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Isolation, culture and genetic manipulation of mouse pancreatic ductal cells

Nature Protocols volume 8, pages 1354–1365 (2013)Cite this article

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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 ductlike 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.

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Figure 1: DBA lectin distribution in the normal pancreas and disease.
Figure 2
Figure 3: In vitro Cre recombination.
Figure 4: Ratio of relative gene expression in DBA lectin–positive and DBA lectin–negative cells.
Figure 5: Cells in 2D culture.
Figure 6: Confocal photomicrograph of cysts.
Figure 7: In vitro branching morphogenesis.
Figure 8: Cells in 3D culture.

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Acknowledgements

This work was supported by the US National Institutes of Health (NIH) (grant no. DK060694 to M.R., S.T., S.H., B.B., A.K.R.), the National Pancreas Foundation (M.R.), the Honjo International Scholarship Foundation (S.T.), Deutsche Krebshilfe (S.H.), the NIH/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (grant no. F30 DK088402-01 to G.P.B.), NIH/NIDDK grant no. P30 DK050306 Center for Molecular Studies in Digestive and Liver Diseases (and Molecular Pathology and Imaging, Molecular Biology/Gene Expression, Cell Culture, Transgenic and Chimeric Mouse Cores) and American Cancer Society grant no. RP-10-033-01-CCE.

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Authors and Affiliations

  1. Division of Gastroenterology, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Maximilian Reichert, Shigetsugu Takano, Steffen Heeg, Basil Bakir, Gregory P Botta & Anil K Rustgi

  2. Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Maximilian Reichert, Shigetsugu Takano, Steffen Heeg, Basil Bakir & Anil K Rustgi

  3. Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Maximilian Reichert, Shigetsugu Takano, Steffen Heeg, Basil Bakir & Anil K Rustgi

  4. Department of Hematology and Oncology, University of Freiburg Medical Center, Freiburg, Germany

    Steffen Heeg

  5. Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA

    Gregory P Botta

  6. Department of Genetics, University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Anil K Rustgi

Authors
  1. Maximilian Reichert
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  2. Shigetsugu Takano
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  3. Steffen Heeg
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  4. Basil Bakir
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  5. Gregory P Botta
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  6. Anil K Rustgi
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Contributions

M.R. and A.K.R. conceived the DBA lectin sorting strategy. M.R., S.T., S.H., B.B., G.P.B. and A.K.R. performed the experiments and wrote the manuscript. M.R., S.T., S.H., B.B., G.P.B. and A.K.R. discussed the results and commented on the manuscript at all stages.

Corresponding author

Correspondence to Anil K Rustgi.

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The authors declare no competing financial interests.

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Reichert, M., Takano, S., Heeg, S. et al. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells. Nat Protoc 8, 1354–1365 (2013). https://doi.org/10.1038/nprot.2013.079

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