Trends in Early Graft Failure Leading to Regrafting After Endothelial Keratoplasty in the United States.

PURPOSE: The purpose of this study was to report trends in the prevalence of early graft failure after endothelial keratoplasty in the United States. METHODS: Descemet membrane endothelial keratoplasty (DMEK) and Descemet stripping automated endothelial keratoplasty (DSAEK) graft volumes were collected from records maintained by 6 major eye banks in the United States from January 1, 2013, to December 31, 2018. The prevalence and presumed cause of early graft failures (defined as a graft with persistent edema or regrafted within 8 weeks after keratoplasty) each year were sourced from surgeon-reported adverse events. Failed graft cases from the 3 eye banks were compared with nonfailures at the donor and recipient levels to perform subset analysis of factors associated with early graft failure. RESULTS: A total of 51,887 endothelial keratoplasty tissues were distributed during the study period; 72% were DSAEK grafts. The total number of early graft failures reported was 168 of 14,284 (1.18%) for DMEK and 322 of 37,603 (0.86%) for DSAEK. Early DMEK failures decreased from 2013 (7.69%) to 2018 (0.68%). In generalized linear mixed model analyses adjusting for donor tissue characteristics, recipient age, and diagnosis, an association of borderline significance was found between higher donor age and early failure [odds ratio (95% confidence interval): 1.03 (1.00-1.05); unit change of 1 yr] and DSAEK [odds ratio 1.02 (1.00-1.04); unit of change 1 yr] cases. CONCLUSIONS: The proportion of early graft failures in DMEK decreased over time and was comparable with failure rates in DSAEK at the end of the study period. The surgical learning curve might have played a role.

Production of Homogeneous Cultured Human Corneal Endothelial Cells Indispensable for Innovative Cell Therapy.

Purpose: Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. The aim of this study was to establish proper culture protocols to successfully obtain a reproducibly homogeneous subpopulation (SP) with matured cHCEC functions and devoid of cell-state transition suitable for cell-injection therapy. Methods: The presence of SPs in cHCECs was investigated in terms of surface cluster-of-differentiation (CD) marker expression level by flow cytometry, as combined analysis of CD markers can definitively specify the SP (effector cells) conceivably the most suitable for cell therapy among diverse SPs. The culture conditions were evaluated by flow cytometry in terms of the proportion (E-ratio) of effector cells designated by CD markers. Results: Flow cytometry analysis identifying CD44-CD166+CD133-CD105-CD24-CD26- effector cells proved convenient and reliable for standardizing the culture procedures. To ascertain the reproducible production of cHCECs with E-ratios of more than 90% and with no karyotype abnormality, the preferred donor age was younger than 29 years. The continuous presence of Rho-associated protein kinase (ROCK)-inhibitor Y-27632 greatly increased the E-ratios, whereas the presence of transforming growth factor-beta/Smad-inhibitor SB431542 greatly reduced the number of recovered cHCECs. The seeding cell density during culture passages proved vital for maintaining a high E-ratio for extended passages. The continuous presence of ROCK-inhibitor Y-27632 throughout the cultures greatly improved the E-ratio. Conclusions: Our findings elucidated the culture conditions needed to obtain reproducible cHCECs with high E-ratios, thus ensuring homogeneous cHCECs with matured functions for the treatment of corneal endothelial dysfunction.

MicroRNA Profiles Qualify Phenotypic Features of Cultured Human Corneal Endothelial Cells.

