Development and Evaluation of Porcine Atelocollagen Vitrigel Membrane With a Spherical Curve and Transplantable Artificial Corneal Endothelial Grafts

Yoshida, Junko; Oshikata-Miyazaki, Ayumi; Yokoo, Seiichi; Yamagami, Satoru; Takezawa, Toshiaki; Amano, Shiro

doi:10.1167/iovs.14-14211

Cited by 42 publications

(35 citation statements)

References 20 publications

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“…Porcine eye may be a good animal model to mimic human ocular conditions because access to human eyes is limited while fresh porcine eyes are readily available; the physical dimensions and anatomic properties of porcine eyeball are similar to those of humans 20,21 ; the electrolyte composition of the AH as well as the characteristics of AH dynamics in pig are similar to those in humans 1,19 ; and the biocompatibility of pig and human eyes has been demonstrated in an artificial corneal graft study. 32 Our recent findings of cAMP-and forskolininduced Isc stimulation in porcine CE 22 are also in good agreement with the results obtained for the human ciliary process, 23 suggesting that pig may be a good animal model for studying AH dynamics.…”

Section: Species Variation In Aqueous Humor Secretionsupporting

confidence: 84%

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium

Shan

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. Gap junctions provide a conduit between the intracellular fluids of the pigmented (PE) and non-pigmented (NPE) ciliary epithelial cells, and are therefore critical in the secretion of the aqueous humor (AH). However, opinions differ concerning the connexin (Cx) composition of the gap junctions. Therefore, we aimed to characterize the expression of Cx in the porcine ciliary epithelium (CE), a favorable model for humans; and determine the contribution of the highest expressed Cx to AH secretion. METHODS.Freshly-harvested porcine CE cells were used. The mRNA and protein expressions of gap junctions were assessed by reverse transcription polymerase chain reaction (RT-PCR) and Western blotting (WB), respectively. The relative gene expressions of various Cx were determined by quantitative PCR. The gap junction permeability of isolated PE-NPE cell couplets was evaluated by Lucifer Yellow dye transfer.RESULTS. Using RT-PCR and WB, Cx43, Cx45, Cx47, Cx50, and Cx60 were present in porcine CE, with Cx43 being the most abundant isoform, having over 200-fold higher expression than other Cx. Cx43 was primarily localized in the PE-NPE interface and the basolateral membranes of PE cells. Knockdown of Cx43 by siRNA significantly reduced gene and protein expressions, resulting in reduction of transcellular fluid flow by 90%.CONCLUSIONS. Cx43 was found to be the major component of gap junctions in porcine CE. Consistent with results from a bovine model, our results support the important role of Cx43 in mediating AH secretion. This finding may shed light on the development of a novel ocular hypotensive agent.

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Section: Species Variation In Aqueous Humor Secretionsupporting

confidence: 84%

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium

Shan

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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“…Identifying the growth conditions that support the growth of cells with a transcriptome profile similar to evHCEnC will provide researchers with a more accurate model of in vivo HCEnC and a potential source of endothelial cells for management of endothelial dysfunction. Optimization of HCEnC culturing techniques should take into consideration several anatomic and physiologic features of in vivo HCEnC (60), such as: 1) adherence to a complex milieu of extracellular matrix proteins (13,18,22,38,40,51,57); 2) contact with physiological proteins and other factors present in aqueous humor (25,33,36,42,53); 3) exposure to appropriate biomechanical forces (35,40,57); and 4) maintenance of a confluent semipermeable layer of cells with strong apicobasal polarity. Replication of each of these features in a single culturing method would be challenging, but would represent a significant advance in the development of cultured HCEnC that closely resemble in vivo HCEnC.…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell Replacement Therapy

