August 2009
Volume 50, Issue 8
Free
Cornea  |   August 2009
Enhancement on Primate Corneal Endothelial Cell Survival In Vitro by a ROCK Inhibitor
Author Affiliations
  • Naoki Okumura
    From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; the
  • Morio Ueno
    From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; the
    Department of Ophthalmology, National Center for Geriatrics and Gerontology, Obu, Japan; the
  • Noriko Koizumi
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan; and the
  • Yuji Sakamoto
    Research Laboratory, Senju Pharmaceutical Co., Ltd., Kobe, Japan.
  • Kana Hirata
    Department of Biomedical Engineering Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, Japan; and the
  • Junji Hamuro
    From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; the
  • Shigeru Kinoshita
    From the Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; the
Investigative Ophthalmology & Visual Science August 2009, Vol.50, 3680-3687. doi:10.1167/iovs.08-2634
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      Naoki Okumura, Morio Ueno, Noriko Koizumi, Yuji Sakamoto, Kana Hirata, Junji Hamuro, Shigeru Kinoshita; Enhancement on Primate Corneal Endothelial Cell Survival In Vitro by a ROCK Inhibitor. Invest. Ophthalmol. Vis. Sci. 2009;50(8):3680-3687. doi: 10.1167/iovs.08-2634.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. The transplantation of cultivated corneal endothelial cells (CECs) has gained attention recently for the treatment of patients with corneal endothelial dysfunction. However, an efficient culturing technique for human (H)CECs has yet to be properly established. The present study was conducted to investigate the applicability of the Rho kinase (ROCK) inhibitor Y-27632 in promoting cultivation of cynomolgus monkey (M)CECs.

methods. MCECs of cynomolgus monkeys were cultured in a medium containing 10 μM Y-27632. The number of viable cells adherent to culture plates were monitored by a luminescent cell-viability assay and colony growth was detected by toluidine blue staining. Proliferating cells were detected by Ki67 expression using flow cytometry and a BrdU-labeling assay for immunocytochemistry. Annexin V-positive apoptotic cells were analyzed by flow cytometry.

results. The number of viable cultivated MCECs was enhanced by Y-27632 addition after 24 hours in culture. The colony area of the culture in the presence of Y-27632 was higher than in the absence of Y-27632 on day 10. In Y-27632-treated cultures, the number of Ki67-positive cells was significantly increased at 24 and 48 hours, and the number of proliferating BrdU-positive cells was increased at 48 hours. The number of Annexin V-positive apoptotic cells was decreased at 24 hours.

conclusions. The inhibition of Rho/ROCK signaling by specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the number of proliferating cells. These results suggest that the ROCK inhibitor may serve as a new tool for cultivating HCECs for transplantation.

