Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle

Loret, Suzanne; Rusu, Dorina; Moualij, Benaı̈ssa El; Taminiau, Bernard; Heinen, Ernst; Dandrifosse, Guy; Mainil, Jacques

doi:10.1016/j.rvsc.2008.12.002

Cited by 15 publications

(22 citation statements)

References 58 publications

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“…However, PoCo83-3 achieved at least 61 PDs beyond the stage at which the original cells entered a proliferative arrest. Our findings are comparable to Loret et al [2009], who reported 53 subcultivations using a split ratio of 1: 2 (equivalent to 53 PDs) for SV40 TAg-transduced bovine jejunocytes and colonocytes. Considering that PoCo83-3 was derived from a single epithelial cell clone, the cumulative PD level of this cell line at P35 is …”

Section: Discussionsupporting

confidence: 89%

“…Although PoCo83-3 cells maintained expression of the epithelial marker KRT18 up to P43, we observed the expression of VIM, commonly described as a marker for mesenchymal cells and epithelial-mesenchymal transition [Lee et al, 2006;Satelli and Li, 2011]. However, VIM expression in epithelial cells has also been detected in primary cultures [Rusu et al, 2005;Stumpff et al, 2011] and in other epithelial cell lines [Loret et al, 2009;Anand et al, 2012;Desmarets et al, 2013;Zakrzewski et al, 2013]. It has been proposed that VIM expression in epithelial cells may represent an essential adaption mechanism to the artificial environment during in vitro growth [Mark et al, 1990;Baer and Bereiter-Hahn, 2012].…”

Section: Characteristics Of Late-passage Poco83-3 Culturesmentioning

confidence: 59%

“…A contamination by mesenchymal cells is a common problem in pCEC cultivation [Petto et al, 2011] and epithelial cell culture establishment in general [Anand et al, 2012;Desmarets et al, 2013]. To generate a homogenous cell line possessing consistent and reproducible characteristics, we selected clonal epithelial cell clusters to ensure that the PoCo83-3 cell line was derived from a single epithelial cell clone [Loret et al, 2009;Miyazawa et al, 2010].…”

Section: Poco83-3 Characteristics Resemble Those Of Colonic Epitheliamentioning

confidence: 99%

“…A common and reliable approach towards overcoming cellular senescence is the use of simian virus 40 large T antigen (SV40 TAg) expression constructs [Telgmann, 2001;Kirchhoff et al, 2004;Loret et al, 2009;Miyazawa et al, 2010;Desmarets et al, 2013;Gleich et al, 2016;Hu et al, 2016;Kim et al, 2016]. A complex signaling network involving the p53 and p16(INK4A)-pRb tumor suppressor pathways plays a predominant role in "replicative senescence" [Hara et al, 1991;Hahn, 2002].…”

Section: Introductionmentioning

confidence: 99%

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Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Kaiser

Böttner²,

Wedel³

et al. 2017

Cells Tissues Organs

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Continuous cell lines have become indispensable tools that have enabled investigations into cellular mechanisms by increasing experimental reproducibility and sample availability, and decreasing the use of experimental animals. To facilitate studies of epithelial barrier function of the porcine colon, we aimed to establish an epithelial cell line with an extended replicative capacity. Cells were isolated from the proximal colon of a 3-week-old piglet and transduced using a recombinant retroviral vector construct containing the simian virus 40 large T antigen (SV40 TAg). We established a clonal epithelial cell line, referred to as PoCo83-3, that stably expressed the SV40 TAg, verified at mRNA and protein levels. PoCo83-3 showed epithelial cell-specific features, such as cobblestone-like morphology, dome structure formation, the presence of apical microvilli, and the expression of keratin 18, E-cadherin and the tight junction-associated proteins zonula occludens-1, occludin, and claudin-1. To validate PoCo83-3 as an in vitro model in epithelial barrier research, proinflammatory cytokine-inducible alterations in barrier integrity were demonstrated by incubating the cells with TNF-α and IFN-γ for 48 h. These cytokine treatments promoted a decreased transepithelial electrical resistance. In summary, PoCo83-3 exhibited an extended life span and a differentiated phenotype while maintaining epithelial characteristics. Based on these results, we present this cell line as a valuable in vitro model for investigations of epithelial barrier function in the porcine colon.

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Section: Discussionsupporting

confidence: 89%

Section: Characteristics Of Late-passage Poco83-3 Culturesmentioning

confidence: 59%

Section: Poco83-3 Characteristics Resemble Those Of Colonic Epitheliamentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Kaiser

Böttner²,

Wedel³

et al. 2017

Cells Tissues Organs

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“…Several successful isolations of enterocytes have been described, using various procedures [9][10][11] . Most of these methods, such as separation using magnetic nano-beads, or separation on the specific density gradient 11 allows only for the isolation of specific cells types.…”