Frausto

Aldave

2016

Cell Transplant

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The corneal endothelium plays a primary role in maintaining corneal homeostasis and clarity, and must be surgically replaced with allogenic donor corneal endothelium in the event of visually significant dysfunction. However, a worldwide shortage of donor corneal tissue has led to a search for alternative sources of transplantable tissue. Cultured human corneal endothelial cells (HCEnC) have been shown to restore corneal clarity in experimental models of corneal endothelial dysfunction in animal models, but characterization of cultured HCEnC remains incomplete. To this end, we utilized next-generation RNA sequencing technology to compare the transcriptomic profile of ex vivo human corneal endothelial cells (evHCEnC) with that of primary HCEnC (pHCEnC) and HCEnC lines, and to determine the utility of cultured and immortalized corneal endothelial cells as models of in vivo corneal endothelium. Multidimensional analyses of the transcriptome datasets demonstrated that primary HCEnC have a closer relationship to evHCEnC than do immortalized HCEnC. Subsequent analyses showed that the majority of the genes specifically expressed in HCEnC (not expressed in ex vivo corneal epithelium or fibroblasts) demonstrated a marked variability of expression in cultured cells compared with evHCEnC. In addition, genes associated with either corneal endothelial cell function or corneal endothelial dystrophies were investigated. Significant differences in gene expression and protein levels were observed in the cultured cells compared with evHCEnC for each of the genes tested except for AGBL1 and LOXHD1, which were not detected by RNA-seq or qPCR. Our transcriptomic analysis suggests that at a molecular level pHCEnC most closely resemble evHCEnC and thus represent the most viable cell culture based therapeutic option for managing corneal endothelial cell dysfunction. Our findings also suggest that investigators should perform an assessment of the entire transcriptome of cultured HCEnC prior to determination of their potential clinical utility for the management of corneal endothelial cell failure.

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“…In recent years, several groups have been using natural [25–27] or recombinant [28] collagen scaffolds for the culture of human CECs, being porcine [29] or bovine [30,31] type I collagen the most widely used.…”

Section: Introductionmentioning

confidence: 99%

Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model

et al. 2016

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Corneal keratoplasty (penetrating or lamellar) using cadaveric human tissue, is nowadays the main treatment for corneal endotelial dysfunctions. However, there is a worldwide shortage of donor corneas available for transplantation and about 53% of the world’s population have no access to corneal transplantation. Generating a complete cornea by tissue engineering is still a tough goal, but an endothelial lamellar graft might be an easier task. In this study, we developed a tissue engineered corneal endothelium by culturing human corneal endothelial cells on a human purified type I collagen membrane. Human corneal endothelial cells were cultured from corneal rims after corneal penetrating keratoplasty and type I collagen was isolated from remnant cancellous bone chips. Isolated type I collagen was analyzed by western blot, liquid chromatography -mass spectrometry and quantified using the exponentially modified protein abundance index. Later on, collagen solution was casted at room temperature obtaining an optically transparent and mechanically manageable membrane that supports the growth of human and rabbit corneal endothelial cells which expressed characteristic markers of corneal endothelium: zonula ocluddens-1 and Na+/K+ ATPase. To evaluate the therapeutic efficiency of our artificial endothelial grafts, human purified type I collagen membranes cultured with rabbit corneal endothelial cells were transplanted in New Zealand white rabbits that were kept under a minimal immunosuppression regimen. Transplanted corneas maintained transparency for as long as 6 weeks without obvious edema or immune rejection and maintaining the same endothelial markers that in a healthy cornea. In conclusion, it is possible to develop an artificial human corneal endothelial graft using remnant tissues that are not employed in transplant procedures. This artificial endothelial graft can restore the integrality of corneal endothelium in an experimental model of endothelial dysfunction. This strategy could supply extra endothelial tissue and compensate the deficit of cadaveric grafts for corneal endothelial transplantation.

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Development and Evaluation of Porcine Atelocollagen Vitrigel Membrane With a Spherical Curve and Transplantable Artificial Corneal Endothelial Grafts

Abstract: We developed a transparent and biocompatible porcine-derived atelocollagen vitrigel membrane with a spherical curvature. A transplantable artificial endothelial graft was created by combining cultured human CECs and the CV-CERT.

Cited by 42 publications

References 20 publications

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium

Characterization and Regulation of Gap Junctions in Porcine Ciliary Epithelium

Transcriptomic Analysis of Cultured Corneal Endothelial Cells as a Validation for Their Use in Cell Replacement Therapy

Human Bone Derived Collagen for the Development of an Artificial Corneal Endothelial Graft. In Vivo Results in a Rabbit Model

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