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Methods Mol Biol. 2018;1842:139-166. doi: 10.1007/978-1-4939-8697-2_10.

Quantitative Analysis of Intestinal Stem Cell Dynamics Using Microfabricated Cell Culture Arrays.

Author information

1
Department of Medicine, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
2
NC State/UNC Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
3
Department of Medicine, Center for Gastrointestinal Biology and Disease, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. magness@med.unc.edu.
4
NC State/UNC Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. magness@med.unc.edu.
5
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. magness@med.unc.edu.

Abstract

Regeneration of intestinal epithelium is fueled by a heterogeneous population of rapidly proliferating stem cells (ISCs) found in the base of the small intestine and colonic crypts. ISCs populations can be enriched by fluorescence-activated cell sorting (FACS) based on expression of combinatorial cell surface markers, and fluorescent transgenes. Conventional ISC culture is performed by embedding single ISCs or whole crypt units in a matrix and culturing in conditions that stimulate or repress key pathways to recapitulate ISC niche signaling. Cultured ISCs form organoid, which are spherical, epithelial monolayers that are self-renewing, self-patterning, and demonstrate the full complement of intestinal epithelial cell lineages. However, this conventional "bulk" approach to studying ISC biology is often semiquantitative, low throughput, and masks clonal effects and ISC phenotypic heterogeneity. Our group has recently reported the construction, long-term biocompatibility, and use of microfabricated cell raft arrays (CRA) for high-throughput analysis of single ISCs and organoids. CRAs are composed of thousands of indexed and independently retrievable microwells, which in combination with time-lapse microscopy and/or gene-expression analyses are a powerful tool for studying clonal ISC dynamics and micro-niches. In this protocol, we describe how CRAs are used as an adaptable experimental platform to study the effect of exogenous factors on clonal stem cell behavior.

KEYWORDS:

Cell raft array; Enteroid; Genetically engineered mouse model; Intestinal stem cell; Organoid; Sox9EGFP

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