title: Gastrospheres of human gastric mucosa cells: an in vitro model of stromal and epithelial stem cell niche reconstruction.
abstract: The molecular characterization of mechanisms involved in the gastrointestinal tract disorders needs an in vitro 3D culture model able to mimic the in vivo gastric microenvironment. Herein, we propose a 3D coculture system where gastric epithelial and stromal cells are grown together building spherical and solid structures using the NASA bioreactor - cell culture system (RCCS), a bioreactor. Epithelial and stromal cells from human antral gastric mucosa were isolated from endoscopic gastric biopsies. Thereafter, these cells were mechanically and enzymatically dispersed by treatment with dispase and collagenase, respectively. Using specific culture procedures, these cells formed 3D structures by using a RCCS, named "gastrospheres". Briefly, gastrospheres were obtained by initial seeding of 2.5x10⁴ cells/well in 96 well culture plates. At 24 h after their formation, they were transferred into RCCS, and maintained for 7, 14, 21, and 28 days. The gastrospheres were morphologically characterized by immunocytochemisty to evaluate extracellular matrix (ECM), and by electron microscopy. These analysis of gastrospheres revealed that the epithelial cells were cytokeratin (CK) and lectin reactive and were arranged in the outer layer; stromal cells presented long cytoplasmic processes and were localized inside the gastrosphere. They were vimentin (VIM) and α-smooth muscle actin (α-SMA) positive and expressed ECM components such as laminin (LN), fibronectin (FN), and type IV collagen (CIV). Electron microscopy revealed groups of cohesive gastric cells surrounded by complex stromal structures, with multiple microvilli, and tight cellular junctions interspersed with extracellular matrix fibrils and fibers. The presence of some nestin-positive cells was observed in the inner region of the gastrospheres, suggesting an intermediary localization between epithelial and stromal cells. Altogether, our data suggest that in vitro gastrospheres recapitulate the in vivo gastric niche microenvironment.
authors.affiliation: Institute National of Metrology, Quality and Technology, INMETRO, Xerém, Rio de Janeiro, Brazil. cansantos.bio@gmail.com.
authors.forename: Carlos A N
authors.initials: CA
authors.lastname: Santos
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil.
authors.forename: Leonardo R
authors.initials: LR
authors.lastname: Andrade
authors.affiliation: Multidisciplinar Laboratory, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil.
authors.forename: Márcia H M
authors.initials: MH
authors.lastname: Costa
authors.affiliation: Multidisciplinar Laboratory, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil.
authors.forename: Heitor S P
authors.initials: HS
authors.lastname: Souza
authors.affiliation: Institute National of Metrology, Quality and Technology, INMETRO, Xerém, Rio de Janeiro, Brazil.
authors.forename: José M
authors.initials: JM
authors.lastname: Granjeiro
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil.
authors.forename: Christina M
authors.initials: CM
authors.lastname: Takiya
authors.affiliation: Institute National of Metrology, Quality and Technology, INMETRO, Xerém, Rio de Janeiro, Brazil.
authors.forename: Radovan
authors.initials: R
authors.lastname: Borojevic
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, Brazil. luiz.nasciutti@histo.ufrj.br, luiz.nasciutti@icb.ufrj.br.
authors.forename: Luiz E
authors.initials: LE
authors.lastname: Nasciutti
mesh_terms: D060833:Cellular Microenvironment / Q000502:physiology; D018920:Coculture Techniques / Q000379:methods*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005753:Gastric Mucosa / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D055153:Stem Cell Niche / Q000502:physiology*; D017154:Stromal Cells / Q000166:cytology* / Q000378:metabolism*