Cryopreservation and long-term culture of transformed murine corneal endothelial cells.

PURPOSE: To characterize the morphology and gene expression of transformed murine corneal endothelial cells. METHODS: Primary immortomouse corneal endothelial cells were continuously cultured before and after cryopreservation. Morphologic assessment, real time-reverse transcriptase polymerase chain reaction ((RT)-PCR) and immunofluorescence studies were performed using newly cultured cells, cells that had been continuously in culture for 1 year, and cryopreserved cells, to assess for structural and functional integrity. The expression of corneal endothelial markers zonula occludens-1 (ZO1), NaK-ATPase and collagen VIII (α2) (COL8A2), and myofibroblast markers Desmin, alpha smooth muscle actin (αSMA), and Vimentin was assessed and compared by both RT-PCR and immunofluorescence. RESULTS: Cells in culture formed a monolayer, and exhibited a polygonal shape after reaching confluence. Cells retained this morphology during the full observation time of 12 months and when reused after cryopreservation. Immunofluorescence experiments exhibited positive staining for NaK-ATPase and COL8A2 with low variability between the three groups. In RT-PCR experiments, ZO1, COL8A2 and Desmin were increased in fresh and thawed cells, αSMA was decreased, and NaK-ATPase and Vimentin remained unchanged, compared to 12-month-old cells. Comparing fresh and thawed cells, COL8A2 was increased in thawed cells, while Desmin was increased in fresh cells. CONCLUSIONS: Using the immortomouse strain, murine corneal endothelial cells can be propagated over a long time period and be used after cryopreservation. Cells retain the expression of NaK-ATPase, but show some decline in ZO1 and COL8A2 over time and after cryopreservation. The expression of myofibroblast markers suggests an endothelial-to-mesenchymal transformation process in culture.

A cellular model for the investigation of Fuchs' endothelial corneal dystrophy.

Fuchs' endothelial corneal dystrophy is the most common corneal endotheliopathy, and a leading indication for corneal transplantation in the US. Relatively little is known about its underlying pathology. We created a cellular model of the disease focusing on collagen VIII alpha 2 (COL8A2), a collagen which is normally present in the cornea, but which is found in abnormal amounts and distribution in both early and late-onset forms of the disease. We performed cellular transfections using COL8A2 cDNAs including both wild-type and mutant alleles which are known to result in early-onset FECD. We used this cell model to explore the cellular production of wild-type and mutant monomeric and trimeric collagen VIII and measured production levels and patterns using Western blotting and immunofluorescence. We studied the thermal stability of the mutated collagen VIII helices using computer modeling, and further investigated these differences using collagen mimetic peptides. The Western blots demonstrated that similar amounts of wild-type and mutant collagen VIII monomers were produced in the cells. However, the levels of trimeric collagen peptide in the mutant-transfected cells were elevated. Intracellular accumulation of trimeric collagen VIII was confirmed on immunofluorescence studies. Both the computer model and the collagen mimetic peptides demonstrated that the L450W mutant was less thermally stable than either the Q455K or wild-type collagen VIII. Thus, although both mutant collagen VIII peptides were retained intracellularly, the biochemical reasons for the retention varied between genotypes. Collagen VIII mutations, which clinically result in Fuchs' dystrophy, are associated with abnormal cellular accumulation of collagen VIII. Different collagen VIII mutations may act via distinct biochemical mechanisms to produce the FECD phenotype.

Comparison of non-viral methods to genetically modify and enrich populations of primary human corneal endothelial cells.

PURPOSE: To compare different techniques of transfection of primary human corneal endothelial cells (HCECs) by non-viral methods and to enrich genetically modified cells to a highly pure population. METHODS: HCECs were cultured following previously published methods. Dissection of the Descemet membrane (DM) was performed by tearing off strips from corneal buttons with forceps or by hydrodissection. Confirmation of HCECs identity was performed by reverse transcriptase polymerase chain reaction (RT-PCR) for alpha2 collagen VIII. For transfection, non-viral methods such as lipid-/liposome-mediated reagents and electroporation techniques were compared. Genetically modified cells were enriched by use of selection antibiotics and flow cytometry. RESULTS: Viability of primary HCECs was lower in hydrodissected corneas. The rate of transfection varied from approximately 5%-30%. Highest rates of transfection were obtained with the Amaxa electroporation method. The next highest rate was yielded by the lipid-mediated reagent GenCarrier2, followed by electroporation with the BTX apparatus. Toxicity was moderate and manageable by adjusting the concentration of reagents, incubation times, and electrical parameters. Selection by flow cytometry was superior to antibiotic selection and produced nearly 100% genetically modified cells. CONCLUSIONS: Electroporation of HCECs yields higher transfection efficiency than chemically mediated methods. It is possible to select genetically modified HCECs to high levels of homogeneity. Techniques to genetically modify and select HCECs as shown in this study could lead to improved success of future endothelial transplant procedures.

