Generating Crude Adenoviral Particles in theCorning® HYPERFlask® Cell Culture VesselApplication Note

Katherine E. Strathearn, Ph.D. andMark E. Rothenberg, Ph.D.Corning Life SciencesKennebunk, Maine

IntroductionAdenoviruses are non-enveloped DNA viruses that havetremendous promise in research and industrial applications.Some of these advantages include: (i) the ability to infect awide range of actively dividing and non-dividing mammaliancells (e.g., primary neurons); (ii) minimum risk of integrationinto the host genome (the virus is epichromosomal); and (iii)the capacity to be amplified to very high titers in tissue cul-ture (1). To allow researchers and vaccine manufacturers theopportunity to produce even higher yields in the same spatialfootprint as a T-175 flask, Corning offers the HYPERFlaskvessel. The HYPERFlask® Cell Culture Vessel featuresCorning’s HYPER (High Yield PERformance) technologywhich utilizes a gas permeable film as the attachment surfaceand eliminates the headspace within a vessel. This allows foran increase in the number of layers and corresponding cellgrowth surface area compared to traditional rigid singlelayer culture vessels. The HYPERFlask Vessel cell growthsurface area is 1,720 cm2 across 10 layers and has the samespatial footprint of a T-175 vessel.

Crude adenoviral particles are produced when cells (e.g.,HEK-293AD) are transfected with DNA encoding the vari-ous genes needed for adenovirus generation. After the crudeadenoviral stocks have been obtained the virus may thenbe amplified by transducing particular cell lines (e.g. HEK-293AD or A549). The focus of this study was to determinethe efficacy of generating crude virus using the uniqueCorning HYPER technology. Standard methodologies uti-lize traditional T-flask vessels for virus generation. Utilizingthe HYPERFlask vessel, researchers can generate highertiters in a smaller spatial footprint saving both time andspace. The results depicted here demonstrate that the exper-imental approach to generate adenovirus in the HYPERFlaskvessel led to higher titers and yields compared to a standardT-175 flask.

Methods and MaterialsCell CultureHEK-293AD cells (Cell BioLabs, Cat. No. AD-100) weremaintained in DMEM without sodium pyruvate (Corningcellgro®, Cat. No. 10-017-CM), 10 % FBS (PAA, Cat. No.A15-201), and 1X MEM Nonessential Amino Acids (Corningcellgro, Cat. No. 25-025-Cl). HEPES (10 mM) (Corningcellgro, Cat. No. 25-060-CI) was added to this mediumduring adenovirus production.

DNA PreparationGC10 competent cells (Sigma-Aldrich,® Cat. No. G2544)were heat shocked with the DNA obtained from the RAPAd®

CMV Adenoviral Bicistronic Expression System (GFP) (CellBioLabs, Cat. No. VPK-354) and cultured in LB-Broth(Sigma-Aldrich, Cat. No. L3022) in a 1L Erlenmeyer flask(Corning, Cat. No. 431403) at 37°C for 16 h at 250 rpm.Plasmid DNA was purified using the AxyPrep™ PlasmidMaxiprep kit (Axygen, Cat. No. AP-MX-P-25) and quan-tified with the EnVision® multimode plate reader (PerkinElmer). Purified plasmid DNA was digested with Pac-I(New England BioLabs, Cat. No. R0547L) at 37°C for atleast 4 hours then heat inactivated at 65°C for 30 minutes.

