Porcine Corneal Endothelial Cells Research Articles

Temperature Dependence of Membrane Permeability Parameters for Five Cell Types Using Nonideal Thermodynamic Assumptions to Mathematically Model Cryopreservation Protocols.

Cryopreservation is the process of preserving biological matter at subzero temperatures for long-term storage. During cryopreservation, cells are susceptible to various injuries that can be mitigated by controlling the cooling and warming profiles and cryoprotective agent (CPA) addition and removal procedures. Mathematical modeling of the changing cell volume at different temperatures can greatly reduce the experiments needed to optimize cryopreservation protocols. Such mathematical modeling requires as inputs the cell membrane permeabilities to water and CPA and the osmotically inactive fraction of the cell. Since the intra- and extracellular solutions are generally thermodynamically nonideal, our group has been incorporating the osmotic virial equation to model the solution thermodynamics that underlie the cell volume change equations, adding the second and third osmotic virial coefficients of the grouped intracellular solute to the cell osmotic parameters that must be measured. In our previous work, we reported methods to obtain cell osmotic parameters at room temperature by fitting experimental cell volume kinetic data with equations that incorporated nonideal solution thermodynamics assumptions. Since the relevant cell volume excursions occur at different temperatures, the temperature dependence of the osmotic parameters plays an important role. In this work, we present a new two-part fitting method to obtain five cell-type-specific parameters (water permeability, dimethyl sulfoxide permeability, osmotically inactive fraction, and the second and third osmotic virial coefficients of the intracellular solution) from experimental measurements of equilibrium cell volume and cell volume as a function of time at room temperature and 0 °C for five cell types, namely, human umbilical vein endothelial cells (HUVECs), H9c2 rat myoblasts, porcine corneal endothelial cells (PCECs), the Jurkat T-lymphocyte cell line, and human cerebral microvascular endothelial cells (hCMECs/D3 cell line). The fitting method in this work is based on both equilibrium and kinetic cell volume data, enabling us to solve some technical challenges and expand our previously reported measurement technique to 0 °C. Finally, we use the measured parameters to model the cell volume changes for a HUVEC cryopreservation protocol to demonstrate the impact of the nonideal thermodynamic assumptions on predicting the changing cell volume during freezing and thawing.

Oxidative Stress Induces a Breakdown of the Cytoskeleton and Tight Junctions of the Corneal Endothelial Cells.

Purpose: To investigate the impact of oxidative stress, which is a hallmark of Fuchs dystrophy, on the barrier function of the corneal endothelial cells. Methods: Experiments were carried out with cultured bovine and porcine corneal endothelial cells. For oxidative stress, cells were supplemented with riboflavin (Rf) and exposed to UV-A (15-30 min) to induce Type-1 photochemical reactions that release H2O2. The effect of the stress on the barrier function was assayed by transendothelial electrical resistance (TER) measurement. In addition, the associated changes in the organization of the microtubules, perijunctional actomyosin ring (PAMR), and ZO-1 were evaluated by immunocytochemistry, which was also repeated after direct exposure to H2O2 (100 μM, 1 h). Results: Exposure to H2O2 led to the disassembly of microtubules and the destruction of PAMR. In parallel, the contiguous locus of ZO-1 was disrupted, marking a loss of barrier integrity. Accordingly, a sustained loss in TER was induced when cells in the Rf-supplemented medium were exposed to UV-A. However, the addition of catalase (7,000 U/mL) to rapidly decompose H2O2 limited the loss in TER. Furthermore, the adverse effects on microtubules, PAMR, and ZO-1 were suppressed by including catalase, ascorbic acid (1 mM; 30 min), or pretreatment with p38 MAP kinase inhibitor (SB-203580; 10 μM, 1 h). Conclusions: Acute oxidative stress induces microtubule disassembly by a p38 MAP kinase-dependent mechanism, leading to the destruction of PAMR and loss of barrier function. The response to oxidative stress is reminiscent of the (TNF-α)-induced breakdown of barrier failure in the corneal endothelium.

