All chemicals in the Materials and Methods section were obtained from Sigma Aldrich unless stated otherwise.
Cell Transfection and Maintenance
The cMycCHO cell line resulted from calcium-mediated stable transfection of CHO-K1 with the DORclaG123 (c-Myc plasmid). The plasmid was kindly donated by Dr. T. Littlewood (Imperial Cancer Research Fund, UK). The surviving cells were pooled separately for each transfection and were maintained in Ham's F12 supplemented with 5% FCS. Using dilution cloning a stable cMycCHO clone was selected and used in subsequent experiments. A detailed transfection and selection procedure is described in a previous publication . Selection pressure was maintained by incubation in DMEM/F12, 4 mM L-glutamine, 5% FBS (Lonza Biologics) and 1 mg/mL geneticin G418 every 10th generation and cell samples were taken before and after incubation to ensure stable overexpression of the c-Myc protein. Selection pressure was removed 2 passages before an experimental run to negate the effects of the presence of antibiotic.
Static Batch Culture and Cell Size of CHO-K1 and cMycCHO
CHO-K1 and cMycCHO were maintained in DMEM/F12 with 5% FBS (Lonza Biologics, UK) and 4 mM L-glutamine at 37°C vented at 5% CO2. Static cultures from each cell line were plated in 6 well plates, 80,000 cells/cm2, with an initial density of 2 × 105 cells/ml allowing triplicate cultures to be sacrificed at regular intervals for each cell line. A duplicate culture was plated from the experimental seeding inoculum for mid-exponential phase harvesting to carry out the western analysis. When harvesting cells, floating cells were collected by centrifugation of the aspirated medium, and adherent cells were detached by Accutase™ and collected by centrifugation, these were pooled together in a media volume equal to the original culture volume before cell counts. Viable cell concentration and viability were monitored using the trypan blue exclusion method . All cell volume measurements were done using the coulter principle of electronic volume on a Quanta SC (Beckman Coulter) flow cytometer which has forward scatter replaced by electronic volume.
Static Spent Media Cultures
The spent media was obtained from each respective culture to replicate conditions of supplement depletion. Each cell line was seeded in T-25 flasks with 10 ml of the spent media at 5 × 105 cells/ml at 37°C vented at 5% CO2. Cells were derived from a mid-exponential phase culture. Samples were taken at 0, 24, 44, and 118 hours for analysis of apoptosis.
Transfection with the hSEAP-hFc gene
The plasmid, pFUSE-SEAP-hFc1, (InvivoGen, USA) was used to transfect cMycCHO and CHO-K1 cell lines using FuGENE® 6 transfection reagent (Roche, UK). Transfected cells were incubated in fresh DMEM/F12 (Sigma-Aldrich) with 5% heat inactivated FBS (PAA, UK) and 4 mM L-glutamine or 4 mM L-alanine-L-glutamine (Ultra-Glutamine, Lonza, UK) at a temperature of 37°C vented at 5% CO2. The culture medium was then replaced with fresh medium containing 400 ug/ml Zeocin™ and incubated at 37°C. After 3 days the transfected cells were selected in fresh medium containing 400 ug/ml Zeocin™ and 1 mg/ml G418 followed by isolating the population of cells by limiting dilution. Selection of stable clones was done by assessing for the highest SEAP activity for each cell line over a period of 6 weeks. To ensure that expression remained stable the cultures were cultured in the presence of G418 and Zeocin™ every 10th generation.
Total cellular protein was prepared from cells washed in 0.35 M sucrose and resuspended in 400 μl of lysis buffer (Ultrapure reagents from Amersham Biosciences, Piscataway, NJ): 2% (w/v) CHAPS, 9.5 M urea, 0.8% (w/v) pharmalyte pH 3-10, 1% (w/v) DTT (Amersham Biosciences, UK) and 1 protease inhibitor tablet per 10 ml (Roche Molecular, Mannheim, Germany) for 30 minutes before subsequent 4°C centrifugation at 17,000 g for 30 minutes. Sample was stored at -80°C for minimum of 24 hours before concentration determination using a modified Bradford assay . 50 μg/well underwent electrophoresis on a 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) mini gel (Pierce, Rockford, IL) after dilution in 1× Laemli buffer prior to denaturation at 95°C for 10 minutes. The gel was stained with Instantblue™ (Novexin Ltd, UK) for visual verification of loading before de-staining for electrotransfer. The protein was then transferred to nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK). Then, the membrane was incubated with the primary antibody, anti-myc 9E10 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), and then incubated with a HRP conjugated anti-mouse whole IgG from goat as the secondary antibody. Bands were developed by chemiluminescent detection (SuperSignal West Pico System, Pierce) according to the manufacturer's instructions. The membrane was stained with Ponceau S to re-verify loading and uniformity of the electrotransfer.
