Establishment of a Long-Term Culture System for Rat Colon Epithelial Cells

Abstract

The aim of this study was to establish a long-term culture system for rat colon epithelial cells. Colonic crypts were isolated by incubating a 4-cm-long rat colon segment cut longitudinally with an ethylenediaminetetraacetic acid [disodium salt]-containing buffer, taken up in conditioned medium from the normal rat kidney fibroblast cell line NRK (i.e., the supernatant of pure NRK cultures), directly plated on mitomycin C-treated NRK cells and subcultured with conditioned medium from NRK cells. Cells started to migrate out of the crypts shortly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The cells proliferated extremely slowly but continuously over a period of 4 mo and were epithelial because they expressed cytokeratin 19 and were stained by crystal violet at pH 2.8. In conclusion, the experimental system described in this study allows to maintain rat colon epithelial cells for up to 4 mo in culture and can be used to study the effects of a variety of tumor-modulating factors on growth and gene expression of normal colon epithelial cells in vitro.

The Plant Hormone Cytokinin Confers Protection against Oxidative Stress in Mammalian Cells

“…NRK, an epithelial rat kidney cell line [20], was obtained from the American Type Culture Collection (ECACC,Salisbury, UK) and cultured at 37°C, 5% (v/v) CO 2 in DMEM high glucose (4.5 g/l) with 10.0% (v/v) FCS, 1.0% (v/v) L-glutamine, 1.0% (w/v) non-essential amino acids and 1.0% (v/v) antibiotics (50 U/ml penicillin, 50 mg/ml streptomycin). They were subcultured twice per week.…”

“…[20] with slight modifications using a fluorescence microscope (Labophot 2; Nikon GmbH, Düsseldorf, Germany) at 200-fold magnification using image analysis software (Komet 5; BFi OPTiLAS, Gröbenzell, Germany). Briefly, cells were treated according to the experimental design with (i) different concentrations (1, 3.2, 10, 32,100 and 500 nM) of kinetin for 24 hr.…”

Synthesis and biological investigations of a ZnPc-antiCEA bioconjugate for imaging of colorectal cancer

“…To investigate this, we designed a co-culture system consisting of HT-29 cells and murine colon epithelial cells obtained from a primary culture of mucosal scrapings [27]. Cells in the co-culture were incubated with ZnPc-CEA MAb 2 then stained for CEA and for mucin-like glycoproteins.…”