title: A method for high purity intestinal epithelial cell culture from adult human and murine tissues for the investigation of innate immune function.
abstract: Intestinal epithelial cells (IECs) serve as an important physiologic barrier between environmental antigens and the host intestinal immune system. Thus, IECs serve as a first line of defense and may act as sentinel cells during inflammatory insults. Despite recent renewed interest in IEC contributions to host immune function, the study of primary IEC has been hindered by lack of a robust culture technique, particularly for small intestinal and adult tissues. Here, a novel adaptation for culture of primary IEC is described for human duodenal organ donor tissue as well as duodenum and colon of adult mice. These epithelial cell cultures display characteristic phenotypes and are of high purity. In addition, the innate immune function of human primary IEC, specifically with regard to Toll-like receptor (TLR) expression and microbial ligand responsiveness, is contrasted with a commonly used intestinal epithelial cell line (HT-29). Specifically, TLR expression at the mRNA level and production of cytokine (IFNγ and TNFα) in response to TLR agonist stimulation is assessed. Differential expression of TLRs as well as innate immune responses to ligand stimulation is observed in human-derived cultures compared to that of HT-29. Thus, use of this adapted method to culture primary epithelial cells from adult human donors and from adult mice will allow for more appropriate studies of IECs as innate immune effectors.
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL, USA; Department of Periodontology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL 32610-0434, USA. Electronic address: cgraves@dental.ufl.edu.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Graves
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL, USA; Department of Neuroscience, College of Medicine, University of Florida, P.O. Box 100244, Gainesville 32610, FL, USA. Electronic address: sharden@ufl.edu.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Harden
authors.affiliation: Department of Periodontology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL 32610-0434, USA. Electronic address: mlapato94@ufl.edu.
authors.forename: Melissa
authors.initials: M
authors.lastname: LaPato
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL, USA; Department of Periodontology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL 32610-0434, USA. Electronic address: nelson@immunext.com.
authors.forename: Michael
authors.initials: M
authors.lastname: Nelson
authors.affiliation: Department of Periodontology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL 32610-0434, USA. Electronic address: bamador@dental.ulf.edu.
authors.forename: Byron
authors.initials: B
authors.lastname: Amador
authors.affiliation: Department of Periodontology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL 32610-0434, USA. Electronic address: hsorenson@dental.ufl.edu.
authors.forename: Heather
authors.initials: H
authors.lastname: Sorenson
authors.affiliation: Department of Neuroscience, College of Medicine, University of Florida, P.O. Box 100244, Gainesville 32610, FL, USA; Department of Pharmacodynamics, College of Medicine, University of Florida, P.O. Box 100244, Gainesville, FL 32610, USA. Electronic address: cjfraz@ufl.edu.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Frazier
authors.affiliation: Department of Oral Biology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL, USA; Department of Periodontology, College of Dentistry, University of Florida, P.O. Box 100434, Gainesville, FL 32610-0434, USA. Electronic address: swallet@dental.ufl.edu.
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Wallet
mesh_terms: D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002469:Cell Separation; D004386:Duodenum / Q000166:cytology*; D005434:Flow Cytometry; D019073:HT29 Cells; D006801:Humans; D007113:Immunity, Innate / Q000276:immunology*; D007371:Interferon-gamma / Q000096:biosynthesis; D007413:Intestinal Mucosa / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D061251:Primary Cell Culture; D012333:RNA, Messenger / Q000096:biosynthesis; D051193:Toll-Like Receptors / Q000096:biosynthesis / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis
chemical_list: D012333:RNA, Messenger; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma
keywords: Human; Innate immunity; Intestinal epithelial cells; Murine; Primary cell culture