Investigation and characterization of the duct cell-enriching process during serum-free suspension and monolayer culture using the human exocrine pancreas fraction.
OBJECTIVE
We aimed to characterize a serum-free culture system resulting in highly enriched duct cells from human exocrine pancreas. In addition, we tested the effect of vascular endothelial growth factor (VEGF) on endothelial cell proliferation and endocrine differentiation of the duct cells.


METHODS
The exocrine pellet fraction was cultivated in suspension followed by monolayer culture. Time course analysis of multiple acinar and duct cell markers was performed using reverse transcription-polymerase chain reaction and immunocytochemistry. The effects of VEGF and placental growth factor on the quantities of endothelial, duct, and endocrine cells and fibroblasts were investigated using computerized imaging analysis.


RESULTS
Suspension culture of the exocrine material efficiently enriched the cultures for duct cells. Frequent acinar cell death as well as cell selective adherence of acinar cells to the culture dish was the underlying cause of the enrichment. Confocal microscopy demonstrated the virtual absence of cells coexpressing duct cell- and acinar cell-specific markers. The endothelial immunoreactivity of the suspension culture system could be increased 2-fold by VEGF treatment, yet no effect was observed on endocrine cell numbers.


CONCLUSIONS
We have characterized a serum-free in vitro culture system to enrich human duct cells and further show that the contribution of acinoductal transdifferentiation to the enrichment of duct cells is negligible.
Klein
Tino
T
Department of Developmental Biology, Hagedorn Research Institute, Gentofte, Denmark. tkle@hagedorn.dk
Heremans
Yves
Y
Heimberg
Harry
H
Pipeleers
Daniel
D
Madsen
Ole D
OD
Serup
Palle
P
Heller
R Scott
RS
D000328:Adult; D017209:Apoptosis; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion; D049109:Cell Proliferation; D054338:Cell Transdifferentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D046790:Pancreas, Exocrine / Q000166:cytology* / Q000378:metabolism; D010183:Pancreatic Ducts / Q000166:cytology* / Q000378:metabolism; D000072483:Placenta Growth Factor; D011257:Pregnancy Proteins / Q000378:metabolism; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D015415:Biomarkers; D016895:Culture Media, Serum-Free; C000608036:PGF protein, human; D011257:Pregnancy Proteins; D042461:Vascular Endothelial Growth Factor A; D000072483:Placenta Growth Factor