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Tej Kalakuntla, Yuntian Xue, Andrew Browne, Bryce McLelland, Gabriel Nistor, Hans Keirstead, William Tang, Magdalene J Seiler; Pre-transplantation analysis of intrinsic fluorophores by 2-photon microscopy to validate suitability of retinal organoids. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3313.
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© ARVO (1962-2015); The Authors (2016-present)
Stem cell derived retinal organoid transplants improved vision in rat retinal degeneration models (McLelland et al, 2018, IOVS). Tissue selection prior to transplantation of retinal organoids include visual inspection using traditional brightfield microscopy. However, the subjectivity of user-dependent visual selection neglects the need for quantitative metrics to objectively discern the quality of tissue. Dual-wavelength excitation induces the intrinsic auto-fluorescence of metabolic marker NADH. Fluorescence Lifetime Imaging Microscopy (FLIM) can identify retinal cell populations as glycolytic or oxidative. In addition to providing detailed structural images which reveal arrangement and lamination of cellular layers, information from intrinsic fluorophores can quantify metabolic activity on a cellular level (Browne et al. 2017, IOVS). Fluorescence Lifetime Imaging Microscopy (FLIM) can indicate cellular metabolism as either glycolytic or oxidative.
Retinal organoids were produced and provided by AIVITA Biomedical. Organoids were analyzed by brightfield microscopy to confirm that all subjects seem viable according to traditional verification methods. Organoids were then analyzed via 2-photon excitation (740nm) to provide structural and metabolic data for analysis.
By brightfield microscopy, all organoids appear to be of retinal composition and likely viable. FLIM imaging demonstrates a difference in the total area of glycolytic metabolic activity between organoids. Additionally, results have shown a difference in the free NADH to bound NADH ratio – a high free NADH to bound NADH ration is indicative of glycolytic activity – among organoids. Retinal photoreceptors cells are glycolytic, so an increased distribution of glycolytic activity can indicate presence of photoreceptors and suitability of the tissue for transplantation.
While all organoids utilized were produced under similar conditions and confirmed to be viable via traditional visual inspection methods, differences in glycolytic activity were seen. Therefore, 2-photon excitation of intrinsic fluorophores is an essential step to confirm suitability of stem cell derived tissues prior to transplantation.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Comparison of Brightfield and FLIM Images. FLIM images demonstrate different total area of autofluorescence, as well as different levels of glycolytic activity.
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