Isolation and culture of fibroblasts from endoscopic duodenal biopsies of celiac patients

Abstract

Background: Fibroblasts are actually considered pivotal in inflammation and tissue remodelling process and for these reasons they are involved in the pathogenesis of autoimmune disorders such as celiac disease. Investigations to define the role of fibroblasts in celiac diseases are obstructed by the absence of specific models. Our objective is to isolate and culture primary fibroblasts from endoscopic duodenal biopsies of celiac and non-celiac subjects, to analyze their growth patterns and the morphometric characteristics.

Methods: 60 duodenal bioptic specimens from 20 celiac patients and 114 from 38 non-celiac subjects were mechanically chopped and enzymatically digested in order to obtain primary cell cultures. Growth patterns, karyotype (Q-banding analysis), expression of typing proteins (fibroblast surface protein and cytokeratin 20) and morphometric parameters (diameters and their ratio, perimeter, area and perimeter/area ratio at computerised image analysis) were investigated on cultured cells.

Results: Primary cells were successfully cultured in 78% of the collected duodenal biopsies. Cultured cells, expressing the fibroblast surface protein, were negative for cytokeratine 20 and maintained a normal kariotype. Cells grew slowly without differences between the celiac and the non celiac group. Morphometric analysis of celiac fibroblasts revealed significantly increased dimensions, with a preserved diameters ratio, and a reduced perimeter/area ratio.

Conclusion: For the first time this study demonstrates the feasibility of culturing primary fibroblast cell from endoscopic duodenal biopsies in celiac and non-celiac subjects, opening a new window of opportunity in studies intended to establish the role of fibroblasts as a possible partaker in the pathogenesis of the celiac mucosal damage.

Figure 1

Left panel: disposable materials used in primary fibroblast cultures; A endoscopic forceps, B and C tubes, D laboratory forceps, E T25 flask, F and G Petri dishes, H cover glasses, I surgical knife. Right Panel: duodenal endoscopic biopsy procedure.

Figure 2

Cellular growth from a chopped and enzymatically digested fragment of endoscopic duodenal biopsy at different times after seeding as visualised at microscopy (10 × magnification, upper panels) and after computer image analysis skeletonizing objects compatible with cells (fibroblasts) evidencing growth pattern radially spreading from the tissue sample.

Figure 3

Fibroblast surface protein immunocytochemistry of primary cells from duodenal endoscopic biopsies from celiac (upper panel) and non celiac (lower panel) patients; DAPI counterstained cellular nuclei.

Figure 4

Contrast microscopy images of celiac and non celiac (control) fibroblasts at the third passage (upper panel) and skeletonized computer image analysis used for morphometric measurements.