Preparation of the DNA: Lipofectamine ComplexOn the day of transfection, a master mix of DNA: Lipofec-tamine solution was prepared based on a total growth areaof 1945 cm2 (1720 cm2 (HYPERFlask) + 175 cm2 (comparisonT-175) + 50 cm2 [extra]). To prepare the mix, Opti-MEM(Life Technologies, Cat. No. 31985070) was added to two150 mL storage bottles (Corning, Cat. No. 431175) at aratio of 0.032 mL/cm2 (~63 mL) per bottle. Lipofectamine2000 (Life Technologies, Cat. No. 11668019) was added toone storage bottle at a ratio of 3 µL of reagent per 1 µg ofDNA (~1.5 mL Lipofectamine 2000) and incubated at roomtemperature for 5 minutes. To the second storage bottle con-taining Opti-MEM the following heat inactivated Pac-Idigested DNA components (see above for preparation)were added at a combined ratio of 0.245 µg/cm2:

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� pacAd5 CMV-Green Fluorescent Protein (GFP)Control Vector (335 µg)

� pacAd5 9.2-100 Vector (145 µg)

Following the 5 minute incubation, the Opti-MEM con-taining DNA was added to the Opti-MEM containingLipofectamine, briefly swirled, incubated for 20 minutes atroom temperature, and transferred to a 1L storage bottle(Corning, Cat. No. 430518) containing 508 mL of mediafor adenovirus production. Each experiment was performedthree individual times.

Transfection of HEK-293AD CellsCells were seeded onto a T-175 Corning® CellBIND®

Surface flask (Corning, Cat. No. 431328) or CorningHYPERFlask® vessel (Corning, Cat. No. 10034) at 40,000cells/cm2 (0.326 mL/cm2) and incubated overnight at 5%CO2, 98% relative humidity, 37°C. The following day themedia was removed and fresh media (0.326 mL/cm2) con-taining the DNA:Lipofectamine complex (see above) wasadded. 16 hours post transfection the media was changedand the cells were incubated at 37°C for 11 to 14 days.Maintaining proper pH conditions (~pH 7.2) is critical foroptimal virus production (2) and therefore half mediachanges were performed as indicated by the color of themedium, an indicator of changes in pH (~ every 2 to 3days). Additionally, to help control the pH, HEPES (10mM) was added to the medium. GFP expression was moni-tored throughout the course of the experiment using theAMG EVOS® Fl microscope.

Adenovirus HarvestWhen approximately half of the cells appeared attachedto the vessels and half were floating in the medium, both

medium and cells were collected (Fig. 1). To collect the cellsstill attached to the vessel, PBS (without Ca2+ and Mg2+)(Corning cellgro,® Cat. No. 21-040-CM) was added to eachvessel (0.029 mL/cm2) and incubated at 37°C for 3 to 5minutes. One or more PBS washes were performed depend-ing on the overall density of the cells that remained attachedto the surface and added to the media containing cells. Tominimize volume, and concentrate the crude adenoviruspreparation, the cells were centrifuged at 500 x g for 10 min-utes at room temperature and the cell pellet was resuspend-ed in 10 mM Tris, pH 8.0, 100 mM NaCl (0.023 mL/cm2).The cell suspension was then subjected to three freeze/thawcycles (-80°C /37°C), and centrifuged at 3000 x g for 10minutes at 4°C to pellet the cell debris. The supernatantcontaining the adenoviruses released from the cells werecollected. The adenovirus encoding GFP was then aliquot-ed and stored at -80°C.

Adenovirus TiterThe QuickTiter™ Adenovirus Titer Elisa kit was purchasedfrom Cell BioLabs (Cat. No. VPK-110). 293AD cells wereseeded on a Poly-D-Lysine coated 96 well plate (Corning,Cat. No. 3841) at 156,250 cells/cm2 (0.3125 mL/cm2) andincubated for 2 hours to allow for cell attachment. Adeno-virus isolated from each vessel was thawed, added to multi-ple wells at various dilutions (10-3 to 10-5), and incubated at37°C for 48 hours. The Elisa assay was performed accord-ing to manufacturer’s instructions and the signal in thewells was measured utilizing a Perkin-Elmer EnVision®

Multilabel Reader.

Transduction of Vero and MDBK CellsTo verify that the crude virus was functional Vero (ATCC,Cat. No. CCL-81) and MDBK (ATCC, Cat. No. CCL-22)

HEK- AD

pacAd5

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Figure 1. Schematicof crude adenovirusproduction.