PURPOSE: To elucidate a noninvasive method to qualify and identify cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition and adaptable for cell-based therapy. METHODS: The variations of cHCECs in their composition of heterogeneous subpopulations (SPs) were verified in relation to their surface cluster-of-differentiation (CD) markers and their morphology. The profiles of microRNA (miRNA) in cultured cells or supernatants were detected by 3D-Gene Human microRNA Chips (Toray Industries, Inc.). The profiles were also analyzed for fresh corneal tissues with distinct endothelial cell densities (ECD) with or without gutatta. To validate the 3D-Gene results, quantitative real-time polymerase chain reaction (PCR) was performed. RNAs were extracted from cHCECs transfected with selected miRNA, and target genes were presumed by PCR array (Qiagen). RESULTS: Among a variety of morphologically different cHCECs, miRNA expression profiles were distinctively revealed. The one miRNA capable of discriminating CD44- SP from SPs with CD44++∼CD44+++ phenotypes was identified as miR34a. The downregulation of miRNAs in the 378 family paralleled the upregulation of surface CD44 on cHCECs. Interestingly, upregulated miRNAs in the 378 family in corneal endothelium dramatically decreased in the tissues with lower ECD with advanced gutatta, providing new insight on the pathogenesis of Fuchs' endothelial corneal dystrophy. CONCLUSIONS: The specified cultured SPs sharing the CD44- surface phenotypes with matured HCECs showed the highest expression of miR-378. Conversely, SPs with upregulated CD44+++ showed a reduction of miR-378. Thus, miRNA in cultured cells may serve as an alternative method to qualify cHCECs.

Metabolic Plasticity in Cell State Homeostasis and Differentiation of Cultured Human Corneal Endothelial Cells.

PURPOSE: To clarify whether cultured human corneal endothelial cells (cHCECs), heterogeneous in their differentiation state, exhibit distinctive energy metabolism with the aim to develop a reliable method to sort cHCECs applicable for regenerative medicine. METHODS: The presence of cHCEC subpopulations (SPs) was verified via surface cluster-of-differentiation (CD) marker expression. Cultured HCEC metabolic extracts or corresponding culture supernatants with distinctive cellular phenotypes in regard to energy-metabolism-related functional markers c-Myc and CD44 were prepared and analyzed via capillary electrophoresis-tandem mass spectrometry. The metabolic requirements of heterogeneous SPs of cHCECs were also investigated. RESULTS: After successfully discriminating SPs, as verified via surface CD markers in terms of their secretory metabolites, we found that the CD44+++ SP with cell-state transition (CST) exhibited disposition for anaerobic glycolysis, whereas the CD44-SP without CST was disposed to mitochondria-dependent oxidative phosphorylation (OXPHOS). These results raised the possibility of establishing effective culture conditions to selectively expand mature cHCECs with a hexagonal cobblestone shape and inclination for mitochondria-dependent OXPHOS. CONCLUSIONS: The findings of this study open a pathway for monitoring the disposition of cHCECs via their energy metabolism, thus leading to safe and stable regenerative medicine by use of metabolically defined cHCECs in cell-suspension form.

Cell Homogeneity Indispensable for Regenerative Medicine by Cultured Human Corneal Endothelial Cells.

PURPOSE: To identify the subpopulation (SP) among heterogeneous cultured human corneal endothelial cells (cHCECs) devoid of cell-state transition applicable for cell-based therapy. METHODS: Subpopulation presence in cHCECs was confirmed via surface CD-marker expression level by flow cytometry. CD markers effective for distinguishing distinct SPs were selected by analyzing those on established cHCECs with a small cell area and high cell density. Contrasting features among three typical cHCEC SPs was confirmed by PCR array for extracellular matrix (ECM). Combined analysis of CD markers was performed to identify the SP (effector cells) applicable for therapy. ZO-1 and Na+/K+ ATPase, CD200, and HLA expression were compared among heterogeneous SPs. RESULTS: Flow cytometry analysis identified the effector cell expressing CD166+CD105-CD44-∼+/-CD26-CD24-, but CD200-, and the presence of other SPs with CD166+ CD105-CD44+++ (CD26 and CD24, either + or -) was confirmed. PCR array revealed three distinct ECM expression profiles. Some SPs expressed ZO-1 and Na+/K+ ATPase at comparable levels with effector cells, while only one SP expressed CD200, but not on effector cells. Human leukocyte antigen expression was most reduced in the effector SP. The proportion of effector cells (E-ratio) inversely paralleled donor age and decreased during prolonged culture passages. The presence of Rho-associated protein kinase (ROCK) inhibitor increased the E-ratio in cHCECs. The average area of effector cells was approximately 200∼220 µm2, and the density of cHCECs exceeded 2500 cells/mm2. CONCLUSIONS: A specified cultured effector cell population sharing the surface phenotypes with mature HCECs in corneal tissues may serve as an alternative to donor corneas for the treatment of corneal endothelial dysfunction.

