Background

Proliferative diabetic retinopathy (PDR) is a late-stage complication of diabetes, responsible for vision loss in diabetic patients. The formation of fibrovascular membranes and scar tissue in the pre-retinal space leads to retinal traction and detachment [1]. Understanding the molecular features of the cells contributing to fibrovascular membrane formation and the cell–cell interactions is crucial for identifying new therapeutic targets that improve treatment approaches and patients’ quality of life.

scRNA-Seq is a powerful technique that allows us to study the molecular profile of each single cell. Since the first paper on scRNA-Seq was published in 2009, studies on this technique have provided insightful information in several fields, followed by exponential growth in the last decade [2–8]. In the context of retinal biology, this approach was fundamental for building a comprehensive transcriptome atlas of fetal and adult retinas and for dissecting the retinal developmental stages [9–11]. scRNA-Seq assays have also revealed the complexity and heterogeneity of the different cell types in the retina and aided in uncovering cell-specific gene expression changes in pathological conditions [12–17]. Single-cell transcriptomics also enables the study of individual pathogenic cell populations in the context of fibrosis at quite high resolution [18–22].

Despite significant improvements, scRNA-Seq is still a challenging technique [23]. Generating high-quality single-cell suspensions from any tissue is critical to preserve their expression profile and ensure meaningful downstream transcriptome data analysis. Several parameters in the dissociation protocol can compromise the viability of the cells and potentially impact the quality of the scRNA-Seq data. Preparing cell suspensions might be difficult for tissue samples, especially for fibrotic tissue, where gentle dissociation might not be sufficient to break the fibers of scar tissue, leading to inefficient cell yield after processing. On the other hand, an excessively harsh digestion might differentially damage cells that are more fragile [23,24].

Papain-based protocols have been shown to successfully dissociate retinal tissue and have been described in several scRNA-Seq studies of retina tissue and organoids [16,23,25]. Another enzyme described for tissue digestion is Collagenase (I and IV), which was described to digest fibrovascular membranes from proliferative diabetic retinopathy patients as well as mouse retinas and other tissues into single-cell suspensions [18,21,26–28]. In our experiments, we used a Collagenase I–based protocol [29].

Another important factor in the scRNA-Seq experiments is the downstream data analyses. Throughout the analysis, it is important to include filtering steps in data analysis processing with the aim of excluding low-quality cells or empty droplets that have very few genes and cell doublets that may exhibit an aberrantly high gene count.

Our protocol is quite efficient in digesting the fibrovascular membranes from PDR patients [29], and we were able to preserve a lot of cell types with a high number of genes detected per cell.