Development and characterization of an in vitro system of the human retina using cultured cell lines

Abstract

Background Previously developed in vitro cultures of the human retina have been solo or dual cell cultures. We developed a triple‐cell culture in vitro model utilizing a membrane system to produce a better representation of a functional and morphological human retina. Methods Retinal microvascular endothelial cells (HRMVEC/ACBRI181, cell systems), retinal pigment epithelium cells (RPE/ARPE‐19, ATCC) and Müller glial cells (Moorfield Institute of Ophthalmology‐Müller 1, UCL) were grown in a triple culture. Our optimized triple‐culture media contained a mix of specific endothelial medium and high glucose Dulbecco's Modified Eagle's medium, where all three layers were viable for up to 5 days. Co‐culture effect on morphological changes (cell staining) and gene expression of functional genes (pigment epithelium derived factor [PEDF] and vascular endothelial growth factor [VEGF]) were measured from RNA via real‐time polymerase chain reaction. Expression of tight junction protein 1 (TJP1) was measured in RNA isolated from ARPE‐19s, to assess barrier stability. Results The triple‐culture promotes certain cell functionality through up‐regulation of TJP1, increasing PEDF and decreasing VEGF expression highlighting its importance for the assessment of disease mechanisms distinct from a solo culture which would not allow the true effect of the native microenvironment to be elucidated. Conclusions This model's novelty and reliability allows for the assessment of singular cellular function within the retinal microenvironment and overall assessment of retinal health, while eliminating the requirement of animal‐based models.

Impact of Culture Medium on Cellular Interactions in in vitro Co-culture Systems

“…Most co-culture studies involve two cell types, owing to an increased complexity in establishing a stable system when more cell types are involved (Goers et al, 2014). There are also studies reporting the use of three (Venter and Niesler, 2018;Churm et al, 2019;Lin et al, 2019) or even four cell types (Zhang et al, 2009;DesRochers et al, 2015). Different strategies to co-culture cells in 3D exist, each allowing for a different degree of contact between the cell types.…”

Tools and Data Towards Supporting Quantitative Systems Pharmacology of the Eye – An Overview

“…Cell systems may include primary human cells of one type or co-cultures in which different cell types of one tissue are cultivated together to account for intracellular, e.g., cytokine-mediated communication (21,22). Using coculture systems, it is possible to replicate complex ocular structures such as the retina for experimental studies in vitro (23,24). The use of polymer-based scaffolds in co-culture systems allows colonisation by the different cells and leads to the formation of cellular interactions within a spatial 3D structure in vitro, allowing the interaction patterns to partially reproduce in vivo situations (25), (21) (26), (27).…”

Tissue engineering in age-related macular degeneration: a mini-review

“…Cell lines are particularly suited for rapid and efficient screening of drug candidates for their toxicity and biocompatibility. Different cell lines that have been used for retinal research include the ARPE-19, RPE-1, D407 (RPE cell type), human WERI-RB1, Y79 retinoblastoma derived cell lines, 661 W (photoreceptor cell type), RGC-5 (ganglion cell type), and MIO-M1(glial Müller cell type) among others [ 66 – 68 ]. Although cell lines are widely accessible and easily cultured, up to 20% of currently used cell lines are contaminated or erroneously categorized, thus, validation of its origins and characteristic properties are of paramount importance prior to initiating work with them [ 69 ].…”