Corneoscleral Rings Research Articles

Culturing Limbal Epithelial Cells of Long-term Stored Corneal Donors (Organ Culture) In Vitro - A Stepwise Linear Regression Algorithm.

To assess various potential factors on human limbal epithelial cell (LEC) outgrowth in vitro using corneal donor tissue following long-term storage (organ culture) and a stepwise linear regression algorithm. Of 215 donors, 304 corneoscleral rings were used for our experiments. For digestion of the limbal tissue and isolation of the limbal epithelial cells, the tissue pieces were incubated with 4.0 mg/mL collagenase A at 37 °C with 95% relative humidity and a 5% CO2 atmosphere overnight. Thereafter, limbal epithelial cells were separated from limbal keratocytes using a 20-µm CellTricks filter. The separated human LECs were cultured in keratinocyte serum-free medium medium, 1% penicillin/streptomycin (P/S), 0.02% epidermal growth factor (EGF), and 0.3% bovine pituitary extract (BPE). The potential effect of donor age (covariate), postmortem time (covariate), medium time (covariate), size of the used corneoscleral ring (360°, 270°180°, 120°, 90°, less than 90°) (covariate), endothelial cell density (ECD) (covariate), gender (factor), number of culture medium changes during organ culture (factor), and origin of the donor (donating institution and storing institution, factor) on the limbal epithelial cell outgrowth was analyzed with a stepwise linear regression algorithm. The rate of successful human LEC outgrowth was 37.5%. From the stepwise linear regression algorithm, we found out that the relevant influencing parameters on the LEC growth were intercept (p < 0.001), donor age (p = 0.002), number of culture medium changes during organ culture (p < 0.001), total medium time (p = 0.181), and size of the used corneoscleral ring (p = 0.007), as well as medium time × size of the corneoscleral ring (p = 0.007). The success of LEC outgrowth increases with lower donor age, lower number of organ culture medium changes during storage, shorter medium time in organ culture, and smaller corneoscleral ring size. Our stepwise linear regression algorithm may help us in optimizing LEC cultures in vitro.

Assessment of Cidofovir for Treatment of Ocular Bovine Herpesvirus-1 Infection in Cattle Using an Ex-Vivo Model.

Bovine herpesvirus-1 (BoHV-1) infection contributes to keratoconjunctivitis, respiratory disease, and reproductive losses in cattle. The objective of this study was to determine the most appropriate ophthalmic antiviral agent for BoHV-1 inhibition using in-vitro culture and novel ex-vivo bovine corneal modeling. Half-maximal inhibitory concentrations of BoHV-1 were determined for cidofovir, ganciclovir, idoxuridine, and trifluridine via in-vitro plaque reduction assays. In-vitro cytotoxicity was compared amongst these compounds via luciferase assays. Trifluridine and cidofovir were the most potent BoHV-1 inhibitors in vitro, while trifluridine and idoxuridine were the most cytotoxic agents. Therefore, cidofovir was the most potent non-cytotoxic agent and was employed in the ex-vivo corneal assay. Corneoscleral rings (n = 36) from fresh cadaver bovine globes were harvested and equally divided into an uninfected, untreated control group; a BoHV-1-infected, untreated group; and a BoHV-1-infected, cidofovir-treated group. Virus isolation for BoHV-1 titers was performed from corneal tissue and liquid media. Histologic measurements of corneal thickness, epithelial cell density, and tissue organization were compared between groups. Substantial BoHV-1 replication was observed in infected, untreated corneas, but BoHV-1 titer was significantly reduced in cidofovir-treated (1.69 ± 0.08 × 103 PFU/mL) versus untreated (8.25 ± 0.25 × 105 PFU/mL, p < 0.0001) tissues by day 2 of culture. No significant differences in histologic criteria were observed between groups. In conclusion, cidofovir warrants further investigation as treatment for BoHV-1 keratoconjunctivitis, with future studies needed to assess in-vivo tolerability and efficacy.

