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HEK 293 Cell Growth and Virus Production in EX-CELL 293 Serum

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HEK 293 Cell Growth and Virus Production in EX-CELL 293 Serum SAFC® 3050 Spruce Street Saint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900 Fax: 1-888-289-8449 Global Email: [email protected] Research Report HEK 293 Cell Growth and Virus Production in EX-CELL® 293 Serum-Free Medium Manisha Sahni, Shelley Wilcox, Pamela Mettner, Cynthia Reiss, Sarah L. Gilliland, Douglas R. Purtle, Karen J. Etchberger Abstract Materials The Human Embryonic Kidney (HEK) 293 cell line is used in Cells the development of viral vaccines, anticancer agents and the • HEK 293 cell line, American Type Culture Collection, production of recombinant adenoviral vectors. EX-CELL 293 Catalog No. CRL-1573 is a serum-free, animal-protein free medium designed and Virus optimized to support high-density suspension culture of HEK • Wild-type Adenovirus Type 5, American Type Culture 293 cells. The HEK 293 cell line easily adapted to EX-CELL Collection, Catalog No. VR-5 293 without forming cellular aggregates and achieved average cell densities of 2.5 x 106 cells/mL in shaker fl asks, with an Serum-Free Media approximate doubling time of 33 hours. HEK 293 cells in • EX-CELL 293, SAFC, Catalog No. 14570C. L-glutamine was EX-CELL 293 were also infected with wild-type Adenovirus type aseptically added to EX-CELL 293 to a fi nal concentration of 10 5 (Ad5) and produced 1-3 x 10 TCID50/mL. We conclude that 6 mM. No antibiotics or other supplements were added to EX-CELL 293 supports rapid HEK 293 cell growth in shaker the medium. fl asks and produces high density, highly viable cultures that are Other Media and Supplements capable of adenovirus production. • 200 mM L-glutamine, SAFC, Catalog No. 59202C Introduction • Dulbecco’s Modifi ed Eagle’s Medium (DMEM)/High The HEK 293 cell line is an attractive instrument in the fi eld of Modifi ed, SAFC, Catalog No. 51444C gene therapy. The cell line is capable of producing glycosylated human proteins and acts as a host for the production of • Fetal Bovine Serum (FBS) - Gamma Irradiated, SAFC, recombinant adenoviral vectors, which have shown promise Catalog No. 12107C in treating diseases such as cystic fi brosis, hypertension and • 0.25% Trypsin (1X) 0.1% EDTA Solution, SAFC, arthritis. Traditionally, HEK 293 cells are grown as attachment Catalog No. 59229C (available for custom development only) cultures in a serum-supplemented basal medium such as Dulbecco’s Modifi ed Eagle’s Medium (DMEM). EX-CELL Methods 293 is a serum-free, animal-protein free medium specifi cally Adaptation and Growth Studies formulated to support large-scale, high-density suspension HEK 293 cells were adapted to EX-CELL 293 by direct culture and virus production in the HEK 293 cell line. The adaptation. As such, adherent cultures of HEK 293 cells medium contains very low levels of recombinant protein in DMEM/High Modifi ed supplemented with 5% FBS were (approximately 1.1 mg/L), facilitating downstream processing trypsinized and counted. Cells were subsequently seeded of expressed products and eliminating regulatory concerns (6 x 105 cells/mL) directly into pre-warmed EX-CELL 293 associated with serum and animal proteins. Additionally, media (35 mL volume per 125 mL shaker fl asks). Flasks the liquid formulation of EX-CELL 293 is formulated without were incubated with loosened caps at 37 C in a 10% CO2 L-glutamine, which avoids problems associated with atmosphere, with an orbital shaking speed of 90 - 100 rpm. L-glutamine degradation and improves product shelf-life. Cultures were passaged twice per week (5 x 106 cells/mL) and Our experiments show EX-CELL 293 supports high-density, were considered fully adapted after 6 passages, as indicated serum-free HEK 293 cell growth and supports the production of by sustained viabilities above 90%, and at least one cell division high yields of adenovirus. per passage. Cell densities and viabilities were determined by trypan blue exclusion. safcglobal.com SAFC® 3050 Spruce Street Saint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900 Fax: 1-888-289-8449 Global Email: [email protected] Growth of HEK 293 cells was monitored for more than Figure 1. HEK 293 cells in EX-CELL 293 15 passages in EX-CELL® 293. Cultures were maintained in shaker fl asks using the same culture conditions as above. Additionally, growth characteristics in unfed cultures were examined in shaker fl