Culture Of Human Corneal Endothelial Cells Research Articles

Effect of Magnetic Microparticles on Cultivated Human Corneal Endothelial Cells.

To investigate effects of magnetic microparticles on movement of magnet controlled human corneal endothelial cells (HCECs). Immortalized HCEC line (B4G12) and primary culture of HCECs were exposed to two commercially available magnetic micro- or nanoparticles, SiMAG (average size 100 nm) and fluidMAG (average size <1000 nm). Cell viability assays and reactive oxygen species production assays were performed. Cellular structural changes, intracellular distribution of microparticles, and expression levels of proteins related to cellular survival were analyzed. Ex vivo human corneas were exposed to microparticles to further evaluate their effects. Magnetic particle-laden HCECs were cultured under the influence of a neodymium magnet. No significant decrease of viability was found in HCECs after exposure to both magnetic particles at concentrations up to 20 µg/mL for 48 hours. However, high concentrations (40 µg/mL and 80 µg/mL) of SiMAG and FluidMAG significantly decreased viability in immortalized HCECs, and only 80 µg/mL of SiMAG and FluidMAG decreased viability in primary HCECs after 48 hours of exposure. There was relative stability of viability at various concentrations of magnetic particles, despite a dose-dependent increase of reactive oxygen species, lactate dehydrogenase, and markers of apoptosis. Ex vivo human cornea study further revealed that exposure to 20 µg/mL of SiMAG and fluidMAG for 72 hours was tolerable. Endocytosed magnetic particles were mainly localized in the cytoplasm. The application of a magnetic field during cell culture successfully demonstrated that magnetic particle-loaded HCECs moved toward the magnet area and that the population density of HCECs was significantly increased. We verified short-term effects of SiMAG and fluidMAG on HCECs and their ability to control movement of HCECs by an external magnetic field. A technology of applying magnetic particles to a human corneal endothelial cell culture and controlling the movement of cells to a desired area using a magnetic field could be used to increase cell density during cell culture or improve the localization of corneal endothelial cells injected into the anterior chamber to the back of the cornea.

Potential Effect of Human Platelet Lysate on in vitro Expansion of Human Corneal Endothelial Cells Compared with Y-27632 ROCK Inhibitor.

PurposeCorneal endothelial cell (CEC) therapy can be used as a promising therapeutic option for patients with various corneal endothelial dysfunctions. In this study, we compared the proliferative effect of human platelet lysate (HPL), as a xeno-free medium supplement, with Y-27632 Rho/rho-associated protein kinase (ROCK) inhibitor, as a well-known proliferative and adhesive agent for CECs, and fetal bovine serum (FBS) as the control, in the culture medium of human corneal endothelial cells (HCECs).MethodsWe isolated HCECs from human donors and treated the cells as three different treatment groups including 20% HPL only, 10 μM Y-27632 ROCK inhibitor, combination of 20% HPL and 10 μM Y-27632 ROCK inhibitor, and 20% FBS as the control group. ELISA cell proliferation assay and cell counting was performed on the treated cells. Finally, HCECs were characterized by morphology and immunocytochemistry (ICC).Results There was no significant proliferative effect of HPL on cell proliferation compared with the cells treated with Y-27632 ROCK inhibitor or the combination of HPL and Y-27632 ROCK inhibitor, but all the respected treatments had significant inducible effect on cell proliferation as compared with FBS-treated cells. The cells grown in all three treatment groups exhibited CEC morphology. Also, there was a higher expression of Na+/K+-ATPase and ZO-1, as CEC characteristic markers, in the culture of HCECs treated with HPL as compared with FBS.ConclusionHPL offers a xenofree and affordable medium supplement for CEC expansion that can be used in clinical applications.

