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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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Cell viability assay. (a) Representative images of preinduced and spontaneously formed GL15 aggregates and spontaneously formed SH-SY5Y aggregates (as indicated). Aggregates were stained with DAPI (blue), Annexin V-Alexa 488 (green), and propidium iodide (PI; red). DAPI-positive/Annexin V-Alexa 488-negative/PI-negative cells are healthy; DAPI-positive/Annexin V-Alexa 488-positive/PI-negative and PI-positive cells are considered apoptotic (Annexin V, arrowheads); DAPI-positive/Annexin V-Alexa 488-negative/PI-positive cells are necrotic (PI, arrowheads). (b) Quantification of apoptotic and necrotic cells in aggregate sections. Data derived from 3 independent experiments.
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fig2: Cell viability assay. (a) Representative images of preinduced and spontaneously formed GL15 aggregates and spontaneously formed SH-SY5Y aggregates (as indicated). Aggregates were stained with DAPI (blue), Annexin V-Alexa 488 (green), and propidium iodide (PI; red). DAPI-positive/Annexin V-Alexa 488-negative/PI-negative cells are healthy; DAPI-positive/Annexin V-Alexa 488-positive/PI-negative and PI-positive cells are considered apoptotic (Annexin V, arrowheads); DAPI-positive/Annexin V-Alexa 488-negative/PI-positive cells are necrotic (PI, arrowheads). (b) Quantification of apoptotic and necrotic cells in aggregate sections. Data derived from 3 independent experiments.

Mentions: The cell viability in the preinduced aggregates and the spontaneously formed aggregates was also assessed after 2 weeks in the RCCS bioreactor, to determine the apoptotic or the necrotic cells (Figure 2). To this aim, the cells were tested to measure early apoptosis by detecting phosphatidylserine expression revealed by Annexin V binding or necrosis by membrane permeability to the propidium iodide (PI) vital dye. Cells positive to Annexin V green fluorescence signal are known to be apoptotic cells, while those positive to PI red fluorescence signal are necrotic cells; the absence of green or red signal and the nuclear staining with DAPI revealed viable cells. The image analyses of stained cells revealed that some apoptotic cells (Figure 2(a), green fluorescence) were evident at a similar extent in preinduced and spontaneous GL15 aggregates (Figure 2(b)). A relevant amount of necrotic cells (Figure 2(a), red fluorescence) was present in preinduced aggregates compared to spontaneous ones in which necrotic cells were nearly absent (Figure 2(b)). These data revealed the presence of possible stress conditions in the preinduced aggregates, while the spontaneously formed aggregates showed cells that were in a more healthy state. This cell stress might be the result of hypoxic processes in the central core of the preinduced aggregates in particular, potentially due to the static conditions encountered in the hanging drops. For this reason, the rest of the investigations used only the spontaneously formed cell aggregates, and those formed by the GL15 cells are henceforth referred to as the G-aggregates.


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)

Cell viability assay. (a) Representative images of preinduced and spontaneously formed GL15 aggregates and spontaneously formed SH-SY5Y aggregates (as indicated). Aggregates were stained with DAPI (blue), Annexin V-Alexa 488 (green), and propidium iodide (PI; red). DAPI-positive/Annexin V-Alexa 488-negative/PI-negative cells are healthy; DAPI-positive/Annexin V-Alexa 488-positive/PI-negative and PI-positive cells are considered apoptotic (Annexin V, arrowheads); DAPI-positive/Annexin V-Alexa 488-negative/PI-positive cells are necrotic (PI, arrowheads). (b) Quantification of apoptotic and necrotic cells in aggregate sections. Data derived from 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Cell viability assay. (a) Representative images of preinduced and spontaneously formed GL15 aggregates and spontaneously formed SH-SY5Y aggregates (as indicated). Aggregates were stained with DAPI (blue), Annexin V-Alexa 488 (green), and propidium iodide (PI; red). DAPI-positive/Annexin V-Alexa 488-negative/PI-negative cells are healthy; DAPI-positive/Annexin V-Alexa 488-positive/PI-negative and PI-positive cells are considered apoptotic (Annexin V, arrowheads); DAPI-positive/Annexin V-Alexa 488-negative/PI-positive cells are necrotic (PI, arrowheads). (b) Quantification of apoptotic and necrotic cells in aggregate sections. Data derived from 3 independent experiments.
Mentions: The cell viability in the preinduced aggregates and the spontaneously formed aggregates was also assessed after 2 weeks in the RCCS bioreactor, to determine the apoptotic or the necrotic cells (Figure 2). To this aim, the cells were tested to measure early apoptosis by detecting phosphatidylserine expression revealed by Annexin V binding or necrosis by membrane permeability to the propidium iodide (PI) vital dye. Cells positive to Annexin V green fluorescence signal are known to be apoptotic cells, while those positive to PI red fluorescence signal are necrotic cells; the absence of green or red signal and the nuclear staining with DAPI revealed viable cells. The image analyses of stained cells revealed that some apoptotic cells (Figure 2(a), green fluorescence) were evident at a similar extent in preinduced and spontaneous GL15 aggregates (Figure 2(b)). A relevant amount of necrotic cells (Figure 2(a), red fluorescence) was present in preinduced aggregates compared to spontaneous ones in which necrotic cells were nearly absent (Figure 2(b)). These data revealed the presence of possible stress conditions in the preinduced aggregates, while the spontaneously formed aggregates showed cells that were in a more healthy state. This cell stress might be the result of hypoxic processes in the central core of the preinduced aggregates in particular, potentially due to the static conditions encountered in the hanging drops. For this reason, the rest of the investigations used only the spontaneously formed cell aggregates, and those formed by the GL15 cells are henceforth referred to as the G-aggregates.

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