Cell Dissociation Techniques in Salamanders

Abstract

Cell dissociation is an important technique for the study of tissue phenotypes. The method chosen to harvest cells from solid tissues profoundly influences the types of cells recovered. Methodology also shapes any biases that are introduced that can act upon cell surface protein phenotypes or gene expression. Here we describe examples of cell surface phenotypic changes and typical yields, under 4 different isolation conditions (enzymatic/non-enzymatic), using the axolotl spleen, and the regenerating limb. We describe simple methods for evaluating the liberation of viable cells and the downstream characterization of cell diversity using a live-cell flow cytometry approach. Of note, the cellular composition of dissociated cells and surface antigen detection vary with each condition. TrypLE and "no enzyme" protocols give the highest surface marker expression, but poor liberation of non-immune cells in the blastema. Liberase-DH and Liberase-TL have alternative neutral proteases and both give acceptable dissociation of diverse cell types in the blastema. Liberase-TL provides the highest yield of all cell sizes and a larger non-immune fraction. Matching dissociation times between limb blastemas and spleens, we demonstrate the effect of "over-digestion" in soft tissues. In the spleen, the Liberase enzyme cocktails produced the lowest yields, worst viability, and the greatest loss of immune cell surface markers, when compared with non-enzymatic and TrypLE dissociation. These examples provide a template for optimizing protocols for individual tissues while achieving the balance between cell recovery and the mitigation of cellular changes appropriate for downstream applications such as single-cell RNA sequencing and flow cytometry.

Keywords: Axolotl; Cell dissociation; Phenotyping; RNAseq; Regeneration; Salamander; Single cells.