Large-scale production of lentiviral vectors using multilayer cell factories
Robinson Research Institute, Adelaide SA 5000, Australia
Adelaide Medical School, University of Adelaide, Adelaide SA 5005, Australia
- HEK 293T cells (American Type Culture Collection, cat. # CRL-3216)
- Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, cat. # 11965-084)
- Fetal calf serum (FCS) (Australian Origin) (Gibco, cat. # 10099-141)
- Penicillin-streptomycin (penicillin 10000 IU/ml, streptomycin 10000 µg/ml) (Gibco, cat. # 15140-122)
- 75 cm2 cell culture flasks, red filter screw cap (T75) (Greiner Bio-one, cat. # 658175)
- Corning tissue-culture treated culture dishes 150 mm × 25 mm (Sigma-Aldrich, cat. # CLS430599-60)
- Phosphate buffered saline (PBS) (Sigma-Aldrich, cat. # D8537)
- TrypLE express cell dissociation reagent (Gibco, cat. # 12604-021)
- NUNC™ EasyFill™ Cell Factory™ System (Thermo Fisher Scientific, cat. # 140400)
- Na2HPO4 (Sigma-Aldrich, cat. # S3264)
- HEPES (Sigma-Aldrich, cat. # H3375)
- NaCl (Sigma-Aldrich, cat. # S3014)
- CaCl2∙2H2O (Sigma-Aldrich, cat. # C8106)
- Lentiviral packaging, envelope, and expression plasmids (e.g., for our second generation, 5-plasmid system we use: pTat, pRev, pGag-Pol, pVSV-G and an expression plasmid. See Table 1 for details). Users should produce the plasmids for their system in the appropriate quantity.
- 225 ml conical tubes (Falcon, cat # 352075)
- L-Glutamine (Gibco, cat. # 21051-024)
- OptiPRO™ SFM (Gibco, cat. # 12309-019)
- Penicillin-streptomycin (penicillin 10000 IU/ml, streptomycin 10000 µg/ml) (Gibco, cat. # 15140-122)
Vector harvest and purification
- Fetal calf serum (FCS) (Australian origin) (Gibco, cat. # 10099-141)
- Bovine serum albumin (BSA) (Sigma-Aldrich, cat. # A7906)
- NaCl (Sigma-Aldrich, cat. # S3014)
- 0.9% saline for injection (Fresenius Kabi, cat. # AUST R 197200)
- Heat-inactivated serum from the species that the vector preparation is intended for (e.g., mouse serum if the vector is to be delivered to mice) to minimize potential immune reaction.
- 500 ml centrifugation bottles (Beckman Coulter, cat. # 355607)
- 50 mm Polydisc AS 0.45 µm Whatman filter (GE Healthcare Life Sciences, cat. # 6724-5045)
- Mustang® Q XT5 capsule (Pall Corporation, cat. # XT5MSTGQPM6)
- Masterflex peroxide-cured silicone tubing L/S 16 (Masterflex, cat. # 46400-16)
- Male luer lock with 3.2 mm (1/8”) hose barb connector (Cole-Parmer, cat. # EW-45505-04)
- Female luer lock with 3.2 mm (1/8”) hose barb connector (Cole-Parmer, cat. # EW-45500-04)
- 14 ml thin wall polyallomer ultracentrifuge tubes (Beckman Coulter, cat. # Z604085CA)
Mustang Q XT5 maintenance
- NaOH (Sigma-Aldrich, cat. # 55881)
- NaCl (Sigma-Aldrich, cat. # S3014)
- Cole Parmer Masterflex® L/S® pump (Cole Parmer, model # 77200-50)
- Optima™ L-100 XP Ultracentrifuge with a SW40 Ti rotor (Beckman Coulter, cat. # 392050 and # 331302)
- Avanti® J-E Centrifuge with a JA-10 rotor (Beckman Coulter, cat. # 369001 and # 369687)
- HEK 293T cell growth media: DMEM, 10% FCS, 10 units/ml Penicillin, 10 µg/ml Streptomycin
- 2× HeBS buffer: 1.5 mM Na2HPO4, 50 mM HEPES, 0.28 M NaCl in water, adjust pH to 7.08 and filter-sterilise