ask cultures by observing cell growth over a period of time (10 days). Ad5 Viral Infection and TCID50 Titration Three independent infections with wild-type Ad5 were performed in EX-CELL 293. Cells were passed on day 4 at a seeding density of 5 x 105 cells/mL, and infected on day 2 at approximately 1 x 106 cells/mL with wild-type Ad5 at a multiplicity of infection (MOI) of 5. Samples were collected at 24, 48, 72 and 96 hours and were subjected to 3 rounds of EX-CELL 293 retards HEK 293 cellular aggregation, freezing/thawing (-70 C to 37 C). Each lysate was passed allowing for easier culture, increased culture viability and through a 0.22 μm sterile syringe fi lter and stored frozen (-70 C) greater product yield. until titered. Figure 2. Typical Growth of HEK 293 Cells in EX-CELL 293 in Titrations were performed in 96-well microtiter plates as follows: Shaker Flasks HEK 293 cells were seeded in DMEM with 6 mM L-glutamine and 5% FBS at 1 x 104 cells/100 μL/well. Duplicate serial Viable Cells/mL -1 -11 4.50E+06 50.00 dilutions (10 to 10 ) of each lysate were made in DMEM with Doubling Time 4.00E+06 45.00 2% FBS and 2 mM L-glutamine, and 100 μL was added to each 40.00 3.50E+06 well. Plates were incubated at 37 C, 10% CO2 for 10 days, then 35.00 3.00E+06 observed on an inverted microscope for cytopathic effect (CPE). 30.00 2.50E+06 The titer (T) was determined as follows: 25.00 2.00E+06 20.00 1+d(S-0.5) 1.50E+06 15.00 T = 10 Cells/mL Viable 1.00E+06 10.00 Doubling Time (Hours) Where d = Log10 of the dilution and S = Sum of the ratios. 5.00E+06 5.00 0.00 Results 0.00E+06 Passage Number HEK 293 cells were adapted to EX-CELL 293 by direct adaptation, i.e. a complete medium exchange from HEK 293 cells were seeded at 4 x 105 cells/mL in 125 mL serum-containing DMEM to 100% serum-free medium. The cells shaker fl asks containing 35 mL of EX-CELL 293. Flasks adapted quickly without experiencing a signifi cant lag phase were incubated in a 10% CO2 atmosphere at 37 C, with a shaker speed of 90 - 100 rpm and were subcultured every and without forming cellular aggregates or clumps (see Figure 1). 3 - 4 days. Figure 2 depicts the growth of HEK 293 cells cultured in Figure 3. Daily Growth of HEK 293 cells in EX-CELL 293 EX-CELL 293 in shaker fl asks over 16 passages. The average 6 cell density reached per passage was 2.5 x 10 cells/mL (range 7.00E+06 Viable Cells/mL 120.00 1.4-4.1 x 106 cells/mL) and the average cell doubling time was Percent Viability 6.00E+06 33 hours (range 24 - 45 hours). Culture viability was typically 100.00 5.00E+06 above 98% and was greater than 95% for all passages. 80.00 4.00E+06 Figure 3 illustrates a typical growth pattern of HEK 293 cells in 60.00 3.00E+06 unfed shaker cultures over a period of 10 days. EX-CELL 293 40.00 Viable Cells/mL Viable 2.00E+06 Percent Viability supported exponential cell growth for 8 days with viabilities above 90%. 1.00E+06 20.00 0.00E+06 0.00 Preliminary studies determined the optimal time of infection and MOI (i.e., 48 hours after seeding at a MOI of 5) for Days in Culture HEK 293 cells in EX-CELL 293. Using these parameters, HEK 293 cells were seeded at 4 x 105 cells/mL in HEK 293 cells grown in EX-CELL 293 supported the production 125 mL shaker fl asks containing 35 mL of EX-CELL 293. 10 Flasks were incubated in a 10% CO2 atmosphere at 37 C, of wild-type Ad5 with yields in the range of -3 x 10 TCID50/mL with a shaker speed of 90 - 100 rpm, for 10 days without refeeding. safcglobal.com SAFC® 3050 Spruce Street Saint Louis, MO 63103 USA Phone: 1-800-244-1173 • 1-314-534-4900 Fax: 1-888-289-8449 Global Email: [email protected] Figure 4. Titration (TCID50/mL) of Ad5 Produced by HEK Conclusions 293 Cells in EX-CELL® 293 • HEK 293 cells easily adapted to suspension culture in EX-CELL 293 with very little cellular aggregation. • EX-CELL 293 produced high-density, highly viable HEK 293 1.0E+11 cultures with up to 5 x 106 viable cells/mL. 1.0E+10 • EX-CELL 293 supports highly viable HEK 293 cultures with 1.0E+09 most cultures above 98% viability. • HEK 293 cells grown in EX-CELL 293 produce high yields of 1.0E+08 10 adenovirus, up to 3 x 10 TCID50/mL. TCID50/mL 1.0E+07 • EX-CELL 293 is serum-free, animal-protein free and Hours Post-Infection contains very low levels of recombinant protein, facilitating downstream purifi cation of products. HEK 293 cells were infected with wild-type Ad5 48 hours • EX-CELL 293 is formulated without L-glutamine, thereby post-seeding at a MOI of 5.
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