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. 2017 Jan 5;7(1):e2088.
doi: 10.21769/BioProtoc.2088.

Ex vivo Culture of Adult Mouse Antral Glands

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

Ex vivo Culture of Adult Mouse Antral Glands

Valeria Fernandez Vallone et al. Bio Protoc. .

Abstract

The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker's protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016 ).

Keywords: 3D; Antral glands; Gastric organoids; Murine gastric epithelium; Single cell; ex vivo.

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Figures

Figure 1.
Figure 1.. Overall procedure schematic of step A1
Figure 2.
Figure 2.. Examples of equipment (a) and dissection tools (b).
a. Binocular and cold light source; b. Dissection tools: micro-dissection scissors, scalpel, straight thin tip forceps, curved serrated forceps and different size scissors.
Figure 3.
Figure 3.. Representative pictures of stomach dissection.
Consecutive steps are detailed (a-l). Ant: antral area, Cor: corpus area, Col: colon, Duo: duodenum, Dia: diaphragm, Eso: esophagus, Fas: fascias, GC: greater curvature, Liv: liver, Mu: mucosa, Ms: muscle, SC: small curvature, SI: small intestine, Sq: squamous area, Sto: stomach. Scale bars = 0.5 cm.
Figure 4.
Figure 4.. Adult glands isolation.
Example of mouse antral glands suspension before plating or single cell dissociation. Scale bars = 100 µm.
Figure 5.
Figure 5.. Overall procedure schematic of step A2
Figure 6.
Figure 6.. Antral organoids at day 5 of the initial seeding: scheme showing side view of the 3D culture (a) and representative picture of culture at day 5 (b).
Scale bars = 100 µm.
See this image and copyright information in PMC

References

    1. Barker N., Huch M., Kujala P., van de Wetering M., Snippert H. J., van Es J. H., Sato T., Stange D. E., Begthel H., van den Born M., Danenberg E., van den Brink S., Korving J., Abo A., Peters P. J., Wright N., Poulsom R. and Clevers H.(2010). Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro . Cell Stem Cell 6(1): 25-36. - PubMed
    1. Fernandez Vallone V., Leprovots M., Strollo S., Vasile G., Lefort A., Libert F., Vassart G. and Garcia M. I.(2016). Trop2 marks transient gastric fetal epithelium and adult regenerating cells after epithelial damage. Development 143(9): 1452-1463. - PMC - PubMed
    1. Sato T., Vries R. G., Snippert H. J., van de Wetering M., Barker N., Stange D. E., van Es J. H., Abo A., Kujala P., Peters P. J. and Clevers H.(2009). Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche . Nature 459(7244): 262-265. - PubMed