TGF-\u03b21 promotes cell barrier function upon maturation of corneal endothelial cells

Human corneal endothelial cells (HCECs) easily become fibroblastic-like when cultured, rendering them unsuitable for tissue engineering of the cornea. Transforming growth factor β (TGF-β) could be a key factor in this phenomenon; however, TGF-β is also known to maintain the endothelium in a quiescent state in vivo. This work aimed to compare the effects of TGF-β1 on the phenotype of HCECs during the proliferation and maturation phases. Our results show that addition of TGF-β1 during the active proliferation phase produced fibroblastic HCECs and loss of the cell junction markers ZO-1 and n-cadherin, independent from the presence of epidermal growth factor (EGF). By contrast, addition of TGF-β1 in maturation media containing few mitogens led to an endothelial phenotype and functional cell junctions as HCECs developed a high trans-endothelial resistance. Furthermore, addition of AG-1478, an epithelial growth factor receptor inhibitor, enhanced the gain of the endothelial phenotype and cell barrier function. Overall, these results show that TGF-β1 can be used to promote the formation of a typical leaky endothelial barrier during the maturation phase of cultured HCECs. A two-phase culture of HCECs using distinct proliferation and maturation media could also be key for developing ideal HCEC culture conditions.

Improving the success rate of human corneal endothelial cell cultures from single donor corneas with stabilization medium

Main objective of this study was to improve the success rate of human corneal endothelial cell (hCEC) cultures from single donor corneas. We could show that the use of stabilization medium prior to cell isolation may have a positive effect on the success rate of hCEC cultures from single research-grade donor corneas by allowing growth of otherwise possibly not successful cultures and by improving their proliferative rate. hCEC were obtained from corneo-scleral rims of 7 discarded human research-grade cornea pairs. The Descemet membrane–endothelium (DM–EC) sheets of each pair were assigned to 2 experimental conditions: (1) immediate cell isolation after peeling, and (2) storage of the DM–EC sheet in a growth factor-depleted culture medium (i.e. stabilization medium) for up to 6 days prior to cell isolation. hCEC isolated by enzymatic digestion were then induced to proliferate on pre-coated culture plates. The success rate of primary cultures established from single donor corneas were higher for DM–EC sheets kept in stabilization medium before cell isolation. All cultures (7/7) initiated from stabilized DM–EC sheets were able to proliferate up to the third passage, while only 4 out of 7 cultures initiated from freshly peeled DM–EC sheets reached the third passage. In addition, for the 4 successful paired cultures we observed a faster growth rate if the DM–EC sheet was pre-stabilized prior to cell isolation (13.8 ± 1.8 vs 18.5 ± 1.5 days, P < 0.05). Expression of the phenotypical markers Na+/K+-ATPase and ZO-1 could be shown for the stabilized cultures that successfully proliferated up to the third passage.

Use of human serum for human corneal endothelial cell culture

Background/aimsTo study human corneal endothelial cells (HCECs) cultured in vitro with human serum (HS) supplemented media (HS-SM) compared with HCEC cultured in fetal bovine serum (FBS) supplemented media (FBS-SM).MethodsOne cornea...

Cultivation of Corneal Endothelial Cells on a Pericellular Matrix Prepared from Human Decidua-Derived Mesenchymal Cells

The barrier and pump functions of the corneal endothelium are essential for the maintenance of corneal transparency. Although corneal transplantation is the only current therapy for treating corneal endothelial dysfunction, the potential of tissue-engineering techniques to provide highly efficient and less invasive therapy in comparison to corneal transplantation has been highly anticipated. However, culturing human corneal endothelial cells (HCECs) is technically difficult, and there is no established culture protocol. The aim of this study was to investigate the feasibility of using a pericellular matrix prepared from human decidua-derived mesenchymal cells (PCM-DM) as an animal-free substrate for HCEC culture for future clinical applications. PCM-DM enhanced the adhesion of monkey CECs (MCECs) via integrin, promoted cell proliferation, and suppressed apoptosis. The HCECs cultured on the PCM-DM showed a hexagonal morphology and a staining profile characteristic of Na+/K+-ATPase and ZO-1 at the plasma membrane in vivo, whereas the control HCECs showed a fibroblastic phenotype. The cell density of the cultured HCECs on the PCM-DM was significantly higher than that of the control cells. These results indicate that PCM-DM provides a feasible xeno-free matrix substrate and that it offers a viable in vitro expansion protocol for HCECs while maintaining cellular functions for use as a subsequent clinical intervention for tissue-engineered based therapy of corneal endothelial dysfunction.