Therapeutic potential of Rho-associated kinase inhibitor Y27632 in corneal endothelial dysfunction: an in vitro and in vivo study

To investigate the effects of a selective inhibitor of Rho-associated kinase (ROCK), Y-27632, on inbred Wuzhishan porcine corneal endothelial cells (PCECs) in vitro and in vivo studies. Primary PCECs were trypsinized from Wuzhishan miniature porcine corneal tissues. The optimal concentration of Y-27632 on PCECs was determined through MTT and 5-ethynyl-2'-deoxyuridine (EdU)-labeling assays. Seven New Zealand rabbits were used as a corneal endothelial dysfunction model, and a PCECs suspension supplemented with Y-27632 was injected into the anterior chamber of the rabbits. The progression of rabbit corneal opacity and edema were observed by slit lamp examination. The rabbits were sacrificed, and rabbit globes were enucleated for trypan blue-alizarin red staining, hematoxylin-eosin staining, and immunofluorescence analysis. Administration of 100 µmol/L Y-27632 facilitated PCECs' proliferation obviously. The rabbit corneas injected with PCECs suspension and 100 µmol/L Y-27632 were restored to transparency significantly after 14d. The 100 µmol/L Y-27632 treatment improves PCECs' proliferation significantly. And our results suggest that Y-27632 and PCECs can be used to treat corneal endothelial dysfunction.

Effects of Perfluorobutylpentane (F4H5) on Corneal Endothelial Cells

ABSTRACTPurpose: To evaluate the effects of perfluorobutylpentane (F4H5) on corneal endothelial cell density (ECD) and morphology using a porcine corneal endothelial organ culture model.Materials and methods: “Split corneal buttons” were cultivated for 15 days (d) after incubation in F4H5 (15, 30, 60, and 120 min) or BSS (controls). ECD was assessed manually on d1, d8, and d15. After histological staining (trypan blue, alizarin red S) on d15 morphological changes (reformation figures, rosette formations, and alizarin red cells) were evaluated.Results: ECD was significantly reduced after incubation in F4H5 for 120 min (median ± 25%/75%-quartile; 3281 ± 43/222 cells/mm2; p = 0.046) on d15 compared to controls (3658 ± 129/296 cells/mm2), but not after shorter incubation times (15, 30, and 60 min). Morphological assessment supports these findings as reformation figures (F4H5 120 min: 10.5 ± 9.3/13.9/mm2 vs. controls: 5.2 ± 2.8/7.2/mm2; p = 0.010), rosette formations (F4H5 120 min 25.566 ± 17.044/36.219/mm2 vs. controls: 8.333 ± 0.000/15.667/mm2; p = 0.002), and alizarin red cells (F4H5 120 min: 38.350 ± 29.827/51.333/mm2 vs. controls: 20.833 ± 10.417/25.000/mm2; p = 0.049) were significantly more prevalent after incubation in F4H5 for 120 min compared to controls. Also, F4H5 60 min showed significantly more rosette formations (25.452 ± 16.968/36.057/mm2; p = 0.006) and alizarin red cells (46.662 ± 42.420/50.903/mm2; p = 0.007), but not reformation figures (7.0 ± 2.2/1.6 %; p = 0.953).Conclusion: Short exposure (≤30 min) of porcine corneal endothelial cells to F4H5 does not have significant effects on ECD or morphological characteristics. Longer exposure times (≥60–120 min) may cause ECD decline and/or induce morphological changes.

Evaluation of the Suitability of Biocompatible Carriers as Artificial Transplants Using Cultured Porcine Corneal Endothelial Cells

ABSTRACTPurpose/Aim: Evaluating the suitability of bioengineered collagen sheets and human anterior lens capsules (HALCs) as carriers for cultivated porcine corneal endothelial cells (pCECs) and in vitro assessment of the cell-carrier sheets as tissue-engineered grafts for Descemet membrane endothelial keratoplasty (DMEK).Materials and Methods: pCECs were isolated, cultured up to P2 and seeded onto LinkCell™ bioengineered matrices of 20 µm (LK20) or 100 µm (LK100) thickness, and on HALC. During expansion, pCEC viability and morphology were assessed by light microscopy. ZO-1 and Na+/K+-ATPase expression was investigated by immunohistochemistry. Biomechanical properties of pCEC-carrier constructs were evaluated by simulating DMEK surgery in vitro using an artificial anterior chamber (AC) and a human donor cornea without Descemet membrane (DM).Results: During in vitro expansion, cultured pCECs retained their proliferative capacity, as shown by the positive staining for proliferative marker Ki67, and a high cell viability rate (96 ± 5%). pCECs seeded on all carriers formed a monolayer of hexagonal, tightly packed cells that expressed ZO-1 and Na+/K+-ATPase. During in vitro surgery, pCEC-LK20 and pCEC-LK100 constructs were handled like Descemet stripping endothelial keratoplasty (DSEK) grafts, i.e. folded like a “taco” for insertion because of challenges related to rolling and sticking of the grafts in the injector. pCEC-HALC constructs behaved similar to the DMEK reference model during implantation and unfolding in the artificial AC, showing good adhesion to the bare stroma.Conclusions: In vitro DMEK surgery showed HALC as the most suitable carrier for cultivated pCECs with good intraoperative graft handling. LK20 carrier showed good biocompatibility, but required a DSEK-adapted surgical protocol. Both carriers might be notional candidates for potential future clinical applications.

Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me2SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me2SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.

Expansion and cryopreservation of porcine and human corneal endothelial cells

Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me2SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me2SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.

Corneal Endothelial Cell Migration and Proliferation Enhanced by Rho Kinase (ROCK) Inhibitors in In Vitro and In Vivo Models.

PurposeTo explore the role of Rho-associated kinases (ROCK) in corneal physiology and regeneration, and the effects of suppressing its activity in stimulating corneal endothelial cell proliferation and migration in vitro and in vivo.MethodsImmunohistochemistry was performed to detect RhoA and ROCK-1 and ROCK-2 in human corneal tissue. Adult porcine corneal endothelial cells (CECs) were isolated, grown to confluence, and further characterized. Under the treatment of ROCK inhibitors, changes in the cellular distribution profile of ZO-1 and F-actin were examined by immunofluorescence staining. Corneal endothelial cells migration was evaluated by scratch assay and analyzed with Axiovision software. Cell proliferation was quantified using Click-iT EdU HCS Assay. In vivo, the corneal endothelia of rabbits were surgically injured and H-1152 was topically applied for 10 days. Progress of wound healing was evaluated daily by monitoring corneal edema, inflammation, and thickness using slit-lamp examination, photography, and pachymetry. Rabbits were euthanized and enucleated for further evaluation.ResultsH-1152 exhibited significant stimulatory effect on CEC migration and proliferation in vitro compared with both untreated and Y-27632–treated cells. Furthermore, topical administration of H-1152 led to marked reduction in corneal edema and formation of multinucleate CECs in vivo suggestive of proliferation associated with healing.ConclusionsH-1152 exhibited a better stimulatory effect on CEC migration and proliferation in vitro than Y-27632. Our findings suggest that topical administration of H-1152 promotes healing of injured corneal endothelium in vivo. These results demonstrate the efficacy of ROCK inhibitors as a potential topical therapy for patients with corneal endothelial disease.

Biophysico-functional compatibility of Seoul National University (SNU) miniature pig cornea as xenocorneal graft for the use of human clinical trial.

Xenocorneal transplantation is one of the solutions for shortage of donor cornea, and remarkable advances have been made in pig-to-rhesus studies from the immunological perspective. Most successful preclinical trials have been carried out with corneas of the Seoul National University (SNU) miniature pig (SNU pig, genetically unmodified) as donor tissues; however, there has been no biophysico-functional evaluation of the SNU pig cornea as a substitute for human cornea. The purpose of this study was to investigate the biophysical and functional compatibility of SNU pig cornea for use in human clinical trials. Ninety-three eyeballs obtained from 51 SNU pigs were used to evaluate the physical properties and changes in porcine corneal endothelial cells (PCECs) depending on preservation time and storage condition before surgery, proliferative and functional characteristics of PCECs, and the microbiologic safety of porcine cornea. Corneal diameters and curvatures, axial length, anterior chamber depth, and central corneal thickness were measured and compared with previously reported human data. Corneal endothelial cell density (ECD) was serially measured with a confocal microscope during 7 days of preservation in the same storage solution used for human corneas. Corneal endothelial cell proliferation and immunofluorescence staining of Na- and K-dependent ATPase in PCECs were evaluated after 7 days of preservation. The corneoscleral rims of SNU pigs were cultured for gram-positive bacteria, gram-negative bacteria, and fungi to evaluate their microbiological safety. Corneal diameter and thickness in SNU pigs was larger than human and corneal curvature was flatter; however, they were within surgically operable ranges. Mean ECD (day 0) and ECD loss after 7 days of preservation were 2625 ± 81 cells/mm(2) and 7.60 ± 1.53%, respectively, which is comparable to human ECD and ECD loss in the same conditions. The ECD of SNU pigs was inversely decreased with aging (R(2) = 0.4034, P = 0.001), and the estimated ages of pigs whose mean ECD would be more than 2500 and 2200 cells/mm(2) or more were 48 and 72 months or less, respectively. Mean doubling time of the endothelial cells was 52 to 96 h depending on the method used. The Na- and K-dependent ATPase pump in SNU pig cornea was well maintained for 7 days. No cultured microorganisms were found upon using the modified European Eye Bank Association protocol, which included additional antiseptic management during the enucleation procedure. In conclusion, SNU pig cornea is feasible for xenocorneal transplantation using the same preservation protocol as human with respect to biophysical and functional properties and can be stored for up to 7 days for transplantation in human clinical trials. An age limitation of donor pigs may be required for qualified corneal products to be used in human trials.

