title: Intestinal crypts reproducibly expand in culture.
abstract: BACKGROUND
  In vitro growth techniques for intestinal crypts and single intestinal stem cells have been recently described, but several questions of translational importance remain unaddressed. The purpose of this study was to first, evaluate if intestinal crypts reproducibly expand in vitro; second, determine the impact of age and region of intestine on crypt growth in vitro; and third, determine the effects of cryopreservation on crypt growth in vitro.
  
  
  METHODS
  Crypts were harvested from 5 cm of proximal, middle, and distal small intestine of C57BL/6J mice aged 4 wk, 6-8 wk, 12-14 wk, and 18-20 wk (n = 4-6 animals) and cultured. For each region, we determined the efficiency of crypts forming enterospheres (day 1) and progressing to enteroids (day 7). Subsequently, enteroids were passaged and cryopreserved to determine if growth was changed by these manipulations.
  
  
  RESULTS
  Forty-three to 99% of intestinal crypts formed enterospheres, with higher efficiency in proximal small intestine and in younger mice. Twenty-five to 64% of enterospheres progressed to budding enteroids within 7 d. In vitro expansion was greater in proximal enteroids. This expansion continued in a logarithmic fashion, with ≈ 97% replating efficiency of isolated enteroid crypt buds. Following cryopreservation, ≈ 90% of enteroids recovered normal proliferative capacity.
  
  
  CONCLUSIONS
  Intestinal crypt culture is efficient and significantly expands intestinal tissue in a reproducible manner. Regional and age growth differences may reflect distinct stem cell characteristics or differences in support cells. The ability to culture and expand intestinal tissue in vitro provides a potential translational approach toward understanding and treating patients with short bowel syndrome.
authors.affiliation: Department of Surgery, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Megan K
authors.initials: MK
authors.lastname: Fuller
authors.forename: Denver M
authors.initials: DM
authors.lastname: Faulk
authors.forename: Nambirajan
authors.initials: N
authors.lastname: Sundaram
authors.forename: Noah F
authors.initials: NF
authors.lastname: Shroyer
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Henning
authors.forename: Michael A
authors.initials: MA
authors.lastname: Helmrath
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology; D000818:Animals; D049109:Cell Proliferation; D015925:Cryopreservation / Q000379:methods; D007413:Intestinal Mucosa / Q000166:cytology*; D007421:Intestine, Small / Q000166:cytology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009924:Organ Culture Techniques / Q000379:methods* / Q000592:standards*; D015203:Reproducibility of Results; D018874:Spheroids, Cellular / Q000166:cytology; D023822:Tissue Engineering / Q000379:methods* / Q000592:standards*; D057170:Translational Research, Biomedical