Experimental techniques for the isolation of pancreatic stellate cells

Abstract

Background: Two decades ago the pancreatic stellate cell (PSC) was identified as the cell type predominantly responsible for the ‘desmoplastic reaction’ associated with pancreatic cancer. PSCs have since been found to exhibit a great deal of interaction with cancer cells in vitro and in vivo. This feature, combined with their contribution to the stromal reaction, makes PSCs fundamental to pancreatic cancer progression. Understanding the mechanisms which mediate the transformation of stellate cells from their non proliferative quiescent state, to their tumour promoting activated state is vital, since inactivation of stellate cells in pancreatic tumours should reduce the mass of fibrotic tissue in and around the tumour, and enable better delivery of chemotherapeutic agents. Methods: Attempts were made to isolate PSCs from pathological human pancreatic tissue using an ‘explant’ method. Cells which grew from the tissue were propagated and characterised according to morphology and the fluorescent expression of stellate cell markers: alpha SMA, GFAP, desmin, vimentin. Results: Primary cells were isolated from 10 patients who possessed a variety of pancreatic diseases. Characterisation revealed three distinct cell types, one of which most closely resembled the PSC due to its morphology and expression of cell markers. However, several difficulties encountered during characterisation, particularly the lack of suitable cell controls, meant that it was not possible to identify these primary cells as PSCs. Alpha SMA and GFAP were expressed in the primary cells, and antibody binding was specific according to isotype control stains. Unfortunately, the immortalised PSCs that were utilised as a positive cell control failed to exhibit alpha SMA. Furthermore, numerous epithelial cancer cell lines unexpectedly expressed GFAP, desmin, and vimentin. Around eighty flasks of primary cells were cryopreserved for use in future experiments. Two of the primary cell samples, along with the immortalised PSC line were taken forward for preliminary investigations of microRNA-29 expression. However, this yielded inconclusive data. Discussion: A subsequent literature search revealed several studies which also demonstrated the mesenchymal characteristics of epithelial cancer cells. This suggests that specific binding may well have taken place in our experiments. As a result of the work described in this thesis, there are now stocks of tumour derived- and inflammatory derived primary pancreatic cells, which following complete characterisation, will be ready to be utilised as an adjunct to the epithelial cancer cell lines frequently used for pancreatic cancer research in this laboratory