Isolation, characterization and cryopreservation of porcine corneal endothelial cells

Isolation, characterization and cryopreservation of porcine corneal endothelial cells

Mechanism of 5-Fluorouracil-Induced Apoptosis on Cultured Corneal Endothelial Cells

Repeated subconjunctival injections with 5-fluorouacil (5-FU) after trabeculectomy are widely used in glaucoma patients for the inhibition of excess scar formation in wound site. The aim of this study was to evaluate the toxic effects of 5-FU and mechanisms of drug-induced apoptosis in cultured porcine corneal endothelial cells. Cellular damage and the caspase pathway were estimated with a MTT assay. The apoptotic characteristics were detected with flow cytometry, a TUNEL test and Western blotting. The results indicated that 5-FU was toxic to corneal endothelial cells in a time- and dose-dependent manner. Pretreatment with a general caspase inhibitor, Z-VAD-FMK, a caspase-8 inhibitor, Z-IETD-FMK, and a caspase-9 inhibitor, Z-LEHD-FMK, reversed 5-FU-induced cellular damage. Following exposure to 5-FU, a flow cytometric assay with MitoLight dye demon-strated the loss of mitochondrial membrane potential. A positive TUNEL test revealed that cellular DNA apoptosis occurred following exposure to 0.05, 0.1, and 0.5 mg/ml 5-FU for 15 h. Annexin V-FITC and negative propidium iodide (PI) staining indicated that the cell membrane underwent apoptosis upon exposure to 0.1 and 0.5 mg/ml 5-FU for 15 h. The Western blot assay demonstrated up-regulation of the Bax, p53 and p21 proteins induced by 5-FU. Taken together, these data reveal that 5-FU-induced cellular apoptosis in corneal endothelial cells may be mediated through caspase-8, caspase-9 and mitochondrial regulated pathways, as well as by up-regulation of Bax-, p53-, and p21-dependent signal transduction pathways.

Safety of Moxifloxacin and Voriconazole in Corneal Storage Media on Porcine Corneal Endothelial Cells

The aim of this study was to evaluate the safety of moxifloxacin and voriconazole as a supplement in corneal storage media for porcine corneal endothelial cells. Twenty-eight eyes were divided into 4 groups. In the control group (the C group), corneal buttons were stored for 3 days in Optisol-GS. In the treatment group, the corneal buttons were preserved for 3 days in Optisol-GS mixed with 250 microg/mL moxifloxacin (M group), 100 microg/mL voriconazole (V group), and 250 microg/mL moxifloxacin plus 100 microg/mL voriconazole (MV group). We evaluated the samples via specular microscopy before and after 3 days of preservation. The mean changes of endothelial cell counts were compared among the 4 groups. Scanning electron microscopy was conducted after 3 days of preservation. Before the preservation, the endothelial cell counts did not differ among the 4 groups (P > 0.05). After 3 days of preservation, the endothelial cell count in the MV group was the lowest among the 4 groups (P < 0.05). After 3 days of preservation, the rate of corneal endothelial cell loss in the M and V groups did not differ significantly from the control group (P > 0.05). The rate of endothelial cell loss in the MV group was significantly higher than that of the control group (P < 0.05). Scanning electron microscopy revealed a normal mosaic pattern for the C, V, and F groups, but hexagonality was not preserved in the MV group. Preservation in Optisol-GS mixed with moxifloxacin (250 microg/mL) plus voriconazole (100 microg/mL) induced significant toxicity on the endothelial cells in porcine corneas, when compared with the control group.

