Isolation, culture and genetic manipulation of mouse pancreatic ductal cells

Abstract

The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). pDac resembles duct cells morphologically and, to some extent, at a molecular level. recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and duct-like cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. this approach is fast (∼4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas.

175 The Ets-Transcription Factor Etv1 Regulates Stromal Expansion and Metastasis in Pancreatic Cancer

“…To examine the potential role of Etv1 in pancreatic ductal morphogenesis, we utilized a 3D organoid culture system. 28 Using KC cells, we noted that these ductal organoids suspended in collagen tend to form regular cysts with hollow lumens ( Figure 3F). However, in Etv1 overexpressing cells, we saw significantly more cysts that are highly irregular/disrupted and demonstrate a spindle-shape morphology ( Figure 3F).…”

“…Three-dimensional (3D) pancreatic ductal cell organoids were performed as described previously 28,29 and as detailed in Supplemental Experimental Procedures.…”

“…Mouse primary pancreatic cells were cultured and maintained as described previously. 19,28 KPfC and KPfC Sparc -/-cells were transduced with dTomato to allow for lineage labeling in vivo. Sorting and Isolation of Ecad + and Ecad-fractions from KPfCY mice is described in detail in Supplemental Experimental Procedures.…”

“…Lentiviral transduction was completed as described previously. 28 Parental cell lines were transduced in parallel with Etv1-Flag constructs or empty vector constructs (Control).…”

Incorporation of polaxamers during tissue dissociation allows isolation of high quality RNA from FACS-sorted pancreatic cells

“…Current methods for avoiding the degradation of intact RNA from pancreatic tissues include the use of perfusion techniques to inactivate RNAses using inhibitors (1), the use of commercially available RNAse inhibitors to store tissues prior to RNA extraction (3) and techniques that require rapid freezing prior to tissue processing. Furthermore, a number of methods have been described to isolate ductal cells (6), centroacinar cells (8) and alpha cells from the pancreas (4). Although these protocols yield material that is adequate for RT-PCR and some microarray applications, its use in whole transcriptome analysis by RNA-Sequencing is limited due to the requirement for purified RNA with RNA Integrity Numbers (RIN) greater than eight.…”