Abstract
Purpose :
In vitro cultures of the human retina have previously been developed as solo or dual cell cultures. Only mixed species cell triple-culture systems have been developed so far. We propose an expansion of current in vitro models to utilize a membrane system for a triple-culture that will be representative of a truer functional and morphological human retina, than currently available.
Methods :
Human retinal microvascular endothelial cells (ACBRI181), human retinal pigment epithelium (RPE/ARPE-19) cells and human Müller glial (MIO-M1) cells were grown in a triple-culture (Figure 1). RPEs were matured for 28 days prior to the addition of Müller glial cells on day 28 and endothelial cells on day 30. Co-culture effect on morphological changes (via cell staining) and gene expression of functional genes (pigment epithelial derived factor (PEDF) and vascular endothelial growth factor (VEGF)) were measured from RNA via real time PCR. Expression of tight junction protein 1 (TJP1) was measured in RNA isolated from RPEs only, to assess barrier stability. All data were analysed using SPSS V25.
Results :
No alterations noted in cell morphologies in any culture formation. Gene expression analysis, PEDF was significantly increased for both RPEs (+5.2Fold Change [FC], p<0.01) and Müller glial cells (+7.5FC, p<0.05) in triple compared to solo-culture. Triple-culture VEGF expression was unaltered in both Müller glial cells (+1.0FC, p=0.49) and RPEs (+0.6FC, p=0.34) however, VEGF expression was decreased within endothelial’s (-3.2FC, p<0.05). A sub-analysis using a dual culture of; Müller glial + endothelial cells and RPEs + endothelial cells implicates Müller glial cells for the change in VEGF expression. There was a significant increase inTJP1 when RPEs are cultured with either Müller glial cells (+11.4FC, p<0.01), endothelial cells (+12.9FC, p<0.01) in dual cultures or within a triple-culture (+7.8FC, p<0.01).
Conclusions :
The triple-culture system promotes certain cell functionality through up-regulation of TJP1 and increasing PEDF and decreasing VEGF expression. Highlighting the importance of using a triple-culture for the assessment of disease mechanisms, as a solo culture would not elucidate the true effect of the native environment. This model allows exploration of singular cellular function within the retinal microenvironment in addition to analysis of overall retinal health, whilst eliminating the need of animal-based models.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.