DNA Microarray-Based Gene Expression Profiling in Porcine Keratocytes and Corneal Endothelial Cells and Comparative Analysis Associated with Xeno-related Rejection

Porcine to rat corneal xenotransplantation resulted in severe inflammation and rejection of the corneal stroma, whereas an allograft showed mainly endothelial cell-associated rejection. We, therefore, investigated and compared the gene expression between porcine keratocytes and corneal endothelial cells. RNA was isolated from primary cultured porcine or human keratocytes and porcine corneal endothelial cells. Gene expression was comparatively analyzed after normalization with microarray method using Platinum pig 13 K oligo chip (GenoCheck Co., Ltd., Ansan, Korea). Real-time polymerase chain reaction (PCR) was performed for C1R, CCL2, CXCL6, and HLA-A in porcine keratocytes and corneal endothelial cells. As a result, upregulated expression more than 2 folds was observed in 1,162 genes of porcine keratocytes versus porcine endothelial cells. Among the immune-regulatory genes, SEMA3C, CCL2, CXCL6, F3, HLA-A, CD97, IFI30, C1R, and G1P3 were highly expressed in porcine keratocytes, compared to porcine corneal endothelial cells or human keratocytes. When measured by real-time PCR, the expression of C1R, CCL2, and HLA-A was higher in porcine keratocytes compared to that in porcine corneal endothelial cells. In conclusion, the increased expression of C1R, CCL2, and HLA-A genes in porcine keratocytes might be responsible for the stromal rejection observed in a porcine to rat corneal xenotransplantation.

Susceptibility of Porcine Keratocytes to Immune-Mediated Damage in Xeno-Related Rejection

We admixed cultured porcine keratocytes or corneal endothelial cells in the presence of human sera or peripheral blood mononuclear cells (PBMCs) for 4 to 72 hours to investigate their immune-related susceptibilities to xeno-related rejection. We evaluated complement deposition at 48 hours by flow cytometry after staining with the C3 anti-goat cy3 antibody. The inhibition of proliferation of porcine corneal cells by human sera was examined using the 3-[4,5-dimethy/thiazol-2,5-dephenyl tetrazolium bromide (MTT) assay over 24 to 72 hours. The amount of 51chromium (Cr)-release was estimated after a reaction between the porcine cells and human PBMCs for 4 hours. There was greater C3 deposition in keratocytes (60.2%) than in endothelial cells (26.9%; P = .05, Mann-Whitney U test). Both keratocytes and endothelial cells showed significant levels of proliferative inhibition over a period of 72 hours. The number of 51Cr-release cells on interleukin-2 addition was significantly higher among keratocytes (88.0%) than endothelial cells (51.4%) at a 1:100 target:effector ratio ( P = .04, Mann-Whitney U test). Our present data suggested that porcine keratocytes might be key target cells in xeno-related rejections when the porcine cornea is transplanted to primates.

Corneal and conjunctival toxicity of disinfectants—Assessing safety for use with ophthalmic surgical instruments

We investigated the corneal toxicity of ortho-phthalaldehyde (Cidex ROPA, Johnson and Johnson K.K.) and its predecessor glutaraldehyde (Cidex R, Johnson and Johnson K.K.). We made primary cultures of porcine and human corneal endothelial cells. Commercially available cell lines were also used including human, bovine, and rabbit corneal epithelium and human conjunctival cells. Following incubation for two days, cell survival was measured using a WST-1 assay for endothelia and a MTT assay for the other cells. Test solutions included 2.25% and 3.5% glutaraldehyde and 0.55% ortho-phthalaldehyde. Cell survival was presented as a percentage of the control value. ortho-phthalaldehyde displayed less toxicity than glutaraldehyde for all cell types tested. As expected 3.5% glutaraldehyde was slightly more toxic than 2.25% glutaraldehyde. When primary human corneal endothelial cultures were exposed to ortho-phthalaldehyde, the survival rates were 88% for 100-fold dilutions and 95% for 500-fold dilutions. The survival rates for all cells tested were greater than 90% when dilutions of 1000-fold or more were used. In conclusion, the corneal toxicity of glutaraldehyde and ortho-phthalaldehyde appears to be within safe levels following washing procedures and therefore the use of these disinfectants may be suitable for selected ophthalmic surgical instruments in urgent or under-equipped circumstances.