The corneal endothelium is essential for the maintenance of corneal transparency. Since human corneal endothelial cells (HCECs) have poor in vivo proliferative potency, corneal endothelial disorders such as Fuchs’ endothelial dystrophy, pseudophakic bullous keratopathy, and trauma lead to a compensatory enlargement of the remaining endothelial cells and irreversible corneal endothelial dysfunction. Penetrating keratoplasty has been widely performed for the improvement of endothelial dysfunction; however, the procedure has several adverse effects such as the potential for irregular astigmatism, suture-induced problems, fragility against trauma, and invasiveness. Alternative methods for replacing the corneal endothelium have been developed, including posterior lamellar keratoplasty, deep lamellar endothelial keratoplasty, and Descemet’s-stripping endothelial keratoplasty. 1 2 3 Although these methods provide considerable benefits clinically, allograft rejection and primary graft failure remain a problem. Moreover, the worldwide shortage of donors is critical. Recently, the transplantation of cultivated CECs has been suggested as an alternative approach to the treatment of corneal endothelial dysfunction. The transplantation of cultured HCECs as a sheet, with 4 5 or without 6 a carrier, and the injection of progenitor cells 7 8 has been explored in animal studies. However, the animal model used in these studies was the rabbit, in which CECs retain high-proliferation ability, and in which residual peripheral CECs proliferate rapidly after injury and regenerate a clear cornea. 9 Aiming to establish a nonhuman primate model with poorly proliferative CECs, we recently reported a cynomolgus monkey model of cultivated CEC sheet transplantation. 10  
However, efficient culture techniques of HCECs need further development before practical application. HCECs are arrested at the G1-phase of the cell cycle, 11 12 and an age-dependent negative regulation of the cell cycle might causally contribute to the poor proliferative activity in vitro. 13 14 15 Several studies have reported the successful cultivation of HCECs by use of an animal-derived extracellular matrix (ECM). 16 17 18  
To establish a clinically applicable efficient way to cultivate HCECs free of animal-origin pathogens, we focused our study on modulating the activity of GTPase Rho, regulating cell-to-substrate and cell-to-cell adhesions. 19 20 21 Rho and Rho-associated kinases (ROCKs) have a critical function in regulating cell adhesion and cell motility. 22 23 ROCKs are essential in regulating focal adhesions in cultured fibroblasts and epithelial cells. 24 The Rho subfamily contributes to the regulation of many different biological processes through actin-myosin–mediated contractile force generation via the phosphorylation of downstream target proteins. 
In terms of other biological effects, Rho GTPases are well known to play a crucial role in cell-cycle progression and in apoptosis. It was initially reported that Rho inactivation blocks G1-S phase progression and that the microinjection of active RhoA into quiescent cells induces G1-S phase progression in Swiss 3T3 fibroblasts. 25 Although the underlying mechanism has yet to be thoroughly revealed, ROCK signaling is thought to promote cell-cycle progression in various cell types, 25 26 including CECs. 27 Unlike these reported findings, we have found that inhibition of the ROCK pathway by a selective inhibitor of ROCK, Y-27632, promotes proliferation as well as adhesion of MCECs and inhibits apoptosis. 
Materials and Methods
Animal Experiment Approval
In all experiments, animals were housed and treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental procedures were approved by the committee for Animal Research at Kyoto Prefectural University of Medicine. 
Primary Cultures
We used eight corneas from four cynomolgus monkeys (3–5 years of age; estimated equivalent human age, 5–20 years) housed at Nissei Bilis Co., Ltd. (Otsu, Japan) and Keari Co., Ltd. (Wakayama, Japan). The corneas were harvested at the time of euthanatization of the monkeys for other research purposes, and the cells were placed in culture within 12 hours. We cultivated MCECs according to a modified protocol of HCEC culture reported previously. 5 Descemet’s membrane was stripped of intact MCECs and transferred to 0.6 U/mL of Dispase II (Roche Applied Science, Penzberg, Germany). After a 60-minute incubation at 37°C, the MCECs obtained from individual corneas were resuspended in culture medium and were plated in 1 well of a 12-well plate. All primary cell cultures and serial passages of MCECs were performed in growth medium composed of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, 50 μg/mL streptomycin, and 2 ng/mL basic fibroblast growth factor (bFGF; Invitrogen Corp., Carlsbad, CA). MCECs were cultured in a humidified atmosphere at 37°C in 5% CO2. The culture medium was changed every 2 days. When cells reached confluence in 10 to 14 days, they were rinsed in Ca2+ and Mg2+-free Dulbecco’s phosphate-buffered saline (PBS), trypsinized with 0.05% trypsin-EDTA (Invitrogen) for 5 minutes at 37°C, and passaged at ratios of 1:2 to 4. 
Determination of the Number of Viable Cells
