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A novel culture system for adult porcine intestinal crypts

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Abstract

Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors.

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Acknowledgments

We sincerely thank Dr. Randal Buddington (University of Memphis) for providing fetal porcine intestinal samples; Drs. JanLee Jensen and Sandra Duarte-Vogel, Sonia Watt, Guillermo Moreno, and Eileen So (University of California, Los Angeles) for providing porcine tissue; Liara Gonzalez (North Carolina State University) for sharing expertise with neonatal piglet culture; Scott Magness (University of North Carolina) for helpful discussions; and Emmanuelle Faure (University of California, Los Angeles Vectorcore) for help and guidance with viral transduction.

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Authors and Affiliations

  1. Department of Surgery, UCLA David Geffen School of Medicine, Los Angeles, CA, USA

    Hassan A. Khalil, Nan Ye Lei, Andrew Scott, Ziyad B. Jabaji, James C. Y. Dunn & Matthias G. Stelzner

  2. Division of Gastroenterology and Nutrition, Department of Pediatrics, Mattel Children’s Hospital and the David Geffen School of Medicine at UCLA, University of California, Los Angeles, Los Angeles, CA, USA

    Garrett Brinkley, Jiafang Wang, Upendra K. Kar & Martín G. Martín

  3. Department of Pathology & Laboratory Medicine, VA Greater Los Angeles Health System, Los Angeles, CA, USA

    Michael Lewis

  4. Department of Surgery, VA Greater Los Angeles Health System, Los Angeles, CA, USA

    Matthias G. Stelzner

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  1. Hassan A. Khalil
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  2. Nan Ye Lei
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  3. Garrett Brinkley
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  4. Andrew Scott
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  5. Jiafang Wang
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  7. Ziyad B. Jabaji
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  8. Michael Lewis
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  9. Martín G. Martín
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  10. James C. Y. Dunn
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  11. Matthias G. Stelzner
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Corresponding author

Correspondence to Matthias G. Stelzner.

Additional information

The UCLA Vectorcore is supported by CURE/P30 DK041301. This research was performed as a project 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 Infections Diseases (DK085535-01 and DK085535-02S2) and was supported in part by the California Institute of Regenerative Medicine (RT2-01985).

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Fig. S1

Characterization of porcine ISEMFs by immunostaining (green) for (a) α smooth muscle actin, (b) vimentin, and (c) desmin. Nuclei are counter-stained with DAPI (blue). Bar 100 μm.(GIF 137 kb)

High Resolution Image (TIF 4.29 mb)

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Khalil, H.A., Lei, N.Y., Brinkley, G. et al. A novel culture system for adult porcine intestinal crypts. Cell Tissue Res 365, 123–134 (2016). https://doi.org/10.1007/s00441-016-2367-0

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