Section: Discussionmentioning

confidence: 99%

Derivation and basic characterization of colorectal carcinoma primary cell lines

Krbal¹,

Soukup²,

John³

et al. 2017

BIOMED PAP

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Background. Colorectal carcinoma is one of the most common malignancies in western countries. Among different approaches to its research, primary cancer cell lines can play an important role. Aim. The main purposes of this study were: 1) to establish an effective and reproducible method of colorectal cancer cell isolation and cultivation from primary tumours and lymph node metastases and 2) to elucidate the biological features of the tumours favouring successful cell cultivation. Materials and Methods. The tumour cells were obtained from colectomy specimens. Primary tumour and lymph node metastasis tissue was used for establishing the tissue cultures. Colectomy samples were further processed for routine histopathological assessment: tumour grade, stage, angioinvasion and perineural spread were evaluated. Features of tissue culture cells were assessed using phase contrast microscopy and immune-histochemical techniques. WST-1 assay and X-CELLigence real time analysis were carried out for viability and proliferation testing before and after treatment with irinotecan and oxaliplatin. Molecular features of the tumour including K-RAS/B-RAF/N-RAS mutations were tested using allele-specific PCR. Results of the cultivation process were compared to the histopathological and molecular features of the tumours. Results. In total, we obtained 33 samples from the primary site of tumours and 20 samples from lymph node metastases; in total, 27 cell lines were successfully isolated. Morphologic features characteristic of tumour cells in primary cell lines and epithelial differentiation (positive for AE1/AE3 cytokeratin) were evaluated. Higher tumour stage, angioinvasion and presence of perineural spread in primary tumour correlated positively with successful cell isolation from lymph node metastasis. All samples tested were NRAS wild-type. No correlation was found between molecular phenotype and the cell culture features. A higher proliferation potential was observed in the primary tumour cells, whereas higher sensitivity to irinotecan was found in the lymph node metastatic cells. Conclusions. Using mechanical dissociation, we successfully derived and cultivated CRC cells from primary tumours and lymph node metastases with success rate 3 % and 70% respectively. Primary tumour features favouring successful establishment of cell cultures were identified.

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The Correlation Between the Expression of Differentiation Markers in Rat Small Intestinal Mucosa and the Transcript Levels of Schlafen 3

Kovalenko

Basson

2013

JAMA Surg

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IMPORTANCE The normal absorptive function and structural maintenance of the intestinal mucosa depend on a constant process of proliferation of enterocytic stem cells followed by progressive differentiation toward a mature phenotype. The mechanisms that govern enterocytic differentiation in the mucosa of the small intestine are poorly understood. OBJECTIVE To determine whether schlafen 3 (but not other schlafen proteins) act in vivo and whether its effects are limited to the small intestine. We have previously demonstrated in nonmalignant rat intestinal IEC-6 cells that schlafen 3 levels correlate with the expression of various differentiation markers in vitro in response to differentiation stimuli. DESIGN Randomized controlled experiment. SETTING Animal science laboratory. PARTICIPANTS Male Sprague-Dawley rats 8 to 13 weeks old. MAINOUTCOMES AND MEASURES Messenger RNA (mRNA) from jejunal and colonic mucosa was isolated, and transcript levels of schlafen proteins 1, 2, 3, 4, 5, 13, and 14; sucrase isomaltase (SI); dipeptidyl peptidase 4 (Dpp4); glucose transporter type 2 (Glut2); and villin were measured by quantitative reverse transcriptase–polymerase chain reaction. We tested parallel variations in protein levels by Western blotting and Dpp4 enzyme activity. RESULTS The transcript level of schlafen 3 (Slfn3) correlated with the levels of the differentiation markers SI, Dpp4, Glut2, and villin. However, the expression of schlafen proteins 1, 2, 4, 5, 13, and 14 did not correlate with the expression of the differentiation markers. The mucosal mRNA levels of Slfn3, SI, Glut2, and Dpp4 were all substantially higher in the rat jejunum than in colonic mucosa by a mean (SE) factor of 51.0 (13.2) for 6 rats (P < .05), 599 (99) for 8 rats (P < .01), 12.5 (5.5) for 8 rats (P < .01), and 14.0 (3.9) for 8 rats (P < .01), respectively. In IEC-6 cells, infection with adenovirus-expressing GFP-tagged Slfn3 significantly increased Slfn3 expression and Dpp4-specific activity compared with GFP-expressing virus (in 6 rats; P < .05). CONCLUSIONS AND RELEVANCE Taken together with our previous in vitro observations, the results suggest that small intestinal enterocytic epithelial differentiation in rats may be regulated by Slfn3 in vivo, as in vitro, and that these effects may be specific to the small intestinal enterocytic phenotype as opposed to that of the mature colonocyte. Slfn3 human orthologs may be targeted to stimulate intestinal differentiation in patients with short bowel syndrome

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Preliminary characterization of jejunocyte and colonocyte cell lines isolated by enzymatic digestion from adult and young cattle

Cited by 15 publications

References 58 publications

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Establishment and Characterization of an SV40 Large T Antigen-Transduced Porcine Colonic Epithelial Cell Line

Derivation and basic characterization of colorectal carcinoma primary cell lines

The Correlation Between the Expression of Differentiation Markers in Rat Small Intestinal Mucosa and the Transcript Levels of Schlafen 3

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