OBJECTIVES: Regulatory mechanisms of cell proliferation have been extensively studied as they represent major challenges when dealing with pathologies such as fibrosis, tumourigenesis or tissue regeneration. Numerous in vitro studies still exploit conventional, two-dimensional cell cultures where cells are forced to adhere to unnaturally stiff and flat surfaces of culture dishes. In the living organism, however, each cell is in contact with components of the extracellular matrix and/or neighbouring cells, thus creating a complex three-dimensional (3D) tissue structure. The current paper describes a native 3D culture of cells, based on the GD25β1 fibroblast cell line, and its use for investigating cell proliferation in in vivo-like conditions. MATERIALS AND METHODS: Four-day post-confluent culture of GD25β1 fibroblasts resulted in formation of a 3D system of cells embedded in naturally synthesized extracellular matrix. Morphological characterization of the culture was performed by histochemistry, immunohistochemistry and immunofluorescence. Viability/proliferation was assayed by MTT testing, FACS analysis and Western blotting for determination of expression levels and activation status of the relevant signalling molecules. RESULTS: GD25b1 fibroblasts, grown as 3D culture, gave rise to tissue-like structures characterized by low level of apoptosis, low senescence and development of 3D matrix adhesions, typical of living tissues. Transition to three-dimensionality led to a switch from exponential to linear culture growth, accompanied by accumulation of activated ERK1/2 into caveolin-containing raft domains. Disruption of raft domains as well as reverse transition from 3D back to monolayer culture led to release of phosphorylated ERK1/2 from rafts, activation of cyclin D1 expression and increase in proliferation levels. CONCLUSIONS: These results imply that under in vivo-like conditions, cells might achieve reduction of their proliferation level by sequestering activated ERK1/2 to lipid rafts.
OBJECTIVES: Regulatory mechanisms of cell proliferation have been extensively studied as they represent major challenges when dealing with pathologies such as fibrosis, tumourigenesis or tissue regeneration. Numerous in vitro studies still exploit conventional, two-dimensional cell cultures where cells are forced to adhere to unnaturally stiff and flat surfaces of culture dishes. In the living organism, however, each cell is in contact with components of the extracellular matrix and/or neighbouring cells, thus creating a complex three-dimensional (3D) tissue structure. The current paper describes a native 3D culture of cells, based on the GD25β1 fibroblast cell line, and its use for investigating cell proliferation in in vivo-like conditions. MATERIALS AND METHODS: Four-day post-confluent culture of GD25β1 fibroblasts resulted in formation of a 3D system of cells embedded in naturally synthesized extracellular matrix. Morphological characterization of the culture was performed by histochemistry, immunohistochemistry and immunofluorescence. Viability/proliferation was assayed by MTT testing, FACS analysis and Western blotting for determination of expression levels and activation status of the relevant signalling molecules. RESULTS: GD25b1 fibroblasts, grown as 3D culture, gave rise to tissue-like structures characterized by low level of apoptosis, low senescence and development of 3D matrix adhesions, typical of living tissues. Transition to three-dimensionality led to a switch from exponential to linear culture growth, accompanied by accumulation of activated ERK1/2 into caveolin-containing raft domains. Disruption of raft domains as well as reverse transition from 3D back to monolayer culture led to release of phosphorylated ERK1/2 from rafts, activation of cyclin D1 expression and increase in proliferation levels. CONCLUSIONS: These results imply that under in vivo-like conditions, cells might achieve reduction of their proliferation level by sequestering activated ERK1/2 to lipid rafts.
Authors: Cherry Ballard-Croft; Adam C Locklar; Gentian Kristo; Robert D Lasley Journal: Am J Physiol Heart Circ Physiol Date: 2006-03-24 Impact factor: 4.733