Mechanism of unoprostone-induced cytosolic Ca 2+ mobility in cultured porcine corneal endothelial cells

The effect of unoprostone isopropyl on the intracellular free Ca 2+ signaling ([Ca 2+] i) in cultured porcine corneal endothelial cells was evaluated by using fura-2-AM as a Ca 2+ probe. In Ca 2+-containing buffer and Ca 2+-free buffer, unoprostone increased [Ca 2+] i concentration dependently from 28.2 to 0.282 μM, diluted from original concentration to 1/100, 1/1000 and 1/10,000. The response was inhibited on extracellular Ca 2+ removal. In Ca 2+-free buffer, pretreatment of the cells with unoprostone inhibited the endoplasmic reticulum Ca 2+ pump inhibitor thapsigargin-induced [Ca 2+] i increase and the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced Ca 2+ release by 96 and 95%, respectively. Pretreatment of the cells with thapsigargin or CCCP also inhibited unoprostone-1-induced [Ca 2+] i rise by 84 and 57%, respectively. The intracellular calcium release caused by unoprostone was partially inhibited by phospholipase C inhibitor (U73122) and by phospholipase A 2 inhibitor aristolochic acid. After incubation of the cells with unoprostone in Ca 2+-free buffer, the addition of 5 mM CaCl 2 increased Ca 2+ influx, implying that release of Ca 2+ from internal stores caused by unoprostone further induced capacitative Ca 2+ entry. These results suggest that unoprostone-induced [Ca 2+] i increase in corneal endothelial cells results from Ca 2+ influx from external buffer and release of Ca 2+ from intracellular stores from the endoplasmic reticulum and mitochondria Ca 2+ stores followed by capacitative Ca 2+ entry. Unoprostone-induced intracellular Ca 2+ release was also modulated by phospholipase A 2 and C-coupled events.

Cold-Induced Injury to Porcine Corneal Endothelial Cells and Its Mediation by Chelatable Iron

During hypothermic storage of the cornea, corneal endothelial damage restricts storage times. We previously reported a new, iron-dependent mechanism of cold-induced injury to cultured liver cells. In this study, we sought to evaluate whether corneal endothelial cells incur a similar kind of injury. Cultured porcine corneal endothelial cells were exposed to 4 degrees C in either cell culture medium, Krebs-Henseleit buffer, Optisol-GS solution, or McCarey-Kaufman medium for 5 hours to 14 days and then rewarmed under cell culture conditions (3 hours). The cultures were assessed for lethal cell injury (LDH release); cellular, nuclear, and mitochondrial morphologic alterations; lipid peroxidation; and mitochondrial membrane potential. Corneal endothelial cells sustained substantial injury following cold storage and rewarming in cell culture medium (47% +/- 8% and 64% +/- 20% cell death after 2 and 5 days cold storage, respectively). The injury displayed some apoptotic features, and cells lost mitochondrial membrane potential before cell death occurred. The iron chelators deferoxamine, 1,10-phenanthroline, and 2,2'-dipyridyl and the antioxidant butylated hydroxytoluene completely inhibited this cell injury. Marked iron-dependent cell injury and lipid peroxidation also occurred during and after cold incubation in Krebs-Henseleit buffer and, most importantly, iron-dependent cell injury was also observed after cold incubation in Optisol solution and in McCarey-Kaufman medium. Cultured porcine corneal endothelial cells incur a strong iron-dependent injury elicited by hypothermia. This cold-induced injury might provide an explanation for the known corneal endothelial susceptibility to hypothermic preservation injury, which thus might be amenable to therapeutic interventions (ie, by iron chelators).

Transplantation of cultured adult human or porcine corneal endothelial cells onto human recipients in vitro. Part II: Evaluation in the scanning electron microscope.