Promoting limbal stem cells proliferation and maintenance using post-thaw human amniotic membranes fortified by platelet lysate

In this study, we aimed to investigate the proliferation and maintenance rate of cultivated human limbal stem cells (hLSCs)-enriched after treating with post-thaw human amniotic membranes (HAMs) fortified by platelet lysate (PL). To prepare PL, three volunteers were selected and platelet rich plasma (PRP) were isolated from their peripheral blood. Thrombin solution was added afterwards at a 1:5 ratio and the produced PL was used to fortify HAMs after a freeze-thaw cycle. hLSCs obtained from corneo-scleral rings of donated cadaveric corneas and cultured on the post-thaw fortified HAMs as the test group. Cell viability as well as genotypic and phenotypic variations in hLSCs-enriched were compared between the test group and the hLSCs cultivated on post-thaw HAMs without PL (as the control group). Cell Viability assessment demonstrated a significant increase of the hLSCs-enriched viability in the test group on days 1 and 3 and a borderline increase after 1 week in comparison to the control group. Tp63 gene expression decreased remarkably on day 7. NGFR gene expression, however, showed a notable increase on days 1, 3, and 7. There was not a significant difference between the hLSCs-enriched in the test and control groups after 1 week, concerning ΔNp63 protein expression. Our findings strongly indicate that applying PL to fortify HAMs after thawing could potentiate hLSCs-enriched proliferation and differentiation to corneal epithelial precursor cells. In this respect, PL-fortified post-thaw HAMs may potentially improve ocular surface wound healing and re-epithelialization.

Identification, quantification and age-related changes of human trabecular meshwork stem cells

BackgroundLoss of cells in the human trabecular meshwork (TM) has been reported with ageing and in glaucoma. This study aims to identify, quantify and determine the age-related changes of human TM stem cells (TMSCs).MethodsIsolation of TM cells/ paraffin sectioning was carried out using human corneoscleral rings and whole globes. The TM cells/ sections were immunostained for the stem cell markers ATP-binding cassette protein G2 (ABCG2), nerve growth factor receptor p75 and AnkyrinG (AnkG). Images were acquired using Leica SP8 confocal microscope. The isolated cells were analyzed for two parameters- ABCG2 expression and nucleus to cytoplasmic ratio (N/C ratio). The total number of TM cells and those positive for ABCG2 and p75 in each section were quantified. Spearman rank order correlation was used to determine the association between age and the cell counts.ResultsThe TMSCs were identified based on two parameters- high ABCG2 expression and high N/C ratio > 0.7. These stem cells were also positive for p75 and AnkG. The TMSC content based on the two parameters was 21.0 ± 1.4% in < 30 years age group, 12.6 ± 6.6% in 30–60 years and 4.0 ± 3.5% in > 60 years. The stem cells with high ABCG2 and p75 expression were restricted to the Schwalbe’s line region of the TM. A significant correlation was observed between the reduction in TMSC content and TM cell count during ageing.ConclusionThe human TMSCs were identified and quantified based on two parameter analysis. This study established a significant association between age-related reduction in TMSC content and TM cell loss.

Prophylactic exposure of human corneal endothelial cells to Rho-associated kinase inhibitor reduced apoptosis rate after phacoemulsification: Ex vivo study

To evaluate whether prophylactic exposure of corneal endothelial cells (CECs) to a selective Rho-associated kinase (ROCK) inhibitor will inhibit CEC apoptosis after phacoemulsification. Laboratory evaluations at the Edith Wolfson Medical Center, Holon, Israel and the Chaim Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel and the Chaim Sheba Medical Center, Tel-Hashomer, Ramat-Gan, Israel. Experimental study. Human donor corneolimbal rings were divided into fragments that were stored in commercial storage media with or without the addition of 10mM ROCK inhibitor for 1week and were then exposed to phacoemulsification energy. Samples were dissociated into single cells by trypsin digestion and CECs were targeted using the antihuman CD166 antibody, a new biomarker. The CEC survival was evaluated for early and late apoptosis rate with flow cytometric analysis of annexin-V and propidium iodide (PI) double staining. Six corneoscleral rings from 4 donors were studied. Afterphacoemulsification, CEC exposed to ROCK inhibitor demonstrated a 37.06% reduction in early apoptosis rate (29.36%±4.33% [SD] versus 46.65%±1.51%, P=.006) and 45.27% reduction in late apoptosis rate (17.6%±16.81% versus 32.16%±26.30%, P=.007), compared with controls. Subsequently, ROCK levels in apoptotic CECs were significantly lower incells incubated with ROCK inhibitor than the control medium. In this exvivo study, ROCK inhibitor reduced endothelial loss and thus, could be used to limit or slow down CEC loss. Rho-associated kinase inhibitor might be used before cataract surgery, especially in high risk patients. This might be a promising new method for preventing pseudophakic bullous keratopathy.