Cultured Human Corneal Endothelial Cell Aneuploidy Dependence on the Presence of Heterogeneous Subpopulations With Distinct Differentiation Phenotypes.

PURPOSE: Cultured human corneal endothelial cells (cHCECs) are anticipated to become an alternative to donor corneas for the treatment of corneal endothelial dysfunction. However, cHCECs reportedly tend to exhibit chromosomal abnormality during in vitro cell division, thereby hampering their use in the clinical setting. The purpose of this study was to clarify whether a specified subpopulation (SP) of heterogeneous cHCECs would exhibit aneuploidy, whereas other SPs would not. METHODS: The presence of SPs in cHCECs was analyzed on the basis of surface cluster of differentiation (CD) antigen CD166, CD105, CD44, CD26, and CD24 expression levels by flow cytometry. Cytogenetic examination was performed for 23 lots of cHCECs, either as whole-cell preparations (bulk) consisting of mixed SPs or as a semipurified SP by magnetic activated cell sorting (MACS). The HCEC donors ranged from 9 to 69 years of age and the culture passages from primary to fifth passage. RESULTS: Flow cytometry analysis demonstrated the presence of at least three cHCEC SPs. One SP, purified by MACS, with surface expression of CD166+, CD105-, CD44-, CD24-, and CD26- did not show any aneuploidy in 50 cells. However, CD166+, CD44+++, CD24-, and CD26+ cHCEC SPs showed sex chromosome loss in all cells (60 cells), whereas CD166+, CD44+++, CD24+, and CD26- SPs exhibited, albeit partly, trisomy on chromosomes 6, 7, 12, and 20. CONCLUSIONS: We found that cHCEC aneuploidy is linked to specified SPs present in cHCECs and that the SP sharing the surface phenotype with mature HCECs in corneal tissues was devoid of the karyotype abnormality.

Gene Signature-Based Development of ELISA Assays for Reproducible Qualification of Cultured Human Corneal Endothelial Cells.

PURPOSE: To develop a method to qualify the function of cultured human corneal endothelial cells (cHCECs) applicable for clinical settings. METHODS: The diversified gene and microRNA (miRNA) signatures in HCECs from a variety of tissue donors were confirmed by three-dimensional (3D) gene human miRNA profiling. These were compared with those of more than 20 cHCECs distinct in their cell morphology or culture lots. Candidate genes were selected after quantitative (q)RT-PCR validation, and gene products were assayed by ELISA. After three additional screening steps, final candidate cytokines for qualification were selected. RESULTS: Gene and miRNA signatures among distinct cHCEC lots were greatly diversified compared with those among fresh tissues from different age donors. By comparing more than 20 lots of cultures, 32 candidate genes were assigned to be seemingly linked to distinct cHCEC morphologic features. The validation of candidate genes by qRT-PCR revealed the genes, either upregulated or downregulated, corresponding to morphologic variances in cHCECs (e.g., epithelial-mesenchymal transition or cell senescence). Further adding the ELISA results by Bio-Plex Human Cytokine 27-Plex Panel, 11 candidate cytokines suitable to qualify cHCEC function were selected. In consideration of the presence of these cytokines in the anterior chamber, IL-8, tissue inhibitors of metalloproteinases 1 (TIMP-1), monocyte chemotactic protein-1 (MCP-1), and platelet-derived growth factor-BB (PDGF-BB) were ultimately selected and applied in practice for the qualification of cHCECs actually used in our clinical cell-injection studies. CONCLUSIONS: The specified cytokines properly discriminating the functional features of cHCECs indicates a correlation between profiling signatures and cell morphology.

The effect of donor age on penetrating keratoplasty for endothelial disease: graft survival after 10 years in the Cornea Donor Study.

