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9.2.3 Culture
Traditionally, human ES cells were cultured on mouse embryonic fi broblast (MEF)
feeder layers, which maintain continuous proliferation in an undifferentiated state.
The growth of ES cells and MEF together needs meticulous care and is often expen-
sive (Amit et al. 2003 ). The dual growth of these cells induces risks of pathogen
transfer and viral infection to ES cells. Moreover, the preparation of these feeder
layers is labour intensive, and variations between different batches of fi broblasts
make the experiments complex and diffi cult to defi ne (Amit et al. 2000 ). For such
reasons human ES cells grown on animal feeder layers are not suitable for applica-
tion in humans (Lee et al. 2004 ).
One of the initial developments made to the original culture methods came about
with the growth of human ES cells under serum-free conditions, using basic fi bro-
blast growth factor (bFGF) instead of serum (Amit et al. 2003 ). Along with com-
pounds benefi cial to human ES cells, serum contains harmful compounds detrimental
to stem cell survival as well, suggested by constantly low cloning effi ciency in the
presence of serum in the medium. The use of serum induces further variability to
experiments as each serum batch shows different capability to support vigorous
undifferentiated proliferation of ES cells. It is unknown though whether the effects
of bFGF on undifferentiated human ES cell growth in such culture conditions are
mediated through the fi broblasts, the ES cells or both (Amit et al. 2000 ). Serum
replacement with defi ned components should reduce variability of experiments and
permit more carefully defi ned differentiation studies (Amit et al. 2000 ). Further,
identifying the factors produced by MEF cells responsible for promoting human ES
cell renewal can be used to culture ES cells without the use of animal products.
Further improvement was made when Xu et al. ( 2001 ) were able to culture
human ES cells under serum- and/or feeder-free culture conditions. In such
feeder- free conditions, a cell adhesion coating supportive of the proliferation and
maintenance of pluripotency of ES cells is necessary. They began to culture
human ES cells on Matrigel or laminin in the medium conditioned by MEF
(MEF-CM); these ES cells grown in feeder-free conditions were reported to have
maintained a normal karyotype, stable proliferation rate and high telomerase
activity (Xu et al. 2001 ). However, the growth of MEF for the production of the
conditioned medium (MEF-CM) was still required, and Matrigel is also an animal
product (Lee et al. 2004 ).
These xenosupport systems are associated with the risk of transferring animal
pathogens from the animal feeder, matrix or conditioned medium to the human ES
cells making them clinically inapplicable. Thus, a culture system based on a human
feeder layer was developed. Richards et al. ( 2002 ) reported that ‘human foetal and
adult fi broblast feeders support prolonged undifferentiated human ES cell growth of
existing cell lines’ (Richards et al. 2002 ). These ES cells cultured with human
feeder layers in the medium supplemented with human serum showed features simi-
lar to cells grown on MEF, including pluripotency, morphology and expression of
cell-surface markers. Amit et al. ( 2003 ) were able to culture ES cells on foreskin
D.M. Kalaskar and S.M. Shahid