To evaluate the morphology of endothelial monolayers, which have been regrafted onto the denuded Descemet's membrane, with scanning electron microscopy (SEM). Material derived from each of the experimental groups described in part I of this investigation was evaluated in the current study. Recipient corneas, denuded of their native endothelium by mechanical, chemical, or physical debridement, were examined to assess the effectiveness of each technique in killing and removing cells. Porcine or human donor corneal endothelial cells maintained in monolayer culture for up to 10 passages then were seeded onto the denuded Descemet's membranes of recipients in the absence or presence of fibroblast growth factor (FGF). The monolayers thereby established were examined in the SEM, and the morphologic status of individual cells compared with that manifested in normal human donor corneas maintained for 4 weeks in organ culture (reference control). Isolated and cultured human keratocytes regrafted onto the denuded Descemet's membranes of recipient corneas served as nonendothelial control specimens. Tissue was processed for examination in the SEM according to standard techniques. Each of the three methods used to strip recipient corneas of their native endothelium was effective and elicited no gross structural damage to Descemet's membrane. Some small focal defects within this latter layer were, however, observed, these being encountered at higher frequency after mechanical debridement than after chemical or physical stripping. Porcine or human endothelial cells seeded onto the denuded Descemet's membranes of recipient corneas formed stable monolayers. The morphologic status of regrafted cells corresponded to that manifested in monolayer cultures before seeding, porcine ones always being more differentiated than their human counterparts. Poorly differentiated human endothelial cells had a slender, elongated, fibroblast-like appearance, whereas more highly differentiated ones manifested broad, flat, polygonal profiles. Monolayers covered the entire corneal surface and impinged to a variable degree onto the trabecular meshwork, at which juncture cells always assumed a less well-differentiated morphology. FGF consistently effected an increase in differentiation status, and as this became augmented, the capacity of monolayers to violate the corneal-trabecular meshwork border was correspondingly repressed. Seeded keratocytes formed dense, multilayered sheaths, resembling retrocorneal membranes, across the entire corneal surface, trabecular meshwork, and iris root. The surface characteristics of the constituent cells were quite distinct from those manifested by endothelial cells, even the least well-differentiated ones. Regrafting of human corneal endothelial cells onto the denuded Descemet's membranes of recipients resulted in the formation of stable monolayers. Because the morphologic status of seeded cells closely mimicked that manifested in monolayer cultures before transplantation, it may be anticipated that efforts to refine and optimize culturing conditions would yield improvements in this parameter after regrafting. If these expectations can be realized, then the possibility of successfully establishing a "new" and functional endothelium on recipient corneas destined for clinical grafting may well be brought to fruition in the not-too-distant future.

Transplantation of adult human or porcine corneal endothelial cells onto human recipients in vitro. Part I: Cell culturing and transplantation procedure.

To develop a method for grafting endothelial cells isolated from organ-cultured adult human corneas onto the denuded Descemet's membrane of human recipients. Adult human or porcine corneal endothelial cells were isolated and maintained in monolayer cultures before seeding. Recipient corneas were stripped of their own endothelium by one of three different methods (mechanical, chemical, or physical) and the completeness of removal assessed after vital staining. The utility of each method was evaluated by monitoring the quality of attachment of the seeded-cell population. The seeding density of transplanted cells required for optimal results also was determined and the final numeric cell density achieved on recipient corneas after culturing for 7-20 days ascertained. The influence of incubating source cells with fibroblast growth factor (FGF), both on this latter parameter and on cell morphology, also was evaluated. The functional integrity of regrafted endothelium was assessed in 24-h perfusion experiments. The seeding of between 150,000 and 700,000 cells onto recipient corneas, followed by gentle centrifugation to improve attachment, yielded maximal final numeric cell densities of 3,450/mm2 and 1,850/mm2 in porcine and human lines, respectively. Recipient corneas were most effectively denuded of their own endothelium by freezing-and-thawing. The newly established endothelial monolayer remained stable for up to 20 days in organ culture (longest period monitored). FGF treatment did not enhance the final numeric density of cells attained on recipient corneas, but it did have a beneficial effect on their morphology. Only those recipient corneas that exhibited a well-differentiated monolayer of seeded endothelial cells underwent stromal deswelling near to physiologic levels. A practical working model has been developed, whereby recipient corneas stripped of their own endothelium can be furnished with a "new," near-normal endothelium by appropriate manipulations of the seeded-cell population. This now paves the way for a realistic tackling of the problem of endothelial cell paucity in donor corneas destined for transplantation.

Fetal Calf Serum Protects Cultured Porcine Corneal Endothelial Cells from Endotoxin-Mediated Cell Damage

In corneal organ culture, a contamination of sterile culture media with endotoxin is frequently found. Thus, we investigated if the presence of endotoxin affects the viability of cultured porcine corneal endothelial cells. Endotoxin in high concentrations caused morphological cell changes in porcine corneal endothelial monolayer cultures, delayed proliferation and decreased cellular esterase activity of porcine corneal endothelial cells in vitro. The toxic effect of endotoxin was modulated by the fetal calf serum content of the medium, the concentration of endotoxin and the incubation time.