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Chromosome analyses of NRCE cells at passage 191. A: The chromosomal aneuploidy of NRCE cells with chromosome numbers ranged from 36 to 47. Among them, NRCE cells with chromosome number of 44 were about 51.06%. B: Chromosomes from a NRCE cell with a number of 44.
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To establish and characterize a novel untransfected corneal endothelial cell line from New Zealand white rabbits (NRCE cell line) for studies on corneal endothelial cells.
Primary culture was initiated with a pure population of NRCE cells from corneal endothelia by successive detachment and reattachment procedure of different durations, and culture...
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The morphometric parameters of the corneal endothelium-cell density (ECD), cell size variation (CV), and hexagonality (HEX)-provide clinically relevant information about the cornea. To estimate these parameters, the endothelium is commonly imaged with a non-contact specular microscope and cell segmentation is performed to these images. In previous...
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... Recently, the establishment of untransfected corneal endothelial cell lines has been reported. These cell lines may be the most ideal cell lines for the treatment of human corneal disease (28). But, the cells may demonstrate some err side pathologies ,like cytogenetic instability, abnormal karyotype, and a transformed phenotype under long-term cultivation. ...
... Gene expression analysis of the harvested cell sheet showed the expression of the Col-IV gene, confirming the cellular activity of the cell sheet. Fan et al. reported on the expression of FLK-1 (vascular endothelial growth factor receptor-2) by corneal endothelial cells (28). Madathil et al. (49) reported in their study that the gene expression of both marker proteins, FLK-1 and Col-IV, along with phase contrast microscopy data, confirmed that cultured cells were corneal endothelial cells. ...
The demand for corneal tissue for transplantation is not fully supplied, forcing the harvesting of corneal endothelial cells within a viable scaffold in demanded tissue supplementation. Recent advances in corneal transplantation surgery have provided new ways to achieve partial tissue transplantation, enabling artificial tissue engineering methods to provide novel materials in the replacement of human corneal tissue. Important hurdles are on this novel path. First, endothelial cells are not proliferative, and new cell lines could be produced if they might regain proliferative capacity and sustain that capacity. Second, the characterization of endothelial cells and their specific markers must be optimized to prove whether cells harvested from regenerative cell processes are genuine corneal endothelial cells. In addition, endothelial cells must be incorporated with artificial tissue scaffolds in order to be implanted into the receptive corneal tissue. In this report, recent developments in these three challenging areas are reviewed, and hidden facets of this intriguing regenerative medicine puzzle are seek out to response.
... and amplified (Genei Red dye PCR kit, Merck-Genei) using their respective primers (Table 1) by PCR. Amplification for the target genes Cytokeratin-12 [22], Vimentin [23] and beta actin [24] was done at 30 cycles at their respective annealing temperatures. Na + K + ATPase target genes, ATP1A1 [24], were amplified using the following parameters: 5 min at 94 °C, 10 cycles of step-down PCR consisting of 1 min at 94 °C, 50 s at 55 °C then decrease 0.5 °C each cycle until 50 °C; 1 min at 72 °C, followed by 27 cycles of 1 min at 94 °C, 50 s at 50 °C, 1 min at 72 °C, with a final extension of 5 min at 72 °C. ...
... Amplification for the target genes Cytokeratin-12 [22], Vimentin [23] and beta actin [24] was done at 30 cycles at their respective annealing temperatures. Na + K + ATPase target genes, ATP1A1 [24], were amplified using the following parameters: 5 min at 94 °C, 10 cycles of step-down PCR consisting of 1 min at 94 °C, 50 s at 55 °C then decrease 0.5 °C each cycle until 50 °C; 1 min at 72 °C, followed by 27 cycles of 1 min at 94 °C, 50 s at 50 °C, 1 min at 72 °C, with a final extension of 5 min at 72 °C. Beta actin was used as a loading control. ...