Identification of Cell Surface Markers Glypican-4 and CD200 That Differentiate Human Corneal Endothelium From Stromal Fibroblasts

There is a lack of definitive cell surface markers to differentiate cultured human corneal endothelial cells (HCECs) from stromal fibroblasts, which could contaminate HCEC cultures. The aim of our study is to discover cell surface antigens on HCECs that can be used to identify and purify HCECs from stromal fibroblasts. RNA sequencing (RNA-seq) was used to find differentially overexpressed genes in HCECs and commercial antibodies against these overexpressed antigens were screened by immunofluorescence assay. Similarly, 242 commercial antibodies against cell-surface antigens also were screened. Selected antibodies were used to sort HCECs from stromal fibroblasts by fluorescence-activated cell sorting (FACS). Two monoclonal antibodies, anti-GPC4 and anti-CD200, were identified to stain HCECs specifically. FACS was used successfully to sort HCECs away from stromal fibroblasts. Recovery efficiency of HCECs after sorting using anti-GPC4 antibody was higher compared to anti-CD200 antibody, but purity of HCECs culture using either antibody was comparable. Taken together, the anti-GPC4 and anti-CD200 antibodies can be useful for purification and identification of HCECs in cultures containing stromal fibroblasts.

Air Toxicity for Primary Human-Cultured Corneal Endothelial Cells

To investigate the possible toxic effect of air exposure for an in vitro model of primary human corneal endothelial cells (HCECs). Primary HCECs were isolated from donor corneal rings and cultivated at 37°C in 5% CO2 and 95% humidified air. Six groups of HCEC cultures were set up, and 4 samples were enclosed in each group: group 1 consisted of samples in which HCECs were exposed to air for 30 minutes. Group 2 consisted of HCECs exposed to air for 1 hour, group 3 for 3 hours, group 4 for 6 hours, group 5 for 12 hours, and group 6 for 24 hours. Three hours after exposure, the morphology of the cells was still intact; however, a few cells within the monolayer appeared enlarged and exhibited characteristics of more senescent cells. Six hours after exposure to air, the endothelial cells started losing their typical hexagonal morphology and appeared enlarged and compromised. Viability was superior to 95% in groups 1 to 3, whereas for groups 4, 5, and 6 was 71%, 22.4%, and 6.3%, respectively. The present study illustrates that the toxic effect of air exposure for the studied in vitro model of primary human-cultured corneal endothelial cells is not significant for the period of 3 hours, whereas after 6 hours it starts to induce major apoptotic mechanisms, leading to reduced viability until the period of 24 hours where the percentage of living cells is drastically decreased.

Impact of photodynamic inactivation (PDI) using the photosensitizer chlorin e6 on viability, apoptosis, and proliferation of human corneal endothelial cells

Photodynamic inactivation (PDI) may be a potential alternative in case of therapy-resistant infectious keratitis. PDI using the photosensitizer chlorin e6 (Ce6) with high photosensitizing efficacy offers a valuable option, also for keratitis. The purpose of our study was to determine the impact of PDI with the photosensitizer Ce6 on viability, apoptosis, and proliferation of human corneal endothelial cells (HCECs), in vitro. Human corneal endothelial cell line was cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. HCECs cultures underwent illumination using red (670 nm) light for 13 min following exposure to 50-500 nM concentrations of Ce6 in the culture medium. Twenty-four hours after PDI, cell viability was evaluated by the Alamar blue assay, total DNA content of the cells and apoptosis using the APO-DIRECT Kit, and cell proliferation by the BrdU Cell Proliferation Assay Kit. Using Ce6 or illumination only, we did not detect significant changes of cell viability, apoptosis, and proliferation. Following PDI, viability and total DNA content of HCECs decreased significantly above 150 nM Ce6 concentration (P < 0.01; P < 0.05). The percentage of apoptotic HCECs increased significantly from 250 nM Ce6 concentration (P < 0.01), and proliferation of endothelial cells decreased significantly (P < 0.05) above 100 nM concentration of Ce6 after PDI. Photodynamic inactivation using Ce6 decreases viability and proliferation, and also triggers apoptosis of HCECs in vitro. PDI using the photosensitizer Ce6 may be a potential treatment alternative in infectious keratitis. However, to avoid endothelial cell damage, the photosensitizer must not penetrate the endothelium.