The number of viable cells was determined by a cell-viability assay (CellTiter-Glo Luminescent Cell Viability Assay; Promega Corp., Madison, WI) using the recommended protocol. Viability assays that generate luminescent signals are based on quantification of the ATP levels. MCECs were plated at a density of 2.0 × 103 cells onto 96-well plates. An equal volume of the chemiluminescent reagent was added to 100 μL of medium containing cells for each 96-well plate. Luminescence in each well was measured by a luminometer (Veritas Microplate Luminometer; Turner Biosystems, Sunnyvale, CA) and was standardized to the luminescence of the control. Analyses were performed on the first day of passage and five samples were prepared for each group. 
Colony-Forming Efficiency
The clonal growth ability of primary MCECs was determined by the colony-forming efficiency (CFE). Cells were plated at a density of 2.0 × 103 cells/cm2. Then, the colonies were fixed on day 11 and stained with 0.1% toluidine blue, and the accumulated area was analyzed by Image J software (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD; available at http://rsb.info.nih.gov/ij/index.html). CFEs were expressed as the multiple of change between the control and treated areas. Five samples were prepared for each group. 
Immunohistochemistry
Cultured MCECs on chamber slides (Laboratory-Tek; NUNC A/S, Roskilde, Denmark) were fixed in 4% formaldehyde for 10 minutes at room temperature (RT), permeabilized for 5 minutes in PBS containing 0.1% Triton X-100, washed, and incubated for 30 minutes with 1% bovine serum albumin (BSA). For actin studies, the MCECs were incubated at 4°C overnight with a 1:400-dilution rhodamine-conjugated phalloidin molecular probe (Invitrogen) and again washed three times with PBS. For Ki67 and actin double-staining studies, after blocking, the MCECs were incubated at RT with 1:400-dilution rhodamine-conjugated phalloidin (Invitrogen), washed with PBS three times, incubated overnight at 4°C with 1:400-dilution anti-mouse Ki67, and washed again three times. They were then incubated at RT with 1:2000-diluted Alexa Fluor 488–conjugated goat anti-mouse IgG (Invitrogen) and washed three times. During all steps, the endothelial side was face up to avoid damage. After they were washed with PBS in the dark, the specimens were mounted on glass slides with antifade mounting medium containing DAPI (Vector Laboratories, Burlingame, CA), and the slides were inspected with a fluorescence microscope (AX70; Olympus, Tokyo, Japan). 
Flow Cytometry Analyses
For Ki67 studies, MCECs prepared as just described were passaged in 1:4 dilutions and cultured for 1 or 2 days. After this, the MCECs were dissociated to single cells by 0.25% trypsin digestion, fixed in 70% (wt/vol) ethanol, washed, and incubated for 20 minutes with 1% BSA. Then, the MCECs were incubated with 1:20-diluted anti-mouse Ki67, washed, and incubated with 1:1000-diluted Alexa Fluor 488–conjugated goat anti-mouse IgG (Invitrogen). For annexin V studies, MCECs were passaged in 1:4 dilutions, and all cells were dissociated to single cells by 0.25% trypsin digestion and recovered, including cells floating in the culture medium, at day 1. They were then subjected to an annexin V assay (Annexin V-FITC Apoptosis Detection Kit Plus; MBL Corp., Nagoya, Japan), according to the manufacturer’s instructions. Flow cytometric analyses were then performed (FACSCalibur; BD Biosciences, Franklin Lakes, NJ). 
BrdU-Labeling Assay
MCECs prepared as just described were passaged in 1:4 dilutions onto chamber slides (Laboratory-Tek; NUNC A/S), and cultured for 24 hours. They were then incubated for a further 24 hours with 1:1000-diluted 5-BrdU-labeling reagent (Amersham Biosciences, Buckinghamshire, UK). MCEC cultures were next washed in PBS, fixed for 30 minutes in acid-ethanol (90% ethanol; 5% acetic acid; 5% distilled water), washed with PBS, and incubated with 1% BSA at 37°C for 30 minutes to block nonspecific binding, followed by a 1-hour incubation at RT with mouse anti-BrdU antibody (Amersham Biosciences). After washing, they were then incubated, again at RT, with 1:2000-diluted Alexa Fluor 488–conjugated goat anti-mouse IgG (Invitrogen), washed three times, and mounted on glass slides with antifade mounting medium containing DAPI (Vector Laboratories). The slides were subsequently inspected with a fluorescence microscope. 
Statistical Analysis
The statistical significance (P) in mean values of the two-sample comparison was determined with Student’s t-test. The statistical significance in the comparison of multiple sample sets was analyzed with the Dunnett multiple-comparisons test. Values shown on graphs represent the mean ± SE. 
Results
Primary Culture of MCECs
The numbers of viable primary MCECs attached on culture plates 24 hours after the start of the culture were monitored by a luminescent cell-viability assay. The number of viable primary MCECs adherent to the culture plate invariably increased over a wide range of concentrations, from 1 to 100 μM, of Y-27632. A commonly used working concentration of Y-27632, 28 10 μM, resulted in the highest (*P < 0.05 vs. control) cell-survival enhancement (Fig. 1)
