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Intestinal stem cells remain viable after prolonged tissue storage

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Abstract

Intestinal stem cells (ISCs) are responsible for renewal of the epithelium both during normal homeostasis and following injury. As such, they have significant therapeutic potential. However, whether ISCs can survive tissue storage is unknown. We hypothesize that, although the majority of epithelial cells might die, ISCs would remain viable for at least 24 h at 4 °C. To explore this hypothesis, jejuna of C57Bl6/J or Lgr5-LacZ mice were removed and either processed immediately or placed in phosphate-buffered saline at 4 °C. Delayed isolation of epithelium was performed after 24, 30, or 48 h storage. At the light microscope level, despite extensive apoptosis of villus epithelial cells, small intestinal crypts remained morphologically intact for 30 h and ISCs were identifiable via Lgr5-LacZ positivity. Electron microscopy showed that ISCs retained high integrity for 24 h. When assessed by flow cytometry, ISCs were more resistant to degeneration than the rest of the epithelium, including neighboring Paneth cells, with higher viability across all time points. Cultured isolated crypts showed no loss of capacity to form complex enteroids after 24 h tissue storage, with efficiencies after 7 days of culture remaining above 80 %. By 30 h storage, efficiencies declined but budding capability was retained. We conclude that, with delay in isolation, ISCs remain viable and retain their proliferative capacity. In contrast, the remainder of the epithelium, including the Paneth cells, exhibits degeneration and programmed cell death. If these findings are recapitulated in human tissue, storage at 4 °C might offer a valuable temporal window for the harvesting of crypts or ISCs for therapeutic application.

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Acknowledgments

The authors wish to thank Dr. Joe Galanko from the UNC Center for Gastrointestinal Biology and Disease for assistance with the statistical analysis, Ashley Ezzel from the Cell and Molecular Physiology Core at UNC for assistance with the histology, Victoria Madden, Steven Ray and Dr. Robert Bagnell of the UNC School of Medicine Microscopy Services Lab for sample preparation and useful input concerning the electron microscopy and Drs. Christopher Dekaney, Scott Magness and P. Kay Lund for useful discussions.

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Authors and Affiliations

  1. Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Megan K. Fuller

  2. Department of Medicine and Cell Biology & Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Kara M. Stout, Richard J. von Furstenberg, Brian J. Smith & Kirk K. McNaughton

  3. Department of Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Denver M. Faulk, Nambirajan Sundaram, Maxime M. Mahe & Michael A. Helmrath

  4. Department of Pediatrics: Division of Gastroenterology, Hepatology & Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    Noah F. Shroyer

  5. University of North Carolina at Chapel Hill, 4341 Medical Biomolecular Research Building (MBRB), CB# 7555, 111 Mason Farm Road, Chapel Hill, NC, 27599-7555, USA

    Susan J. Henning

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  1. Megan K. Fuller
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  2. Denver M. Faulk
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  3. Nambirajan Sundaram
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  4. Maxime M. Mahe
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  5. Kara M. Stout
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  6. Richard J. von Furstenberg
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  7. Brian J. Smith
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  8. Kirk K. McNaughton
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  9. Noah F. Shroyer
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  10. Michael A. Helmrath
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Correspondence to Susan J. Henning.

Additional information

This work was supported an AGA Student Research Fellowship Award (K.M.S.) and by the National Institutes of Health Grants R01-DK083325 (M.A.H.), P30 DK034987 (S.J.H., M.A.H.) and U01-DK085547 (S.J.H., M.A.H.). The last-mentioned grant is part of the Intestinal Stem Cell Consortium, a collaborative research project funded by the National Institute of Diabetes and Digestive and Kidney Diseases and the National Institute of Allergy and Infectious Diseases. Additionally, this work received support from the UNC Department of Surgery (M.K.F.). The UNC Flow Cytometry Core Facility is supported in part by an NCI Center Core Support Grant (P30CA06086) to the UNC Lineberger Comprehensive Cancer Center.

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Fuller, M.K., Faulk, D.M., Sundaram, N. et al. Intestinal stem cells remain viable after prolonged tissue storage. Cell Tissue Res 354, 441–450 (2013). https://doi.org/10.1007/s00441-013-1674-y

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