Establishment and Characterization of Six Primary Pancreatic Cancer Cell Lines

Research Article

Austin J Cancer Clin Res 2015;2(7): 1055.

Establishment and Characterization of Six Primary Pancreatic Cancer Cell Lines

Rückert F¹*#, Werner K¹#, Aust D², Hering S³, Saeger H-D¹, Grützmann R¹ and Pilarsky C¹

¹Department of Visceral, Thoracic and Vascular Surgery, Dresden University of Technology, Germany

²Institute of Pathology, Dresden University of Technology, Germany

³Institute of Legal Medicine, Dresden University of Technology, Germany #Both Authors Contributed Equally

*Corresponding author: Felix Rückert, Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Dresden University of Technology, Fetscherstr, 74, 01307 Dresden, Germany

Received: May 18, 2015; Accepted: August 08, 2015; Published: August 11, 2015


Background: Pancreatic ductal adenocarcinoma is an aggressive tumor; treatment remains a challenge because of the lack of effective therapeutic strategies. Basic research in this field is dependent on the availability of model systems. New pancreatic cancer cell lines are therefore important for the study of its biology. In the present study, we report the establishment and characterization of six new pancreatic cancer cell lines (PaCaDD-141, -159, -161, -165, -183, -188).

Material and Methods: All cell lines were derived from pancreatic ductal adenocarcinomas by the Dresden outgrowth protocol. The six cell lines originated from different sample locations. We characterized the cell lines by examining their morphology and their cytostructural and functional profiles.

Results: All cell lines were cultured in optimized Dresden-Medium. The doubling time ranged from 20 to 43 hours. KRAS mutations were detected in four of the six cell lines. Immunohistochemical staining showed cytoplasmic expression of CK8/18, mostly membrane and partially cytoplasmic expression of E-cadherin and strong expression of ezrin in all cell lines. Three cell lines showed nuclear p53 accumulation and heterogeneous expression of vimentin. SMAD4 was heterogeneously expressed in the cell lines.

Conclusions: We were able to establish six new primary pancreatic carcinoma cell lines. As applicable tools for basic research, these cell lines might contribute to a better understanding and treatment of this aggressive tumor.

Keywords: Cell lines; Pancreatic cancer; Outgrowth method


Pancreatic ductal adenocarcinoma (PDAC) is by far the most common type of tumor in the exocrine pancreas [1]. Biological models are needed for the study of pancreatic cancer, as better knowledge about the pathophysiology, molecular biology, and functional characteristics might help to better treat this neoplasm. Without cell lines of human pancreatic cancer, investigation of the biological and especially the functional properties of this tumor is not possible. As well, there can be no statement about possible therapeutic responses of this tumor towards new therapeutic agents without new cell lines. In recent years, we and others have reported the isolation of different human pancreatic cancer cell lines [2]. To obtain a greater phenotypic heterogeneity of the disposable cell lines, and to circumvent the use of “old” cell lines, it is recommended for research labs that focus on pancreatic cancer to establish their own primary carcinoma cell lines. Therefore, we have attempted to establish additional carcinoma cell lines from surgical specimens of pancreatic adenocarcinomas.

In the present study we report the successful isolation of six new primary human pancreatic carcinoma cell lines, designated as PaCaDD-141, PaCaDD-159, PaCaDD-161, PaCaDD-165, PaCaDD-183 and PaCaDD-188, and will describe the phenotypes of the cell lines, including the histopathology and the in vitro growth characteristics.

Material and Methods

Culture procedure and patient cohort

Primary tumor tissues were taken from primary pancreatic tumors, malignant ascites/pleural effusions, metastatic liver tumors, or metastases to lymph nodes, all of which were obtained surgically from patients with ductal adenocarcinomas of the pancreas. All patients were treated at the Department of Visceral, Thoracic and Vascular Surgery, University Hospital Dresden, Germany and gave informed consent prior to operation. After the surrounding connective tissues and hemorrhagic regions were removed, the tumor tissues were minced finely using scalpels into cubes of approximately 1 mm3. Neither enzymatic nor mechanical dissociation of the tumor cells was performed. For a detailed report of our technique see Rückert et al., 2011 [3]. For the primary culture, we used Dresden-medium, consisting of CP medium and KSF medium at a ratio of 2:1 as described previously [3]. All media were supplemented with penicillin (100 U/ mL) and gentamicin (2.5 mg/mL) (Invitrogen, Karlsruhe, Germany). The cells were maintained at 37oC in a humidified atmosphere of 5% CO2 in air, and the medium was replaced every 3 days. All cell lines showed an absence of mycoplasma. Cell lines were named Pancreatic Cancer Dresden (PaCaDD). All experiments for this study were performed on cell lines between the 4th and 9th passage.

Morphology and infiltration assay

Growth patterns and cell morphologies were determined in vitro using a Zeiss phase-contrast microscope.

Cell doubling time

Cell doubling time was determined by counting the number of viable cells derived from freshly trypsinized monolayers in duplicate. Seven 6-well plates (9.6 cm²/well) with 5 x 105 cells plated per well were used. Cells were counted at 24 h intervals for 7 days. The culture medium was changed every 3 days. The doubling time of the cell population was calculated from the logarithmic growth curve by the following formula:

υ= lgN - lgN0/ lg2 (t- t0), with doubling time= 1/ υ.


Cell line purity and clonality were verified by microsatellite analyses using the commercially available multiplex PCR kits Mentype® NonaplexQS (Biotype AG, Germany) and genRES® MPX-2 (Serac, Germany). Amplicons were detected by capillary electrophoresis in the denaturing polymer POP4 in an ABI 310 sequencer (Perkin-Elmer, USA) according to the manufacturer’s instructions.


For immunohistochemical analysis, cells from each cell line were centrifuged into a cell pellet, embedded in paraffin and then cut for immunohistochemical staining. Additionally, a representative slide of the corresponding primary tumors was used for comparison. All cell lines and sections were examined by one observer (D.A.) blinded to both clinical and pathological data. Table 1 shows the details of the antibodies used. Immunohistochemical staining for CK8/18, vimentin, ezrin, E-cadherin and p53 was done using a Lab-Vision 480- 2D immunostainer (Thermo Fisher, Fremont, CA, USA); staining for SMAD4 was done by hand. All reactions were visualized with DAB as a chromogen. Positive and negative controls were included in each run for all the antibodies used. Isotype controls for all antibodies were negative.