Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion

Abstract

Abstract:The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is prerequisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid (EDTA) digestion method to disassociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRP1-positive cells in cell suspensions prepared through cold digestion was consistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells.

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“…This was despite the fact that the overall viability of cells after dispersal from gross tumour material was unexpectedly low (< 40%), presumably due to injury caused by the enzymatic (collagenase IV, DNase II) and mechanical (mincing of gross tissue using scalpel blades) process of dissociation. The high level of cell death observed in the current study is in contrast to Li et al [ 34 ] who provided a mean cell viability of 65.66% (±4.96) using trypan blue staining following disaggregation of murine pancreatic fragments using collagenase IV. As malignant tumour tissue is marked by regions of necrosis owing to rapid oncogene-driven proliferation not supported by an adequate vascular bed [ 35 ], it is a possibility that presence of necrotic cells contributed to the lower overall viability in the current study.…”

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“…Preparation of single-cell suspensions from the mouse pancreas and pancreatic ductal cell isolation was performed as described (37,38,60). Briefly, mouse pancreata were removed and soaked overnight in cold 0.25% trypsin-EDTA (Thermofisher Scientific).…”

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“…We tested different procedures to increase the yield of single-dissociated cells. For example, overnight digestion with cold trypsin at 4°C, as described by Li et al ( 2013 ), produced an insufficient number of viable dissociated cells (~8 000 000 cells/pancreas) (data not shown). When we performed a collagenase (Col) digestion at 37°C with either EGTA- or Ca 2+ - buffer, throughout the protocol, we obtained either a low number of dissociated cells or a high number of cells still organized in clusters, respectively.…”