Isolation, Culture, and Characterization of Human Pancreatic Duct Cells

Trautmann, B; Schlitt, Hans-Jürgen; Hahn, E. G.; Löhr, Matthias

doi:10.1097/00006676-199303000-00017

Cited by 34 publications

(32 citation statements)

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“…2B), cytokeratin 7, carbonic anhydrase-II, and carbohydrate antigen 19-9 (not shown). These four markers have been previously shown to be specifically localized in pancreatic duct cells (9,(11)(12)(13)(14).…”

Section: Resultsmentioning

confidence: 99%

“…This culture condition was indeed found to stimulate the mitotic activity in human islet cell preparations, but the effect was almost exclusively located in the ductal cells, which are consistently associated with isolated human endocrine islet cells (unpublished observations). These ductal cells can be identified by specific markers such as cytokeratin 19, cytokeratin 7, carbonic anhydrase-II, or carbohydrate antigen 19-9 (9,(11)(12)(13)(14). In sections from detached cell cultures, we noticed the formation of a ductal cell monolayer on top of clusters of insulin-positive cells.…”

Section: Discussionmentioning

confidence: 99%

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Culture of Adult Human Islet Preparations With Hepatocyte Growth Factor and 804G Matrix Is Mitogenic for Duct Cells but Not for β-Cells

et al. 1998

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I slet transplantation represents a potential treatment for insulin-dependent diabetes. Development and application of this technique is hampered by the limited availability of human donor tissue. In vitro expansion of human p-cells may provide an adequate source for p-cell grafts. So far, attempts to expand adult rodent p-cells have been unsuccessful. In different experimental conditions, this cell type has a low rate of replication both in vivo and in vitro (1,2). Nutrients and growth factors can stimulate fetal or neonatal P-cell replication, but most adult p-cells appear unresponsive to growth factor stimulation in vitro (1). In Address correspondence and reprint requests to Veronique H. Lefebvre, Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, B-1090 Brussels, Belgium. E-mail: velefeb@mebo.vub.ac.be.Received for publication 29 August 1997 and accepted in revised form 9 October 1997.BrdU, 5-bromo-2'-deoxyuridine; HGF, hepatocyte growth factor .vivo, the main mechanism leading to increased p-cell numbers appears to involve neogenesis from ductal progenitor cells (2,3). Human p-cells exhibit a very low proliferative activity both during fetal development (9) and in adult life (4). According to a recent report, the proliferation of fetal human p-cells can be stimulated by hepatocyte growth factor (HGF) (5). Furthermore, also adult human p-cells appear to proliferate when cultured in the presence of HGF and an extracellular matrix prepared from rat 804G cells (6). The present study compares the mitogenic effect of this condition on the different cell types that compose a human islet preparation. Our findings contradict the conclusion of Hayek et al. (6), but indicate, instead, that HGF stimulates the proliferation of adult pancreatic duct cells. RESEARCH DESIGN AND METHODSIslet isolation and culture. Human islets from ten heart-beating organ donors were isolated at the Central Unit of p-cell Transplant, Medical Campus, Vrije Universiteit, Brussels. The mean age of the organ donors was 41 ± 5 years (means ± SE [range 19-64]) and the mean organ preservation time 11 ± 2 h (range 4-20 h). Pancreases were processed by ductal distension with collagenase, gentle dissociation, and Ficoll gradient purification of islets. Islet-enriched fractions were cultured in serum-free Ham's F10 medium (Gibco, Life Technologies, Paisley, Scotland, U.K.) with 7.5 mmol/1 glucose, 1% bovine serum albumin, 0.075 ing penicillin/ml, 0.1 mg streptomycin/ml, and 2 mmol/1 glutamine (7). After 4-16 days of culture, these preparations contained <5% damaged cells, no acinar cells, 25 ± 5% ductal cells, 63 ± 5% insulin-positive cells, and 12 ± 2% glucagon-positive cells. They were recovered within this culture period and distributed into 35-mm dishes for either suspension culture or monolayer culture on 804G matrix (6) for a subsequent 1-7 days culture in RPMI1640 medium (Gibco, Life Technologies) with 5.5 mmol/1 or 11.1 mmol/1 glucose, 10% pooled AB human serum (BioWhittaker, Walkersville, MD, or Finnish Red Cross, Helsin...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Culture of Adult Human Islet Preparations With Hepatocyte Growth Factor and 804G Matrix Is Mitogenic for Duct Cells but Not for β-Cells

et al. 1998

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“…Batches of the first expansion were frozen down in order to obtain large stocks for all further experiments. The purity of tumour cell lines with regard to contamination by fibroblasts was monitored by phasecontrast microscopy, haematoxylin and eosin (H&E) staining and immunofluorescence for cytokeratin (Trautmann et al, 1993;Rafiee et al, 1992).…”