Corneal Stiffening by a Bacteriochlorophyll Derivative With Dextran and Near-Infrared Light: Effect of Shortening Irradiation Time up to 1 Minute.

The aim of this study is to determine the effect of variation of the exposure time of near-infrared irradiation on corneal stiffening after a bacteriochlorophyll derivative (WST11) with dextran (WST-D) application. One hundred four paired eyes of 3-month-old New Zealand White rabbits were included in this study. Fifty-two eyes (ex vivo n = 34, in vivo n = 18) were mechanically deepithelialized, treated topically with WST-D, and irradiated at 10 mW/cm using a diode laser at 755 nm for 1, 5, or 30 minutes. Untreated fellow eyes served as controls. Corneoscleral rings were removed immediately after treatment (ex vivo), or 1 month after treatment (in vivo). Corneal strips were cut and underwent biomechanical stress-strain measurements. Ex vivo, the mean tangent elastic modulus was significantly higher in the treatment groups than in the control groups for 1, 5, and 30 minutes of irradiation, respectively, 6.06 MPa, 95% confidence interval (CI, 4.5-7.6) versus 14.02 MPa, 95% CI (10.2-17.8), n = 11, 4.8 MPa, 95% CI (3.9-5.7) versus 15.03 MPa, 95% CI (12-18.1), n = 11, and 7.8 MPa, 95% CI (5.6-10.02) versus 16.2 MPa, 95% CI (13.6-18.9), n = 11; P < 0.001 for all comparisons. In vivo, the mean elastic moduli in the treatment groups were significantly higher for 5 and 30 minutes of irradiation but not for 1 minute of irradiation, respectively, 11.4 MPa, 95% CI (8.5-14.2), versus 17.1 MPa, 95% CI (14.5-19.7), n = 5; P < 0.001, and 9.4 MPa, 95% CI (5.1-13.8) versus 16 MPa, 95% CI (13.1-19), n = 5; P < 0.01, and 11.3 MPa, 95% CI (6-16.6) versus 12.2 MPa, 95% CI (7.5-16.8), n = 5; P = 0.7. WST-D/near-infrared treatment using shortened irradiation time (1 minute ex vivo and 5 minutes in vivo) results in significant corneal stiffening, and this might provide an alternative to the currently applied riboflavin/ultraviolet A cross-linking.

Growth Factor Concentration in Keratocyte Supernatant after Incubation with Human Serum In Vitro

Application of serum eye drops is an alternative treatment option for therapy-resistant corneal epithelial defects. In case of persisting epithelial defects, cytokines, which are secreted from stromal keratocytes, may play a role in epithelial wound healing. Our aim was to analyze fibroblast growth factor basic (FGFb), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and transforming growth factor β1 (TGF-β1) concentration in the supernatant of keratocytes, after incubation with human serum (HS). Serum eye drops of 10 patients were prepared using the standards of the LIONS Eye Bank Saar-Lor-Lux, Trier/Westpfalz, and were stored at - 80 °C. Primary human keratocytes were isolated from human corneoscleral rings using collagenase A (1 mg/ml) (n = 1) and were cultured in DMEM/Ham's culture medium with 10% fetal bovine serum (FBS). Thereafter, keratocyte cultures were incubated in 15 or 30% HS (in DMEM/F14 without FBS) and FGFb, HGF, KGF and TGF-β1 concentration was determined with an enzyme-linked immunoabsorbent assay (ELISA) from the supernatant of the culture after 24 hours. We used 15 or 30% HS without keratocytes (under the same storage conditions) as controls. HGF concentration was, for both HS concentrations, significantly higher in the supernatant of keratocytes, than in HS controls (without keratocytes) following 24 hours (p < 0.01). FGFb concentration was significantly increased in 30% HS with keratocytes compared to 30% HS without keratocytes after 24 hours (p < 0.01). TGF-β1 and KGF concentrations remained unchanged through keratocytes. HGF and FGFb concentrations increase in the supernatant of keratocytes, 24 hours after incubation with human serum. These concentration changes may play a role in wound healing of epithelial defects.