Tissue engineering intends to develop three dimensional living substitutes to restore, maintain and improve tissue functions. In addition, these biological substitutes also find application in in- vitro toxicity studies. Cell sheet technology enables the development of scaffold free tissue constructs from thermoresponsive culture surfaces. In this study we demonstrate the feasibility of spin coated copolymer, N-Isopropylacrylamide-co-Glycidyl methacrylate (NGMA-SC) as a thermoresponsive substrate to create an over layered construct from native corneal cell types. The corneal cells were independently cultured and characterized by immunostaining. The multi-layered cornea was developed by sequential culture technique. Rabbit corneal endothelial cell monolayer on NGMA-SC was overlayered with corneal stromal fibroblasts. Corneal epithelial cell sheet was transferred on the endothelial-fibroblast bilayer to get three dimensional culture systems. The positive expression of characteristic genes such as Na+K+ATPase, CK12 and vimentin in the construct confirmed the presence of functionally active corneal endothelial, epithelial and fibroblast cells respectively. The layered three dimensional culture system was retrieved from NGMA-SC as an intact tissue construct by modulating thermal stimuli. We propose the sequential cell seeding and layering technique as a potential approach to develop in vitro corneal constructs.
... Gene expression analysis of the harvested cell sheet shows the expression of Col-IV gene confirming the cellular activity of the cells sheet. Fan et al. have reported on the expression of FLK-1 (Vascular endothelial growth factor receptor-2) by corneal endothelial cells [15]. The gene expression of both marker proteins FLK-1 and Col-IV along with the phase contrast microscopy data confirmed that the in vitro cultures were of corneal endothelial cells. ...
Endothelial keratoplasty is a recent shift in the surgical treatmentof corneal endothelial dystrophies, where the dysfunctional endothelium is replaced whilst retaining the unaffected corneal layers. To overcome the limitation of donor corneal shortage, alternative use of tissue engineered constructs is being researched. Tissue constructs with intact extracellular matrix are generated using stimuli responsive polymers. In this study we evaluated the feasibility of using thethermoresponsive poly(Nisopropylacrylamide-co-glycidylmethacrylate) polymer as a culture surface to harvest viable corneal endothelial cell sheets. Incubation below the lower critical solution temperature of the polymer allowed the detachment of the intact endothelial cell sheet. Phase contrast and scanning electron microscopy revealed the intact architecture, cobble stone morphology, and cell-tocell contact in the retrieved cell sheet. Strong extracellular matrix deposition was also observed. The RT-PCR analysis confirmed functionally active endothelial cells inthe cell sheet as evidenced by the positive expression of aquaporin 1, collagen IV, Na+-K+ATPase, and FLK-1. Na+-K+ATPase protein expression was also visualized by immunofluorescence staining. These results suggest that the in-house developed thermoresponsive culture dish is a suitable substrate for the generation of intact corneal endothelial cell sheet towards transplantation for endothelial keratoplasty.
... Cells established by a retrovirus carry a potential risk of promoting carcinogenesis [30], and direct transplantation to humans of cell sheets composed of such cells may lead to complex problems. Recently, to resolve this problem, several studies have reported the establishment of untransfected corneal endothelial cell lines [31,32,33], which are the most ideal cell lines for the treatment of human corneal disease. Meanwhile, alternative bioengineering approaches, including lipofection of p27kip1 siRNA [34], proteomics technology analyzing the difference between younger and older HCEC [35] and drug usage of promyelocytic leukemia zinc finger protein, a cell cycle transcriptional repressor and negative regulator [36], have also been introduced. ...
Hexagonal-shaped human corneal endothelial cells (HCEC) form a monolayer by adhering tightly through their intercellular adhesion molecules. Located at the posterior corneal surface, they maintain corneal translucency by dehydrating the corneal stroma, mainly through the Na(+)- and K(+)-dependent ATPase (Na(+)/K(+)-ATPase). Because HCEC proliferative activity is low in vivo, once HCEC are damaged and their numbers decrease, the cornea begins to show opacity due to overhydration, resulting in loss of vision. HCEC cell cycle arrest occurs at the G1 phase and is partly regulated by cyclin-dependent kinase inhibitors (CKIs) in the Rb pathway (p16-CDK4/CyclinD1-pRb). In this study, we tried to activate proliferation of HCEC by inhibiting CKIs. Retroviral transduction was used to generate two new HCEC lines: transduced human corneal endothelial cell by human papillomavirus type E6/E7 (THCEC (E6/E7)) and transduced human corneal endothelial cell by Cdk4R24C/CyclinD1 (THCEH (Cyclin)). Reverse transcriptase polymerase chain reaction analysis of gene expression revealed little difference between THCEC (E6/E7), THCEH (Cyclin) and non-transduced HCEC, but cell cycle-related genes were up-regulated in THCEC (E6/E7) and THCEH (Cyclin). THCEH (Cyclin) expressed intercellular molecules including ZO-1 and N-cadherin and showed similar Na(+)/K(+)-ATPase pump function to HCEC, which was not demonstrated in THCEC (E6/E7). This study shows that HCEC cell cycle activation can be achieved by inhibiting CKIs even while maintaining critical pump function and morphology.