- 2.5 M CaCl2∙2H2O in water and filter-sterilise
- Harvest media: OptiPRO™ SFM, 10 units/ml Penicillin, 10 µg/ml Streptomycin, 4 mM L-glutamine
- 2% heat inactivated serum: use serum from the species that the vector preparation is intended for to minimize potential immune reaction. Dilute serum in H2O and incubate at 65°C for 30 min
- 200 mM L-Glutamine in water and filter-sterilise
- 2.5% (w/v) BSA in PBS
- 1.5 M NaCl in water and filter sterilise
- Mustang Q XT5 column equilibration buffer: 1 M NaOH in water
- Mustang Q XT5 column storage buffer: 0.1 M NaOH and 1 M NaCl in water
1.Culture of sufficient HEK 293T cells
1.1.Pre-warm PBS, DMEM/10%FCS/1%Pen-Strep, and 10% TrypLE-Express in PBS to 37°C.
1.2.Thaw a vial of frozen HEK 293T cells rapidly in a 37°C water bath and transfer to a 50 ml conical tube and make up to 50 ml with PBS. Centrifuge at 450× g for 5 min at 4°C. Aspirate the supernatant, re-suspend the cell pellet in DMEM/10% FCS/1% Pen-Strep and transfer to a T75 flask. Incubate at 37°C in 5% CO2.
1.3.When the cells are 90% confluent, split cells (1:4) by aspirating media and washing cells with 4 ml PBS. Aspirate PBS and trypsinize cells using 4 ml of 10% TrypLE-Express in PBS, incubate for 5 min at room temperature, or until cells start to detach. Add 4 ml of media, spritz cells and transfer 2 ml to 3 × T75 flasks containing 12 ml of media.
1.4.When cells are 90% confluent (approximately 48 h after seeding), repeat step two to achieve a total of 11 × T75 flasks.
1.5.When the 11 × T75 flasks are 90% confluent, harvest cells as previously and transfer one T75 flask into one 150 mm round culture dish with a final volume of 32 ml.
2.Cell factory seeding
2.1.Pre-warm PBS, DMEM/10%FCS/1%Pen-Strep, and 10% TrypLE-Express in PBS to 37°C.
2.2.When the HEK 293T cells in the 150 mm round culture dishes are 90% confluent, harvest by first aspirating the media and washing cells with 6 ml of PBS. Aspirate PBS and detach cells by incubating them with 6 ml of 10% TrypLE-Express for 5 min. Add 6 ml of media to neutralize TrypLE-Express, spritz cells and transfer to a 1 L bottle.
2.3.Rinse each round culture dish with fresh media and add to the 1 L bottle containing the cell suspension.
2.4.Perform a viable cell count on the cell suspension and determine the volume of cells required to achieve a final concentration of 3.476 × 105 cells/ml in a total of 1500 ml. Transfer this volume to a sterile 2 L bottle and bring up to a total of 1500 ml with media.
2.5.Pour the cell suspension into a NUNC™ EasyFill™ Cell Factory™ System and equilibrate the chambers as per the manufacturer’s instructions.
2.6.Seed a T75 flask at a density of 6.14 × 106 cells/ml to use as a reference of confluency for the cell factory. The reference flask is used to assess cell confluency because the contents of the cell factory cannot be observed under the microscope.
3.1.We advise that users optimize the plasmid ratios for their system using small-scale preparations. Here we use example ratios for a second-generation, 5-plasmid LV vector system.
3.2.Prepare plasmid mix (with all reagents at room temperature) in a 50 ml conical tube as per Table 1.