Role of human corneal endothelial cells in T-cell-mediated alloimmune attack in vitro.

PURPOSE: Human corneal endothelial cells (HCEC) are a potential target of immune attack after corneal transplantation. The aim of this in vitro study was to investigate the role of HCEC during the alloimmune response of T-cells by examining cytokine profiles, function of the immunosuppressive enzyme indoleamine 2,3-dioxigenase (IDO), major histocompatibility complex (MHC-I/-II), T-cell proliferation, and the induction of cell death. METHODS: Real-time PCR and RP-HPLC were used to determine IDO expression and activity. Multiplex assay was performed for quantification of cytokine levels. T-cell proliferation was assessed by thymidine incorporation, and HCEC cell death was measured by flow cytometry. RESULTS: Human corneal endothelial cells induce strong proliferation of allogeneic T-cells and an increase of proinflammatory cytokines such as interleukin-1α (IL-1α), IL-1ß, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Tumor necrosis factor-alpha (and to a lesser extent IFN-γ) induces apoptosis. Moreover, IFN-γ strongly upregulates MHC-II molecules and IDO activity in HCEC as reflected by high kynurenine (Kyn) concentrations. Interestingly, the T-cell response was not affected by increased IDO activity, since blocking of IDO did not affect the proliferation rate. Indoleamine 2,3-dioxigenase-induced Kyn levels did not exceed concentrations of 175 ± 20 µM. Concentrations of ≥400 µM Kyn were required to suppress T-cell proliferation. CONCLUSIONS: Our data show that T-cell attack on HCEC leads to increased concentrations of proinflammatory cytokines. Inflammatory cytokines induce apoptosis and upregulate MHC-II molecules and IDO in HCEC. Although increased IDO activity does not influence the T-cell response, it constitutes an inflammatory marker of the alloimmune response toward HCEC.

Evaluation of Irradiated Corneas Using Scatterometry and Light and Electron Microscopy.

OBJECTIVE: To determine if sterilization of donor corneas by irradiation alters optical, histologic, or ultrastructural tissue characteristics. METHODS: Blinded assessment of 10 irradiated and 10 nonirradiated donor corneas by a scatterometer used to objectively measure backscattered corneal light. Light and electron microscopy were performed on samples from both groups. RESULTS: The mean scattering of the nonirradiated (control) corneas was significantly lower (0.0060 ± 0.0034, mean ± SD) than the scattering of the irradiated corneas (0.023 ± 0.0078; P = 0.0001). There was no statistical difference between ages, days in storage, or central corneal thickness of the 2 groups. Light microscopic evaluation revealed attenuation of the irradiated corneal epithelium with superficial stromal clefting. Transmission electron microscopy demonstrated similar fibril diameter in the nonirradiated and irradiated corneas. Although the mean interfibrillar distance was similar between the nonirradiated and irradiated groups, the interfibrillar distance in the superficial stroma was increased in the nonirradiated compared with the irradiated groups (15.4 ± 2.4 vs. 11.8 ± 0.98 nm; P ≤ 0.05). CONCLUSIONS: Our data quantitatively support increased light scatter after irradiation, which cannot be attributed to increased corneal thickness. Irradiation alters the histology of the epithelium and the ultrastructure of the superficial corneal stroma. Increased light scatter because of epithelial changes would not be expected to impact postoperative visual function. However, increased light scatter because of stromal changes may affect postoperative visual function if used for optical corneal rehabilitation. Further investigation is warranted to determine whether attenuated epithelium or superficial stromal changes are the cause of increased light scatter in the irradiated corneal tissue.

Transforming growth factor-ß signaling pathway activation in Keratoconus.