OBJECTIVE: To determine whether the 10-year success rate of penetrating keratoplasty for corneal endothelial disorders is associated with donor age. DESIGN: Multicenter, prospective, double-masked clinical trial. PARTICIPANTS: A total of 1090 participants undergoing penetrating keratoplasty at 80 sites for Fuchs' dystrophy (62%), pseudophakic/aphakic corneal edema (34%), or another corneal endothelial disorder (4%) and followed for up to 12 years. METHODS: Forty-three eye banks provided corneas from donors aged 12 to 75 years, using a randomized approach to assign donor corneas to study participants without respect to recipient factors. Surgery and postoperative care were performed according to the surgeons' usual routines. MAIN OUTCOME MEASURES: Graft failure defined as a regraft or, in the absence of a regraft, a cloudy cornea that was sufficiently opaque to compromise vision for 3 consecutive months. RESULTS: In the primary analysis, the 10-year success rate was 77% for 707 corneas from donors aged 12 to 65 years compared with 71% for 383 donors aged 66 to 75 years (difference, +6%; 95% confidence interval, -1 to +12; P = 0.11). When analyzed as a continuous variable, higher donor age was associated with lower graft success beyond the first 5 years (P<0.001). Exploring this association further, we observed that the 10-year success rate was relatively constant for donors aged 34 to 71 years (75%). The success rate was higher for 80 donors aged 12 to 33 years (96%) and lower for 130 donors aged 72 to 75 years (62%). The relative decrease in the success rate with donor ages 72 to 75 years was not observed until after year 6. CONCLUSIONS: Although the primary analysis did not show a significant difference in 10-year success rates comparing donor ages 12 to 65 years and 66 to 75 years, there was evidence of a donor age effect at the extremes of the age range. Because we observed a fairly constant 10-year success rate for donors aged 34 to 71 years, which account for approximately 75% of corneas in the United States available for transplant, the Cornea Donor Study results indicate that donor age is not an important factor in most penetrating keratoplasties for endothelial disease.

Impact of the cornea donor study on acceptance of corneas from older donors.

PURPOSE: To evaluate retrospectively whether the findings from the Cornea Donor Study (CDS) led to changes in the transplantation of corneas from older donors. METHODS: Eye banks in United States provided complete data on donor age and placement (domestic or international) for 86,273 corneas from 1998 to 2009. The data were analyzed by 3 periods, preceding CDS (1998-1999), during CDS (2000-2007), and after publication of CDS 5-year results (2008-2009), and separately for corneas placed within versus outside the United States. RESULTS: For corneal tissues transplanted in the United States, the percentage of donors who were 66 years or older increased from 19% before CDS to 21% during CDS and 25% after CDS (P<0.001). Corresponding median (25th-75th percentile) donor ages were 53 (39-63), 54 (41-64), and 57 (46-66), respectively (P<0.001). The opposite trend was observed for corneas distributed outside the United States, with the percentage of donors 66 years and older decreasing from 56% to 42% to 34%, respectively. Donor age trends over time varied by eye bank. CONCLUSIONS: There was a modest overall increase in the donor age of corneas transplanted in the United States from 1998 to 2009, but the retrospective nature of the study limits our ability to attribute this change to the CDS. The modest increases in the donor age of corneas transplanted is a positive finding, but wider acceptance of older corneal donor tissue should be encouraged based on the 5-year evidence generated by the CDS.

Preparation of corneal donor eyes comparing 1% versus 5% povidone-iodine.

