Paired Donor Corneas Research Articles

Thickness Profile of Donated Corneas Preserved in Optisol-GS versus Sinasol: An Ex-vivo Study.

This study aimed to compare the thickness profile and the endothelial cell density (ECD) of donated corneas maintained in Optisol-GS with those preserved in Sinasol over seven days. Twenty paired donor corneas were received from the Central Eye Bank of Iran. After recording the osmolarity of each medium, one of each of the cornea pairs was preserved in either Optisol-GS or Sinasol media. Then, slit-lamp biomicroscopy and specular microscopic examinations were performed at the baseline and on day seven. Visante optical coherence tomography (V-OCT) was also performed at 1 hour (h), 24h, 72h, and one week post-preservation. The specular microscopic and V-OCT values were then compared between the two groups. The mean osmolarity of the Sinasol group was significantly less than the Optisol-GS group (296 vs. 366 mOsm/L, = 0.0008). The mean central corneal thickness at the measurement points was comparable between the two groups. However, the increase of thickness one week post-preservation in the Sinasol group was remarkably lower than those in the Optisol-GS group ( = 0.027). The mean ECD was comparable between the groups at the baseline and on day seven. However, the mean change of ECD from baseline to day seven was considerably higher in the Optisol-GS group than in the Sinasol group ( = 0.019). Corneal storage in Sinasol over seven days provides better and superior maintenance and preservation of corneal tissue deturgescence and a lower rate of ECD loss over Optisol-GS.

Endothelial cell viability after DMEK graft preparation

AbstractPurposeTo evaluate the effect of graft preparation and organ‐culture storage on endothelial cell density (ECD) and viability of Descemet membrane endothelial keratoplasty (DMEK) grafts.MethodsDMEK grafts (n = 27) were prepared at Amnitrans EyeBank Rotterdam from 27 corneas (15 donors) that were eligible for transplantation but could not be allocated due to the Covid‐19‐related cancellation of elective surgeries. Cell viability (by Calcein‐AM staining) and ECD of 5 grafts originally scheduled for transplantation, were evaluated on the originally planned surgery day, whereas 22 grafts from paired donor corneas were evaluated either directly post‐preparation or after 3–7 days of storage. ECD was analyzed by light microscopy (LM ECD) and Calcein‐AM staining (Calcein‐ECD)ResultsLight microscopy (LM) evaluation of all grafts showed an unremarkable endothelial cell monolayer directly after preparation. However, median Calcein‐ECD for the 5 grafts initially allocated for transplantation was 18% (range 9–73%) lower than median LM ECD. For the paired DMEK grafts, Calcein‐ECD determined by Calcein‐AM staining on the day of graft preparation and after 3–7 days of graft storage showed a median decrease of 1% and 2%, respectively. Median percentage of central graft area populated by viable cells after preparation and after 3‐7 days of graft storage was 88% and 92%, respectively.ConclusionsCell viability of most of the grafts will not be affected by preparation and storage. Endothelial cell damage may be observed for some grafts within hours after preparation with insignificant additional ECD changes during 3–7 days of graft storage. Implementing an additional post‐preparation step in the eye bank to evaluate cell density before graft release for transplantation may help to reduce postoperative DMEK complications

Endothelial Cell Viability after DMEK Graft Preparation

ABSTRACT Aim: To evaluate the effect of graft preparation and organ-culture storage on endothelial cell density (ECD) and viability of Descemet membrane endothelial keratoplasty (DMEK) grafts. Materials and methods: DMEK grafts (n = 27) were prepared at Amnitrans EyeBank Rotterdam from 27 corneas (15 donors) that were eligible for transplantation but could not be allocated due to the Covid-19-related cancellation of elective surgeries. Cell viability (by Calcein-AM staining) and ECD of five grafts originally scheduled for transplantation were evaluated on the originally planned surgery day, whereas 22 grafts from paired donor corneas were evaluated either directly post-preparation or after 3–7 days of storage. ECD was analyzed by light microscopy (LM ECD) and Calcein-AM staining (Calcein-ECD). Results: Light microscopy (LM) evaluation of all grafts showed an unremarkable endothelial cell monolayer directly after preparation. However, median Calcein-ECD for the five grafts initially allocated for transplantation was 18% (range 92–73%) lower than median LM ECD. For the paired DMEK grafts, Calcein-ECD determined by Calcein-AM staining on the day of graft preparation and after 3–7 days of graft storage showed a median decrease of 1% and 2%, respectively. Median percentage of central graft area populated by viable cells after preparation and after 3–7 days of graft storage was 88% and 92%, respectively. Conclusion: Cell viability of most of the grafts will not be affected by preparation and storage. Endothelial cell damage may be observed for some grafts within hours after preparation, with insignificant additional ECD changes during 3–7 days of graft storage. Implementing an additional post-preparation step in the eye bank to evaluate cell density before graft release for transplantation may help to reduce postoperative DMEK complications.

