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. 2017 Jan 5;7(1):e2089.
doi: 10.21769/BioProtoc.2089.

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors

Valeria Fernandez Vallone  1 , Morgane Leprovots  1 , Gilbert Vassart  1 , Marie-Isabelle Garcia  1
Affiliations

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors

Valeria Fernandez Vallone et al. Bio Protoc. .

Abstract

Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016 ).

Keywords: 3D; Mouse fetal epithelium; Progenitors; Single cells; Spheroids; ex vivo.

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Figures

Figure 1.
Figure 1.. Overall procedure schematic of step A1
Figure 2.
Figure 2.. Example of equipment (a) and material and dissection tools (b).
a. Binocular and cold light source; b. Material and dissection tools needed for embryos dissection: tubes with DPBS, Petri dish with embryos media, scalpels and straight thin tip forceps.
Figure 3.
Figure 3.. Representative pictures of embryonic stomach dissection at E18.5.
Successive steps are detailed (a-o). Duo: duodenum, Em: embryo, Eso: esophagus, Liv: liver, Pan: pancreas, Per: peritoneum, SI: small intestine, Spl: spleen, Sto: stomach, Uc: umbilical cord, Ut: uterus, Ys: yolk sac. Scale bars: 0.5 cm (l-m); 0.1 cm (n-o).
Figure 4.
Figure 4.. Overall procedure schematic of step A2
Figure 5.
Figure 5.. Spheroids initial seeding.
a. Scheme showing side and upper view of the 3D culture; b. Representative pictures of gastric spheroids culture at day 6, arrows show dead cells inside the spheroids (b). Scale bars = 100 µm.
Figure 6.
Figure 6.. Procedure for maintenance of gastric spheroids.
Fully grown spheroids are isolated from the Matrigel, mechanically dissociated into small groups of cells, which are then mixed with a new Matrigel aliquot and seeded. The ENR seeding medium is added after Matrigel polymerization.
See this image and copyright information in PMC

References

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