Air Toxicity for Primary Human-Cultured Corneal Endothelial Cells

To investigate the possible toxic effect of air exposure for an in vitro model of primary human corneal endothelial cells (HCECs).Primary HCECs were isolated from donor corneal rings and cultivated at 37°C in 5% CO2 and 95% humidified air. Six groups of HCEC cultures were set up, and 4 samples were enclosed in each group: group 1 consisted of samples in which HCECs were exposed to air for 30 minutes. Group 2 consisted of HCECs exposed to air for 1 hour, group 3 for 3 hours, group 4 for 6 hours, group 5 for 12 hours, and group 6 for 24 hours.Three hours after exposure, the morphology of the cells was still intact; however, a few cells within the monolayer appeared enlarged and exhibited characteristics of more senescent cells. Six hours after exposure to air, the endothelial cells started losing their typical hexagonal morphology and appeared enlarged and compromised. Viability was superior to 95% in groups 1 to 3, whereas for groups 4, 5, and 6 was 71%, 22.4%, and 6.3%, respectively.The present study illustrates that the toxic effect of air exposure for the studied in vitro model of primary human-cultured corneal endothelial cells is not significant for the period of 3 hours, whereas after 6 hours it starts to induce major apoptotic mechanisms, leading to reduced viability until the period of 24 hours where the percentage of living cells is drastically decreased.

Culture of human corneal endothelial cells isolated from corneas with Fuchs endothelial corneal dystrophy

The purpose of this study was to assess the feasibility of initiating primary cultures of corneal endothelial cells from patients suffering from Fuchs endothelial corneal dystrophy (FECD; MIM# 1036800). We also evaluated which conditions yielded the best results for culture. Twenty-nine patients undergoing Descemet stripping automated endothelial keratoplasty consented to the use of their excised Descemet's membrane for this study. Out of the 29 specimens, 18 successfully initiated a culture. Cell morphology varied between endothelial (rounded, slightly elongated cells, n = 12) and fibroblastic-like (thin and very elongated cells, n = 6). These differences in cell morphology were also observed with the normal human corneal endothelial cell cultures. The cultures that initially presented an endothelial morphology maintained their shape in subcultures. Clusterin expression was similar in FECD and normal endothelial cells. Transmission electron microscopy of FECD Descemet's membranes showed a high degree of various abnormalities generally found in this disease, such as a thickened Descemet's membrane, presence of a posterior banded layer, presence of a fibrillar layer and striated bodies of various sizes and periodicities. Patient's age was predictive of culture success, all younger FECD donors generating cultures of endothelial morphology. The absence of a fibrillar layer was also a factor associated with greater success. Culture success was not dependent on specimen size, specimen pigmentation, or patient's preoperative central corneal thickness. In conclusion, this paper shows for the first time that central Descemet's membranes of patients suffering from FECD possess proliferative endothelial cells that can be isolated and cultured without viral transduction, opening the way for new in vitro studies of this disease.

Pseudotyping and Culture Conditions Affect Efficiency and Cytotoxicity of Retroviral Gene Transfer to Human Corneal Endothelial Cells

To evaluate retroviral vectors as a tool to transduce normal human corneal endothelial cells (HCECs) and to optimize transduction to increase gene transfer efficiency. Enhanced green fluorescent protein (EGFP) encoding retroviral vectors based on HIV-1 or murine leukemia virus (MLV), pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or a modified foamy virus envelope protein (FV Env), and prototype foamy virus (PFV) were produced. Transduction was performed in four HCEC culture media that were previously described for specific cultivation of HCECs or organ culture of donor corneas, namely enriched HCEC growth medium F99(HCEC), its unsupplemented basal medium F99, MEM + 2% fetal calf serum (FCS) (MEM), and Human Endothelial-SFM (SFM). Transduction efficiency was evaluated by marker gene transfer assay, and cytotoxic effects of virus infection were evaluated by means of resazurin conversion assay. PFV- and HIV-1-based vectors showed superior transduction efficiency compared with MLV-based vectors. Pseudotyping with a modified FV Env increased transduction efficiency compared with pseudotyping with VSV-G. In medium SFM, transduction efficiency of PFV, HIV-1-/FV Env, and MLV-based vectors was markedly reduced compared with the other culture media. When cells were cultured in F99-based media, cell viability was reduced by retroviral transduction compared with uninfected or mock infected controls, but remained unaffected when cells were cultured in SFM and was even increased when cells were cultured in MEM. HIV-1-based vectors pseudotyped with FV Env can efficiently be used to transduce primary HCECs in vitro. However, transduction efficiency is dependent on culture conditions and impairs metabolic activity and viability of HCECs in vitro.