Sinasol versus Optisol-GS for cold preservation of human cornea: a prospective ex vivo and clinical study.

To compare ex vivo results of donor corneas maintained in Sinasol with those stored in Optisol-GS and reporting clinical outcomes of grafted Sinasol-versus Optisol-GS-stored corneas. In phase I, paired donor corneas were maintained in Sinasol or Optisol-GS. Afterward, the corneas were subjected to slit-lamp biomicroscopic and specular microscopic examinations on days 1 and 7, and then to trypan blue staining on day 7. The same examinations were performed on the corneas that were kept in Sinasol or Optisol-GS for 14days. In phase II, the post-operative reports of 72 consecutive corneal transplantations were recorded using Sinasol- or Optisol-GS-preserved corneas. In phase I, 128 corneas from 64 donors and 59 corneas from 33 donors were investigated for 7 and 14days, respectively. The EC indices were comparable between the groups at the measurement periods. The EC losses over 7 and 14days were 3.7% and 19.9% in Sinasol against 4.6% and 20.8% in Optisol-GS. Although fair quality corneas were more common in Optisol-GS group after 7 (P = 0.04) and 14days (P = 0.034), changes of stromal edema, Descemet's fold, and other quality ratings during 14days were not different between the groups. In phase II, all the transplanted corneas were postoperatively clear with no adverse reactions. The overall results indicate that Sinasol is a safe, effective, and affordable intermediate cold storage medium for preservation of corneas.

Ultra-thin DSAEK using an innovative artificial anterior chamber pressuriser: a proof-of-concept study

To report the impact of establishing and maintaining a high intracameral pressure (ICP) of 200mmHg on UT-DSAEK graft preparation using an artificial anterior chamber pressuriser (ACP) control unit (Moria SA, Antony, France). Retrospective laboratory and clinical study. Four paired donor corneas were mounted on an artificial anterior chamber and subjected to 70mmHg ("low") and 200mmHg ("high") ICP using an ACP system. The central corneal thinning rate was measured after 5min using AS-OCT and the endothelial cell viability was analysed using trypan blue and live/dead staining following 70mmHg and 200mmHg ICP. Visual outcomes and complications in a clinical case series of nine patients with bullous keratopathy who underwent UT-DSAEK using 200mmHg ICP during graft preparation are reported. Laboratory outcomes showed 2 ± 1% and 2 ± 2% dead cells following 70mmHg and 200mmHg ICP respectively. Percentage viability in the 70mmHg group (52.94 ± 5.88%) was not found to be significantly different (p = 0.7) compared to the 200mmHg group (59.14 ± 10.43%). The mean corneal thinning rate after applying 200mmHg ICP was 27 ± 13μm/min centrally (7.2%/min). In the clinical case series, two cases were combined with cataract surgery. Re-bubbling rate was 11%. At the last follow-up (259 ± 109days), graft thickness was 83 ± 22μm centrally, endothelial cell density was 1175 ± 566 cell/mm2 and the BCVA of 0.08 ± 0.12 logMAR was recorded with no episodes of rejection. ACP control unit for UT-DSAEK graft preparation helps in consistently obtaining UT-DSAEK grafts without compromising endothelial cell viability.

Recombinant human serum albumin for corneal preservation.

To study the performance of a completely synthetic organ culture (OC) preservation system containing recombinant human serum albumin (rHSA) for preservation of human donor corneas. Twenty-four paired donor corneas were randomly collected, and one cornea from each donor was preserved in synthetic (experimental) and serum-based media (control). The tissues were assessed at day 0; after 6days of preservation at room temperature (RT) in Cornea Trans® and Cornea Prep II® ; after 28days at 31°C in Cornea Syn® [with rHSA] and Cornea Max® [with foetal calf serum (FCS)] and; 4-day post deswelling in Cornea Trans® and Cornea Jet® . Thickness was determined with optical coherence tomography (OCT) and transparency with a validated, custom device. Morphology, endothelial cell density (ECD) and mortality were observed after treating the tissues with Trypan blue and sucrose. Glucose uptake by the cells was analysed. Data were compared using non-parametric paired Wilcoxon tests with p<0.05 deemed significant. Histology using periodic acid-schiff (PAS), expressions of p63, CK12, αSMA and ZO-1 were analysed, and cell apoptosis postpreservation was studied. Corneas stored in synthetic media showed a higher and statistically significant value as compared to serum-based media in terms of viable endothelial cell density (VECD), mortality, morphology and glucose uptake postpreservation. Histology showed presence of all the layers, all the markers were expressed, and no apoptosis was observed in either media. The new synthetic preservation system containing rHSA (and other confidential constituents) showed better preservation effects than traditional media containing serum.