PURPOSE: To assess the presence of transforming growth factor-ß (TGFß) pathway markers in the epithelium of keratoconus patient corneas. DESIGN: Retrospective, comparative case series of laboratory specimens. METHODS: Immunohistochemistry results for TGFß2, total TGFß, mothers against decacentaplegic homolog (Smad) 2, and phosphorylated Smad2 was performed on formalin-fixed, paraffin-embedded sections of keratoconus patient corneas and normal corneas from human autopsy eyes. Keratoconus patient corneas were divided in two groups, depending on their severity based on keratometer readings and pachymetry. Autopsy controls were age-matched with the keratoconus cases. Immunohistochemistry signal quantification was performed using automated software. Real-time reverse-transcriptase polymerase chain reaction was performed on total ribonucleic acid of epithelium of keratoconus patient corneas and autopsy control corneas. RESULTS: Immunohistochemistry quantification showed a significant increase in mean signal in the group of severe keratoconus cases compared with normal corneas for TGFß2 and phosphorylated Smad2 (P < .05). Immunohistochemistry analysis using antibodies against total TGFß and Smad2 did not show any significant increase in the keratoconus cases versus the autopsy controls. Reverse-transcriptase polymerase chain reaction exhibited elevated messenger ribonucleic acid levels of Smad2 and TGFß2 in severe keratoconus corneal epithelium. CONCLUSIONS: This work shows increased TGFß pathway markers in severe keratoconus cases and provides the rationale for investigating TGFß signaling further in the pathophysiology of keratoconus.

Unfolded protein response in fuchs endothelial corneal dystrophy: a unifying pathogenic pathway?

PURPOSE: To assess for activation of the unfolded protein response in corneal endothelium of Fuchs endothelial corneal dystrophy patients. DESIGN: Retrospective, comparative case series of laboratory specimens. METHODS: Corneal specimens of patients with Fuchs dystrophy and controls with corneal pathologic features other than Fuchs dystrophy were evaluated by transmission electron microscopy (TEM) to evaluate for structural changes of the rough endoplasmic reticulum in corneal endothelium. TEM images were evaluated for alterations of rough endoplasmic reticulum as a sign of unfolded protein response. Normal autopsy eyes, Fuchs dystrophy corneas, and keratoconus corneas were used for immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of patient corneas for 3 unfolded protein response markers (GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein) and 2 apoptosis markers (caspase 3 and 9). Immunohistochemistry signal quantitation of corneal endothelium for evaluation of marker expression was performed using automated software. Corneal sections were assessed quantitatively for levels of immunohistochemistry marker expression. RESULTS: TEM showed enlargement of rough endoplasmic reticulum in corneal endothelium of all Fuchs dystrophy specimens. Immunohistochemistry quantitation demonstrated a significant increase in mean signal in corneal endothelium from Fuchs dystrophy patients for markers GRP78, the alpha subunit of eukaryotic initiation factor 2, C/EBP homologous protein, and caspase 9 compared with non-Fuchs dystrophy corneas (P < .05). CONCLUSIONS: Results of both TEM and immunohistochemistry indicate activation of unfolded protein response in Fuchs dystrophy. Unfolded protein response activation leads to endothelial cell apoptosis in Fuchs dystrophy and may play a central pathogenic role in this disease.

Evaluation of phacoemulsification-induced oxidative stress and damage of cultured human corneal endothelial cells in different solutions using redox fluorometry microscopy.

PURPOSE: To describe the basic concept of redox fluorometry microscopy and investigate its efficacy in evaluating the state of cultured human corneal endothelial cells in different solutions when ultrasonic energy was applied in vitro. METHODS: Human corneal endothelial cells from human donor tissue not suitable for transplantation were cultured. A phacoemulsification probe with a 30° round, 1.1-mm TurboSonics(®) ABS™ Tip (Alcon, Fort Worth, Texas) was introduced into culture dishes filled with balanced salt solution (BSS) and BSS plus (Alcon, Fort Worth, Texas). Cellular autofluorescence images were obtained using a Zeiss inverted microscope. The redox fluorometric ratio, which can be related to cellular metabolism, mitochondrial distribution patterns, which can shift in reaction to environmental changes, and cell size were analysed with a software program. RESULTS: Human corneal endothelial cells exposed to increasing phacoemulsification times and ultrasonic energy displayed dose-dependent decreases in redox ratios. At a lower ultrasonic power and time, BSS plus showed significantly less change in redox ratio than BSS and control (p < 0.05, Mann-Whitney test). As ultrasonic power and time increased, BSS plus had no more significance. CONCLUSION: Redox fluorometry, with further technological improvement, might be an interesting and potentially useful tool for evaluation of phacoemulsification-induced corneal endothelial damage and screening of protective agents in vitro.

Assessment of attachment factors for primary cultured human corneal endothelial cells.

