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Development and characterization of an in vitro system of the human retina using cultured cell lines
Author(s) -
Churm Rachel,
Dunseath Gareth J.,
Prior Sarah L.,
Thomas Rebecca L.,
Banerjee Sanjiv,
Owens David R.
Publication year - 2019
Publication title -
clinical and experimental ophthalmology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.3
H-Index - 74
eISSN - 1442-9071
pISSN - 1442-6404
DOI - 10.1111/ceo.13578
Subject(s) - pedf , microbiology and biotechnology , retina , cell culture , retinal pigment epithelium , in vitro , retinal , endothelial stem cell , vascular endothelial growth factor a , cell , vascular endothelial growth factor , biology , medicine , neuroscience , cancer research , biochemistry , genetics , vegf receptors
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.