Randomized, prospective, single-masked clinical trial of endothelial keratoplasty performance with 2 donor cornea 4°C storage solutions and associated chambers.

The aim of this study was to compare endothelial cell loss and graft success 6 months after endothelial keratoplasty (EK) with paired donor corneas stored in Optisol GS and Life4°C solutions and their associated storage chambers. Donor pairs were stored, one in Optisol GS and the other in Life4°C, and prepared for Descemet stripping automated EK or Descemet membrane EK. Matched pairs of recipients with Fuchs dystrophy were randomized to 1 member of each donor pair. Clarity of recipient stroma, intraocular pressure, and complications were followed for 6 months. Central endothelial images of the donor cornea at screening and 3 and 6 months after EK were analyzed by a masked central reading center. Dual grading of endothelial cell density was performed using the center method. Differences in endothelial cell density and cell loss were examined by paired analysis. Thirty-two pairs were enrolled, and 27 were analyzed (5 had improper matching or loss to follow-up). Donor age was 59 ± 14 years, median death-to-surgery time was 4 days (range, 2-9 days), 6 recipient pairs had Descemet membrane EK, and 21 had Descemet stripping automated EK. Recipient age was comparable in the Optisol GS and Life4°C groups (70 vs. 68 years, respectively, P = 0.46). Six-month central endothelial cell loss did not differ significantly between the Life4°C and Optisol GS groups (18 ± 18% vs. 20 ± 20%, respectively, P = 0.55). All recipient corneas were clear at 6 months in both groups. Endothelial cell loss and graft success were comparable at 6 months for paired donor corneas stored in Optisol GS and Life4°C.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT01657500.

Deformation response of paired donor corneas to an air puff: Intact whole globe versus mounted corneoscleral rim

To evaluate the influence of ocular shell biomechanical characteristics on corneal deformation response to an air puff. The Ohio State University, Columbus, Ohio, USA. Experimental study. Twenty-four eyes of 12 human donors were obtained in matched pairs. One eye was secured in a purpose-designed whole globe mount (whole-globe group). The cornea from the fellow eye was placed in a Barron artificial anterior chamber (artificial-chamber group). The corneas were mounted sequentially and connected to a pressure-control system. Deformation data were acquired using the Corvis ST, a dynamic Scheimpflug analyzer. Internal pressure was set to 10, 20, 30, 40, and 50 mm Hg; at least 4 examinations were performed at each pressure. Statistically significantly higher maximum deformation amplitude was observed in the whole-globe group than in the artificial-chamber group at all pressures. The mean amplitude differences were 1.006 mm ± 0.238 [SD], 0.614 ± 0.137 mm, 0.384 ± 0.099 mm, 0.229 ± 0.087 mm, and 0.133 ± 0.068 mm at 10, 20, 30, 40, and 50 mm Hg, respectively (P<.0001, P<.0001, P<.0001, P<.0001, and P<.0002, respectively). Nonlinear regression of the deformation amplitude differences between pairs showed a significant decrease with increasing pressure (P<.0001, R(2) = 0.8385). The deformation response to an air puff was affected by the type of mount used, with a stiffer shell producing a stiffer corneal response and decreasing differences at higher internal pressures. In vivo air-puff examinations may be affected by scleral stiffness in addition to the cornea. Dr. Roberts is a consultant to Oculus Optikgeräte GmbH and Ziemer Ophthalmic Systems AG and has received research funding from Carl Zeiss Meditec AG and travel funds from Sooft Italia. No other author has a financial or proprietary interest in any material or method mentioned.

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro

BackgroundGlobal shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology.ResultsEstablished primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 (‘LOW’); 5,000 cells per cm2 (‘MID’); 10,000 cells per cm2 (‘HIGH’); and 20,000 cells per cm2 (‘HIGH×2’), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology.ConclusionsOur results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Evaluation of interface quality in organ-cultured lamellar corneal transplants.