PURPOSE: To compare the effect of 1% versus 5% polyvinylpyrrolidone-iodine (PVP-I) chemical preparation (prep) of the eye on the recovery of organisms from donor globes before in situ recovery of donor corneal tissue. METHODS: One hundred consecutive pairs of donor corneas (200 eyes) were randomized to receive either 1% or 5% PVP-I drops applied to the conjunctival cul-de-sac, which was left in place for 2 minutes. Limbal cultures were obtained before and after prepping of the eye. RESULTS: Twenty-five different species of organisms were recovered. Native flora of the eye included coagulase-negative staphylococci (62%), Corynebacterium species (27%), streptococcal species (9.5%), gram-negative bacilli (14.5%), Staphylococcus aureus (5%), anaerobes (10%), and yeast (2%). After PVP-I instillation of the donor eye, 74 isolates were recovered from the 1% P-I group and 76 isolates from the 5% PVP-I group. Cultures were sterile after PVP-I prep in 49 eyes and 47 eyes in the 1% PVP-I group and 5% PVP-I group, respectively. Microorganism colony forming units were similar among post-prep cultures from both PVP-I groups. The effect of the PVP-I prep on the number of negative cultures and on the reduction in the number of isolates was highly significant for both the 1% PVP-I group and the 5% PVP-I group when compared with the limbal cultures taken before PVP-I instillation. CONCLUSIONS: This study found that 1% and 5% PVP-I solutions are equally effective for chemical prep of the donor eye. Because PVP-I is known to be toxic to the corneal endothelium and corneal fibroblasts, this study suggests that 1% PVP-I should be the preferred disinfectant for the recovery of corneal donors.

Donor risk factors for graft failure in the cornea donor study.

PURPOSE: The purpose of this study was to assess the relationship between donor factors and 5-year corneal graft survival in the Cornea Donor Study. METHODS: Donor corneas met criteria established by the Eye Bank Association of America, had an endothelial cell density of 2300 to 3300/mm, and were determined to be of good to excellent quality by the eye banks. Donor corneas were assigned using a random approach and surgeons were masked to information about the donor cornea including donor age. Surgery and postoperative care were performed according to the surgeons' usual routines and subjects were followed for 5 years. Donor and donor cornea factors were evaluated for their association with graft failure, which was defined as a regraft or a cloudy cornea that was sufficiently opaque to compromise vision for a minimum of 3 consecutive months. RESULTS: Graft failure was not significantly associated with the type of tissue retrieval (enucleation versus in situ), processing factors, timing of use of the cornea, or characteristics of the donor or the donor cornea. Adjusting for donor age did not affect the results. CONCLUSION: Donor and donor cornea characteristics do not impact graft survival rates for corneas comparable in quality to those used in this study.

The effect of donor age on corneal transplantation outcome results of the cornea donor study.

OBJECTIVE: To determine whether graft survival over a 5-year follow-up period using corneal tissue from donors older than 65 is similar to graft survival using corneas from younger donors. DESIGN: Multicenter prospective, double-masked, controlled clinical trial. PARTICIPANTS: One thousand ninety subjects undergoing corneal transplantation for a moderate-risk condition (principally Fuchs' dystrophy or pseudophakic corneal edema); 11 subjects with ineligible diagnoses were not included. METHODS: Forty-three participating eye banks provided corneas from donors in the age range of 12 to 75 with endothelial cell densities of 2300 to 3300 cells/mm(2), using a random approach without respect to recipient factors. The 105 participating surgeons at 80 sites were masked to information about the donor cornea including donor age. Surgery and postoperative care were performed according to the surgeons' usual routines. Subjects were observed for 5 years. MAIN OUTCOME MEASURES: Graft failure, defined as a regraft or a cloudy cornea that was sufficiently opaque as to compromise vision for a minimum of 3 consecutive months. RESULTS: The 5-year cumulative probability of graft survival was 86% in both the <66.0 donor age group and the >/=66.0 donor age group (difference = 0%, upper limit of 1-sided 95% confidence interval = 4%). In a statistical model with donor age as a continuous variable, there was no significant relationship between donor age and outcome (P = 0.11). Three graft failures were due to primary donor failure, 8 to uncorrectable refractive error, 48 to graft rejection, 46 to endothelial decompensation (23 of which had a prior, resolved episode of probable or definite graft rejection), and 30 to other causes. Distributions of the causes of graft failure did not differ between donor age groups. CONCLUSIONS: Five-year graft survivals for cornea transplants at moderate risk for failure are similar using corneas from donors >/= 66.0 years and donors < 66.0. Surgeons and patients now have evidence that corneas comparable in quality to those used in this study from donors through age 75 are suitable for transplantation.

