Dataset Information

An in vitro 3-dimensional Collagen-based Corneal Construct with Innervation Using Human Corneal Cell Lines.

ABSTRACT:

Purpose

To develop a 3-dimensional corneal construct suitable for in vitro studies of disease conditions and therapies.

Design

In vitro human corneal constructs were created using chemically crosslinked collagen and chondroitin sulfate extracellular matrix and seeded with 3 human corneal cell types (epithelial, stromal, and endothelial) together with neural cells. The neural cells were derived from hybrid neuroblastoma cells and the other cells used from immortalized human corneal cell lines. To check the feasibility and characterize the constructs, cytotoxicity, cell proliferation, histology, and protein expression studies were performed.

Results

Optimized culture condition permitted synchronized viability across the cell types within the construct. The construct showed a typical appearance for different cellular layers, including healthy appearing, phenotypically differentiated neurons. The expected protein expression profiles for specific cell types within the construct were confirmed with western blotting.

Conclusions

An in vitro corneal construct was successfully developed with maintenance of individual cell phenotypes with anatomically correct cellular loci. The construct may be useful in evaluation of specific corneal disorders and in developing different corneal disease models. Additionally, the construct can be used in evaluating drug targeting and/or penetration to individual corneal layers, testing novel therapeutics for corneal diseases, and potentially reducing the necessity for animals in corneal research at the early stages of investigation.

Financial disclosures

Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

SUBMITTER: Islam MM

PROVIDER: S-EPMC11321308 | biostudies-literature | 2024 Nov-Dec

REPOSITORIES: biostudies-literature

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Publications

An in vitro 3-dimensional Collagen-based Corneal Construct with Innervation Using Human Corneal Cell Lines.

Islam Mohammad Mirazul MM Saha Amrita A Trisha Farzana Afrose FA Gonzalez-Andrades Miguel M Patra Hirak K HK Griffith May M Chodosh James J Rajaiya Jaya J

Ophthalmology science 20240506 6

<h4>Purpose</h4>To develop a 3-dimensional corneal construct suitable for in vitro studies of disease conditions and therapies.<h4>Design</h4>In vitro human corneal constructs were created using chemically crosslinked collagen and chondroitin sulfate extracellular matrix and seeded with 3 human corneal cell types (epithelial, stromal, and endothelial) together with neural cells. The neural cells were derived from hybrid neuroblastoma cells and the other cells used from immortalized human corneal ...[more]

PMID: 39139547

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Project description:PurposeTo study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection.MethodsHuman monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFNγ), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively.ResultsCorneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1α and MIP-1β, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation.ConclusionsHuman macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.

Dataset Information

An in vitro 3-dimensional Collagen-based Corneal Construct with Innervation Using Human Corneal Cell Lines.

Purpose

Design

Results

Conclusions

Financial disclosures

Publications

An in vitro 3-dimensional Collagen-based Corneal Construct with Innervation Using Human Corneal Cell Lines.

Similar Datasets

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