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Adaptation of NS cells growth and differentiation to high-throughput screening-compatible plates.

Garavaglia A, Moiana A, Camnasio S, Bolognini D, Papait R, Rigamonti D, Cattaneo E - BMC Neurosci (2010)

Bottom Line: NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.This optimized system has also been exploited in homogeneous and high-content assays.Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dialectica S.r.l., c/o Nerviano Medical Sciences, V.le Pasteur 10, 20014 Nerviano MI, Italy.

ABSTRACT

Background: There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.

Results: We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96- and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays.

Conclusions: Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

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Related in: MedlinePlus

Adaptation of culture conditions of aNS-1 cells to microplates. aNS-1 cells can be cultured in 96- (A) and 384-well (B) plates, maintaining the expression of proper NS cells markers (Nestin, Vimentin, BLBP, Olig2), without exiting the cell cycle (Phospho-Histone3 immunoreactivity, P-H3), without differentiation (beta III-tubulin, MAP2 and GFAP absence) or cell death (cleaved caspase-3 absence).
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Figure 1: Adaptation of culture conditions of aNS-1 cells to microplates. aNS-1 cells can be cultured in 96- (A) and 384-well (B) plates, maintaining the expression of proper NS cells markers (Nestin, Vimentin, BLBP, Olig2), without exiting the cell cycle (Phospho-Histone3 immunoreactivity, P-H3), without differentiation (beta III-tubulin, MAP2 and GFAP absence) or cell death (cleaved caspase-3 absence).

Mentions: Mouse NS cells are regularly grown in 25 cm2 flasks and tested for their proliferation rate and differentiation capability in 24-well dishes. In these conditions, cells grow stably and homogenously while maintaining their pluripotency and neurogenic capacity [15,19]. In order to adapt their growth and differentiation potential to plate formats compatible with high-throughput screening, we have performed experiments in which seeding density and medium volume per well have been optimized to 96- and 384-well formats. After harvesting, aNS-1 cells, an adult SVZ-derived NS cell line [15], were plated at a density of 4 × 103, 8 × 103 or 16 × 103 cells/well in 96-well and at a density of 1 × 103, 2 × 103 or 4 × 103 cells/well in 384-well microplates. 80 μl and 45 μl respectively of growth medium containing FGF and EGF were added to the two formats. The day after plating and for the following three days, cells did not show any evident morphological change. After staining, they maintained expression of neural progenitor cell markers, such as Nestin, Vimentin, BLBP and Olig2 (Figure 1A and 1B). Analyses of Phospho Histone3 (P-H3) immunoreactivity shows that in both 96- and 384-well microplates cells undergo self-renewal in absence of unwanted differentiation as they do not express antigens proper of neurons such as beta III-tubulin and MAP2, nor of glial cells, such as GFAP (Figure 1A and 1B). Finally, the use of 96- or 384-well microplates did not affect the survival of the cells, as shown by the absence of active caspase-3 immunoreactivity (Figure 1A and 1B). Filling the wells on the border of the microplates with PBS, allowed the added media volume in each well to last for three days after plating. These growth conditions in 96- and 384-well formats have been successfully adapted also for LC1, a NS cell line derived from ESCs [13] (Additional file 1).


Adaptation of NS cells growth and differentiation to high-throughput screening-compatible plates.

Garavaglia A, Moiana A, Camnasio S, Bolognini D, Papait R, Rigamonti D, Cattaneo E - BMC Neurosci (2010)

Adaptation of culture conditions of aNS-1 cells to microplates. aNS-1 cells can be cultured in 96- (A) and 384-well (B) plates, maintaining the expression of proper NS cells markers (Nestin, Vimentin, BLBP, Olig2), without exiting the cell cycle (Phospho-Histone3 immunoreactivity, P-H3), without differentiation (beta III-tubulin, MAP2 and GFAP absence) or cell death (cleaved caspase-3 absence).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823757&req=5

Figure 1: Adaptation of culture conditions of aNS-1 cells to microplates. aNS-1 cells can be cultured in 96- (A) and 384-well (B) plates, maintaining the expression of proper NS cells markers (Nestin, Vimentin, BLBP, Olig2), without exiting the cell cycle (Phospho-Histone3 immunoreactivity, P-H3), without differentiation (beta III-tubulin, MAP2 and GFAP absence) or cell death (cleaved caspase-3 absence).
Mentions: Mouse NS cells are regularly grown in 25 cm2 flasks and tested for their proliferation rate and differentiation capability in 24-well dishes. In these conditions, cells grow stably and homogenously while maintaining their pluripotency and neurogenic capacity [15,19]. In order to adapt their growth and differentiation potential to plate formats compatible with high-throughput screening, we have performed experiments in which seeding density and medium volume per well have been optimized to 96- and 384-well formats. After harvesting, aNS-1 cells, an adult SVZ-derived NS cell line [15], were plated at a density of 4 × 103, 8 × 103 or 16 × 103 cells/well in 96-well and at a density of 1 × 103, 2 × 103 or 4 × 103 cells/well in 384-well microplates. 80 μl and 45 μl respectively of growth medium containing FGF and EGF were added to the two formats. The day after plating and for the following three days, cells did not show any evident morphological change. After staining, they maintained expression of neural progenitor cell markers, such as Nestin, Vimentin, BLBP and Olig2 (Figure 1A and 1B). Analyses of Phospho Histone3 (P-H3) immunoreactivity shows that in both 96- and 384-well microplates cells undergo self-renewal in absence of unwanted differentiation as they do not express antigens proper of neurons such as beta III-tubulin and MAP2, nor of glial cells, such as GFAP (Figure 1A and 1B). Finally, the use of 96- or 384-well microplates did not affect the survival of the cells, as shown by the absence of active caspase-3 immunoreactivity (Figure 1A and 1B). Filling the wells on the border of the microplates with PBS, allowed the added media volume in each well to last for three days after plating. These growth conditions in 96- and 384-well formats have been successfully adapted also for LC1, a NS cell line derived from ESCs [13] (Additional file 1).

Bottom Line: NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.This optimized system has also been exploited in homogeneous and high-content assays.Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dialectica S.r.l., c/o Nerviano Medical Sciences, V.le Pasteur 10, 20014 Nerviano MI, Italy.

ABSTRACT

Background: There is an urgent need of neuronal cell models to be applied to high-throughput screening settings while recapitulating physiological and/or pathological events occurring in the Central Nervous System (CNS). Stem cells offer a great opportunity in this direction since their self renewal capacity allows for large scale expansion. Protocols for directed differentiation also promise to generate populations of biochemically homogenous neuronal progenies. NS (Neural Stem) cells are a novel population of stem cells that undergo symmetric cell division in monolayer and chemically defined media, while remaining highly neurogenic.

Results: We report the full adaptation of the NS cell systems for their growth and neuronal differentiation to 96- and 384-well microplates. This optimized system has also been exploited in homogeneous and high-content assays.

Conclusions: Our results show that these mouse NS cells may be suitable for a series of applications in high-throughput format.

Show MeSH
Related in: MedlinePlus