Human Corneal Endothelial Cell Expansion for Corneal Endothelium Transplantation: An Overview

The monolayer of cells forming the human corneal endothelium is critical to the maintenance of corneal transparency and is not known to regenerate in vivo. Thus, dysfunction of these cells constitutes the most often cited reasons for the 150,000 or so corneal transplants performed yearly. Although current corneal transplantation is more than 90% successful at 1 year, longer term results are not as encouraging with approximately 70% success at 5 years. Nonimmunologic graft failure and allograft endothelial rejection are the main problems. Furthermore, the global shortage of donor corneas greatly restricts several corneal transplantations performed. With advances in understanding corneal endothelial cell biology, it is now possible to cultivate human corneal endothelial cells (HCECs) in vitro, thus providing new opportunities to develop novel tissue-engineered human corneal endothelium. This review will provide an overview of (a) the characteristics of human corneal endothelium; (b) past and present HCECs isolation and culture protocols; (c) various potential carriers for the generation of tissue-engineered corneal endothelium, together with some of the functional studies reported in various animal models; and (d) the current rapid advancements in surgical techniques for keratoplasty. A successful combination of tissue-engineered human corneal endothelium coupled with innovative and groundbreaking surgical procedures will bridge basic research involving cultured HCECs, bringing it from bench to bedside.

Adhesion, Migration, and Proliferation of Cultured Human Corneal Endothelial Cells by Laminin-5

To investigate the expression of laminin-5 (LM5) and its receptors by human corneal endothelial cells (HCECs) and whether recombinant human LM5 influences adhesion, proliferation, and migration of cultured HCECs. The expression of LM5 and its receptors was examined in human donor corneas by immunohistochemistry, reverse transcription-polymerase chain reaction, and flow cytometry. HCECs cultured under serum-free conditions were used for analysis of the biological effects of LM5. Changes in HCEC adhesion and proliferation due to LM5 were evaluated by counting the number of cells. HCEC migration was assessed by quantifying the percentage of wound closure in the wound-healing assay with an image-processing and -analysis software program. Adult HCECs expressed the LM5 receptor α3β1 integrin, but not LM5 itself. Significantly more cells became adherent to recombinant LM5 (1.0 μg/mL)-coated dishes than to uncoated dishes in the cell adhesion assay. The proliferation of cultured HCECs was moderately promoted by LM5 (1.0 μg/mL) and soluble LM5 (20 ng/mL and 50 ng/mL) in the cell proliferation assay. A significantly higher percentage of wound closure was obtained with medium containing soluble LM5 than with control medium in the wound-healing assay. HCECs express the LM5 receptor α3β1 integrin. Recombinant LM5 promotes adhesion, migration, and moderate proliferation of cultured HCECs. It may be a critical factor in promoting HCEC culture and may contribute to the practical use of tissue-engineered HCECs.

