Automated 3D Culture of Neural Stem Cells · 2014. 6. 30. · Compared with 2-D, higher yields and...
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Automated 3D Culture of Neural Stem Cells A. Keric 1 , S. Haupt 1 , C. Cavelier 2 , Brad Justice 3 & O. Brüstle 1 , 4 [1] Life & Brain GmbH, Bonn, Germany; [2] Hamilton Bonaduz AG, Bonduz, Switzerland; [3] Global Cell Solutions Inc., Charlottesville, USA ; [4] Institute of Reconstructive Neurobiology, Bonn, Germany Fig. 1: Description of the 3-D cell culture system. Cells are grown on GEMs, a microcarrier uniquely designed for cell culture. The BioLevitator is a bench- top cell culture device that supports the culture of cells on GEMs. It ensures homogenisation of the cells / GEMs suspension and provides all functions of a cell culture incubator. Increased demand for cells that more closely mimic in vivo functions Many researchers have embraced 3-D biology as the latest paradigm HAMILTON Bonaduz AG & Global Cell Solutions ™ are developing a com- plete 3-D cell culture system that streamlines the cell culture process (Fig. 1) - GEMs (Global Eucaryotic Microcarriers ™) is a microcarrier uniquely designed for cell culture - BioLevitator supports the complete cell culture process from inoculation to harvesting Introduction Results Conclusion The automated 3-D culture system - facilitates cultivation of human ES cell-derived neural stem cells (It-hESNSC) with higher cell yields per cm 2 (fig. 2) - supports undifferentiated growth of It-hESNSC (Fig. 3) - maintains morphological characteristics of lt-hESNSCs, thus demonstrating its biocompatibility (Fig.4). Automated 3-D cell culture opens the path towards using cells as reagent: - uncouple cell culture from downstream assays - from freezer to assay in one step Fig. 5: Flowchart of the cells as reagent model. Automated 3-D cell culture eliminates the need for seed-split cycles and streamlines the cell cul- ture process. It also enables the uncoupling of cell culture from downstream assays and supports the direct use of cells from the freezer into an assay in a single step. A. GEM TM BioLevitator TM GEM TM BioLevitator TM hESCs hESCs lt-hESNSCs lt-hESNSCs guided differentiation B. 100 μm 100 μm 100 μm 100 μm C. 2-D 3-D Fig.3: It-hESNSCs remain undifferentiated after expansion in the BioLevitator I3 lt-hESNSC, grown on laminin coated GEMs for 9 days, maintain neural stem cell marker expression (Nestin, Sox2). The quantities of spontaneously differentiated cells and con- ventional 2-D cultures are comparable as shown by immunocytochemical analysis for the early neuronal differentiation marker TUJI. 3-D 2-D Fig.4: It-hESNSCs keep their typical rosette-like architecture. lt-hESNSC, cultured on laminin coated GEMs, exhibit a rosette- like morphology in a conventional 2-D culture centred around tight- junction protein ZO-1+ lumens (A). lt-hESNSC, grown on GEMs substrate maintain their typical rosette-like structure marked by white dotted lines (B). ZO-1 DAPI ZO-1 DAPI 2-D 100 μm 100 μm DAPI DAPI A. B. 3-D Fig.2: 3-D cultivation of a rosette-type, self-renewing human embryonic stem (ES) cell-derived neural stem cell (lt-hESNSCs) population. lt-hESNSC exhibit extensive self-renewal, clonogenicity (A) and a constant neuro- and gliogenic potential. Proliferating cells express high levels of the neural stem cell markers Nestin, Sox2, Sox1 and Pax6. Under optimal conditions, few cells differentiate spontaneously (Koch et al., 2009). Considering their amenability to large-scale expansion lt-hESNSCs provide an attractive tool for pharmacological screening and bio- medical applications. We used two different lt-hESNSC lines derived from H9.2 and I3 hESCs to test their behavior in 3-D cultures. After inoculating 25.000 cells/cm 2 with laminin coated GEMs for 6h, >80% of the cells attached to the GEMs (B). Subsequently, cells were cultured in the BioLevitator for 9 (I3) and 6 (H9.2) days. Mean growth (counting the viable cells at the last day of culture) and mean doubling times for both cell lines were determined and comparable to the conventional 2-D cultures (C). Compared with 2-D, higher yields and cell densities on the GEMs were observed under 3-D culture conditions (D,E). Shown are representative experiments performed as duplicates. 1. Evaluation of 3-D human ES cell-derived natural stem cell cultures 2. Characterization of 3-D human ES cell-derived neural stem cell cultures Reference Koch P., Opitz T., Steinbeck J.A., Ladewig J. and Brüstle O. (2009) A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. PNAS USA 106(9):3225-30 E. D. R ™
Transcript of Automated 3D Culture of Neural Stem Cells · 2014. 6. 30. · Compared with 2-D, higher yields and...
