The Effects of Different Culture Media on Human Corneal Endothelial Cells

Kim, Eunbi; Kim, Jin Joo; Hyon, Joon Young; Chung, Eun‐Sung; Chung, Tae Young; Yi, Kayoung; Wee, Won Ryang; Shin, Young Joo

doi:10.1167/iovs.14-14564

Cited by 26 publications

(25 citation statements)

References 40 publications

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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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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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“…Increased cell proliferation is achieved using insulin, growth factors (nerve growth factor, epithelial growth factor, and basic fibroblast growth factor), bovine pituitary extract, ascorbic acid, serum, and mitogens [55–57]. Chondroitin sulfate, one of the GAGs in the human cornea, is also used in the media [57], since it acts as a scaffold material of extracellular matrix (ECM) template that is needed for proper cell growth and organization [58]. Kim et al [57] compared four different media for corneal endothelial cell culture.…”

Section: Human Corneal Cell Modelsmentioning

confidence: 99%

“…Chondroitin sulfate, one of the GAGs in the human cornea, is also used in the media [57], since it acts as a scaffold material of extracellular matrix (ECM) template that is needed for proper cell growth and organization [58]. Kim et al [57] compared four different media for corneal endothelial cell culture. Traditional human corneal endothelial cell medium with FBS, epidermal growth factor, basic fibroblast growth factor, and chondroitin sulfate was the best medium in terms of stem cell-associated protein expression, cell proliferation, and migration.…”

Section: Human Corneal Cell Modelsmentioning

confidence: 99%

“…However, the cells become elongated and more fibroblast-like than in the stem cell media that was more appropriate for maintaining the correct cell shape and functionality. Fibronectin-collagen, laminin, and collagen type I are often used as a substrata for the endothelial cells [57, 59–61]. These compounds have influence on cell adhesion, proliferation, and migration.…”

Section: Human Corneal Cell Modelsmentioning

confidence: 99%

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Human corneal cell culture models for drug toxicity studies

Rönkkö

Vellonen

Järvinen

et al. 2016

Drug Deliv. and Transl. Res.

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In vivo toxicity and absorption studies of topical ocular drugs are problematic, because these studies involve invasive tissue sampling and toxic effects in animal models. Therefore, different human corneal models ranging from simple monolayer cultures to three-dimensional models have been developed for toxicological prediction with in vitro models. Each system has its own set of advantages and disadvantages. Use of non-corneal cells, inadequate characterization of gene-expression profiles, and accumulation of genomic aberrations in human corneal models are typical drawbacks that decrease their reliability and predictive power. In the future, further improvements are needed for verifying comparable expression profiles and cellular properties of human corneal models with their in vivo counterparts. A rapidly expanding stem cell technology combined with tissue engineering may give future opportunities to develop new tools in drug toxicity studies. One approach may be the production of artificial miniature corneas. In addition, there is also a need to use large-scale profiling approaches such as genomics, transcriptomics, proteomics, and metabolomics for understanding of the ocular toxicity.

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“…Corneal endothelial cells of the two species were characterized with respect to their elongation ratios. 11 To quantify this value, the long and short axes of 30 cells from three fields were measured using ImageJ software (http:// imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and divided between themselves.…”

Section: Morphology Analysis Of Corneal Endothelial Cellsmentioning

confidence: 99%

Combined PI3K/Akt and Smad2 Activation Promotes Corneal Endothelial Cell Proliferation

Sabater

Andreu

García-Guzmán

et al. 2017

Invest. Ophthalmol. Vis. Sci.

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PURPOSE. The purpose of this study was to develop a culture method for expansion of corneal endothelial cells (CEC) based on the combined activation of PI3K/Akt and Smad2.METHODS. Morphology, proliferation, and migration of cultured rabbit and nonhuman primate CEC were examined in the presence of the PI3K/Akt activators IGF-1 and heregulin beta in combination with the Smad2 activator activin A. Phenotypic characterization of CEC was performed at the RNA and protein levels. Cell pump function and transepithelial electric resistance were used for in vitro functional assessment of CEC. Finally, ex vivo-expanded rabbit CEC were transplanted into a model of endothelial damage in rabbit corneas.RESULTS. Treatment of rabbit and nonhuman primate CEC in vitro with IGF-1, heregulin beta, and activin A induced an upregulation of PI3K/Akt and Smad2 signaling pathways and an increase in proliferation and migration of CEC expressing ZO-1, connexin-43, and Na þ /K þ -ATPase. Cell pump function evaluation revealed the complete functionality of cultured CEC. Injection of rabbit CEC successfully produced recovery of normal corneal thickness in a rabbit model of endothelial dysfunction.CONCLUSIONS. We demonstrated that the combined activation of PI3K/Akt and Smad2 results in in vitro expansion of phenotypic and functional CEC. Expanded cells were able to contribute to restoration of corneal endothelium in a rabbit model. These findings may represent a new therapeutic approach for treating corneal endothelial diseases.

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The Effects of Different Culture Media on Human Corneal Endothelial Cells

Abstract: Culture medium D was superior to the other media with regard to the expression of stem cell-associated proteins, proliferation, and cell migration. However, medium N was more appropriate than the other three media with regard to maintaining the proper cell shape and function.

Cited by 26 publications

References 40 publications

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

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

Human corneal cell culture models for drug toxicity studies

Combined PI3K/Akt and Smad2 Activation Promotes Corneal Endothelial Cell Proliferation

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