3.3.Add the plasmid mix to a 50 ml conical tube containing 4.5 ml of 2.5 M CaCl2 and make up to 45 ml with sterile water.
3.4.Aliquot 22.5 ml of room temperature 2× HeBS into two 225 ml conical tubes.
3.5.Begin vortexing the first 225 ml conical tube, and using a 25 ml serological pipette add 22.5 ml of plasmid solution dropwise over 30–40 s. Continue to vortex for a further 20–30 s and set aside at room temperature.
|DNA mass (μg)||DNA length (bp)||DNA copy number||Plasmid volume (μl)|
|Expression vector||2252.58||8986||2.32E + 14||DNA mass (μg)/plasmid concentration (μg/μl)|
|pcDNATat||39.98||5668||6.53E + 12|
|pHCMV-Rev||39.98||5789||6.40E + 12|
|pHCMV-gagpol||25.29||9771||2.40E + 12|
|pVSV-G||100.08||6363||1.46E + 13|
3.6.After a total of 90 s has elapsed from initial vortexing of the first 2× HeBS in the 225 ml conical tube, begin vortexing the second 225 ml conical tube containing 2× HeBS and again add 22.5 ml of plasmid solution dropwise over 30–40 s. Continue to vortex for a further 20–30 s and set aside.
3.7.Retrieve the NUNC™ EasyFill™ Cell Factory™ System from the incubator, remove both caps and gently pour ~400 ml of media into a 1 L bottle via the small port, ensuring that cell disturbance is minimized. After four minutes has elapsed from initial vortexing of the first 225 ml conical tube, add the DNA complexes from the first 225 ml conical tube to the ~400 ml of media. After a total of five and a half minutes has elapsed, add the second 225 ml conical tube.
3.8.With care to minimize cell disruption, gently pour the media containing the plasmid solution back into the 10-layer cell factory via the large port, equilibrate again as per the manufacturer’s instructions, and place back in the 37°C incubator with 5% CO2 for eight hours.
3.9.During the eight-hour incubation, pre-warm 1.5 L of OptiPRO SFM supplemented with 4 mM L-glutamine and 1% Pen-Strep to 37°C.
3.10.At eight hours post-transfection, perform a media change by pouring out all of the media from the NUNC™ EasyFill™ Cell Factory™ System via the small port (remove as much excess media as possible) and subsequently add 1.5 L of pre-warmed OptiPRO™ SFM into the large port. Pour gently to ensure minimal disturbance to adhered cells and equilibrate media amongst the layers.
3.11.Incubate at 37°C and 5% CO2 for a further 40 h.
4.LV vector harvest
4.1.Pre-weigh 2 × 1 L bottles.
4.2.Remove the NUNC™ EasyFill™ Cell Factory™ System from the incubator and carefully pour the viral supernatant into the pre-weighed 1 L bottles.
4.3.Add 200 ml of PBS to the large port of the NUNC™ EasyFill™ Cell Factory™ System and equilibrate to rinse any remaining viral supernatant. Pour the PBS rinse into the pre-weighed 1 L bottles.
4.4.Weigh the bottles again to determine the volume of supernatant collected and add BSA (stored on ice) to achieve a final concentration of 0.1% BSA to the supernatant.
4.5.Transfer the supernatant to 500 ml centrifuge bottles. Rinse each 1 L bottle with 20 ml PBS and add to 500 ml centrifuge bottles.
4.6.Centrifuge supernatant at 400× g for five minutes at 4°C to remove cellular debris. Pour supernatant back into the 1 L bottles and keep refrigerated at 4°C until required.
5.Lentiviral purification and concentration
5.1.To set up the Mustang Q XT5 column purification system (Fig. 2), connect a 70 cm piece of MasterFlex tubing to the Watson Marlow 323 pump. Pump PBS through the tubing at a rate of 10 ml/min, ensuring no bubbles are present within the tubing.
