Cells were routinely grown and maintained in 250 mL (50 mL working volume) shake flasks (Corning Inc, Suzhou, China) a in serum-free medium (SFM) at 28 °C and 110 rpm. The recombinant baculoviruses were amplified by infecting Sf9 cells and harvesting the cells for 4 days post-infection, as previously described by Nawagitgul et al. (2000 (link)). The virus stock solution titres were determined by a plaque assay, as previously described by O’Reilly et al. (LaBarre and Lowy 2001 (link)). The trypan blue exclusion test is used to determine the number of viable cells present in a cell suspension and the cell density and viability was visually examined in haemocytometer (Strober 2001 ). Cell concentration was determined by haemocytometer cell counts (Anxin Optical, Shanghai, China) and cell viability was evaluated by trypan blue dye exclusion (Sigma Aldrich, CA, USA) in 0.4% PBS.
Shake flasks
Manufactured by Corning
Sourced in United States
Shake flasks are laboratory equipment used for culturing and growing cells, microorganisms, or other biological samples. They provide a controlled environment for agitated cultivation, facilitating aeration and mixing of the contents.
Automatically generated - may contain errors
Lab products found in correlation
Inova 44r, by Eppendorf (3 mentions)
Orbital shaker incubator, by Infors (2 mentions)
Cedex hires analyzer, by Roche (2 mentions)
High five insect cells, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Trypan blue dye exclusion, by Merck Group (1 mentions)
24 well or 96 well plates, by Corning (1 mentions)
Heracell 240 co2 incubator, by Thermo Fisher Scientific (1 mentions)
Shake flasks, by Thermo Fisher Scientific (1 mentions)
T175 flask, by Corning (1 mentions)
Insect xpress medium, by Sartorius (1 mentions)
Acticho sm medium, by GE Healthcare (1 mentions)
Sfm4hek293, by Cytiva (1 mentions)
Ex cell 293, by Merck Group (1 mentions)
Hek293a cells, by American Type Culture Collection (1 mentions)
Kolliphor, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)
Freestyle 293 expression medium, by Thermo Fisher Scientific (1 mentions)
Cellfectin 2, by Thermo Fisher Scientific (1 mentions)
24 protocols using shake flasks
1
Baculovirus Infection and Cell Viability
2
Stable Cell Line Generation for Quality Evaluation
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Using electroporation, individual vector constructs were transfected into the CL1 cell line. The cells were cultured in a selective medium containing puromycin and methotrexate (MTX) 3 days post-transfection. Cells were cultured in either 24-well or 96-well plates (Corning, NY), 125 mL shake flasks (Corning, NY), or 50 mL TubeSpin® tubes (TPP, Trasadingen, Switzerland) at 37°C, 5% CO2, and 85% humidity. Cells were passaged twice a week at 4 × 106–5 × 106 cells/mL concentrations. Once pools achieved >90% viability and maintained consistent doubling times, they were evaluated in fed-batch cultures to evaluate critical quality attributes.
3
Modular Ambr®250 CHO Cell Cultivation
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The first steps of the process, namely the vial thaw and inoculum expansion were performed as described by Boehl et al. [18 (link)]. The production process step was performed in the modular Ambr®250 system (Sartorius, Germany), using proprietary and chemically defined production medium (PM) and two additional feed media (feed medium A; feed medium B) for macro nutrients (e.g., glucose) and micro nutrients (e.g., amino acids) respectively (Sartorius Stedim Cellca GmbH, Germany) [23 (link)]. One Ambr®250 vessel was used to cultivate the cells for the DoE, while the second vessel was used as an internal reference standard.
The cultivation was conducted over 9 days with daily feeds (1% feed medium A, 0.1% feed medium B) from day 3 and additional glucose feeds (400 g/L stock solution) to a culture glucose concentration of 5 g/L from day 5. At peak cell density from day 7 to 9 the cells were cultivated in 250 ml shake flasks (Corning, USA) in a Heracell 240 CO2 incubator at 36.8°C and 7.5% CO2 (Thermo Scientific, USA) using a MaxQ CO2 Plus shaker platform (Thermo Scientific, USA).
The cultivation was conducted over 9 days with daily feeds (1% feed medium A, 0.1% feed medium B) from day 3 and additional glucose feeds (400 g/L stock solution) to a culture glucose concentration of 5 g/L from day 5. At peak cell density from day 7 to 9 the cells were cultivated in 250 ml shake flasks (Corning, USA) in a Heracell 240 CO2 incubator at 36.8°C and 7.5% CO2 (Thermo Scientific, USA) using a MaxQ CO2 Plus shaker platform (Thermo Scientific, USA).
4
Chemically Defined Fed-Batch Antibody Production
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Pools were cultured in one‐liter shake‐flasks under a 12‐day fed‐batch production process using Pfizer's proprietary chemically defined medium and feeds and maintained at 36.5°C, 5% (v/v) CO2 in air using a humidified incubator. Counts and feeds were completed daily. The HC fusion (HCF)‐LC‐HC pools were passaged in Corning shake flasks (431147) while in the experiment assessing different 2xLC vector configurations, pools were passaged in ThermoFisher shake flasks (4113–1000). Daily viabilities and viable cell concentrations were determined using a Vi‐CELL™ automated cell viability analyzer. Antibody concentration in the medium was determined by Protein A HPLC.