Primary MCECs from 4-year-old cynomolgus monkeys prepared as described earlier were plated at a density of 2.0 × 104 cells/well in a 12-well plate in the presence or absence of 10 μM Y-27632, and phase-contrast images were analyzed. MCECs treated with Y-27632 showed better coverage on day 4 than did the nontreated groups (Fig. 2A) . To further confirm the adhesion-improving effect, primary MCECs were seeded at a higher density (2.0 × 105 cells/well) in the presence or absence of 10 μM Y-27632. On day 3, untreated MCECs were proliferating and were enlarged, but were not confluent or homogeneously hexagonal. In contrast, Y-27632-treated MCECs exhibited a confluent monolayer of homogeneously hexagonal cells with smaller sizes (Fig. 2B) . For an examination of the distinction of colony growth of primary MCECs between two culture groups with or without Y27632, MCECs were plated at a lower density of 2.0 × 103 cells/well in a 96-well microplate. On day 10, MCECs treated with Y-27632 demonstrated a markedly enhanced increase in colony growth, detected by toluidine blue staining (Fig. 2C) . The colony area of Y-27632-treated cells was significantly higher than the control (1.6-fold, *P < 0.01). 
Subculture of MCECs
The number of viable primary MCECs adherent to the culture plate was invariably increased in the presence of Y-27632, indicating that there is a possibility that the application of Y-27632 may also improve the CFE of HCECs. To confirm the effect of Y-27632 during the culture passage, we next investigated the effect of Y-27632 on subcultured MCECs. Cultivated cells passaged 4 to 6 times in the presence of Y-27632 were diluted at three dilutions (1:2, 1:4, and 1:8), and the number of viable cells attached on the noncoated culture plate at 24 hours of subculture was then determined. It was found that the number of viable cells recovered was enhanced in the presence of Y-27632 at all three dilutions (P < 0.01; Fig. 3 ). It is of note that the enhancement ratio of adhesion to the plate tends to be higher at higher dilutions during passage. MCECs treated with Y-27632 during subculture showed enhanced cell adhesion both in phase-contrast images and in images of actin fibers immunostained with phalloidin antibody (Fig. 4)
The Effect of Y-27632 on Cell-Cycle Progression
Studies were conducted to determine whether Y-27632 might play a role in cell-cycle progression in MCECs. To answer this question, we first used immunostaining with the cell-cycle population marker Ki67. MCECs at confluence were passaged in 1:4 dilutions and subcultured for 1 or 2 days and were dissociated to single cells by trypsin digestion. MCECs subcultured in the culture medium with Y-27632 showed the presence of a larger number of Ki67-positive cells than was present in the controls (Fig. 5A) . Actin immunostaining was also performed to determine whether there is a relationship between Ki67-positive cells and enhanced actin-fiber progression. It turned out that Ki67 expression had no direct relation to actin fibers. Further quantitative flow cytometric analysis revealed the increased presence of Ki67-positive cells in MCECs cultured with Y-27632, 2 days after subculture (Fig. 5B) . A BrdU-labeling assay of 48-hour subcultures is shown in Figure 5C , and it shows a larger number of BrdU-positive MCECs among cell populations cultured with Y-27632 compared with control cells. Thus, it was demonstrated that Y-27632 plays a relevant role in the cell-cycle progression of MCECs. 
The Effect of Y-27632 on Apoptosis
We next studied the involvement of Y-27632 in apoptosis. MCECs were passaged in 1:4 dilutions at the time of culture confluence. All cells were then dissociated to single cells by trypsin digestion and recovered, including floating cells, 1 day after subculture in the presence of 10% FBS. The flow cytometry patterns of the annexin V assay revealed that Y-27632 treatment significantly decreased apoptosis (P < 0.01; Fig. 6A ). The inhibition of apoptosis during culture without any stress load was lowered from 12.4% ± 4.6% in the control group to 2.0% ± 1.6% in the subculture group containing Y-27632, thus implicating the contribution of the apoptosis-reducing activity of Y-27632 to the efficient subculture of MCECs (Fig. 6B)
Discussion
Inhibition of Rho/ROCK signaling by Y-27632 clearly promoted MCEC adhesion and inhibited apoptosis during culture. It also increased the proliferating cell population. These findings were confirmed with cynomolgus monkey-derived MCECs, which have poor proliferation potential, as do HCECs. MCECs are considered to be the preferable experimental tool for investigating nonproliferative HCECs, compared with rodent CECs or those derived from a rabbit in which the CECs retain a high proliferative ability both in vivo and in vitro. 7 8 9  
Although we found that MCECs tended to show a cell-senescence phenotype after only a few passages, we successfully cultivated and passaged MCECs in the presence of the ROCK inhibitor Y-27632. There have been several reports of successful CEC cultivation in which both explants 4 17 and cell suspensions 15 were used. There is almost no mitotic activity in the HCECs throughout the lifespan, and proliferation of adult HCECs cannot be achieved with standard cell-culturing techniques. 29 However, adult HCECs have been reported to proliferate when cultured with ECM derived from animals. 4 15 16 The fact that adult HCECs proliferate in the presence of ECM suggests that the interaction with the substratum is indispensable for the efficient growth and maintenance of MCECs. 