5.2.Attach a 50 mm Polydisc AS 0.45 µm Whatman filter at the end of the tubing, secure with a cable-tie, and continue to flush PBS until the filter is saturated.
5.3.Attach a 10 cm length of MasterFlex tubing with a tubing clamp to the outlet end of the 50 mm Polydisc AS 0.45 µm Whatman filter, secure with a cable-tie, and flush with PBS, ensuring no bubbles are present within the tubing.
5.4.Attach a male M6 thread with a hose barb connector to the Mustang Q XT5 column and connect the inlet port to the 10 cm piece of MasterFlex tubing, securing with a cable-tie. Connect a 40 cm piece of MasterFlex tubing to the outlet port with a male M6 thread with a hose barb connector, securing with a cable-tie, and place the Mustang Q XT5 column in a retort stand with the outlet facing upwards. Place the end of the tubing into a large container to collect the waste.
5.5.Continue to pump PBS through the system at 10 ml/min to flush out the buffer that the column is stored in. Once the pH is ~7, the system is ready to use (this can be assessed by using pH indicator strips or a pH meter).
5.6.Place the 70 cm length of tubing into a 1 L bottle of vector supernatant that has been stored at 4°C. Pump the supernatant through at 10 ml/min until system has been completely flushed with supernatant and then increase the pump rate to 30 ml/min.
5.7.Once all but a few ml of the vector supernatant has been pumped through the system, rinse 1 L bottles with 150 ml of PBS containing 0.1% BSA and pump through the system.
5.8.Turn off the pump before the 10 cm length of tubing has air introduced into it to ensure air bubbles do not enter the Mustang Q XT5 column. Close the tubing clamp and cut off the lower 7 cm of tubing. Attach a female luer fitting with a hose-barb connector to the end of the tubing (that is attached to the column), and secure with a cable-tie.
5.9.Using a 50 ml male luer lock syringe, flush the column with 60 ml of PBS.
5.10.Cut off all but the last 10 cm of the 40 cm piece of tubing connected to the Mustang Q XT5 outlet port. Using a 50 ml syringe, elute the vector with 25 ml of 1.5 M NaCl (stored on ice), and capture into 25 ml of 2% heat inactivated animal serum (stored on ice).
5.11.Transfer the elution equally into 4 × 14 ml thin wall polyallomer tubes and place into chilled SW 40 Ti buckets. Centrifuge at 20000 rpm for 90 min at 4°C.
5.12.Following ultracentrifugation, gently pour off the supernatant, ensuring that the vector pellet is not disturbed, and blot tubes on tissue paper to remove as much residual liquid as possible.
5.13.Resuspend the vector pellets in 0.9% saline (stored on ice) by pipetting up and down, while ensuring that no bubbles form and clumps are no longer visible. Rinse tubes to maximize vector recovery. Once resuspended, add 0.1% (v/v) heat inactivated animal serum and keep on ice when not re-suspending.
5.14.Aliquot vector into screw cap tubes and store at −80°C. Avoid freeze-thawing as this can decrease titers up to 10-fold /cycle.
5.15.Test virus functionality using an appropriate method based on the expression plasmid used (Fig. 3).
6.Mustang Q XT5 column equilibration and storage
6.1.After elution, equilibrate the column by pumping 25 ml of equilibration buffer at room temperature through the column at 10 ml/min. Stop the pump, close the tubing clamp, and allow to rest for 30 min.
6.2.Pump through 25 ml of Mustang Q XT5 column storage buffer at room temperature at 10 ml/min, stop the pump, disconnect the column, and cap the inlet and outlet ports. Store at room temperature until next use.
|2.3||Low cell yield||
|3.5||HeBS/plasmid complex solution is not cloudy||
|3.5||HeBS/plasmid complex solution has precipitated out||Incorrect calcium chloride used||Make sure to use Cacl2∙2H2O (MW-147.01 g/mol)|
|5.7||Clogged filer||Too much debris in supernatant||
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