BackgroundWith increasing numbers of lamellar keratoplasties, eye banks are challenged to deliver precut lamellar donor tissue. In Europe, the most common technique of corneal storage is organ culture which requires a deswelling process before surgical processing. The aim of this study was to investigate the influence of different deswelling times on the cutting plane quality after microkeratome-assisted lamellar dissection.MethodsEight paired donor corneas (16 specimens) not suitable for transplantation were organ cultured under standard conditions at the Eye Bank of the Ludwig-Maximilians Universität, Munich, Germany. Pairs of corneal buttons were analyzed during the deswelling process in dextrane-containing medium. While one cornea was cut at an early time point during the deswelling process and put back into deswelling medium thereafter, the partner cornea was completely deswollen and dissected after 72 hours. Specimens were then further processed for scanning electron microscopy. Surface quality was assessed both digitally using Scanning Probe Imaging Processing software, and manually by three blinded graders.ResultsThe corneal buttons processed at the beginning of the deswelling process had a smoother surface when compared to the partner cornea that was cut at the end of the deswelling process. In our setting, no relevant difference was detectable between manual and automated microkeratome dissection.ConclusionFor lamellar keratoplasty, organ-cultured corneas should be processed at an early stage during the deswelling process. We interpret the smoother dissection plane during early deswelling as a result of mechanical properties in a highly hydrated cornea.

Cultivation of Human Corneal Endothelial Cells Isolated from Paired Donor Corneas

Consistent expansion of human corneal endothelial cells (hCECs) is critical in the development of tissue engineered endothelial constructs. However, a wide range of complex culture media, developed from different basal media have been reported in the propagation of hCECs, some with more success than others. These results are further confounded by donor-to-donor variability. The aim of this study is to evaluate four culture media in the isolation and propagation of hCECs isolated from a series of paired donor corneas in order to negate donor variability.Isolated primary hCECs were cultured in four previously published medium coded in this study as: M1-DMEM; M2-OptiMEM-I; M3-DMEM/F12, & M4-Ham's F12/M199. Primary hCECs established in these conditions were expanded for two passages and analyzed for (1) their propensity to adhere and proliferate; (2) their expression of characteristic corneal endothelium markers: Na+K+/ATPase and ZO-1; and (3) their cellular morphology throughout the study. We found that hCECs isolated in all four media showed rapid attachment when cultured on FNC-coated dishes. However, hCECs established in the four media exhibited different proliferation profiles with striking morphological differences. Corneal endothelial cells cultured in M1 and M3 could not be propagated beyond the first and second passage respectively. The hCECs cultured in M2 and M4 were significantly more proliferative and expressed markers characteristics of human corneal endothelium: Na+K+/ATPase and ZO-1. However, the unique morphological characteristics of cultivated hCECs were not maintained in either M2 or M4 beyond the third passage.The proliferative capacity and morphology of hCECs are vastly affected by the four culture media. For the development of tissue engineered graft materials using cultured hCECs derived from the isolation methodology described in this study, we propose the use of proliferative media M2 or M4 up to the third passage, or before the cultured hCECs lose their unique cellular morphology.

Regeneration of the epithelium in organ‐cultured donor corneas with extended post‐mortem time

The maximum post-mortem time limit for obtaining donor corneas varies between eye banks. It is not known for how long a time the epithelial cells survive post-mortem, nor is it known if donor corneas with extended post-mortem time are able to regenerate the epithelium. Therefore, we wanted to examine the epithelium in donor corneas for regenerative ability during storage in an eye bank organ culture system. Twenty-four paired donor corneas with post-mortem time from 28 to 163 hr were obtained. One cornea of a pair was fixed immediately to serve as a control, and the second was cultured in eye bank medium at 32 degrees C for 3 days. Examination of the specimens was performed with light and scanning electron microscopy. Immunohistochemical staining methods with antibodies against K 3, K 19, vimentin and p63 were used to further characterize the cells. The control corneas showed decreasing amounts of epithelial cells with increasing post-mortem time. All the cultured corneas demonstrated rapid regeneration of the epithelium. After 3 days in organ culture, 10 of 12 donor corneas were covered with epithelium. Even up to 7 days post-mortem, viable cells reside in the corneal epithelium. The study demonstrates the hardiness and enormous regenerative potential of peripheral corneal cells. Donor corneas processed in an eye bank organ culture storage system will obtain an intact epithelial layer within a few days.

Keratoplasty results of paired donor corneas.

Keratoplasty results of paired donor corneas.