Erratum to: Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells

In regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population. Fibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student’s unpaired t test and considering p < 0.05 as statistically significant. The isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of “the stem gene profile”; 84 were differentially expressed and were involved in structural activity. The fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good proliferative capability, and long-term maintenance of stem cell properties, independently of donor age and long-term culture conditions. Our findings confirm that limbal fibroblast-like stem cells are highly promising for application in regenerative medicine and that in-vitro culture steps do not influence their stem cell properties. Moreover, the proteomic data enrich our knowledge of fibroblast-like stem cells.

Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells.

BackgroundIn regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population.MethodsFibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student’s unpaired t test and considering p < 0.05 as statistically significant.ResultsThe isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of “the stem gene profile”; 84 were differentially expressed and were involved in structural activity.ConclusionsThe fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good proliferative capability, and long-term maintenance of stem cell properties, independently of donor age and long-term culture conditions. Our findings confirm that limbal fibroblast-like stem cells are highly promising for application in regenerative medicine and that in-vitro culture steps do not influence their stem cell properties. Moreover, the proteomic data enrich our knowledge of fibroblast-like stem cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0342-z) contains supplementary material, which is available to authorized users.

Human limbal mesenchymal stem cells express ABCB5 and can grow on amniotic membrane.

To isolate and characterize limbal mesenchymal stem cells (LMSCs) from human corneoscleral rings. Cells were isolated from corneoscleral rings and cultured in a mesenchymal stem cell (MSC)-selective media and examined for differentiation, phenotyping and characterization. LMSCs were capable of trilineage differentiation, adhered to tissue culture plastic, expressed HLA class I and cell surface antigens associated with human MSC while having no/low expression of HLA class II and negative hematopoietic lineage markers. They were capable for CXCL12-mediated cellular migration. LMSCs adhered, proliferated on amniotic membrane and expressed the common putative limbal stem cell markers. Limbal-derived MSC exhibited plasticity, could maintain limbal markers expression and demonstrated viable growth on amniotic membrane.

Top-hat Shaped Corneal Trephination for Penetrating Keratoplasty Using the Femtosecond Laser: A Histomorphological Study

To evaluate a novel technique for penetrating keratoplasty (PK) with the use of a new software algorithm for the femtosecond laser, designed to create penetrating cuts in a top hat configuration. Consecutive histomorphological case series. Twelve eyes of 12 patients underwent penetrating keratoplasty by means of a 60-kHz femtosecond laser (IntraLase, Irvine, California) with a software specifically developed for corneal surgery. Of the 12 patients, the reason for keratoplasty was keratoconus in 4 patients, bullous keratopathy in 6 patients, keratotorus in 1 patient, and status post chemical burn in 1 patient. A new software was used to create penetrating cuts in a top-hat-shaped configuration. In all cases, cutting parameters were identical in all donor and corresponding host corneas: 7.0 mm diameter of the anterior side cut, 8.5-8.7 mm diameter of the posterior side cut, and a depth of 300 microm for the lamellar cut. In all cases, a complete penetrating cut with the laser in the host cornea was not intended intraoperatively. Complete penetration was performed subsequently and manually with a diamond knife because of logistic conditions. Trephined corneoscleral rings and button corneas were analyzed macroscopically and histologically to determine cut quality. All procedures were performed without any complications. With application of appropriate combinations of pulse energy and spacing, trephination took less than 200 seconds. Macroscopic examination and histology of donor and recipient specimens showed a straight, smooth cut with perpendicular edges in all donor buttons. No corneal edema and no visible damage to the keratocyte nuclei were found. At the region of manual dissection, a small stromal tissue-tag was present in parts of the circumference in all donor buttons. No evidence of any cut complication was noted. Top-hat-shaped penetrating keratoplasty using the IntraLase femtosecond laser enables a quick and sufficient trephination of both human donor and host corneas. It creates favorable histomorphological results with regard to cut quality of the donor and host corneas.

