Long-term Quantitative Analysis of Intrinsic Fluorophores in Retinal Organoids by 2-Photon Excitation Microscopy

Abstract

Purpose : Retinal organoids transplantation has shown the potential to restore visual function of retinal degeneration rats (McLelland et al, 2018, IOVS). Advanced imaging techniques can help with the quality control of transplantable tissues. The fluorescence lifetime imaging microscopy (FLIM) and hyperspectral imaging (HSpec) modules integrated with 2-photon excitation microscopy (2PE) can provide metabolic information of intrinsic fluorophore – NADH – and retinol distribution inside organoids at the cellular level (Browne et al, 2017, IOVS). We applied 2PE to monitor retinal organoids' development up to 6 months.

Methods : Retinal organoids were produced and provided by AIVITA Biomedical. Twenty-Eight retinal organoids from four GMP-compatible lots were imaged from 40~50 to 180~190 days of differentiation by 2PE. 10% FBS and 1uM retinoic acid were added to the retinal development media after day 42 of differentiation for organoids long-term maintenance. Images were taken by Zeiss LSM 710 using 740nm pulsed excitation. HSpec fluorescence emissions in the range of 420 nm to 690 nm. Data were analyzed by SimFCS (Global Software) via the phasor approach.

Results : Long-term imaging data from two organoids batches (>180 days) were evaluated. All imaged organoids demonstrated metabolic activities confirming overall cellular viability. As the organoids continued their development in culture, a shift from glycolytic to oxidative metabolic activities was observed initially. As time progressed, a predominance of glycolysis on the surface of the organoids emerged. The quantitative free/bound NADH ratio also demonstrated this trend. HSpec images showed retinol distribution on the surface, which demonstrated the usefulness of retinal organoids in mimicking certain developmental aspects of the photoreceptors of the retina.

Conclusions : In this study, the cultured retinal organoids experienced a similar trend of natural retinal development in metabolic shift in the long term, from glycolytic (proliferative) to oxidative (differentiated) state, and back to the glycolytic surface (a marker of photoreceptor layer). FLIM and HSpec were used as the non-invasive imaging tools for long-term in vitro retinal organoids characterization. Multimodal imaging, therefore, warranted further exploration as a tool to help with the quality control of transplantable tissues.

This is a 2020 ARVO Annual Meeting abstract.

 

Long-term imaging.