In this context, it is of note that inhibition of the Rho/ROCK pathway by Y-27632 promotes cell adhesion to substrates of cultured THP-1 monocytes 19 and human Tenon fibroblast. 30 The actin cytoskeleton plays a critical role in cell adhesion, 19 30 which coincides well with our observation that the adhesion of cultured MCECs is promoted by Y-27632. We suspect that the enhanced adhesion by Y-27632 may be ascribable to the promotion of membrane protrusion by actin reconstitution 19 30 and to cell-to-cell adhesion by cadherin. 21 Further investigations on the underlying mechanism of the improved cell adhesion and the possible combined effect of Y-27632 with coated substrates such as ICAM-1, VCAM, collagen, fibronectin, and laminin 15 16 30 are likely to contribute much to further improvements in the efficacy of the culture of MCECs and HCECs. 
Although the underlying mechanisms have yet to be thoroughly revealed, ROCK plays an important role in cell-cycle progression. ROCK activity is required for the formation of actin stress fibers that contribute to the sustained activation of Ras and the ERK mitogen-activated protein kinase (MAPK) after ligand stimulation. 31 32 33 In addition, RhoA promotes cell-cycle progression to S phase by regulating p27 degradation through its effect on cyclinE/CDK2 activity. 34 In HCECs, p27 is known to play a pivotal role in the negative regulation of cell-cycle progression. 11 12 35 36 37 However, and surprisingly, ROCK inhibition with Y-27632 promoted MCEC proliferation in our study (Fig. 5) . Our finding that ROCK inhibition promotes the cell-cycle progression contrast with previously reported results. 25 31 32 33 This unexpected promotion of the cell-cycle progression of MCECs may be partially explained by the previous findings that the Rho/ROCK signaling, including cell proliferation, are cell-type-dependent. 38 39 It was illustrated that the blockade of sustained ERK/MAPK activity by inhibiting Rho and ROCK led to rapid cyclin-D1 induction through activation of Rac1 and Cdc42 in murine fibroblasts. 31 33 Although further studies are needed, cell adhesion and motility, which are enhanced by ROCK inhibition, 40 may have a positive effect on MCEC proliferation. Further investigations are necessary to determine whether the increased in cell proliferation observed in our studies can be ascribed to an effect on the molecular modules regulating cell-cycle progression. 
ROCK is involved in the regulation of apoptosis. 38 41 Significant morphologic changes including contraction, membrane blebbing, and nuclear disintegration during apoptosis are driven by the ROCK-mediated actin-myosin contractile force. 42 ROCK inhibition has been reported to have an antiapoptotic effect in some models, such as a spinal cord injury model, 43 dissociated human embryonic stem cells, 44 and grafted neural precursors. 45 Recent research has highlighted the prosurvival effect of ROCK inhibitors for clinical use. 38 In this line, we confirmed the antiapoptotic effect of Y-27632 in cultivated MCECs. Reducing apoptotic cells during primary culture and passage procedures is beneficial, because a higher number of viable HCECs could be gained from a limited number of HCECs obtained from a donor in a clinical setting. However, ROCK inhibitors may induce apoptosis in specialized cell types, 46 including corneal epithelial cells. 47 Further research is needed to determine whether ROCK is a crucial target for these effects. The use of a ROCK I−/− and ROCK II−/− mouse model may clarify the contribution of kinase to apoptosis. HCECs with poor cell adhesion in the primary culture and in subcultures tend to show cell senescence with fibroblastic cell contamination. 16 18 Our preliminary observations indicate that MCECs assume the delayed cell-senescence phenotype during passage in the presence of Y-27632 (data not shown). Effective culture of HCECs from a limited number of donors is crucial for clinical use. Moreover, autologous CEC transplantation, obtained from a patient’s fellow eye, will overcome allograft rejection. Our studies will be expanded to HCECs before clinical application, in combination with the previously reported CEC-sheet transplantation technique. 10 We predict that Y-27632 may be useful for the improved cultivation of HCECs. Of importance, no modification of the chromosome in Y-27632-treated cells has been reported, 44 48 and Y-27632, along with Fasudil, is already used clinically in cardiovascular therapies, 49 thus suggesting its safety in the clinical setting. Y-27632 is also reportedly effective in preventing fibroproliferation in glaucoma surgery 30 and as such may be an effective antiscarring agent after ocular surgery. 
In summary, our results indicate that inhibition of Rho/ROCK signaling by the specific ROCK inhibitor Y-27632 promoted the adhesion of MCECs, inhibited apoptosis, and increased the frequency of proliferating cells. Our results with nonhuman primate CECs which have a low proliferative ability, similar to HCECs, raises the possibility that the ROCK inhibitor may serve as a new tool in cultivating HCECs for newly emerging transplantation therapies. 
 
