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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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Localisation of cell-cell interaction markers in the GS-aggregates. Representative confocal images of GS-aggregates cultured under modelled microgravity, for 2 weeks, and immunostained with anti-N-CAM and anti-Cx43 antibodies (as indicated). The GS-aggregate sections were also stained with propidium iodide (PI; right). Insets show image magnification. Scale bars, 10 μm.
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fig9: Localisation of cell-cell interaction markers in the GS-aggregates. Representative confocal images of GS-aggregates cultured under modelled microgravity, for 2 weeks, and immunostained with anti-N-CAM and anti-Cx43 antibodies (as indicated). The GS-aggregate sections were also stained with propidium iodide (PI; right). Insets show image magnification. Scale bars, 10 μm.

Mentions: The GS-aggregates were double-stained for N-CAM and Cx43. These N-CAM-specific and Cx43-specific fluorescent signals revealed a particular distribution of these proteins, whereby even if colocalisation of the N-CAM and Cx43 patterns was not evident, possible heterotypic cell-cell interactions could not be excluded. In particular, within the GS-aggregates, N-CAM localised to the peripheral areas of the cells, while Cx43-specific fluorescent spots appeared to be sparsely distributed, which indicated a low level of cell-cell functional interactions (Figure 9). In addition, in the same GS-aggregates, there were also evident N-CAM-negative and/or Cx43-negative cells, which indicated potential different cell activities due to different protein expression levels.


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)

Localisation of cell-cell interaction markers in the GS-aggregates. Representative confocal images of GS-aggregates cultured under modelled microgravity, for 2 weeks, and immunostained with anti-N-CAM and anti-Cx43 antibodies (as indicated). The GS-aggregate sections were also stained with propidium iodide (PI; right). Insets show image magnification. Scale bars, 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig9: Localisation of cell-cell interaction markers in the GS-aggregates. Representative confocal images of GS-aggregates cultured under modelled microgravity, for 2 weeks, and immunostained with anti-N-CAM and anti-Cx43 antibodies (as indicated). The GS-aggregate sections were also stained with propidium iodide (PI; right). Insets show image magnification. Scale bars, 10 μm.
Mentions: The GS-aggregates were double-stained for N-CAM and Cx43. These N-CAM-specific and Cx43-specific fluorescent signals revealed a particular distribution of these proteins, whereby even if colocalisation of the N-CAM and Cx43 patterns was not evident, possible heterotypic cell-cell interactions could not be excluded. In particular, within the GS-aggregates, N-CAM localised to the peripheral areas of the cells, while Cx43-specific fluorescent spots appeared to be sparsely distributed, which indicated a low level of cell-cell functional interactions (Figure 9). In addition, in the same GS-aggregates, there were also evident N-CAM-negative and/or Cx43-negative cells, which indicated potential different cell activities due to different protein expression levels.

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