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. Author manuscript; available in PMC: 2019 Feb 1.
Published in final edited form as: Biomaterials. 2017 Oct 26;154:60–73. doi: 10.1016/j.biomaterials.2017.10.038

Figure 1.

Figure 1

Schematic of the 2D BLT Sandwich culture system. Intestinal crypts isolated from Lgr5-GFP donor mice or human intestinal biopsies are directly seeded onto Col IV-coated transwells. Alternatively, the isolated crypts can be pre-expanded in Matrigel to form Lgr5+ ISCs-enriched organoids following established methods [1]. The organoids are then mechanically dissociated into ISC-containing IEC clusters and seeded onto Col IV-coated transwells. Next, a thin layer of type I collagen solution is gently overlaid onto the settled cells and gelled, resulting in a sandwich configuration comprising Col IV coating/IECs/Col I gel overlay. Chemically defined ISC maintenance media containing essential signaling modulators (i.e. CV + BMP inhibitor LDN) is then added into the transwell insert. An epithelial monolayer containing high levels of Lgr5+ ISCs is rapidly generated via the combination of potent signaling factors and unique presentation of defined ECM cues.