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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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Neuronal marker localisation in SH-SY5Y cells. Representative confocal images of SH-SY5Y cells cultured as a monolayer (2D, (a), (c), and (e)) and under the modelled microgravity (RCCS bioreactor, (b), (d), and (f)) and immunostained with anti-N-CAM ((a) and (b)), anti-GAP43 ((c) and (d)), and anti-tyrosine hydroxylase (TH) ((e) and (f)) antibodies (as indicated). All of the cells were also stained with propidium iodide (PI). Insets show image magnification. Scale bars, 20 μm.
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fig4: Neuronal marker localisation in SH-SY5Y cells. Representative confocal images of SH-SY5Y cells cultured as a monolayer (2D, (a), (c), and (e)) and under the modelled microgravity (RCCS bioreactor, (b), (d), and (f)) and immunostained with anti-N-CAM ((a) and (b)), anti-GAP43 ((c) and (d)), and anti-tyrosine hydroxylase (TH) ((e) and (f)) antibodies (as indicated). All of the cells were also stained with propidium iodide (PI). Insets show image magnification. Scale bars, 20 μm.

Mentions: We characterised the phenotype expressed by the SH-SY5Y cells in the S-aggregates maintained in the dynamic 3D culture in the RCCS bioreactor for 2 weeks, by determining the expression of the neuronal specific markers N-CAM, GAP43, and tyrosine hydroxylase. Immunofluorescence analysis revealed that N-CAM in the S-aggregates was localised towards the plasma membrane and near cell-cell contact areas, thus resembling its distribution in the SH-SY5Y cells cultured in 2D monolayers (Figures 4(a) and 4(b)), which showed cell-cell adhesion interactions. GAP43 is involved in neurite outgrowth and neuronal plasticity [26], and in SH-SY5Y cell monolayers it was localised into neurite-like processes (Figure 4(c)). In the S-aggregates, GAP43 was localised in the cytoplasmic compartment (Figure 4(d)). The distribution of tyrosine hydroxylase (TH), which is a rate-limiting enzyme in dopamine/norepinephrine synthesis [27], was in the cytoplasm under both of these cell-culture conditions (Figures 4(e) and 4(f)).


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)

Neuronal marker localisation in SH-SY5Y cells. Representative confocal images of SH-SY5Y cells cultured as a monolayer (2D, (a), (c), and (e)) and under the modelled microgravity (RCCS bioreactor, (b), (d), and (f)) and immunostained with anti-N-CAM ((a) and (b)), anti-GAP43 ((c) and (d)), and anti-tyrosine hydroxylase (TH) ((e) and (f)) antibodies (as indicated). All of the cells were also stained with propidium iodide (PI). Insets show image magnification. Scale bars, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Neuronal marker localisation in SH-SY5Y cells. Representative confocal images of SH-SY5Y cells cultured as a monolayer (2D, (a), (c), and (e)) and under the modelled microgravity (RCCS bioreactor, (b), (d), and (f)) and immunostained with anti-N-CAM ((a) and (b)), anti-GAP43 ((c) and (d)), and anti-tyrosine hydroxylase (TH) ((e) and (f)) antibodies (as indicated). All of the cells were also stained with propidium iodide (PI). Insets show image magnification. Scale bars, 20 μm.
Mentions: We characterised the phenotype expressed by the SH-SY5Y cells in the S-aggregates maintained in the dynamic 3D culture in the RCCS bioreactor for 2 weeks, by determining the expression of the neuronal specific markers N-CAM, GAP43, and tyrosine hydroxylase. Immunofluorescence analysis revealed that N-CAM in the S-aggregates was localised towards the plasma membrane and near cell-cell contact areas, thus resembling its distribution in the SH-SY5Y cells cultured in 2D monolayers (Figures 4(a) and 4(b)), which showed cell-cell adhesion interactions. GAP43 is involved in neurite outgrowth and neuronal plasticity [26], and in SH-SY5Y cell monolayers it was localised into neurite-like processes (Figure 4(c)). In the S-aggregates, GAP43 was localised in the cytoplasmic compartment (Figure 4(d)). The distribution of tyrosine hydroxylase (TH), which is a rate-limiting enzyme in dopamine/norepinephrine synthesis [27], was in the cytoplasm under both of these cell-culture conditions (Figures 4(e) and 4(f)).

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