In Vitro Assessment of the Cytotoxicity of Anti-allergic Eye Drops Using 5 Cultured Corneal and Conjunctival Cell Lines

The aim of this study was to evaluate the cytotoxicity of anti-allergic eye drops for human corneal endothelial cells (HCEC) and commercially available ocular surface cells. A primary HCEC culture was derived from human eye bank specimens. SIRC (rabbit corneal epithelium), BCE C/D-1b (bovine corneal epithelial cells), RC-1 (rabbit corneal epithelium), and Chang (human conjunctival cells) were obtained commercially. The WST-1 assay was used to measure HCEC viability, and the viability of other cells was measured using the MTT assay. Cells were treated with 7 commercially available anti-allergic eye drops for 48 h and cell viability was measured and calculated as a percentage of control. The degree of toxicity for each eye-drop solution was based on the cell viability score (CVS). HCECs treated with a 1000-fold dilution of the eye-drop solution had a viability score of 67% for Rizaben and ≥80% for the other solutions with Zepelin being the least toxic. Cell viability in response to eye-drop solutions preserved with benzalkonium chloride (BAK) was dependent on the concentration of the drug solution and exposure time. Treatment of ocular surface cells with a 20-fold dilution of the eye-drop solution resulted in the following order of cell viability as determined by their CVS: Zepelin > Ketas = Zaditen ≥ Tramelas PF = Patanol ≥ Rizaben ≥ Livostin. This order was similar to that observed for HCECs, and cell viability was found to be concentration-dependent. Based on the penetration of the drug into eye tissues, HCECs are only likely to be pharmaceutically damaging in rare cases. Epithelial cell viability depends primarily on the concentration of BAK rather than on the action of the active component in the eye-drop solution. CVS values were useful for comparison of toxicity.

The M100: Face categorization begins within 100 ms of stimulus presentation

To determine whether mouse embryonic stem cell conditioned medium (ESC-CM) increases the proliferative capacity of human corneal endothelial cells (HCECs) in vitro. Primary cultures of HCECs were established from explants of the endothelial cell layer, including the Descemet's membrane. Cells were cultured in human corneal endothelium medium (CEM) containing 25% ESC-CM for the experimental group and CEM alone for the control group. Phase-contrast microscopy and reverse-transcription polymerase chain reaction (RT-PCR) were used to identify HCECs. The eruption time and HCEC morphology were observed under phase-contrast microscopy. We detected the protein expression of zona occludens protein-1 (ZO-1; a tight junction protein) and the Na(+)-K(+)-ATPase by western blot analysis and immunocytochemistry. The mRNA expression of the Na(+)-K(+)-ATPase, voltage-dependent anion channel 3 (VDAC3), solute carrier family 4, sodium bicarbonate cotransporter member 4 (SLC4A4), and chloride channel protein 3 (CLCN3) were detected by RT-PCR. To explore the proliferation capacity of HCECs, the colony forming efficiency (CFE) was determined by Giemsa staining and the cellular proliferation marker of Ki-67 protein (Ki-67) positive cells were detected by immunocytochemistry and flow cytometry. Progression of the cell cycle and apoptosis were analyzed by flow cytometry. Negative regulation of the cell cycle, as measured by cyclin-dependent kinase inhibitor p21 (p21) levels, was detected by western blot analysis and immunocytochemistry. In primary culture, HCECs in the 25%ESC-CM group erupted with polygonal appearance on day 2, while those in the CEM group erupted with slightly larger cells on day 3-4. HCECs in the 25%ESC-CM group could be subcultured until passage 6 without enlargement of cell volume, while those in the CEM group were enlarged and lost their polygonal appearance by passage 2. HCECs in both the 25%ESC-CM and CEM groups expressed ZO-1, Na(+)-K(+)-ATPase, VDAC3, SLC4A4, and CLCN3. The number of Ki67 positive cells, CFE, and percentage of cells entering the S and G(2) phases were higher in the 25%ESC-CM group than in the CEM group. The number of apoptotic cells and p21 protein expression both decreased in the 25%ESC-CM group. Use of 25%ESC-CM significantly increased the number of proliferating cells. These effects may be achieved through inhibition of p21 expression and apoptosis. These results suggested that 25%ESC-CM may be a new tool for cultivating HCECs for transplantation.