Static Cell Cycle Data
Flow cytometry was used to determine the percentage of cells in various phases of the cell cycle. Every 24 hours both CHO-K1 and cMycCHO were dissociated from the wells using Accutase™, centrifuged at 160 g for 5 minutes and were resuspended in 500 μl of Nuclear Isolation Media (NIM)-DAPI staining solution (NIM-DAPI, NPE Systems, Pembroke Pines, FL, USA) at a density of 1 × 106 cells/ml for 1 minute at room temperature. The suspension was then filtered through a 25 μm Filter Tip (NPE Systems). NIM-DAPI stained samples were run on a Beckman Coulter Quanta SC flow Cytometer fitted with a mercury arc lamp, a 357/22 nm exciter and a 465/30 nm emission filter. The G1 peak was fixed at a specific channel number and a minimum of 15,000 nuclear signals was collected. The distribution of cells in the G1, S, and G2/M phases was analyzed with the Multicycle software program (Phoenix Flow Systems, San Diego, CA).
Analysis of Apoptosis
CHO-K1 and cMycCHO cells removed at different times during the batch cultures and cells incubated in spent media were analysed for the percentage of viable, apoptotic and necrotic cells. Dual staining for both Annexin-V binding to phosphatidylserine (PS) surface proteins by Annexin-V- Fluorescein isothiocyanate (FITC) (BD Biosciences, UK) and cellular DNA using Propidium Iodide (PI) was performed as follows (Ishaque and Al-Rubeai, 1999): 1 × 106 cells/ml were washed in ice cold PBS (0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25°C) and centrifuged at 300 g for 5 minutes at 4°C for resuspension in 1× binding buffer ((10 mM HEPES solution) pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2). 1 μl of Annexin-V FITC was added to 100 μl of cell suspension and incubated for 10 minutes at 20-25°C before adding 400 ul of 1× binding buffer and 10 μl of PI (50 ug/ml) which was incubated for 1 minute before analysis. The sample was analysed with a Beckman flow cytometer equipped with a 488 nm diode laser. A 525 band pass detection filter was used to obtain FITC fluorescence and a 670 nm long pass filter is used to collect the maximal emission of PI fluorescence . Fluorescence compensation was carried out before taking final readings.
Cell Cycle Kinetics Labelling
A BrdU (5-Bromo-2'-deoxyuridine) pulse labelling and staining procedure for kinetic analysis of the cell cycle was initiated the 2nd day after initial seeding of the cultures in flasks. 20 μM of BrdU (BD Biosciences, UK) was added to the exponentially growing cells. After a labelling pulse of 1 hour at 37°C, cells were washed with fresh medium and then incubated in the corresponding medium for 1, 3, and 5 hours with samples taken from triplicate wells. After the indicated BrdU-free chase time, cells were harvested and washed twice in ice-cold PBS and centrifuged at 500 g for 5 minutes. The cells were then resuspended in 500 μl ice-cold PBS and 5 ml 70% ethanol was added drop-wise before leaving the suspension on ice for 15 minutes. The samples were stored in a -20°C for 1 week after fixation. The staining procedure to determine the DNA and BrdU content was a modified procedure from Istfan N.W et al. . Briefly, the fixed samples were centrifuged for 10 minutes at 500 g and then washed twice with cold PBS. To this 3 ml of 2N HCl was added and left at room temperature in the dark on a shaker for 30 minutes before washing with PBS and then adding 3 ml of 0.1 M Borax to neutralize the acid at room temperature for 2-4 minutes. 3 ml of 0.5% Triton X-100 was added and left for 10 minutes at room temperature on a shaker after a PBS wash. Afterwards, the pellet was resuspended in 0.1 ml of PBS and 20 μl of anti-BrdU FITC (BD Bioscience) was added directly before incubation for 30 minutes at room temperature in the dark. The sample was then washed with PBS and the pellet resuspended in 0.5-1 ml of PBS before adding 50 μl of RNase A solution per tube and incubated for 30 minutes at 37°C. From a 1 mg/ml stock, 25 μl of PI stain solution was added per tube and left on ice for 30 minutes before bivariate DNA-BrdU analysis was carried out on the flow cytometer.
