1. Academic Validation
  2. In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels

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

  • Nat Protoc. 2021 Jan;16(1):352-382. doi: 10.1038/s41596-020-00419-8.
Samuel S Hinman 1 Yuli Wang 1 Raehyun Kim 1 Nancy L Allbritton 2
Affiliations

Affiliations

  • 1 Department of Bioengineering, University of Washington, Seattle, WA, USA.
  • 2 Department of Bioengineering, University of Washington, Seattle, WA, USA. nlallbr@uw.edu.
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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