Toxic Effects of Recombinant Tissue Plasminogen Activator on Cultured Human Corneal Endothelial Cells

Human corneal endothelial cells (HCECs) are nonmitotic cells. Any intracameral application of a drug requires evaluation of the potential apoptotic and toxic effects. Intracameral recombinant tissue plasminogen activator (rtPA) is successfully used for the treatment of severe and prolonged postoperative fibrin reaction. This study was undertaken to investigate the toxicity of rtPA on cultured HCECs to determine its safety for clinical use. Cell cultures of HCECs were harvested from human donor eyes and exposed to various concentrations of rtPA (10-200 microg/mL). For cytotoxicity testing, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and the live/dead viability/cytotoxicity assay were performed. Annexin V binding combined with propidium iodide (PI) costaining was used for the distinction of viable, early, and late apoptotic cells. Odds ratios (ORs) and confidence intervals (CIs) were calculated for 50 microg/mL, 100 microg/mL, and 200 microg/mL. Cell morphology was studied after 24 hours of exposure to rtPA to identify cellular damage. Immunolocalization of zonula occludens 1 (ZO1) was performed to analyze intercellular barrier disturbance in the presence of rtPA. Reduction of mitochondrial dehydrogenase activity after rtPA exposure was dose dependent and suggested comparable toxicity with the data obtained from the live/dead assay. The mean number of Annexin V-FITC and PI-positive cells was not significantly increased at concentrations of 50 microg/mL and 100 microg/mL. At 200 microg/mL, however, the ORs were 5.082 +/- 0.213 (95% CI, 3.962-6.203; P < 0.001) for apoptosis and 6.154 +/- 0.196 (95% CI, 5.123-7.181; P < 0.001) for necrosis. In addition, increasing concentrations of rtPA resulted in a fading immunopositive staining for ZO1. These data suggest a dose-dependent toxic effect of rtPA on HCECs in vitro. Although intracameral rtPA concentrations up to 100 mug/mL seem to be clinically safe, the use of concentrations higher than 125 microg/mL might induce irreversible cell death and should be restricted to selected cases.

Intrinsic cytotoxic effects of fluoroquinolones on human corneal keratocytes and endothelial cells

ABSTRACTObjective: To determine the intrinsic cytotoxicity of five fluoroquinolones (ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, ofloxacin) on human corneal keratocytes (HCK) and human corneal endothelial cells (HCE).Research design and methods: Cultures of replicating HCK and HCE were exposed to ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, or ofloxacin concentrations of 1 mg/mL, 100 μg/mL, 10 μg/mL, 1 μg/mL, 100 ng/mL, or 10 ng/mL for 15, 30, 60, or 240 min. Each of the 24 fluoroquinolone concentration–time exposures was tested against its own serum-free minimal essential medium (MEM) control. Cell number was quantified with a fluorescence bioassay.Main outcome measure: Cytotoxicity was defined as a significant ( p < 0.05) difference in cell number measured as mean calcein fluorescence product versus control for each fluoroquinolone concentration–time exposure.Results: Fluoroquinolone-induced cytotoxicity was concentration- and time-dependent in HCK and HCE cultures. The number of cytotoxic concentration–time exposures was highest with ciprofloxacin (23 of 24 exposures in HCK and 24 of 24 exposures in HCE) and lowest with levofloxacin (10 of 24 exposures in both HCK and HCE).Conclusions: In vitro cell cultures are useful for evaluating cell response to potentially toxic insults, although cell cultures may lack tissue components that may prevent or ameliorate damage in vivo. In this assay, fluoroquinolones displayed the potential to be cytotoxic to human corneal keratocytes and endothelial cells, depending on drug concentration and duration of exposure. The potential for cytotoxicity may differ among fluoroquinolones.

P27kip1 siRNA Induces Proliferation in Corneal Endothelial Cells from Young but Not Older Donors