Figure 1.
 
The enhanced survival of primary cultured MCECs by Y-27632. MCECs separated from Descemet’s membrane were plated at a density of 2.0 × 103 cells/well in DMEM with 2 ng/mL bFGF. The number of viable MCECs attached to the plate at 24 hours was evaluated by luminescence assay of the ATP levels. Y-27632 at 10 μM resulted in significant cell-survival enhancement (*P < 0.05 vs. control). Data are expressed as the ratio to control cells and as the mean ± SE (n = 5).
Figure 1.
 
The enhanced survival of primary cultured MCECs by Y-27632. MCECs separated from Descemet’s membrane were plated at a density of 2.0 × 103 cells/well in DMEM with 2 ng/mL bFGF. The number of viable MCECs attached to the plate at 24 hours was evaluated by luminescence assay of the ATP levels. Y-27632 at 10 μM resulted in significant cell-survival enhancement (*P < 0.05 vs. control). Data are expressed as the ratio to control cells and as the mean ± SE (n = 5).
Figure 2.
 
The improved culture efficacy of primary MCECs by Y-27632. (A, B) Primary MCECs from 4-year-old cynomolgus monkeys, prepared as shown in Figure 1 , were plated at a density of 2.0 × 104 (A) or 2.0 × 105 (B) cells/well in the presence or absence of 10 μM Y-27632, and phase-contrast images were analyzed. Insets: higher magnification. Scale bar, 250 μm. (C) Colony growth of primary cultured MCECs. Top: the MCECs, prepared as above, were seeded at a density of 2.0 × 103 cells/cm2 and stained with 0.1% toluidine blue on day 10. Bottom: colony areas of Y-27632–treated cells were elevated compared with those in control cultures (1.6-fold; *P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 2.
 
The improved culture efficacy of primary MCECs by Y-27632. (A, B) Primary MCECs from 4-year-old cynomolgus monkeys, prepared as shown in Figure 1 , were plated at a density of 2.0 × 104 (A) or 2.0 × 105 (B) cells/well in the presence or absence of 10 μM Y-27632, and phase-contrast images were analyzed. Insets: higher magnification. Scale bar, 250 μm. (C) Colony growth of primary cultured MCECs. Top: the MCECs, prepared as above, were seeded at a density of 2.0 × 103 cells/cm2 and stained with 0.1% toluidine blue on day 10. Bottom: colony areas of Y-27632–treated cells were elevated compared with those in control cultures (1.6-fold; *P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 3.
 
Y-27632 augmented the adherence of MCECs during subculture. MCECs, passaged four to six times, were diluted 1:2, 1:4, and 1:8 in the presence of 10 μM Y-27632. Viable cells attached 24 hours after the subculture were determined. The number of viable cells recovered was enhanced significantly in the presence of Y-27632 at all 3 dilutions (*P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 3.
 
Y-27632 augmented the adherence of MCECs during subculture. MCECs, passaged four to six times, were diluted 1:2, 1:4, and 1:8 in the presence of 10 μM Y-27632. Viable cells attached 24 hours after the subculture were determined. The number of viable cells recovered was enhanced significantly in the presence of Y-27632 at all 3 dilutions (*P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 4.
 
The morphologic change of passaged MCECs induced by Y-27632. Top: phase-contrast images of cells 24 hours after passage. Bottom: Immunostaining of actin in the same cells. Actin (red) and DAPI (blue). Scale bar: (A) 250 μm; (B) 50 μm.
Figure 4.
 
The morphologic change of passaged MCECs induced by Y-27632. Top: phase-contrast images of cells 24 hours after passage. Bottom: Immunostaining of actin in the same cells. Actin (red) and DAPI (blue). Scale bar: (A) 250 μm; (B) 50 μm.
Figure 5.
 
