Abstract
Purpose :
Single-cell technologies are limited by loss of biological information and confounding variables introduced by tissue dissociation. To more accurately reconstruct the cellular networks that underly ocular biology, novel methods are needed that preserve in vivo states while capturing more biological data, such as phospho-signaling.
Methods :
Mouse and human tissues were preserved using a deep eutectic solvent (DES) fixative (vivoPHIX™) and dissociated with an ultrasonication waterbath. A variety of molecular techniques were used including qPCR, western blotting (WB), flow cytometry (FC), confocal microscopy, and imaging FC. Single-cell RNA-sequencing (scRNA-seq) was performed on human retina and mouse bone marrow. Intracellular Cellular Indexing of Transcriptomes and Epitopes (inCITE-seq) was performed on mouse Th17 T cells.
Results :
DES-based fixation of mouse and human tissues allowed the long-term preservation of RNA quality and quantity, as assessed by qPCR and gel electrophoresis. DES fixation of human retina followed by ultrasonic dissociation retained the morphology of individual neurons, including dendritic arbors and other complex structures. Head-to-head comparisons of scRNA-seq from paired viable vs DES-fixed mouse bone marrow resulted in similar RNA recovery without transcript skewing. Viable bone marrow displayed activation signatures in immune populations, such as STAT1 and STAT3 upregulation, and showed markers of cellular stress, such as upregulation of ubiquitin genes, which was confirmed with WB. scRNA-seq from DES-fixed human retina revealed successful recovery of target populations and the identification of novel pericyte heterogeneity. Lastly, we show that DES preserves post-translational modifications, including phosphorylation, using WB and FC in human retina and mouse tissues. We developed an optimized method for inCITE-seq that allowed the simultaneous quantification of 4 phospho-targets in stimulated vs control mouse Th17 T cells. Compared to gold standard FC measurements from identical samples, inCITE-seq values for phos-ERK1/2, phos-FOS, phos-p65, and phos-STAT3 had R2 correlations of 0.97, 0.96, 0.98, and 0.719 respectively.
Conclusions :
The use of DES-based fixation in single-cell technologies improves the reconstruction of in vivo cellular networks and allows the parallel integration of transcriptomes and intracellular signaling pathways in single-cells.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.