5
Recombinant Monoclonal Antibody Production
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Cell lines were generated by transfection of an Amgen proprietary clonal CHO host cell lines with plasmid DNA encoding for a heavy chain and light chain from a human monoclonal antibody. Following transfection, stably expressing pool populations were created through the repeated passaging in selective growth media until cells reach percent viabilities greater 90% and maintain consistent doubling times. Further selection using increasing levels methotrexate is also applied to improve productivity. Cells were cultured in either 96‐well, 24‐well, or 24‐deepwell microtiter plates (Corning, Corning, NY), 125 mL shake flasks (Corning, Corning, NY), T‐175 flasks (Corning, Corning, NY), or 50 mL TubeSpin (TPP, Trasadingen, Switzerland) in Amgen proprietary media at 37°C, 5% CO2, and 85% humidity. Cells were maintained by passaging multiple times a week at a targeted seed density.
6
Insect High Five Cell Culture Protocol
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Insect High Five cells (B85502, ThermoFisher Scientific) were routinely sub-cultured in 500 ml shake flasks (10% working volume) (Corning) to 0.3 × 106 cells/ml inoculum, every 3–4 days, when cell concentration reached 2–3×106 cells/ml, using Insect X-press medium (BEBP12-730Q, Sartorius). An Innova 44R incubator (New Brunswick) at 27°C and 100 rpm (orbital motion diameter of 2.54 cm) was used.
7
CHO-K1 Cell Line Stability Monitoring
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The clone stability of the CHO-K1/CN54gp140 and CHO-K1/PG9 recombinant cell lines was monitored in shake flasks (Corning) over a period of 20 passages (71–73 days) after thawing in ActiCHO SM medium (GE Healthcare Life Sciences) without selection pressure. Cells were passaged into fresh medium every 3–4 days to 2×105 cells/ml. The cell concentration, viability as well as product concentration was monitored before each splitting.
8
HEK 293SF Cell Culture and Adenoviral Transduction
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The HEK 293SF cell line was derived from HEK 293S cells, and adapted to grow in suspension and serum-free media through multiple steps of adaptation and clone selection [22 (link)]. Cells were grown in suspension in shake flasks (Corning) with an agitation of 120 rpm using orbital shakers (Infors HT, Anjou, QC, Canada), 5% CO2 at 37 °C. Cells were passaged regularly when reaching densities ~1 × 106 cells/mL.
The following culture media were utilized in this study: Pro293s-CDM (Lonza, Walkersville, MD, USA), SFM4Transfx-293 and SFM4HEK293 (both from Cytiva, Logan, UT, USA), Ex-Cell 293 (Sigma-Aldrich, St. Louis, MO, USA) and an in-house-developed serum-free medium (HEK SFM).
The adenovirus used for infection was an Ad5-containing green fluorescence protein (Ad5-GFP) under the control of the CMV promoter. Viral stock titer was 1.7 × 1010 IVP/mL and aliquots were stored at −80 °C for subsequent analysis.
The following culture media were utilized in this study: Pro293s-CDM (Lonza, Walkersville, MD, USA), SFM4Transfx-293 and SFM4HEK293 (both from Cytiva, Logan, UT, USA), Ex-Cell 293 (Sigma-Aldrich, St. Louis, MO, USA) and an in-house-developed serum-free medium (HEK SFM).
The adenovirus used for infection was an Ad5-containing green fluorescence protein (Ad5-GFP) under the control of the CMV promoter. Viral stock titer was 1.7 × 1010 IVP/mL and aliquots were stored at −80 °C for subsequent analysis.
9
CHO DG44 Cell Line Production
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Two different recombinant CHO DG44 production clones (Clones A and B) expressing an identical monoclonal antibody (IgG4 isotype) were routinely cultivated in shake flasks (Corning) unless otherwise stated. Both cell lines were generated in the scope of a cell line development program and thus derived from the same transfection pool of suspension adapted Boehringer Ingelheim (BI) proprietary DHFR deficient (DHFR–/–) CHO DG44 host cells (for more details on the cell line development process please refer to Section 2.3 ). Cells were cultivated at 37°C, 5% CO2 and with agitation at 125 rpm (50 mm orbit) in an orbital shaker incubator (Infors). BI proprietary serum‐free, chemically defined and animal component free cell culture media were used for cultivation. Seeding cell density of stock cultures was set to 0.3 × 106 viable cells per milliliter and cells were passaged every 3–4 days. Cell concentration and viability during routine stock culture cultivation was assessed using a Cedex HiRes Analyzer™ (Roche Diagnostics) by means of trypan blue exclusion.
10
Cultivation of Diverse 293-Derived Cell Lines
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The 293SF-3F627 (link) and the 293SF-CymR cells were cultured in SFM4-Transfx-293 medium (Hyclone, South Logan, UT) supplemented with 6 mM L-glutamine (Hyclone). 293SF-CymR/λR-GyrB cells were generated using low-calcium-serum-free medium (LC-SFM) (Gibco, Life Technology Corporation, Grand Island, NY), supplemented with 6 mM L-glutamine and 10 mg/mL rTransferin (Biogems, Westlake Village, CA) and expanded in suspension culture in SFM4-Transfx-293. The packaging cells (293SF-PacLVIIIb) and producer clones were generated using a mixture of 50% LC-SFM supplemented with 6 mM L-glutamine and 10 mg/mL rTransferin and 50% HyCell-Transfx-H (Hyclone) supplemented with 4 mM L-glutamine and 0.1% kolliphor (Sigma-Aldrich, St. Louis, MO). They were maintained in suspension culture in 100% HyCell-Tranfx-H. For suspension culture, the cells were grown in shake flasks (Corning, Oneonta, NY) at 120 rpm with an orbital diameter of 25 mm. The HEK293A cells (ATCC, Manassas, VA) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone) supplemented with 5% fetal bovine serum (FBS; Hyclone) and 2 mM L-glutamine using tissue culture-treated dishes. All the cell lines were maintained at 37°C in a 5% CO2 humidified atmosphere.
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