Cell Cycle Kinetic Analysis
Dual stained samples were analysed with a Beckman flow cytometer equipped with a 488 nm diode laser, A 525 band pass detection filter was used to obtain green fluorescence from FITC-labeled anti-BrdU and a 670 nm long pass filter is used to collect the maximal emission of PI red fluorescence. The red fluorescence was calibrated by setting the G1 peak to a fixed channel number and green fluorescence was calibrated using an unlabeled control. The cell size was also taken simultaneously at 48 and 53 hours (exponential phase) with these samples to determine the relative cell size in each phase. Cell Lab Quanta SC software (Beckman Coulter, UK) and the computer program WinMDI V2.8 (The Scripps Research Institute) was used to analysis the cytometric histograms. Calculations was carried out using the equations previously described [16–18]. Briefly, the bivariate BrdU-DNA contour plot obtained with WinMDI V2.8 had the cells separated on the x-axis according to DNA content and the y-axis according to BrdU incorporation. Gating was established for G1, G2/M, labeled undivided (flu) cells, and labeled divided (fld) cells. First, the relative movement (RM) at each pulse time point of the cells during the S-phase to G1 and G2 was calculated using the following equation:
Where FL(t) is the mean red fluorescence at time (t) while FG1 and FG2+M are the mean red fluorescence of cells in each respective phase. This can be related to the following linear relationship:
Second, the function v  a quantity (= cTS) represented by the following equations is obtained:
Equation 3a is used when the times are less than the time for the G2+M phase (TG2+M) and equation 3b otherwise. Here the fraction of labeled undivided (flu), labeled undivided in G2+M phase (fluG2+M), and labeled divided (fld) are obtained from the bivariate BrdU-DNA contour plot.
The potential cell cycle time (Tpot) can be calculated using the value for v and TS in the following equation:
To determine the calculated time for the cell spent in G2 and M phase an analytical procedure described in White et al, 2000 was used. This relies on the calculation of the solution to a cubic equation where the cell production rate (c) is obtained through a relation between RM(t), v, and flu:
After determining the cell production rate the following equation is used to determine TG2+M:
After determining TG2+M and TS the time spent in G1 could be calculated by subtraction of both calculated times from Tpot.
Determination of Extracellular Glucose, Lactate, Ammonia and Amino Acid Concentrations
Glucose was measured with an Ascencia contour (Bayer Diagnostics, Ireland) glucose meter with the provided pre-calibrated Microfill test strips (Bayer Diagnostics, Ireland). Lactate was measured using the Accutrend Lactate Meter together with BM-Lactate Strips (Roche Diagnostics GmbH, Germany). Briefly, The BM-Lactate calibration strip was used to calibrate the instrument to the accompanied BM-Lactate strips. Lactate was determined by reflectance photometry at a wave length of 657 nm via a colorimetric-oxidase mediator reaction. L-Glutamine and ammonia were measured with a Megazyme kit K-ASNAM (Megazyme, Ireland) using supernatant from each day of the culture. The amino acids samples were taken at the end of exponential phase by centrifuging 1.5 ml of culture for 5 minutes at 180 g and storing the supernatant at -20°C for 1 week before analyses. Amino acids were analysed by Alphalyse (Denmark) and performed on a Biochrom™ amino acid analyzer.
The hSEAP-hFc reporter constructs containing the SEAP gene were analyzed using the QUANTI™-Blue™ SEAP Reporter System (Invivogen), which detects SEAP enzyme activity. Instead of a single reading after a determined period of time, multiple reads were taken at 1 min intervals for 20 minutes, after 30 minutes of incubation at 37°C, and used to express SEAP activity as absorbance units per second (abs/sec). The SEAP activity was normalized for integral cell area to generate relative specific productivity.