Increased frequency of proliferating MCECs by Y-27632. (A) Double immunostaining of Ki67 and actin fibers; the passaged MCECs were cultured for 48 hours and stained successively with Ki67 and phalloidin. Ki67 (green), actin (red), and DAPI (blue). Inset: higher magnification. (B) Ki67-positive cells were analyzed by flow cytometry. MCECs were subcultured for 1 or 2 days and stained successively with Ki67. The number of Ki67-positive cells was significantly elevated in the presence of Y-27632 on days 1 and 2 (†P < 0.05, *P < 0.01). Data are expressed as the mean ± SE (n = 6). (C) Y-27632 increased the frequency of BrdU-labeled MCECs. The MCECs were subcultured for 24 hours and incubated further for 24 hours with a 5-BrdU-labeling reagent, then stained with mouse anti-BrdU antibody. DAPI (blue). Scale bar: (A) 250 μm; (C) 100 μm.
Figure 5.
 
Increased frequency of proliferating MCECs by Y-27632. (A) Double immunostaining of Ki67 and actin fibers; the passaged MCECs were cultured for 48 hours and stained successively with Ki67 and phalloidin. Ki67 (green), actin (red), and DAPI (blue). Inset: higher magnification. (B) Ki67-positive cells were analyzed by flow cytometry. MCECs were subcultured for 1 or 2 days and stained successively with Ki67. The number of Ki67-positive cells was significantly elevated in the presence of Y-27632 on days 1 and 2 (†P < 0.05, *P < 0.01). Data are expressed as the mean ± SE (n = 6). (C) Y-27632 increased the frequency of BrdU-labeled MCECs. The MCECs were subcultured for 24 hours and incubated further for 24 hours with a 5-BrdU-labeling reagent, then stained with mouse anti-BrdU antibody. DAPI (blue). Scale bar: (A) 250 μm; (C) 100 μm.
Figure 6.
 
(A) Subcultured MCECs were dissociated to single cells by trypsin 24 hours after the subculture and subjected to annexin V assay. (B) The inhibition of apoptosis was lowered significantly from 12.4% ± 4.6% (control) to 2.0% ± 1.6% in the presence of Y-27632 (*P < 0.01). Data are expressed as the mean ± SE (n = 4).
Figure 6.
 
(A) Subcultured MCECs were dissociated to single cells by trypsin 24 hours after the subculture and subjected to annexin V assay. (B) The inhibition of apoptosis was lowered significantly from 12.4% ± 4.6% (control) to 2.0% ± 1.6% in the presence of Y-27632 (*P < 0.01). Data are expressed as the mean ± SE (n = 4).
The authors thank Yoshiki Sasai and Masatoshi Ohgushi for their assistance and invaluable advice about ROCK inhibitors and Hisako Hitora for technical assistance. 
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Figure 1.
 
The enhanced survival of primary cultured MCECs by Y-27632. MCECs separated from Descemet’s membrane were plated at a density of 2.0 × 103 cells/well in DMEM with 2 ng/mL bFGF. The number of viable MCECs attached to the plate at 24 hours was evaluated by luminescence assay of the ATP levels. Y-27632 at 10 μM resulted in significant cell-survival enhancement (*P < 0.05 vs. control). Data are expressed as the ratio to control cells and as the mean ± SE (n = 5).
Figure 1.
 
The enhanced survival of primary cultured MCECs by Y-27632. MCECs separated from Descemet’s membrane were plated at a density of 2.0 × 103 cells/well in DMEM with 2 ng/mL bFGF. The number of viable MCECs attached to the plate at 24 hours was evaluated by luminescence assay of the ATP levels. Y-27632 at 10 μM resulted in significant cell-survival enhancement (*P < 0.05 vs. control). Data are expressed as the ratio to control cells and as the mean ± SE (n = 5).
Figure 2.
 
The improved culture efficacy of primary MCECs by Y-27632. (A, B) Primary MCECs from 4-year-old cynomolgus monkeys, prepared as shown in Figure 1 , were plated at a density of 2.0 × 104 (A) or 2.0 × 105 (B) cells/well in the presence or absence of 10 μM Y-27632, and phase-contrast images were analyzed. Insets: higher magnification. Scale bar, 250 μm. (C) Colony growth of primary cultured MCECs. Top: the MCECs, prepared as above, were seeded at a density of 2.0 × 103 cells/cm2 and stained with 0.1% toluidine blue on day 10. Bottom: colony areas of Y-27632–treated cells were elevated compared with those in control cultures (1.6-fold; *P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 2.
 
