Characterization of Gastrospheres Using 3D Coculture System

Abstract

To understand the molecular mechanisms involved in gastric disorders and regeneration, we need an in vitro tridimensional (3D) culture model, which can mimic the in vivo gastric microenvironment. A 3D coculture system named gastrosphere is proposed herein, composed of primary human gastric epithelial and stromal cells. The primary cultures were obtained from endoscopic gastric biopsies, and after mechanical and enzymatic dispersion, epithelial (HGE3) and stromal (HGS12) cells were expanded. After extensive immunocytochemical characterization, cells were seeded onto 96-well round bottom plates previously covered with 1% agarose. Cells were cultured in KM-F12 culture medium with 10% fetal bovine serum (FBS), antibiotics, and antimycotics, in humidified air at 37 °C and atmosphere containing 5% CO₂ for 72 h or until spheres formation. Then gastrospheres were carefully transferred to a rotary cell culture system (RCCS-4), and maintained for 07, 14, 21, and 28 days. Gastrospheres were morphologically characterized by immunocytochemistry [cytokeratins (CK), vimentin, α-smooth muscle actin (α-SMA), laminin (LN), fibronectin (FN), and type IV collagen (CIV), proliferating cell nuclear antigen (PCNA)], and electron microscopy. In gastrospheres, the cytokeratin-positive epithelial cells were found in the outer layer, while vimentin-positive stromal cells were localized in the center of the gastrospheres. PCNA+ cells were mainly seen at the peripheral and in the intermediary region while nestin+ cells were also depicted in the latter zone. Scanning electron microscopy revealed groups of cohesive gastric cells at the periphery, while transmission electron microscopy demonstrated some differentiated mucous-like or zymogenic-like cells in the periphery and stromal structures located at the center of the 3D structures. Extracellular matrix was deposed between cells. Our data suggest that in vitro gastrospheres recapitulate the in vivo gastric microenvironment.