Cell Banking
Evaluation of Methods for the Inactivation of Feeder Cells
Effective inactivation of feeder cells is an essential
pre-requisite for banking hES Cells. The current protocols used to
inactivate feeders were evaluated for their effectiveness using by
Ki67-antibody staining and monitoring the lack of proliferation of
the feeders. The protocol currently in use in the bank (using
mitomycin C to crosslink the DNA) effectively inhibits
proliferation. However, this method is carried out on cells
attached to the surface of plastic flasks and requires large
quantities of the inactivating agent. Furthermore, there is some
concern over the use of mitomycin C in the context of the
preparation of therapeutic cell banks. The Bank is therefore
investigating the inactivation of feeders in suspension using
irradiation. This will help improve efficiency in the bulk
processing of feeders, cut down on reagents and consumables and
provide a lead for development of more acceptable methods for human
feeder cells proposed for use in clinical grade cell banking.
Optimising feeder cell density for undifferentiated growth of
hESCs
The feeder cells that are used to support the growth of hESCs in
co-culture systems play a major role in regulating hESC phenotype.
We have investigated the effects of varying the density of cells in
the feeder layer on morphological differentiation of co-cultured
hESC colonies. Using a live cell imaging system we were able to
demonstrate the sensitivity of hESCs to subtle variations in feeder
cell density, highlighting the need to optimise and standardise
this aspect of hESC co-culture.
Developing a Generic ‘Feeder' Cell Line & Optimising hESC
Culture
Assessment of a number of alternative feeder lines, both mouse
& human, has been undertaken and a number of lines have been
selected for further investigation. These include a qualified bank
of murine 3T3 cells (currently used by the UK skin & corneal
transplant groups), as well as MRC-5 cells (human foetal lung cells
used for many years in the production of human viral vaccines) and
a number of human dermal fibroblast lines which have been
successfully used for the maintenance of undifferentiated hESCs. We
have recently initiated a study using 4 hESC lines to investigate
the capacity of these selected feeder cells to support the
undifferentiated expansion of the hESCs. This study will monitor
karyotypic, genetic and phenotypic changes over a period of 20
passages. The results generated by this study will enable the bank
to make an informed judgement based on both qualitative and
quantitative data as to the best feeders to be used in the banking
process. This will, in turn aid the standardisation of cell culture
methodologies used by the Bank.
Developing Feeder-Free Methods for hESCs Culture
Within the stem cell community, a number of researchers have
moved from feeders to a feeder-free system that utilise the
basement membrane proteins derived from a mouse tumour (Matrigel).
This is not ideal, but it does provide a more readily defined
culture substrate and also eliminates the arduous task of preparing
large numbers of feeders for the passaging of hESCs.
The Bank is looking at the use of this mode of hESC expansion as
an adjunct to the cells grown on feeders. However, before the Bank
adopts this feeder-free expansion of hESCs, it needs to validate
this culture method alongside the growth of cells on feeders. This
validation is running in tandem with the feeder maintenance project
and uses the same set of assessment parameters. If the results show
that cells grown feeder-free are of similar or higher quality than
those produced on feeder cells, then this methodology also is also
likely be used operationally to produce banks of cells and provide
users of the Bank with a choice of cells grown under different
culture conditions. This may prove useful for specific
research applications (e.g. development of automated culture
systems).
Automation of Stem Cell Culture
The ESNATS project, funded by the European commission for five
years under FP7, is aimed at the development of enhanced in
vitro test methods for drug discovery and toxicology using
hESC lines. The Bank is involved in providing advice on protocol
development and evaluation of methods for automated scale up of
hESCs. The project, in collaboration with expert hESC groups and
The Automation Partnership who have considerable expertise in
automate cell culture systems, is intended to deliver more
reproducible cell culture procedures and also provide critical
savings on technical time in the banking of stem cell lines.