DMEM:F12 medium and stored in the incu-
bator. Intestinal organoids were cultured in
Matrigel following previously published pro-
tocols ( 37 ). For cell loading in the microwells,
concentrated suspension of dissociated in-
testinal stem cells was pipetted on top of the
hydrogel surface. Cells were allowed to sedi-
ment within the microcavities of the gels for
5 to 7 min, and nonadherent cells were gently
washed with medium. Partially gelled collagen
“lid”was placed on top of the cell arrays and
the two layers of collagen were allowed to seal
for additional 15 min in the incubator. ISC
expansion medium supplemented with 2.5mM
thiazovivin was added. The cells were allowed
to self-organize into lumenized epithelial stem
cell colonies adopting the geometry of the
hydrogel microwells (typically 36 hours). To
induce differentiation, budding, and organ-
oid formation, the medium was replaced with
fresh medium containing EGF, Noggin, and
R-spondin.
Microfabricated crypt arrays and crypt-villi–
shaped epithelium were produced in an iden-
tical manner, but the patterns were not sealed
with a second layer of hydrogel and therefore
cells were permitted to grow both within the
cavities and atop the external surfaces.
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ACKNOWLEDGMENTS
We thank F. Radtke and U. Koch for providing intestine from
Hes1-GFP mice and S. Hoehnel for help with establishing intestinal
crypt surfaces.Funding:This work was funded by support from
the Swiss National Science Foundation (SNSF) research grant
310030_179447, the EU Horizon 2020 Project INTENS (#668294-2),
the PHRT - PM/PH Research Project Proposal 2017, Ecole
Polytechnique Fédérale de Lausanne (EPFL), and NIH (R01DK120921).
N.G. was supported in part by an EMBO Long-Term Postdoctoral
Fellowship. T.B. was supported by fellowships from the NSF GRFP
and NIH (T32 GM-065103). F.D. was supported in part by the
Center for Integrated Nanotechnologies, a US Department
of Energy Office of Basic Energy Sciences user facility. Sandia
National Laboratories is a multimission laboratory managed and
operated by National Technology and Engineering Solutions of
Sandia LLC, a wholly owned subsidiary of Honeywell International
Inc., for the US Department of Energy National Nuclear Security
Administration under contract DENA0003525.Author contributions:
N.G. and M.P.L. conceived the study, designed experiments,
analyzed data, and wrote the manuscript. N.G. was involved in
performing and analyzing all experiments in the manuscript except
for those involving macroscopic intestinal surfaces. M.N. designed,
performed, and analyzed experiments with microtissues and
engineered intestinal surfaces and helped in figure preparation.
T.B. designed, performed, and analyzed gel photopatterning
experiments. O.M. designed, performed, and analyzed experiments
with human intestinal microtissues. N.B. developed initial
microfabrication approaches for engineered intestinal surfaces.
F.W.D. performed mechanical characterization of photopatternedGjorevskiet al.,Science 375 , eaaw9021 (2022) 7 January 2022 8of9
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