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In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels

Nature Protocols volume 16, pages 352–382 (2021)Cite this article

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Abstract

The large intestine, with its array of crypts lining the epithelium and diverse luminal contents, regulates homeostasis throughout the body. In vitro crypts formed from primary human intestinal epithelial stem cells on a 3D shaped hydrogel scaffold replicate the functional and architectural features of in vivo crypts. Collagen scaffolding assembly methods are provided, along with the microfabrication and soft lithography protocols necessary to shape these hydrogels to match the dimensions and density of in vivo crypts. In addition, stem-cell scale-up protocols are provided so that even ultrasmall primary samples can be used as starting material. Initially, these cells are seeded as a proliferative monolayer over the shaped scaffold and cultured as stem/proliferative cells to expand them and cover the scaffold surface with the crypt-shaped structures. To convert these immature crypts into fully polarized, functional units with a basal stem cell niche and luminal differentiated cell zone, stable, linear gradients of growth factors are formed across the crypts. This platform supports the formation of chemical gradients across the crypts, including those of growth and differentiation factors, inflammatory compounds, bile and food metabolites and bacterial products. All microfabrication and device assembly steps are expected to take 8 d, with the primary cells cultured for 12 d to form mature in vitro crypts.

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Fig. 1
Fig. 2
Fig. 3: Monolayer hydrogel maintenance of primary intestinal epithelial cells.
Fig. 4: Design of an in vitro crypt photomask.
Fig. 5: Microfabrication and cell culture insert modification for in vitro crypts.
Fig. 6: Formation and characterization of in vitro crypts.

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Data availability

The data that support the findings of this study are reported in previous publications (refs. 20,24,37).

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Acknowledgements

The authors gratefully acknowledge financial support from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (R01 DK109559) and the University of Washington (start-up funding). We additionally wish to thank S. T. Magness for procurement of the intestinal biopsy specimen, and J. S. Dutton, J. C. Huling and A. Proctor for assistance in assembling the working versions of these protocols that are used within our research group.

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Authors and Affiliations

  1. Department of Bioengineering, University of Washington, Seattle, WA, USA

    Samuel S. Hinman, Yuli Wang, Raehyun Kim & Nancy L. Allbritton

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  1. Samuel S. Hinman
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Contributions

N.L.A. and Y.W. conceived and designed the culture platform. S.S.H., R.K. and Y.W. collected and analyzed the data. S.S.H. prepared the figures. All authors contributed to the writing of the manuscript. S.S.H. and N.L.A. revised the manuscript.

Corresponding author

Correspondence to Nancy L. Allbritton.

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Competing interests

N.L.A. and Y.W. have a financial interest in Altis Biosystems, Inc. The remaining authors declare no competing financial interests.

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Peer review information Nature Protocols thanks Gretchen Mahler, Jun Sun and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Wang, Y. L. et al. Cell. Mol. Gastroenterol. Hepatol. 4, 165–182.e7 (2017): https://doi.org/10.1016/j.jcmgh.2017.02.011

Wang, Y. et al. Cell. Mol. Gastroenterol. Hepatol. 5, 113–130 (2018): https://doi.org/10.1016/j.jcmgh.2017.10.007

Wang, Y. L. et al. Biomaterials 128, 44–55 (2017): https://doi.org/10.1016/j.biomaterials.2017.03.005

Supplementary information

Supplementary Information

Supplementary Figs. 1–3 and Supplementary Table 1.

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Supplementary Data 1

Sheet 1: collagen neutralization volumes; Sheet 2: subcultivation ratios

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Hinman, S.S., Wang, Y., Kim, R. et al. In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels. Nat Protoc 16, 352–382 (2021). https://doi.org/10.1038/s41596-020-00419-8

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