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

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.

Fig. 1

Scheme for the isolation of single cells from adult mouse pancreas through warm collagenase digestion, warm digestion with combined collagenase and trypsin-EDTA, or cold digestion with trypsin-EDTA

Fig. 2

Comparison of the single-cell suspensions prepared by warm collagenase digestion, warm digestion with combined collagenase and trypsin-EDTA, or cold digestion with trypsin-EDTA (a) The cell suspension prepared by warm collagenase digestion; (b) The cell suspension prepared by warm digestion with combined collagenase and trypsin-EDTA; (c, d) The cell suspension prepared via cold digestion with trypsin-EDTA; (e) Yields of single cells prepared by warm collagenase digestion (Co), warm digestion with combined collagenase and trypsin-EDTA (C+T), or cold digestion with trypsin-EDTA for 16 h (T16 h) (mean±SD, n=5; ^** P<0.01); (f) The ratio of viable single cells to total single cells in suspensions prepared by warm collagenase digestion (Co), warm digestion with combined collagenase and trypsin-EDTA (C+T), or cold digestion with trypsin-EDTA for 16 h (T16 h) (mean±SD, n=5; ^** P<0.01)

Fig. 3

Effect of incubation time on cell yield and viability in cold trypsin-EDTA digestion and flow cytometry analysis of single cells (a) Yields of single cells and ratios of viable single cells to total single cells after incubation of pancreas fragments in trypsin-EDTA fluid for 2, 6, 10, 14, 18, 22, or 24 h at 4 °C (n=5). (b) Flow cytometry analysis of the percentages of the whole cell population (upper left), isotype control (upper right), and ductal (lower left), and BCRP1-positive (lower right) cells in single-cell suspensions prepared by cold trypsin-EDTA digestion for 16 h

Fig. 4

Proliferation capacity of epithelial cells isolated by cold trypsin-EDTA digestion Colonies grown from the isolated cells through cold trypsin-EDTA digestion (a) were similar to those grown from the isolated cells through warm collagenase digestion (b). Single cells isolated through cold trypsin-EDTA digestion formed a colony after being cultured for 5 d (c) or 10 d (d); The colonial cells (e) were E-cadherin-positive (f) epithelial cells identified by immunofluorescent staining