Donor age and corneal endothelial cell loss 5 years after successful corneal transplantation. Specular microscopy ancillary study results.

OBJECTIVE: To determine whether endothelial cell loss 5 years after successful corneal transplantation is related to the age of the donor. DESIGN: Multicenter, prospective, double-masked clinical trial. PARTICIPANTS: Three hundred forty-seven subjects participating in the Cornea Donor Study who had not experienced graft failure 5 years after corneal transplantation for a moderate-risk condition (principally Fuchs' dystrophy or pseudophakic corneal edema). TESTING: Specular microscopic images of donor corneas obtained before surgery and postoperatively at 6 months, 12 months, and then annually through 5 years were submitted to a central reading center to measure endothelial cell density (ECD). MAIN OUTCOME MEASURE: Endothelial cell density at 5 years. RESULTS: At 5 years, there was a substantial decrease in ECD from baseline for all donor ages. Subjects who received a cornea from a donor 12 to 65 years old experienced a median cell loss of 69% in the study eye, resulting in a 5-year median ECD of 824 cells/mm(2) (interquartile range, 613-1342), whereas subjects who received a cornea from a donor 66 to 75 years old experienced a cell loss of 75%, resulting in a median 5-year ECD of 654 cells/mm(2) (interquartile range, 538-986) (P [adjusted for baseline ECD] = 0.04). Statistically, there was a weak negative association between ECD and donor age analyzed as a continuous variable (r [adjusted for baseline ECD] = -0.19; 95% confidence interval, -0.29 to -0.08). CONCLUSIONS: Endothelial cell loss is substantial in the 5 years after corneal transplantation. There is a slight association between cell loss and donor age. This finding emphasizes the importance of longer-term follow-up of this cohort to determine if this relationship affects graft survival.

An evaluation of image quality and accuracy of eye bank measurement of donor cornea endothelial cell density in the Specular Microscopy Ancillary Study.

PURPOSE: The Specular Microscopy Ancillary Study was designed to examine donor corneal endothelial specular image quality, compare the central endothelial cell density determined by eye banks with the endothelial cell density determined by a central specular microscopy reading center, and evaluate donor factors that may have an impact on specular image quality and endothelial cell density accuracy. DESIGN: Nonrandomized comparative trial. PARTICIPANTS: Endothelial specular images of donor corneas assigned in the Cornea Donor Study. METHODS: Certified readers assessed donor image quality (analyzable from fair to excellent vs. unanalyzable) and determined the central endothelial cell density. Independent adjudication was performed if there was a difference in the quality of grading or if the endothelial cell density varied by > or =5.0% between readers. Average reading center-determined endothelial cell density was compared with the endothelial cell density determined by each eye bank. MAIN OUTCOME MEASURES: Evaluation of image quality and accuracy of endothelial cell density. RESULTS: Of 688 donor endothelial images submitted by 23 eye banks, 663 (96%) were analyzable (excellent, 40 [6%]; good, 302 [44%]; fair, 321 [47%]), and 25 (4%) were unanalyzable by reading center standards. In situ retrieval and greater epithelial exposure correlated with a higher image quality grading. The eye bank-determined endothelial cell density of 434 of the 663 (65%) analyzable images were within 10% of the endothelial cell density determined by the reading center, whereas 185 (28%) were more than 10% higher and 44 (7%) were more than 10% lower. Greater variation in endothelial cell density between the eye banks and the reading center was observed with shorter time of death to preservation, presence of an epithelial defect, folds in Descemet's membrane, lower image quality, and the use of fixed-frame or center method endothelial cell density analysis. CONCLUSIONS: Overall, donor endothelial specular image quality and accuracy of endothelial cell density determination were good. However, the data suggest that factors that may affect image quality and contribute to variation in interpretation of the endothelial cell density should be addressed, because the donor endothelial cell density is an important parameter for assessing long-term corneal graft survival.