present, relatively small, pedigree. The geno-phenotypic attributes of this heterozygosity suggest that gain-of-function mechanism could give rise to PM via a degenerative cell-cell remodeling of the retinal structures.


PubMed | University of Padua, Fondazione Banca degli Occhi del Veneto and University of Naples Federico II
Type: Case Reports | Journal: Stem cells translational medicine | Year: 2016

: Ectrodactyly-ectodermal dysplasia-clefting (EEC) syndrome is a rare autosomal dominant disease caused by mutations in the p63 gene. To date, approximately 40 different p63 mutations have been identified, all heterozygous. No definitive treatments are available to counteract and resolve the progressive corneal degeneration due to a premature aging of limbal epithelial stem cells. Here, we describe a unique case of a young female patient, aged 18 years, with EEC and corneal dysfunction, who was, surprisingly, homozygous for a novel and de novo R311K missense mutation in the p63 gene. A detailed analysis of the degree of somatic mosaicism in leukocytes from peripheral blood and oral mucosal epithelial stem cells (OMESCs) from biopsies of buccal mucosa showed that approximately 80% were homozygous mutant cells and 20% were heterozygous. Cytogenetic and molecular analyses excluded genomic alterations, thus suggesting a de novo mutation followed by an allelic gene conversion of the wild-type allele by de novo mutant allele as a possible mechanism to explain the homozygous condition. R311K-p63 OMESCs were expanded in vitro and heterozygous holoclones selected following clonal analysis. These R311K-p63 OMESCs were able to generate well-organized and stratified epithelia in vitro, resembling the features of healthy tissues. This study supports the rationale for the development of cultured autologous oral mucosal epithelial stem cell sheets obtained by selected heterozygous R311K-p63 stem cells, as an effective and personalized therapy for reconstructing the ocular surface of this unique case of EEC syndrome, thus bypassing gene therapy approaches.This case demonstrates that in a somatic mosaicism context, a novel homozygous mutation in the p63 gene can arise as a consequence of an allelic gene conversion event, subsequent to a de novo mutation. The heterozygous mutant R311K-p63 stem cells can be isolated by means of clonal analysis and given their good regenerative capacity, they may be used to successfully correct the corneal defects present in this unique case of ectrodactyly-ectodermal dysplasia-clefting syndrome.

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