title: A novel in vitro survival assay of small intestinal stem cells after exposure to ionizing radiation.
abstract: The microcolony assay developed by Withers and Elkind has been a gold standard to assess the surviving fraction of small intestinal stem cells after exposure to high (≥8 Gy) doses of ionizing radiation (IR), but is not applicable in cases of exposure to lower doses. Here, we developed a novel in vitro assay that enables assessment of the surviving fraction of small intestinal stem cells after exposure to lower IR doses. The assay includes in vitro culture of small intestinal stem cells, which allows the stem cells to develop into epithelial organoids containing all four differentiated cell types of the small intestine. We used Lgr5-EGFP-IRES-CreERT2/ROSA26-tdTomato mice to identify Lgr5(+) stem cells and their progeny. Enzymatically dissociated single crypt cells from the duodenum and jejunum of mice were irradiated with 7.25, 29, 101, 304, 1000, 2000 and 4000 mGy of X-rays immediately after plating, and the number of organoids was counted on Day 12. Organoid-forming efficiency of irradiated cells relative to that of unirradiated controls was defined as the surviving fraction of stem cells. We observed a significant decrease in the surviving fraction of stem cells at ≥1000 mGy. Moreover, fluorescence-activated cell sorting analyses and passage of the organoids revealed that proliferation of stem cells surviving IR is significantly potentiated. Together, the present study demonstrates that the in vitro assay is useful for quantitatively assessing the surviving fraction of small intestinal stem cells after exposure to lower doses of IR as compared with previous examinations using the microcolony assay.
authors: Radiation Safety Research Center, Nuclear Technology Research Laboratory, Central Research Institute of Electric Power Industry (CRIEPI), 2-11-1 Iwado Kita, Komae, Tokyo 201-8511, Japan.
authors: Motohiro
authors: M
authors: Yamauchi
authors: Kensuke
authors: K
authors: Otsuka
authors: Hisayoshi
authors: H
authors: Kondo
authors: Nobuyuki
authors: N
authors: Hamada
authors: Masanori
authors: M
authors: Tomita
authors: Masayuki
authors: M
authors: Takahashi
authors: Satoshi
authors: S
authors: Nakasono
authors: Toshiyasu
authors: T
authors: Iwasaki
authors: Kazuo
authors: K
authors: Yoshida
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002470:Cell Survival / Q000502:physiology* / Q000528:radiation effects*; D002478:Cells, Cultured; D004307:Dose-Response Relationship, Radiation; D007421:Intestine, Small / Q000166:cytology* / Q000502:physiology* / Q000528:radiation effects; D051379:Mice; D011829:Radiation Dosage; D013234:Stem Cells / Q000166:cytology* / Q000502:physiology* / Q000528:radiation effects
keywords: Lgr5; in vitro culture; ionizing radiation; organoid; small intestinal stem cells; survival assay