... Diverse types of cells and various scaffold carriers have been combined previously and a therapeutic efficiency of about one week of maintenance of corneal transparency has been attained in rabbit (Aboalchamat et al., 1999; Ishino et al., 2004; Koizumi et al., 2008; Fan et al., 2009b; Proulx et al., 2009). Unfortunately, all the previous trials fell well short of a clinical therapy for PCEP; the immortalized seed cells were either potentially tumorigenic or limited in number after primary culture or 4–5 rounds of subculture (Fan et al., 2007; 2009a ). Since 2009, we have successfully constructed TE-HCEs using a non-transfected HCE cell line as the source of seed cells and modified denuded amniotic membranes (mdAMs) as the carrier frames. ...
To evaluate the therapeutic efficiency of tissue-engineered human corneal endothelia (TE-HCEs) on rabbit primary corneal endotheliopathy (PCEP), TE-HCEs reconstructed with monoclonal human corneal endothelial cells (mcHCECs) and modified denuded amniotic membranes (mdAMs) were transplanted into PCEP models of New Zealand white rabbits using penetrating keratoplasty. The TE-HCEs were examined using diverse techniques including slit-lamp biomicroscopy observation and pachymeter and tonometer measurements in vivo, and fluorescent microscopy, alizarin red staining, paraffin sectioning, scanning and transmission electron microscopy observations in vitro. The corneas of transplanted eyes maintained transparency for as long as 200 d without obvious edema or immune rejection. The corneal thickness of transplanted eyes decreased gradually after transplanting, reaching almost the thickness of normal eyes after 156 d, while the TE-HCE non-transplanted eyes were turbid and showed obvious corneal edema. The polygonal corneal endothelial cells in the transplanted area originated from the TE-HCE transplant. An intact monolayer corneal endothelium had been reconstructed with the morphology, cell density and structure similar to those of normal rabbit corneal endothelium. In conclusion, the transplanted TE-HCE can reconstruct the integrality of corneal endothelium and restore corneal transparency and thickness in PCEP rabbits. The TE-HCE functions normally as an endothelial barrier and pump and promises to be an equivalent of HCE for clinical therapy of human PCEP.
... During logarithmic phase, the culture supernatant of HCS cells was collected and centrifuged at 2,000× g for 30 min. In vitro culture of HCE cells: In vitro culture of HCE cells was initiated as described previously [16]. Donated fresh human corneas were placed flatly in a 35 mm culture dish. ...
... Although numerous attempts have been made to cultivate HCE cells for protracted periods in vitro [7,8,10,11], cultured HCE cell lines have only been developed by transfection with viral oncogenes coding for Ha-ras, SV40 large T antigen and HPV16 E6/E7 [12][13][14][15]. The effectiveness of these transfected cell lines as potential research models has been hampered by genetic instability, abnormal phenotypes, and tumorigenicity, precluding their effective use in studies of normal endothelial cell biology and clinical corneal endothelial cell replacement [16]. No untransfected HCE cell line has been established before this study, except for the two untransfected rabbit corneal endothelial cell lines that we established previously [16,17]. ...
... The effectiveness of these transfected cell lines as potential research models has been hampered by genetic instability, abnormal phenotypes, and tumorigenicity, precluding their effective use in studies of normal endothelial cell biology and clinical corneal endothelial cell replacement [16]. No untransfected HCE cell line has been established before this study, except for the two untransfected rabbit corneal endothelial cell lines that we established previously [16,17]. ...
To establish an untransfected human corneal endothelial (HCE) cell line and characterize its biocompatibility to denuded amniotic membrane (dAM).
Primary culture was initiated with a pure population of HCE cells in DMEM/F12 media (pH 7.2) containing 20% fetal bovine serum and various supplements. The established cell line was characterized by growth property, chromosome analysis, morphology recovery, tumorigenicity assay, and expression of marker proteins, cell-junction proteins, and membrane transport proteins. The biocompatibility of HCE cells to dAM was evaluated by light microscopy, alizarin red staining, immunofluorescence assay, and electron microscopy.