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cells were transduced. Cells were seeded onto a 6 well plate(Corning, Cat. No. 3506) at 10,000 cells/cm2 and incubatedovernight at 5% CO2, 98% relative humidity, 37°C. Thefollowing day the adenovirus encoding GFP were added tothe cells at a multiplicity of infection (MOI) of 200 (basedon previous results). The amount of each virus (mL) addedto each well was calculated using the following formula:

(Cells/cm2 [cm2 of well]) × (MOI 200 [IFU/cells])IFU/mL

The cells were harvested 72 hours later and the GFPexpression was analyzed by flow cytometry.

Flow CytometryTo assess GFP expression Vero and MDBK cells transducedwith adenovirus encoding GFP were harvested, centrifugedto remove trypsin/media, and resuspended in 1 mL of PBS(Corning® cellgro®, Cat. No. 21-040-CM). Cell suspen-sions were analyzed using the MACSQuant® Analyzerinstrument (Miltenyi Biotec).

ResultsGFP Expression Throughout ProductionTo assess adenoviral production on a T-175 flask comparedto a HYPERFlask® vessel, 293AD cells were transfected

with DNA obtained from the RAPAd CMV AdenoviralBicistronic Expression System (GFP) and GFP expressionwas monitored throughout the course of the experiment.After 72 hours post transfection, similar GFP expression wasobserved in both vessels (Fig. 2). However, ~10 days posttransfection, differences in GFP expression became visiblebetween the two vessels. 293AD cells cultured/transfectedin the T-175 flask had disproportionate GFP expressionthroughout the vessel. Some areas had little observed GFPexpression (Fig. 3, Panel A) whereas other areas had higherexpression indicating either the beginning (Fig. 3, Panel B),or mid-late stages of plaque formation (Fig. 3, Panel C). Inthe HYPERFlask vessel, the GFP distribution was more uni-form. In areas where plaque formation had not yet begun,GFP expression appeared higher than the expression leveldetected in the T-175 flask (Panel D versus Panel A [Fig. 3]).Additionally, there appeared to be a higher amount of plaqueformations occurring in the beginning (Panel E) and late(Panel F) stages compared to the T-175 flask. Lastly, afterapproximately 11 to 14 days post transfection, the 293AD cellswere harvested from the vessels. The cells cultured on theT-175 flask were ready to be collected on ~day 13, whereascells cultured on the Corning® HYPERFlask® vessel were,on average, collected 1 to 2 days earlier compared to the T-175 flask (Fig. 4).

Figure 2. Similar GFP expression between vessels was observed 72 hours post transfection. Representative images from the same experiment demon-strating similar GFP expression in both vessels. Scale bar represents 1000 µM. These trends were observed with all experiments. Images were capturedusing the AMG EVOS® Fl microscope.

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Figure 3. GFP expression in 293AD cells ~10 days post transfection. Representative images from the same experimentdemonstrating various GFP expression in each vessel type. (A-C) T-175 vessel (A) Areas where GFP expression was low(B) Beginning stages of plaque formation (C) Mid-late plaque formation (D-F) HYPERFlask® vessel (D) High GFP expres-sion was detected throughout the vessel (E) Beginning stages of plaque formation (F) Late plaque formation.Whitearrow points to plaque formations. Scale bar represents 1000 µM. Images were captured using the AMG EVOS® Flmicroscope. These trends were observed in all experiments.

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Adenoviral ProductionOnce collected, the adenovirus’ encoding GFP from eachvessel was then titered using the QuickTiter Elisa kit todetermine infectious forming units (IFU)/mL. Adenovirusobtained from the HYPERFlask vessel yielded higherIFU/mL (Fig. 5A), IFU/cm2 (Fig. 5B), and greater than 20times more IFU (Fig. 5C) compared to adenovirus obtainedfrom the T-175 flask. The average titers produced on theHYPERFlask vessel were 1.8 x 109 IFU/mL and on the T-175

flask were 6.4 x 108 IFU/mL. These results demonstratethat higher infectious adenoviral particles/cm2 occurred inthe HYPERFlask vessel compared to a traditional T-175flask.