Organ culture, but not hypothermic storage, facilitates the repair of the corneal endothelium following mechanical damage

To evaluate the reparative capacity of the mechanically injured endothelium of corneas stored under organ culture (OC) or hypothermic conditions. The central endothelium of 12 pairs of human corneas with similar endothelial parameters was damaged to create a 1 mm(2) lesion. One cornea from each pair was stored under OC and one under hypothermic conditions. The endothelial cell density (ECD), coefficient of variation, hexagonality and percentage of dead cells were assessed before and after damage and on days 7, 14, 21 and 28 of storage. The mean ECD of corneas subsequently stored under OC or hypothermic conditions was 2764/mm(2). Immediately after damage, a denuded Descemet's membrane with a few remaining dead cells was observed at the injured area. After 7 days of storage under OC conditions, almost no dead cells were observed at the place of injury. A non-significant worsening of the qualitative parameters (polymegatism and pleomorphism) was found. After 14 days, ECD was 1933/mm(2) and 2478/mm(2) centrally and pericentrally, respectively. Similar values were found after 21 and 28 days of storage. The lesions with remnant dead cells persisted throughout hypothermic preservation. From day 14 the corneas became cloudy and in poor condition, while the pericentral ECD was 2523/mm(2). The reparative capacity of the cornea is maintained under OC but not under hypothermic conditions. For corneas containing dead endothelial cells, OC is therefore the method of choice because it may improve the quality of the stored tissue.

A Simple Organ Culture Model for Assessing the Effects of Growth Factors on Corneal Re-epithelialization

The effects of growth factors on re-epithelialization of wounded human and bovine corneas were studied in a simple organ culture system. Excisional trephine and epithelial scrape wounds were created on bovine and human corneo-scleral rings in which the endothelial corneal concavity was then filled with an agar–collagen mixture. Organ culture was undertaken at 37°C in a humidified 5% CO 2incubator with serum-free Medium 199 maintained at the level of the conjunctival epithelium. Rates of re-epithelialization in response to addition of exogenous epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and transforming growth factor type β 1(TGF-β 1) were assessed by image analysis. Corneal cultures could be maintained for up to 3 weeks without significant stromal oedema or keratocyte deterioration and with little loss of epithelial architecture. Following wounding the cornea re-epithelialized in a similar fashion to that observed in vivo i.e. a lag phase followed by migration/proliferation and the reformation of an intact multilayered epithelium. EGF accelerated, basic FGF had no effect on, and TGF-β 1inhibited the rate of corneal re-epithelialization. Our organ culture model maintains corneal integrity and provides a practical system in which to study factors that modulate corneal re-epithelialization following wounding.

Proliferation of Corneal Epithelial and Endothelial Cells in the Trabecular Region of Human Donor Corneas in Organ Culture

Thirty corneoscleral rings obtained after trephination of a 4- to 7.5-mm graft and 8 whole corneas with scleral rims were cultured up to 6 weeks. The corneal epithelium grew over the scleral rim and trabecula, and, after 3-4 weeks, was slowly invading the endothelial sheet. Endothelial cells did not show any evidence of migration unless the trabecula was removed. Even then, however, the endothelial growth was very limited and was soon suppressed by fast-growing epithelium.

Low oxygen prevents epithelial overgrowth of corneas in long-term organ culture.

Forth human corneo-scleral rings and 40 whole corneas with scleral rim were preserved in organ culture for up to six weeks in either normal (20%) or low (6%) oxygen concentration in the gas phase of the culture. Decreasing the oxygen prevented epithelial invasion of the inner side of the cornea without adversely affecting the condition of the endothelium. It is suggested that lower oxygen level might improve corneal preservation in organ culture.