Abstract
Purpose :
Progress in genomics allows us to investigate molecular activity at the single cell level. Development of mouse models for retinal degeneration offers a valuable tool to investigate molecular mechanisms underlying specific gene disruptions causing photoreceptor death. As a whole transcriptome or candidate gene approach, automation of cell sorting for downstream application is key in the fast and systematic processing of individual cells. The purpose of this study is to optimize methods for micro-fluidic single photoreceptor capture for gene analysis.
Methods :
Photoreceptor cells were dissociated following previous protocols with minor changes (Wahlin et al, Mol Vis, 2004). 5 U/ml papain containing L-cysteine (2.7 mM) and EDTA (2 mM) was pre-activated for 5 min at 37°C. Retinas from wild-type mouse were collected, cut into 8-10 pieces and transferred in pre-activated papain for 5 min at room temperature. Retina pieces were rinsed without disturbing the pellet then resuspended in 700 µL DMEM containing 10% fetal bovine serum. Tissue was dissociated by trituration and cell suspension allowed to settle for 5 min. Presence of photoreceptor cells from an aliquot of the supernatant was evaluated with bright field microscopy. Once PRC was established, supernatant was transferred to a fresh tube and allowed to settle for 5 min. The cell suspension was loaded into a micro-fluidic chamber and Hoechst, Calcein and Ethidium Homodimer were used to evaluate live/dead status of captured material.
Results :
Cell load on the micro-fluidic chamber showed capture of biological material ranging from single to multiple (corresponding to whole cell) segment composed of 1) outer and inner segment, 2) outer segment only, or 3) cell body with inner segment without outer segment. In some cases, multiple cells corresponding to cell aggregates were also captured in individual capture sites. Chemistry for transcript analysis was run and cDNA concentration for each capture site evaluated. We determined that cDNA collected from each individual capture site is in low abundance due to material size, thus requiring low dilution when harvesting cDNA.
Conclusions :
This study establishes a method to isolate and capture individual photoreceptor cells in an automated manner that will allow investigation and analysis of gene expression with downstream applications such as RNA sequencing or targeted gene expression.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.