Multi-Scale Strategies for Embryonic Stem Cell Culture

M.M. Diogo1, T.G. Fernandes1,2, A.M. Fernandes1, C.A.V. Rodrigues1, R.P. Baptista1,

C. Lobato da Silva1, J. Dordick2 and J.M.S. Cabral1

(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

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)

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

SCALE-UP OF MOUSE EMBRYONIC STEM CELL EXPANSION

PhD Thesis Ana Fernandes

Cultispher S: Macroporous Microcarriers

Cross-linked gelatin

Pore diameter < 10-20 m

± 0.4-0.7 m2 surface area per gram

SEM 46C mESC on Cultispher S

Microcarriers

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

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)

ESC expansion in a fully-controlled Bioreactor

BioFlo 110 (V = 1.3L)

Gases (N2, CO2, air)+ 10% (w/v) NaHCO3

Parameter T pH DO2 Agitation

“Set point” 37ºC 7.2 20% 60 rpm

Medium: Knockout ® serum-free medium + LIF

Feeding regimen: 50% per day (24h/24h)

Duration: 10 days

0.E+00

1.E+06

2.E+06

3.E+06

4.E+06

5.E+06

6.E+06

0 2 4 6 8 10

Time (Day)

Nº

cells

/mL

Bioreactor Spinner-flask

0.0

20.0

40.0

60.0

80.0

100.0

0 1 2 3 4 5 6 7 8 9 10Time (Day)

FI

Bioreactor Spinner-Flask

Direct comparison between Bioreactor and Spinner-flask

Bioreactor Yield: ± 3x109 cells in 8 days

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)

EXPLORING STEM CELL FATE USING 3-D CELLULAR MICROARRAYSIn collaboration with Jonathan Dordick, RPI, USA

PhD Thesis Tiago Fernandes

Lee et al. Proc. Nat. Acad. Sci. USA, 105, 59-63 (2008)

Cells are spotted onto functionalized glass slidesSpatially addressable pattern of cells encapsulated in a 3-D hydrogel matrixVolumes as low as 20 nL

3-D Cell Culture Microarray Platform

Scale-bar: 800 µm

Cell PrintingCell Expansion

Cell Staining

Expansion of ESC in a Microarray Cell ChipExpansion of ESC in a Microarray Cell Chip

Knockout® serum-free medium + LIF

ESGRO® serum-free medium

Ex-vivo expansion of ESC

ESGRO®

Knockout® + LIF

Culture System Volume (day-1)

Culture Plate 5.0 mL 1.0 ±0.3

Spinner Flask 30 mL 1.0 ±0.2

Microarray Cell Chip 20-60 nL 1.0 ±0.3

Critical to quantify levels of protein expression Develop on-chip, in-cell Western analysis

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

Neural Stem Cells (NSC)

ESC NSCNeural

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

EXPANSION OF ESC-DERIVED NEURAL STEM CELLS (NSC) UNDER HYPOXIC CONDITIONS

PhD Thesis Carlos Rodrigues

NSC growth rate is enhanced under 2% O2

2-5% is the optimal O2 tension range for NSC expansion

sucessive passaging

Multipotency of NSC under 2% O2

Nestin expression is maintained above 95% during successive passaging

After expansion under 2% O2 NSC maintain the potential to differentiate into Neurons and Astrocytes Neurons

Astrocytes

NSC Growth Kinetics

without passaging

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% O2 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

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