The improved culture efficacy of primary MCECs by Y-27632. (A, B) Primary MCECs from 4-year-old cynomolgus monkeys, prepared as shown in Figure 1 , were plated at a density of 2.0 × 104 (A) or 2.0 × 105 (B) cells/well in the presence or absence of 10 μM Y-27632, and phase-contrast images were analyzed. Insets: higher magnification. Scale bar, 250 μm. (C) Colony growth of primary cultured MCECs. Top: the MCECs, prepared as above, were seeded at a density of 2.0 × 103 cells/cm2 and stained with 0.1% toluidine blue on day 10. Bottom: colony areas of Y-27632–treated cells were elevated compared with those in control cultures (1.6-fold; *P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 3.
 
Y-27632 augmented the adherence of MCECs during subculture. MCECs, passaged four to six times, were diluted 1:2, 1:4, and 1:8 in the presence of 10 μM Y-27632. Viable cells attached 24 hours after the subculture were determined. The number of viable cells recovered was enhanced significantly in the presence of Y-27632 at all 3 dilutions (*P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 3.
 
Y-27632 augmented the adherence of MCECs during subculture. MCECs, passaged four to six times, were diluted 1:2, 1:4, and 1:8 in the presence of 10 μM Y-27632. Viable cells attached 24 hours after the subculture were determined. The number of viable cells recovered was enhanced significantly in the presence of Y-27632 at all 3 dilutions (*P < 0.01). Data are expressed as the mean ± SE (n = 5).
Figure 4.
 
The morphologic change of passaged MCECs induced by Y-27632. Top: phase-contrast images of cells 24 hours after passage. Bottom: Immunostaining of actin in the same cells. Actin (red) and DAPI (blue). Scale bar: (A) 250 μm; (B) 50 μm.
Figure 4.
 
The morphologic change of passaged MCECs induced by Y-27632. Top: phase-contrast images of cells 24 hours after passage. Bottom: Immunostaining of actin in the same cells. Actin (red) and DAPI (blue). Scale bar: (A) 250 μm; (B) 50 μm.
Figure 5.
 
Increased frequency of proliferating MCECs by Y-27632. (A) Double immunostaining of Ki67 and actin fibers; the passaged MCECs were cultured for 48 hours and stained successively with Ki67 and phalloidin. Ki67 (green), actin (red), and DAPI (blue). Inset: higher magnification. (B) Ki67-positive cells were analyzed by flow cytometry. MCECs were subcultured for 1 or 2 days and stained successively with Ki67. The number of Ki67-positive cells was significantly elevated in the presence of Y-27632 on days 1 and 2 (†P < 0.05, *P < 0.01). Data are expressed as the mean ± SE (n = 6). (C) Y-27632 increased the frequency of BrdU-labeled MCECs. The MCECs were subcultured for 24 hours and incubated further for 24 hours with a 5-BrdU-labeling reagent, then stained with mouse anti-BrdU antibody. DAPI (blue). Scale bar: (A) 250 μm; (C) 100 μm.
Figure 5.
 
Increased frequency of proliferating MCECs by Y-27632. (A) Double immunostaining of Ki67 and actin fibers; the passaged MCECs were cultured for 48 hours and stained successively with Ki67 and phalloidin. Ki67 (green), actin (red), and DAPI (blue). Inset: higher magnification. (B) Ki67-positive cells were analyzed by flow cytometry. MCECs were subcultured for 1 or 2 days and stained successively with Ki67. The number of Ki67-positive cells was significantly elevated in the presence of Y-27632 on days 1 and 2 (†P < 0.05, *P < 0.01). Data are expressed as the mean ± SE (n = 6). (C) Y-27632 increased the frequency of BrdU-labeled MCECs. The MCECs were subcultured for 24 hours and incubated further for 24 hours with a 5-BrdU-labeling reagent, then stained with mouse anti-BrdU antibody. DAPI (blue). Scale bar: (A) 250 μm; (C) 100 μm.
Figure 6.
 
(A) Subcultured MCECs were dissociated to single cells by trypsin 24 hours after the subculture and subjected to annexin V assay. (B) The inhibition of apoptosis was lowered significantly from 12.4% ± 4.6% (control) to 2.0% ± 1.6% in the presence of Y-27632 (*P < 0.01). Data are expressed as the mean ± SE (n = 4).
Figure 6.
 
(A) Subcultured MCECs were dissociated to single cells by trypsin 24 hours after the subculture and subjected to annexin V assay. (B) The inhibition of apoptosis was lowered significantly from 12.4% ± 4.6% (control) to 2.0% ± 1.6% in the presence of Y-27632 (*P < 0.01). Data are expressed as the mean ± SE (n = 4).
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