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RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

Morabito C, Steimberg N, Mazzoleni G, Guarnieri S, Fanò-Illic G, Mariggiò MA - Biomed Res Int (2015)

Bottom Line: We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions.Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation).In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Imaging and Clinical Sciences, Unit of Functional Biotechnology, Aging Research Center (Ce.S.I.), "G. d'Annunzio" University of Chieti-Pescara, Via dei Vestini 29, 66100 Chieti, Italy ; Interuniversity Institute of Myology, Italy.

ABSTRACT
We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

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Expression of glial cell markers. Representative Western blotting and quantification of the levels of GFAP, S100B, and Cx43 in GL15 cells cultured in 2D monolayers and in the RCCS bioreactor for 24 h, 48 h, 96 h, and 2 weeks (2 w). Data are from densitometric ratio analyses as means ± SEM from 3 independent experiments. *P < 0.05 versus 2D monolayers.
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fig5: Expression of glial cell markers. Representative Western blotting and quantification of the levels of GFAP, S100B, and Cx43 in GL15 cells cultured in 2D monolayers and in the RCCS bioreactor for 24 h, 48 h, 96 h, and 2 weeks (2 w). Data are from densitometric ratio analyses as means ± SEM from 3 independent experiments. *P < 0.05 versus 2D monolayers.

Mentions: The differentiation status of cells is characterised not only by marker localisation but also by marker expression levels. To evaluate potential quantitative differences between the G-aggregate modelled microgravity-exposed cultures and the GL15 cells as 2D static monolayer cultures, the expression levels of the GFAP, S100B, and Cx43 proteins were determined by Western blotting (Figure 5). The G-aggregates showed increased levels of GFAP, S100B, and Cx43 after the first 48 h of culture. These levels gradually decreased over the following 2 weeks, when those of S100B and Cx43 were similar to those observed in the GL15 cells as 2D static cultures, while those of GFAP remained increased in the G-aggregates (Figure 5).


RCCS bioreactor-based modelled microgravity induces significant changes on in vitro 3D neuroglial cell cultures.

Morabito C, Steimberg N, Mazzoleni G, Guarnieri S, Fanò-Illic G, Mariggiò MA - Biomed Res Int (2015)

Expression of glial cell markers. Representative Western blotting and quantification of the levels of GFAP, S100B, and Cx43 in GL15 cells cultured in 2D monolayers and in the RCCS bioreactor for 24 h, 48 h, 96 h, and 2 weeks (2 w). Data are from densitometric ratio analyses as means ± SEM from 3 independent experiments. *P < 0.05 versus 2D monolayers.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4309310&req=5

fig5: Expression of glial cell markers. Representative Western blotting and quantification of the levels of GFAP, S100B, and Cx43 in GL15 cells cultured in 2D monolayers and in the RCCS bioreactor for 24 h, 48 h, 96 h, and 2 weeks (2 w). Data are from densitometric ratio analyses as means ± SEM from 3 independent experiments. *P < 0.05 versus 2D monolayers.
Mentions: The differentiation status of cells is characterised not only by marker localisation but also by marker expression levels. To evaluate potential quantitative differences between the G-aggregate modelled microgravity-exposed cultures and the GL15 cells as 2D static monolayer cultures, the expression levels of the GFAP, S100B, and Cx43 proteins were determined by Western blotting (Figure 5). The G-aggregates showed increased levels of GFAP, S100B, and Cx43 after the first 48 h of culture. These levels gradually decreased over the following 2 weeks, when those of S100B and Cx43 were similar to those observed in the GL15 cells as 2D static cultures, while those of GFAP remained increased in the G-aggregates (Figure 5).

Bottom Line: We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions.Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation).In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience, Imaging and Clinical Sciences, Unit of Functional Biotechnology, Aging Research Center (Ce.S.I.), "G. d'Annunzio" University of Chieti-Pescara, Via dei Vestini 29, 66100 Chieti, Italy ; Interuniversity Institute of Myology, Italy.

ABSTRACT
We propose a human-derived neuro-/glial cell three-dimensional in vitro model to investigate the effects of microgravity on cell-cell interactions. A rotary cell-culture system (RCCS) bioreactor was used to generate a modelled microgravity environment, and morphofunctional features of glial-like GL15 and neuronal-like SH-SY5Y cells in three-dimensional individual cultures (monotypic aggregates) and cocultures (heterotypic aggregates) were analysed. Cell survival was maintained within all cell aggregates over 2 weeks of culture. Moreover, compared to cells as traditional static monolayers, cell aggregates cultured under modelled microgravity showed increased expression of specific differentiation markers (e.g., GL15 cells: GFAP, S100B; SH-SY5Y cells: GAP43) and modulation of functional cell-cell interactions (e.g., N-CAM and Cx43 expression and localisation). In conclusion, this culture model opens a wide range of specific investigations at the molecular, biochemical, and morphological levels, and it represents an important tool for in vitro studies into dynamic interactions and responses of nervous system cell components to microgravity environmental conditions.

Show MeSH
Related in: MedlinePlus