title: Co-culture with intestinal epithelial organoids allows efficient expansion and motility analysis of intraepithelial lymphocytes.
abstract: BACKGROUND
  Intraepithelial lymphocytes (IELs) in the intestine play important roles in the regulation of local immune responses. Although their functions have been studied in a variety of animal experiments, in vitro studies on spatiotemporal behaviors of IELs and their interaction with intestinal epithelial cells (IECs) have been hampered due to the lack of a suitable culture system. In this study, we aimed at developing a novel co-culture system of IELs with IECs to investigate dynamic interaction between these two populations of cells in vitro.
  
  
  METHODS
  We optimized experimental conditions under which murine IELs can be efficiently maintained with IECs cultured as three-dimensional organoids. We then tested the effect of IL-2, IL-7, and IL-15 on the maintenance of IELs in this co-culture system. By time-lapse imaging, we also examined the dynamic behaviors of IELs.
  
  
  RESULTS
  IELs can be expanded with epithelial organoids in the presence of IL-2, IL-7, and IL-15. IELs were efficiently maintained within and outside of organoids showing a ~four-fold increase in both αβT and γδT IELs for a period of 2 weeks. Four-dimensional fluorescent imaging revealed an active, multi-directional movement of IELs along the basolateral surface of IECs, and also their inward or outward migration relative to organoid structures. Cell tracking analysis showed that αβT and γδT IELs shared indistinguishable features with regard to their dynamics.
  
  
  CONCLUSIONS
  This novel co-culture method could serve as a unique tool to investigate the motility dynamics of IELs and their temporal and spatial interaction with IECs in vitro.
authors.affiliation: Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Nozaki
authors.affiliation: Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Wakana
authors.initials: W
authors.lastname: Mochizuki
authors.affiliation: Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Yuka
authors.initials: Y
authors.lastname: Matsumoto
authors.affiliation: Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Fukuda
authors.affiliation: Department of Advanced Therapeutics for GI Diseases, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Mizutani
authors.affiliation: Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Department of Advanced Therapeutics for GI Diseases, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519, Japan. nakamura.gast@tmd.ac.jp.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000502:physiology; D018920:Coculture Techniques; D004847:Epithelial Cells / Q000502:physiology; D007413:Intestinal Mucosa / Q000166:cytology*; D008214:Lymphocytes / Q000502:physiology*; D008822:Mice, Transgenic; D009940:Organoids / Q000502:physiology*; D059008:Time-Lapse Imaging / Q000379:methods; D057705:Transendothelial and Transepithelial Migration / Q000502:physiology*
keywords: In vitro culture; Intestinal epithelial cells; Intraepithelial lymphocytes; Lymphocyte migration; Time-lapse imaging