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)
1300
and trypsin were purchased from Gibco. All
chemicals were of analytical grade, and were
purchased from Sigma-Aldrich (St. Louis, MO).
Cell line: The rhFVIII expressing CHO cell line
obtained from the National Institute of Genetic
Engineering and Biotechnology (26) was used
as the rCHO cell line.
Culture conditions: The rCHO cells were
cultured in a basal medium containing DMEM
and Ham’s F12 nutrient mixture at a ratio of 1:1
(v/v), supplemented with 10% FBS. The rCHO
cells were grown at different concentrations of
glucose (1.2, 3.5, 7 g/l) in a T-75 flask so as to
obtain an appropriate glucose concentration in
the basal medium, for the purpose of obtaining
higher amounts of cell density.
The basal medium was prepared at different
concentrations of NH 4
Cl (2, 4, 6, 8, 10, 12 mM)
to determine the effect of ammonium stress on
rCHO cell growth and rhFVIII production. The
control flask contained basal medium without
excess ammonium.
The amino acid-supplemented T-flasks
contained basal medium plus 20 mM of each of
the appropriate amino acids and 10 mM NH 4
Cl.
The negative control flask contained the basal
medium plus 10 mM NH 4
Cl, and the positive
control flask contained only basal medium. This
experiment was carried out at low and high
glucose concentrations (1.2, 3.5 g/l). The
precursor-supplemented T-flasks contained basal
medium plus 10 -3 M of each of the dNTPs (dATP,
dCTP, dTTP, dGTP). The control flask did not
contain any precursors.
For studying effects of temperature on cell
growth and protein production, the rCHO cells
were cultivated at 37 o C until the culture reached
the exponential phase of growth. The temperature
was then reduced to 30 o C., but the control flask
was maintained at 37 o C. The osmolality of the
medium supplemented with 10% FBS was 320
mOsmol/kg. To determine the effects of
osmolality on cell growth and protein production,
NaCl stock solution was added to the basal
medium increasing osmolality by up to 410
mOsmol/kg (1 mg NaCl/ml = 32 mOsm
increase). The control flask had no excess sodium
chloride.
In order to investigate the effects of certain
additives on cell growth, the rCHO cells were
cultivated in basal medium with 10% FBS. After
48 h of cultivation, the medium was replaced
with fresh medium without FBS. Appropriate
amino acids and biotin were added to the serumfree
medium. Basal medium was supplemented
with 10 mg/l biotin. The serum-free control flask
had no additives.
Bioreactor: The batch bioreactor experiment was
performed in a 5 l (3.5 l working volume)
Celligen bioreactor (New Brunswick Scientific,
USA) maintained at 37 o C, pH 7.2, and stirred at
50-100 rpm. The pH level was controlled by
acidic and basic solutions (2N HCl and 80g/l
NaHCO 3
). Dissolved oxygen (DO) was adjusted
to 50% of air saturation, as manipulated by the
bioreactor control program. The polyester disks
(134 g) purchased from New Brunswick
Scientific Co., were used as microcarriers for the
attachment of the rCHO cells in the packed-bed
bioreactor. The appropriate conditions obtained
in the T-flask were also employed in packed-bed
bioreactor. Glutamine was added after 48 h of
cultivation at 20 mM and other additives (proline,
glycine, threonine, glutamine, biotin, dNTP and
glucose) were prepared in the basal medium. The
concentrations of the additives were; amino acids
(proline, glycine, threonine) 20 mM, biotin 2 mg/
l, dNTP 10 -3 M, and glucose 3.5 g/l. After 120 h
of cultivation, the polyester microcarriers were
removed from the bioreactor and the rCHO cells
were separated by the EDTA-Trypsin solution.
Effect of culture conditions on rCHO cell growth