Section: Methodsmentioning

confidence: 99%

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins

Löhr

Trautmann²,

Göttler³

et al. 1994

Br J Cancer

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Summary Pancreatic ductal adenocarcinomas are characterised by a dense connective tissue reaction. To test the hypothesis that stroma components are synthesised and produced by the tumour cells themselves, eight cell lines as well as six xenografted tumours from human ductal adenocarcinomas of the pancreas were examined for the expression of extracellular matrix proteins (ECM), using cDNA probes and antibodies to collagen types I, III and IV, vitronectin, fibronectin, undulin and laminin. All tumour cell lines (CAPAN-1, CAPAN-2, AsPC-1, BxPC-3, PANC-1, PaCa-2, PaCa-3, PaCa-44) and xenografted human pancreatic tumours expressed at least one of the examined ECM at the RNA (collagen type IV> laminin = fibronectin = vitronectin> collagen type III> undulin> collagen type I) or protein level (collagen type IV = collagen type III> vitronectin> laminin> collagen type I = fibronectin> undulin). In nude mouse tumours expression of laminin and collagen I was most pronounced in well-differentiated carcinomas. In a few tumours, collagen type III, vitronectin and undulin were expressed on the luminal side of the neoplastic glands, suggesting loss of normal polar differentiation. Incubation with fetal calf serum modulated ECM RNA levels to a varying extent in all but one cell line (AsPC-1). The results suggest that human pancreatic ductal adenocarcinomas cells are capable of synthesising and producing extracellular matrix proteins in vitro and in vivo, but that the extent and pattern of ECM expression differs between the various tumours and conditions tested.

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“…32 The primary antibodies used were directed against SV40 large T (Calbiochem, . Secondary and tertiary antibodies used for immunocytochemical staining were HRP-conjugated rabbit-anti-mouse, rabbit-anti-goat and swineanti-rabbit IgGs (all from Dako, 1:100) using 3-amino-9-ethylcarbazole (AEC) (Chemicon) as color substrate.…”

Section: Western Blot Analysis and Immunocytochemistrymentioning

confidence: 99%

Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine

Jesnowski

Fürst

Ringel

et al. 2005

Laboratory Investigation

132

134

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Tissue fibrosis is one of the characteristics of chronic pancreatitis and pancreatic adenocarcinoma. Activated pancreatic stellate cells (PSC) play a central role in this process. However, analysis of the molecular mechanisms leading to PSC activation is hampered by the lack of an established human PSC line. To overcome this problem, we immortalized and characterized primary human PSC. The cells were isolated by the outgrowth method and were immortalized by transfection with SV40 large T antigen and human telomerase (hTERT). Primary human PSC served as controls. An immortalized line, RLT-PSC, was analyzed for the expression of stellate cell markers. Moreover, the effects of transforming growth factor b 1(TGFb1) or platelet-derived growth factor stimulation and of cultivation on basement membrane components or N-acetylcysteine (NAC) treatment on gene and protein expression and proliferation were analyzed. Immortal RLT-PSC cells retained the phenotype of activated PSC proven by the expression of a-smooth muscle actin (aSMA), vimentin, desmin and glial fibrillary acidic protein (GFAP). TGFb1 treatment upregulated the expression of aSMA, collagen type I (Col I), fibronectin and TGFb1. Incubation of RLT-PSC cells and primary human activated PSC on Matrigel plus NAC treatment resulted in a deactivated phenotype as evidenced by a decrease of aSMA, connective tissue growth factor and Col I expression and by a decreased proliferation of the cells. Moreover, this treatment restored the ability of the cells to store vitamin A in cytoplasmic vesicles. In conclusion, we have established an immortal pancreatic stellate cell line, without changing the characteristic phenotype. Importantly, we were able to demonstrate that besides soluble factors, the matrix surrounding PSC plays a pivotal role in the maintenance of the activation process of PSC. Cultivation of activated PSC on a reconstituted basement membrane plus treatment with NAC was able to deactivate the cells, thus pointing to the possibility of an antifibrosis therapy in chronic pancreatitis.

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Isolation, Culture, and Characterization of Human Pancreatic Duct Cells

Cited by 34 publications

References 0 publications

Culture of Adult Human Islet Preparations With Hepatocyte Growth Factor and 804G Matrix Is Mitogenic for Duct Cells but Not for β-Cells

Culture of Adult Human Islet Preparations With Hepatocyte Growth Factor and 804G Matrix Is Mitogenic for Duct Cells but Not for β-Cells

Human ductal adenocarcinomas of the pancreas express extracellular matrix proteins

Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine

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