PURPOSE: To assess the ability of various attachment factors to promote attachment of primary cultured human corneal endothelial cells. MATERIALS AND METHODS: Primary cultured human corneal endothelial cells (HCEC) were incubated for 2 hours in 24-well plates. Wells had been precoated with commercially available cell attachment improvement media (FNC coating mix), human collagen I, human fibronectin, fibronectin/collagen I, or poly-d-lysine. Ratios of cell count before and after rinsing with culture medium and ratios of cells showing morphological signs of spreading to total cells were calculated to measure effectiveness of attachment factors. RESULTS: Incubation of HCEC for 2 hours in wells without precoating of attachment factors led to a rate of 41 +/- 16% (mean +/- SD) of cells showing signs of spreading. FNC coating mix, collagen I, and fibronectin/collagen I increased significantly the percentage of cells showing morphological features of attachment at 2 hours. Total cell loss was highest with poly-d-lysine and no pretreatment with attachment factor. Without the use of any attachment factor, 67 +/- 19% of cells remained after rinsing. The lowest cell loss was observed with FNC coating mix where 108 +/- 5% of cells remained after rinsing. CONCLUSION: Collagen I, collagen I/fibronectin, and FNC coating mix significantly enhance the spreading of human corneal endothelial cells to tissue culture plates after 2 hours. FNC coating mix significantly reduces cell loss due to rinsing. Without the use of any attachment factor, 67% of the cells remained in situ after rinsing.

A comprehensive analysis of eye bank-prepared posterior lamellar corneal tissue for use in endothelial keratoplasty.

PURPOSE: The purpose of this study was to assess eye bank-prepared corneal tissue with regards to the accuracy of postcut tissue thickness, endothelial cell loss, and rate of successful processing. METHODS: Details of all 913 corneal tissues processed with an automated microkeratome for use in posterior lamellar transplantation, over a 1-year period, were obtained from a large eye bank. The number and success rate of all attempted cutting procedures were analyzed. The thickness of the corneal button obtained after cutting was compared with the graft thickness requested by the operating surgeon. Changes in endothelial cell density (ECD) during tissue processing were evaluated. RESULTS: The rate of successful tissue preparation increased over the time period examined, from 95% in the first quarter to 99.5% in the fourth quarter. Graft material was frequently slightly thicker than requested by the operating surgeon with 28.3% of tissues cut thicker than requested. Postcut ECD over the entire period increased by an average of 4.7% and was closely related to the starting ECD. CONCLUSIONS: There was a very high rate of successful tissue preparation (98.5%), and early failed attempts at tissue cutting were likely the result of the initial learning curve of the involved technicians. Practical considerations resulted in tissue being cut marginally thicker than requested; this is an issue about which the operating surgeon should be aware, because it may possibly influence tissue handling. The quality of the obtained material, as measured by ECD, was excellent, although the calculated ECD may be prone to measurement artifact.

The effect of phacoemulsification energy on the redox state of cultured human corneal endothelial cells.

OBJECTIVE: To evaluate the effects of phacoemulsification energy on the redox state and mitochondrial distribution of cultured human endothelial cells. METHODS: Human corneal endothelial cells from fresh banked human donor tissue not suitable for transplantation were harvested and cultured. Cellular autofluorescence images were obtained using an inverted microscope. The redox fluorometric ratio, which can be related to oxidative stress, was calculated as the net value of fluorescence from the 4,6-diamidino-2-phenylindole channel divided by the net value of fluorescence from the fluorescein isothiocyanate-conjugated channel after subtraction of background. For determining the mitochondrial distribution patterns, the cell area was divided by drawing a line halfway between the nuclear and cell membranes. The average fluorescence in the central area was divided by the average fluorescence in the peripheral area. This ratio was compared. RESULTS: Human corneal endothelial cells exposed to increasing phacoemulsification times and increasing ultrasonic energy levels displayed dose-dependent decreases in measured redox ratios. Lower redox ratios in response to phacoemulsification did not associate with decreases in cell size or altered patterns of mitochondrial localization. CONCLUSION: Redox fluorometry may serve as a useful indicator for the in vitro study of human corneal endothelial cell physiological response to ultrasonic stressors and potentially other nonoxidative stressors. CLINICAL RELEVANCE: Redox fluorometry in combination with human corneal endothelial cell morphometric measurements has potential to serve as an indicator of human corneal endothelial cell injury resulting secondary to ultrasound phacoemulsification.