HCE cells proliferated to confluence 6 weeks later in primary culture and have been subcultured to passage 224 so far. A continuous untransfected HCE cell line with a population doubling time of 26.20 h at passage 101 has been established. Results of chromosome analysis, morphology, combined with the results of expression of marker protein, cell-junction protein and membrane transport protein, suggested that the cells retained HCE cell properties and potencies to form cell junctions and perform membrane transport. Furthermore, HCE cells, without any tumorigenicity, could form confluent cell sheets on dAMs. The single layer sheets that attached tightly to dAMs had similar morphology and structure to those of HCE in situ and had an average cell density of 3,413±111 cells/mm².
An untransfected and non-tumorigenic HCE cell line has been established, and the cells maintained positive expression of marker proteins, cell-junction proteins and membrane transport proteins. The cell line, with excellent biocompatibility to dAM, might be used for in vitro reconstruction of HCE and provides a promising method for the treatment of diseases caused by corneal endothelial disorders.
Human corneal endothelium (HCE) is vital in maintaining corneal transparency and thickness, and damages to it may result in endotheliopathy and even corneal blindness, which can only be cured by keratoplasty. Due to the shortage of donor corneas, tissueengineered HCE (TE-HCE) has become an equivalent to HCE. This study constructs a high endothelial cell density (ECD) TE-HCE and evaluates its functions by corneal transplantation in rabbits. A TE-HCE was constructed by culturing DiI-labeled nontransfected HCE cells on modified denuded amniotic membrane (mdAM) using collagen IV, fibronectin, and laminin, and rabbit corneas were characterized both in vivo and ex vivo after TE-HCE transplantation. Our results indicated that the constructed TE-HCE with a high ECD of 3611 ± 56.66 cells/mm2 had similar morphology and structure to a native HCE. In vivo postsurgery detections showed that the TE-HCE-transplanted cornea gained transparency and recovered its thickness gradually during a monitoring period of 358 days, while the mdAM-transplanted cornea remained opaque and edematous. Ex vivo examinations revealed that a native-like corneal endothelium with an ECD of 2703 ± 70.37 cells/mm2 was reconstructed by the transplanted TE-HCE. Our findings suggested that the TE-HCE might be used as a HCE equivalent and has promising applications in therapy of corneal endotheliopathy.
Background: How to harvest the purified corneal endothelial seed cells is very important for the corneal tissue engineering technology. Herein, to establish a good culture method and effective identification method of corneal endothelial cells (CECs) is the key. Objective: Present study was to establish the cultivating and identifying approach of the rabbit CECs and detect the biological characteristics of passaged cells. Methods: Rabbits CECs were isolated from Descemet's membrane peeled off completely in 30 New Zealand white rabbits and then digested with 0.25% trypsin-0.02% EDTA and primarily cultured in CECs medium containing 15% fetal bovine serum. The growth situate and cellular morphology of rabbit CECs were observed under the inverted phase-contrast microscope following the alizarin red staining. Rabbit CECs were identified by cellular morphology as well as in gene and protein level, including the detection of expressions of collagen type IV α2(COL4A2), vascular endothelial growth factor receptor 2 (FLK1), Na +-K + ATPase alpha 1 subunit (ATP1A1), aquaporin 1 (AQP1), voltage-dependent anion channels (VDACs) by reverse transcription-polymerase chain reaction (RT-PCR). The expression and distribution of neurone specific enolase (NSE), Na +-K + ATPase, zonula occludens-1 (ZO-1) were also detected by immunocytochemistry under the fluorescence microscope. The proliferation activity of the passage cells was dynamically observed by MTT assay, and Na +-K + ATPase activity of different generations cells was detected by ATPase kit. Results: Majority of the primarily cultured cells adhered in 24 hours, infused in 2-3 days with the hexagon shape in appearance. The morphology of the cells was very varied with passage. Alizarin red staining showed a well-defined and well-lined cellular morphology similar to the corneal cells in vivo. Target genes of COL4A2, FLK1, ATP1A1, AQP1 and VDACs were positively expressed in the cells. However, the expression of CK12 was absent in the cells. NSE, Na +-K + ATPase, ZO-1 positive cells were respectively observed under the laser confocal scanning microscopy. MTT results showed a gradually low growth curve with the passage. Quantitative results also revealed that the Na +-K + ATPase activity was gradually declined in different generations of cells (F=77.174, P=0.000). Conclusion: The enzyme digestion is a better approach of isolating and culturing corneal endothelial cells. Cultured cells can be identified by cells staining, gene expression and protein level. Earlier generation of CECs are ideal seed cells for cornea tissue engineering.