Automated 3D Culture of Neural Stem CellsA. Keric 1, S. Haupt 1, C. Cavelier 2, Brad Justice 3 & O. Brüstle 1, 4
[1] Life & Brain GmbH, Bonn, Germany; [2] Hamilton Bonaduz AG, Bonduz, Switzerland; [3] Global Cell Solutions Inc., Charlottesville, USA ; [4] Institute of Reconstructive Neurobiology, Bonn, Germany
Fig. 1: Description of the 3-D cell culture system. Cells are grown on GEMs, a microcarrier uniquely designed for cell culture. The BioLevitator is a bench-top cell culture device that supports the culture of cells on GEMs. It ensures homogenisation of the cells / GEMs suspension and provides all functions of a cell culture incubator.
Increased demand for cells that more closely mimic in vivo functions Many researchers have embraced 3-D biology as the latest paradigm HAMILTON Bonaduz AG & Global Cell Solutions ™ are developing a com-plete 3-D cell culture system that streamlines the cell culture process (Fig. 1)
- GEMs (Global Eucaryotic Microcarriers ™) is a microcarrier uniquely designed for cell culture - BioLevitator supports the complete cell culture process from inoculation to harvesting
Introduction
Results
Conclusion The automated 3-D culture system
- facilitates cultivation of human ES cell-derived neural stem cells (It-hESNSC) with higher cell yields per cm2 (fig. 2) - supports undifferentiated growth of It-hESNSC (Fig. 3) - maintains morphological characteristics of lt-hESNSCs, thus demonstrating its biocompatibility (Fig.4).
Automated 3-D cell culture opens the path towards using cells as reagent:
- uncouple cell culture from downstream assays - from freezer to assay in one step
Fig. 5: Flowchart of the cells as reagent model. Automated 3-D cell culture eliminates the need for seed-split cycles and streamlines the cell cul-ture process. It also enables the uncoupling of cell culture from downstream assays and supports the direct use of cells from the freezer into an assay in a single step.
A.
GEMTM BioLevitatorTMGEMTM BioLevitatorTM
hESCshESCs
lt-hESNSCslt-hESNSCs
guided differentiation
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100 μm100 μm 100 μm100 μm
C. 2-D 3-D
Fig.3: It-hESNSCs remain undifferentiated after expansion in the BioLevitatorI3 lt-hESNSC, grown on laminin coated GEMs for 9 days, maintain neural stem cell marker expression (Nestin, Sox2). The quantities of spontaneously differentiated cells and con-ventional 2-D cultures are comparable as shown by immunocytochemical analysis for the early neuronal differentiation marker TUJI.
3-D 2-D
Fig.4: It-hESNSCs keep their typical rosette-like architecture.lt-hESNSC, cultured on laminin coated GEMs, exhibit a rosette-like morphology in a conventional 2-D culture centred around tight-junction protein ZO-1+ lumens (A). lt-hESNSC, grown on GEMs substrate maintain their typical rosette-like structure marked by white dotted lines (B).
ZO-1DAPIZO-1DAPI
2-D
100 μm100 μm DAPIDAPI
A. B.
3-D
Fig.2: 3-D cultivation of a rosette-type, self-renewing human embryonic stem (ES) cell-derived neural stem cell (lt-hESNSCs) population.lt-hESNSC exhibit extensive self-renewal, clonogenicity (A) and a constant neuro- and gliogenic potential. Proliferating cells express high levels of the neural stem cell markers Nestin, Sox2, Sox1 and Pax6. Under optimal conditions, few cells differentiate spontaneously (Koch et al., 2009). Considering their amenability to large-scale expansion lt-hESNSCs provide an attractive tool for pharmacological screening and bio-medical applications. We used two different lt-hESNSC lines derived from H9.2 and I3 hESCs to test their behavior in 3-D cultures. After inoculating 25.000 cells/cm2 with laminin coated GEMs for 6h, >80% of the cells attached to the GEMs (B). Subsequently, cells were cultured in the BioLevitator for 9 (I3) and 6 (H9.2) days. Mean growth (counting the viable cells at the last day of culture) and mean doubling times for both cell lines were determined and comparable to the conventional 2-D cultures (C). Compared with 2-D, higher yields and cell densities on the GEMs were observed under 3-D culture conditions (D,E). Shown are representative experiments performed as duplicates.
1. Evaluation of 3-D human ES cell-derived natural stem cell cultures
2. Characterization of 3-D human ES cell-derived neural stem cell cultures
ReferenceKoch P., Opitz T., Steinbeck J.A., Ladewig J. and Brüstle O. (2009) A rosette-type, self-renewing human ES cell-derived neural stem cell with potential for in vitro instruction and synaptic integration. PNAS USA 106(9):3225-30
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