full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and Pharmacy
Vol. 5 (3) 1298 -1308 July 2011, ISSN 0973-8916 (Print), 2230-7303 (Online)
1301
Analysis: All T-flask samples were cultivated in
a humidified incubator at 37 o C and 5% CO 2
. After
proliferation and cell growth, the medium was
removed from the flasks and eluted with
phosphate buffer solution (PBS). The cells were
removed from the surface of the flasks by trypsin-
EDTA solution (0.25%). After the incubation
period (ca. 5 min), trypsin was inhibited by
adding FBS. The cell suspensions were
centrifuged at 1000 rpm for 5 min at 4 o C. The
supernatants were then removed and the cell
pellets were resuspended in 1 ml of medium and
used for subsequent counting of cells. Cell
viability was determined by the trypan blue
exclusion method.
Deficient human coagulation factor VIII
plasma was purchased from Diagnostica
Stago,France. Biological activity of rhFVIII was
determined using a one-stage activated partial
thromboplastein time (aPTT) assay. Briefly, the
samples were mixed with deficient plasma as
substrate. The clotting time was measured,
following addition of CaCl 2
(0.025 M). The
activity of rhFVIII was determined from a
standard curve constructed by the pooled plasma.
Data was presented as relative specific activity,
and computed as the specific activity of samples
against the specific activity of the pooled plasma.
Glucose, ammonium and lactate
concentrations were enzymatically measured by
glucose, ammonium and lactate assay kits
(ChemEnzyme co., Iran)
Results and Discussion
Effect of glucose on cell density and protein
production: Figure 1 shows that the rCHO cell
density significantly decreased by 17% in
medium containing 7 g/l glucose (ANOVA, p <
0.05) in comparison with the medium consisting
3.5 g/l glucose in which the cell density reached
3.5×10 6 cells/ml after 96 h of cultivation. It has
been reported that glucose is vital for cell growth
and productivity, but the excess glucose
concentrations inhibit cell growth in the rCHO
cells which are cell line- and culture modedependent.
On the other hand, decreasing glucose
concentrations to 1.2 g/l led to a reduction in cell
density by more than 20% (ANOVA, p < 0.05)
when compared to 3.5 g/l glucose concentration.
High glucose concentrations reduce cyclic AMP
levels both in prokaryotes and eukaryotes (27).
Furthermore, formation of ammonium and
lactate as the toxic by-products in the mammalian
cell’s metabolic pathway depends on glucose
concentration. To explain the effects of glucose
on cell density, both by-products were measured
simultaneously. While glucose concentration was
elevated from 1.2 to 3.5 g/l, ammonium
formation was reduced from 37 mg/l to 29 mg/l
after 96h. But ammonium formation was no
longer decreased by increasing glucose
concentration. Furthermore, higher amounts of
ammonium were formed at earlier time of culture
at the low glucose concentration (Figure 2). It
can be due to the glucose depletion at 48 h when
glucose concentration was 1.2 g/l (data not
shown).
Ammonium formation in rCHO cells was
increased under glucose limitation. The main
source of ammonium formation is glutamine.
Fig. 1. The rCHO cell density at different
concentrations of glucose after 96 h of cultivation
Shakibaie et al