• AIM: To investigate biological functions of in vitro reconstructed tissue-engineered human corneal endothelia (TE-HCE) by animal corneal endothelium transplantation. • METHODS: TE-HCEs, reconstructed by using untransfected human corneal endothelialcells (HCE cells, labeled with CM-Dil) as seed cells and modified denuded amniotic membrane (mdAM) as scaffold carriers, were used for penetrating corneal endothelium transplantation in New Zealand white rabbits whose corneal endothelium along with Descemet's membrane (DM) was ripped off before transplantation. The corneal transparency was monitored with a slit-lamp biomicroscope, and the CM-Dil label of seed cells was checked with a fluorescent microscope. The morphology of seed cells, formation of cell junctions, integrality of endothelial monolayer and its integrated status to DM were investigated by Alizarin red staining, freeze-section's hematoxylin-eosin (HE) staining and scanning electron microscopy. The ultrastructure of seed cells, DM and corneas were examined by transmission electron microscopy. • RESULTS: Slit-lamp biomicroscopic observation of transplanted eyes showed that the TE-HCEs could maintain corneal transparency of the transplanted New Zealand white rabbits for more than 39 days. Fluorescent observations showed that all the seed cells in the transplanted area had positive CM-Dil labels. Alizarin red staining, freeze-section 's HE staining and scanning electron microscopic detections showed that most of seed cells were in hexagonal morphology, integral endothelial monolayer was reconstructed with tight intercellular junctions, and endothelial monolayer integrated tightly to DM, secreted from seed cells. Transmission electron microscopic examination showed that a continuous endothelial monolayer was reconstructed by transplanted TE-HCE, and the ultrastructures of seed cells, DM and corneas were almost the same with those from control eyes. • CONCLUSION: The cell morphology, status of continuous monolayer, cell junction and ultrastructure of transplanted TE-HCEs are almost the same with those of rabbit corneal endothelia from control eyes. The TE-HCEs, with similar structures and functions to those of rabbit corneal endothelia, have abilities of maintaining long term cornea transparency of New Zealand white rabbits, and may be used promisingly as HCE equivalents for clinical corneal endothelium transplantation.
• AIM: To reconstruct tissue-engineered human corneal endothelia (TE-HCE) in vitro and characterize them in morphology and structure. • METHODS: Monoclonal HCE cells (mcHCE cells) were cloned from untransfected HCE cell line by limited dilution, and their karyotypes were analyzed by routine methods of chromosomal preparing and karyosystematics. Modified denuded amniotic membranes (mdAMs) were prepared from amniotic membrane by inverted trypsin denudation and coated with extracellular matrix proteins. TE-HCEs were in vitro reconstructed by using mcHCE cells at logarithmic phase as seed cells and mdAMs tiled on well bottoms of a 24-well culture plate as scaffold carriers, which were cultured in 200mL/L fetal bovine serum (FBS)-containing DMEM/F12 medium at 37°C in a 50mL/L CO2 incubator. The morphology of seed cells, formation of cell junctions, integrality of endothelial monolayer and its integrated status to mdAM were investigated by Alizarin red staining, freeze-section's hematoxylin-eosin (HE) staining, inverted microscopy and scanning electron microscopy. The ultrastructure of seed cells on mdAM and formation of cell junctions were examined by transmission electron microscopy. The expression patterns of different cell junction proteins of TE-HCE seeder cells were detected by immunofluorescent techniques. • RESULTS: Seven mcHCE cell strains with normal karyotype (2n =46) were screened out from the untransfected HCE cell line. About 30 hours after reconstruction initiation, mcHCE seeder cells formed an integrated monolayer on mdAM with a cell density as high as 3 413/mm2. Most of seed cells were in polygonal morphology, integral endothelial monolayer was reconstructed with various cell-cell and cell-mdAM junctions. And the ultrastructure of seed cells was similar to that of HCE cells in vivo, with a lot of mitochondria scattered in cytoplasm. Besides, the seed cells maintained positive expression of cell junction proteins such as zonula occludens protein 1, N-cadherin, connecxin-43 and integrin áv/β5. • CONCLSUION: The TE-HCEs, with similar morphology and structure to those of HCE in vivo, were successfully reconstructed, and can be used promisingly as HCE equivalents for clinical corneal endothelium transplantation.
