A General Introduction to MicroRNAs,Their Investigation and Exploitation in CHO Cell Lines - MicroRNAs as Tools in Biopharmaceutical Production - page 7

resulted in improved cell lines that can grow to very high densities in suspension cell
cultures (for review see Fussenegger and Bailey 1998 ; Kim et al. 2011 ). To improve
the foreign protein productivity, operators can rely on biphasic cultures whereby
cells are encouraged to proliferate at 37 ◦ C until the culture reaches the critical
biomass and then the temperature is lowered (28-33 ◦ C) to promote a longer and
more viable stationary/production phase (Trummer et al. 2006 ; Yoon et al. 2003a , b ).
The link of microRNA expression and control with cancer (Calin et al. 2002 ) and
subsequently the regulation of apoptosis (Brennecke et al. 2003 ) prompted several
microRNA hybridization array analyses of bioprocessing cell lines, with the aim to
identify growth-phase dependent microRNA markers. For example, Gammel et al
took advantage of the fact that microRNAs are highly conserved and used non-
hamster arrays on their CHO model (Gammell et al. 2007 ). Using this approach, they
showed that miR-21 and miR-24 were up-regulated in CHO-K1 growth-arrested cell
lines, induced either by temperature shift or during normal batch culture. An ensuing
publication into microRNA expression in CHO-K1 cells showed that lowering the
temperature from 37 ◦ Cto31 ◦ C led to increased levels for: miR-219, miR-518d,
miR-126, miR-30e, miR-489 and miR-345, when measured after 24 h, while miR-7,
miR-320, miR-101 and miR-199 were down-regulated (Barron et al. 2011 ). Similar
lines of investigation were carried out by Koh et al in human embryonic kidney 293
(HEK293) cells who examined the microRNA expression profiles in batch cultures at
different growth phases (Koh et al. 2009 ). They reported that 13 out of 14 microRNAs,
including for example hsa-let-7b and hsa-miR-16, were up-regulated in the transition
from exponential to stationary phase indicative of translation slowing down for a large
number of proteins.
Whilst such studies have their merits, they also emphasize that the microRNA
signature greatly fluctuates with the culturing conditions as well as the type of produc-
ing cells. Interestingly, proliferating cells appear to express mRNAs with shortened
3 UTRs and UTRs that contain less miRNA target sites (Sandberg et al. 2008 ), in-
dicating that some mRNA isoforms may evade post-transcriptional regulation in a
growth-phase dependent manner. The cell density has also been reported to impact
on microRNA biogenesis (Hwang et al. 2009 ) with confluent cells showing an in-
crease in Drosha processing. These observations show that careful monitoring of all
aspects of cell culturing are crucial for the accurate analysis of miRNA expression
and suggest that many miRNAs may only have a short time span to fulfil their action.

1.6 Validation of MicroRNAs Target Predictions in

Bioprocessing Applications

Chinese hamster ovary (CHO) cell lines are presently the gold standard mam-
malian expression system for the production of recombinant proteins in the
biopharmaceutical industry. The majority of mammalian expressed and approved
biotechnology-derived biopharmaceuticals are produced in CHO cells and it is ex-
pected that this will remain the case for the foreseeable future. Despite being at the