GFP Expression in Vero and MDBK CellsTo verify that the virus obtained from the HYPERFlask®

vessel was as functional as virus obtained from the T-175flask, Vero and MDBK cells were transduced with adeno-virus encoding GFP. Each cell type was transduced with

Figure 4. Cell morphology and GFP expression on day of harvest. Representative images from the same experiment demonstrating morphology/GFPexpression on the day of harvest. On average, cells were harvested from the T-175 flask on day 13, whereas cells were harvested on day 12 from theHYPERFlask vessel. Scale bar represents 1000 µM. Images were captured using the AMG EVOS® Fl microscope.

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Figure 5. The Corning HYPERFlask vessel leads to higher viral production per cm2 compared to a T-175 flask. (A) Titers obtained from the QuickTiter ElisaAdeno kit. (B)When normalized on a per cm2 basis the HYPERFlask vessel yields greater than 2.5 times more infectious adenoviral particles. (C) TheHYPERFlask vessel generates a significantly higher amount of total infectious adenoviral particles. Paired t-test, * p<0.05, ** p<0.01, N=3.

2.5 x 109

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virus obtained from either the HYPERFlask vessel or T-175flask at an MOI of 200. After 72 hours, the cells were col-lected and analyzed via flow cytometry. The average GFPfluorescence in each cell line with each adenovirus wasgreater than 99.0% (Fig. 6, three independent experiments).Cells were transduced at a MOI 200 to ensure maximumexpression. Previous results also depicted equal GFPexpression amongst the vessels when transduced at lowerMOIs (10, 50, and 100) for shorter time periods (24 and48 hours). GFP expression from each experiment variedbetween 40% and 95% depending on a) MOI and b) time(data not shown). Taken together, these results indicate thatthe HYPERFlask vessel produces more functional adenovi-ral particles per cm2 compared to the traditional T-175flask.

SummaryIn summary, this study demonstrates the utility of theHYPER Technology in adenovirus production as a goodalternative to traditional cell culture flasks for obtaininghigher virus yields. These higher yields could be explainedby the improved gas exchange in the HYPERFlask vesselcompared to standard cell culture flasks. Even though thetransfection efficiency appears similar within the first 72

hours (Fig. 2) the cells are then continuing to propagateover the next 1 to 2 weeks. Over the course of this timea large oxygen gradient may be forming throughout themedia leading to a deprivation of oxygen on standardT-flasks. Cells cultured with HYPER technology are con-tinuously exposed to the same amount of oxygen eliminatingthe oxygen gradient. As a result, the health of the cells overthe course of this study (11 to 14 days) may be significantlyimproved leading to higher viral yields.

� Adenoviral particles can be produced in the CorningHYPERFlask vessel at higher titers than traditional tissueculture vessels, resulting in greater virus production ina similar spatial footprint.

� Furthermore, the HYPER technology platform ofproducts includes larger vessels with higher surface areasproviding a researcher with the ability to further amplifyadenoviral particles (refer to CLS-AN-214).

References1. Vemula, S.V. and Mittal, S. K. Expert Opin Biol Ther. 2010.

10:1469-1487.2. Silva, A.C., Peixoto, C., Lucas, T., Kuppers, C., Cruz, P.E.,

Alves, P.M., and Kochanek, S. Current Gene Therapy. 2010.10:437-455.

Figure 6. Vero and MDBK cells transduced with adenovirus exhibit similar GFP expression levels. Representative flow cytometry data. Expression of GFP(green) compared to a negative control of non-transduced cells (black). After three independent experiments, the GFP expression in both the Vero orMDBK cells was greater than 99%, regardless in which vessel the virus was generated.

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