Article
Extended Data Fig. 3 | Establishment of long-term culture and in vitro
tissue homeostasis. a, Bright-field (middle) and LGR5–eGFP f luorescence
(right) images of mini-gut progression on days 7 and 10, compared to organoids
(left). b, Long-term propagation of mini-guts (up to 30 days) shown in
bright-field (left) and LGR5–eGFP f luorescence (right). Data are represent
ative of at least four independent experiments. EDF of bright-field images,
calculated for a z-stack of 80 μm; f luorescence confocal images correspond to
a maximal intensity projection of a z-stack of around 60 μm. Scale bars, 50 μm.
c, Bright-field and LGR5–eGFP f luorescence of mini-gut deterioration due to
the massive accumulation of dead cells within the lumen in the absence of
perfusion. Scale bars, 100 μm. d, Tubular mini-guts maintain epithelial
integrity and morphology in different cell culture media used for lumina
perfusion. No difference in epithelium morphology and stability was detected
when tissues were apically exposed to organoid culture medium (ENR) or
minimal media lacking growth factors (BMGF, BM). Similar results were
obtained in at least two independent experiments with n = 2 samples per each
condition. Scale bars, 50 μm. e, Frequency map showing the localization of
LGR5–eGFP-expressing ISCs in 7-10-day-old tissues (left) and 30-day-old
tissues (right). Average of the maximal intensity projection of a z-stack of
around 60 μm for n = 20 tissues (7–10-day-old) and n = 8 tissues (30-day-old).