To determine whether small interfering (si)RNA downregulation of the cyclin-dependent kinase inhibitor p27kip1 overcomes G(1)-phase arrest and promotes cell-cycle progression in human corneal endothelial cells (HCECs) from young (<30 years old) and older (>60 years old) donors. Transfection of siRNA was confirmed by incubating confluent cultures of HCECs with FITC-labeled nonsilencing siRNA. Confluent cultures were transfected for 48 hours with p27kip1 siRNA (2.5, 5, 25, or 100 nM) or nonsilencing siRNA, with a lipid transfection reagent. As a comparison, cultures were also transfected for 48 hours with p27kip1 antisense (AS) or missense (MS) oligonucleotides (oligo). At various times after transfection, cells were fixed for immunocytochemical localization of p27kip1 or extracted for Western blot analysis to assess relative p27kip1 protein levels. Cultures were also prepared for ZO-1 immunolocalization, to assess the effect of transfection on the morphology of the monolayer. The number of cells was counted at 0, 48, 96, 144, and 192 hours after incubation, and a cell-viability assay was performed. A dose-dependent decrease in p27kip1 protein level was observed in Western blot analysis, and nuclear staining for p27kip1 was greatly reduced in HCECs incubated with p27kip1 siRNA. No change in p27kip1 levels or in nuclear staining was observed in the nonsilencing control. p27kip1 siRNA (25 nM) appeared to be quantitatively more efficient than antisense oligonucleotide (500 nM) in reducing p27kip1 protein levels. Viability was less affected by siRNA treatment than by AS oligo transfection. ZO-1 staining showed no effect on morphology of the monolayer. The number of HCECs from young donors (<30 years old) transfected with p27kip1 siRNA increased up to 144 hours after incubation, whereas no change in the number of cells was observed in HCECs transfected with nonsilencing siRNA. In contrast to the results from young donors, no change in the number of cells was observed at any time point tested in HCECs from older donors (>60 years old) after p27kip1 siRNA transfection. Transfection of p27kip1 siRNA was sufficient to promote proliferation in confluent cultures of HCECs from younger, but not older donors. These results suggest that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27kip1.

Age Differences in Cyclin-Dependent Kinase Inhibitor Expression and Rb Hyperphosphorylation in Human Corneal Endothelial Cells

Human corneal endothelial cells (HCECs) are considered to be nonreplicative in vivo; however, isolated HCECs can be cultured and grown successfully, indicating that they retain proliferative capacity. This capacity to replicate tends to decrease with donor age. Cyclin-dependent kinase inhibitors (CKIs) are important negative regulators of the cell cycle. Of those CKIs, p16INK4a, p21WAF1/Cip1, and p27Kip1 are expressed in corneal endothelium. To help reveal the mechanism of this age-related difference, the relative expression of those CKIs and the kinetics of hyperphosphorylation of the retinoblastoma protein, Rb, were analyzed in HCECs from various aged donors. Fresh-frozen sections of corneas from an 18-year-old and a 74-year-old donor were immunostained to reveal the expression and localization of the three CKIs in corneal endothelium in situ. HCECs from eight donors of various ages were isolated and cultured until they reached passage 4. After the cells reached confluence, total protein was extracted, and the relative expression of p16(INK4a), p21WAF1/Cip1, and p27Kip1 was determined by Western blot analysis. A parallel analysis was performed with primary cultures of HCECs obtained from eight different donors. Subconfluent passage 2 HCECs from eight donors were serum starved and, at different times after growth factor stimulation, protein was extracted, and Western blot analysis was used to compare the overall expression of Rb protein and the kinetics of Rb hyperphosphorylation. Immunocytochemistry confirmed the expression and nuclear localization of p16(INK4a), p21WAF1/Cip1, and p27Kip1 in HCECs in situ. Western blot studies revealed an age-related increase in p16INK4a and p21WAF1/Cip1 protein expression in cultured HCECs. Expression of p27Kip1 tended to decrease with the donor's age in passage-4 cells; however, there was no significant difference in p27Kip1 expression level between young and older donors in primary cultured HCECs. No age-related difference in total Rb protein was observed in the Western blots; however, the rate of Rb hyperphosphorylation was significantly slower in HCECs from older donors. p16(INK4a), p21WAF1/Cip1, p27Kip1, and Rb were all expressed in HCECs, regardless of donor age. Age-related differences in the relative expression of p16INK4a and p21WAF1/Cip1 and in the kinetics of Rb hyperphosphorylation led to the conclusion that, in addition to the normal inhibitory activity of p27Kip1, there is an age-dependent increase in negative regulation of the cell cycle by p16INK4a and p21WAF1/Cip1. This additional molecular mechanism may be responsible, at least in part, for the reduced proliferative response observed in HCECs from older donors.