1. Multi-Scale Strategies for Embryonic Stem Cell Culture M.M. Diogo 1, T.G. Fernandes 1,2, A.M. Fernandes 1, C.A.V. Rodrigues 1, R.P. Baptista 1, C. Lobato da Silva 1, J. Dordick 2 and J.M.S. Cabral 1 (1) Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, Lisboa, Portugal (2) Rensselaer Polytechnic Institute, Department of Chemical and Biological Engineering, Troy, NY, USA Ciência 2009

2. EMBRYONIC STEM CELLS (ESC) Undifferentiated cells isolated from the inner cell mass of the blastocyst Self-renewal Capacity Can be cultivated indefinitely and produce an unlimited number of cells Pluripotency Capability to differentiate into many different mature cell types (blood cells, skin cells, neurons, etc)

3. Applications for Stem Cells and Stem Cell Progeny Embryonic Stem Cells & Progeny Drug Discovery Cellular Therapy and Tissue Engineering Toxicological Screening Critical to develop efficient bioprocesses

4. SCALE-UP OF MOUSE EMBRYONIC STEM CELL EXPANSION PhD Thesis Ana Fernandes

5. Cultispher S: Macroporous Microcarriers Cross-linked gelatin Pore diameter < 10-20  m ± 0.4-0.7 m 2 surface area per gram SEM 46C mESC on Cultispher S Microcarriers

6. ESC expansion in Spinner-Flask Serum-containing medium supplemented with leukemia inhibitory factor (LIF) Serum-free medium supplemented with LIF and bone morphogenetic protein-4 (BMP-4) Enhance Proliferation Prevent Differentiation Serum-free medium potentially leads to higher cell densities

7. I. Influence of the medium: serum-containing vs serum-free II. Influence of the agitation rate 40, 60, 80, 100 rpm III. Comparison of two serum-free media Knockout ® + LIF VS ESGRO ® IV. Influence of the feeding regimen 50% per day (24h/24h) vs 25% per day (12h/12h) ESC expansion in Spinner-Flask StemSpan (V= 50 mL)

8. ESC expansion in a fully-controlled Bioreactor BioFlo 110 (V = 1.3L) Gases (N 2, CO 2, air) + 10% (w/v) NaHCO 3 ParameterTpHDO 2 Agitation “Set point”37ºC7.220%60 rpm Medium: Knockout ® serum-free medium + LIF Feeding regimen: 50% per day (24h/24h) Duration: 10 days

9. Direct comparison between Bioreactor and Spinner-flask Bioreactor Yield: ± 3x10 9 cells in 8 days

10. Oct4 SSEA-1 Nanog > 95% expression of pluripotent markers Pluripotency Markers (Flow Cytometry) Characterization of ESC after expansion ± 90% Before commitment After commitment Sox1-GFP + Neural Commitment Potential (Flow Cytometry)

11. EXPLORING STEM CELL FATE USING 3-D CELLULAR MICROARRAYS In collaboration with Jonathan Dordick, RPI, USA PhD Thesis Tiago Fernandes

12. Lee et al. Proc. Nat. Acad. Sci. USA, 105, 59-63 (2008) Cells are spotted onto functionalized glass slides Spatially addressable pattern of cells encapsulated in a 3-D hydrogel matrix Volumes as low as 20 nL 3-D Cell Culture Microarray Platform Scale-bar: 800 µm

13. Cell Printing Cell Expansion Cell Staining Expansion of ESC in a Microarray Cell Chip Knockout ® serum-free medium + LIF ESGRO ® serum-free medium

14. Ex-vivo expansion of ESC ESGRO ® Knockout ® + LIF Culture SystemVolume  (day -1 ) Culture Plate5.0 mL1.0 ±0.3 Spinner Flask30 mL1.0 ±0.2 Microarray Cell Chip20-60 nL1.0 ±0.3 Critical to quantify levels of protein expression  Develop on-chip, in-cell Western analysis

15. The immunostaining assay was able to distinguish between different cell populations in terms of Oct-4 and Nanog levels in ES cells Cell-Based Microarray Immunostaining Assay Oct-4 Immunostaining Expansion for 5 days in undifferentiating conditions (ESGRO ® serum-free medium) (I) Bright field (II) Fluorescence

16. Neural Stem Cells (NSC) ESCNSC Neural Progenitors Oligodendrocytes Astrocytes Neurons From Embryonic Stem Cells to Neural Stem Cells Long-term Self-renewal capacity Multipotency (differentiate into Neurons, Astrocytes and Oligodendrocytes) Advantage of NSC avoid contamination with non-neural cells and pluripotent ESC

17. EXPANSION OF ESC-DERIVED NEURAL STEM CELLS (NSC) UNDER HYPOXIC CONDITIONS PhD Thesis Carlos Rodrigues

18. NSC growth rate is enhanced under 2% O 2 2-5% is the optimal O 2 tension range for NSC expansion sucessive passaging

19. Multipotency of NSC under 2% O 2 Nestin expression is maintained above 95% during successive passaging After expansion under 2% O 2 NSC maintain the potential to differentiate into Neurons and Astrocytes Neurons Astrocytes

20. NSC Growth Kinetics without passaging

21. A fully controlled bioreactor was successfully established for the scaling-up of mouse ESC expansion, under serum-free conditions. Cells expanded under stirred conditions maintained their pluripotency and neural commitment potential. The 3-D microarray supports the expansion of mouse ESC under serum-free conditions, while maintaining their pluripotent and undifferentiated state. ESC proliferation inside the spots was comparable with cell proliferation in culture plates and spinner flasks. Hypoxic conditions favor mouse ESC-derived NSC expansion. 2% O 2 leads to a higher NSC growth rate, while multipotenty is not affected. Culture under hypoxic conditions may be used for a more efficient large-scale production of ESC-derived NSC. CONCLUSIONS

22. ACKNOWLEDGEMENTS IBB - Stem Cell Bioengineering Lab @ IST, Lisboa, Portugal www.ibb.pt/scbl Collaborations: Jonathan Dordick Financial Support: Domingos Henrique Fundação para a Ciência e a Tecnologia