Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Kimberly L. Pietersz; Paul J.H. Nijhuis; Matthijs H.M. Klunder; Jo&#235;lle van den Herik; Barbara Hobo; Fred de Winter; Joost Verhaagen

doi:10.3791/66532

JoVE Journal > Neuroscience

Neurosciences

Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells

Published: April 26, 2024

doi:

10.3791/66532

Kimberly L. Pietersz¹, Paul J.H. Nijhuis¹, Matthijs H.M. Klunder¹, Joëlle van den Herik¹, Barbara Hobo¹, Fred de Winter¹, Joost Verhaagen^1,2

¹Laboratory for Regeneration of Sensorimotor Systems, Netherlands Institute for Neuroscience,Royal Netherlands Academy of Arts and Sciences (KNAW), ²Centre for Neurogenomics and Cognitive Research, Amsterdam Neuroscience,Vrije Universiteit Amsterdam

Summary

Here, a suspension HEK293 cell-based AAV production protocol is presented, resulting in reduced time and labor needed for vector production using components that are available for research purposes from commercial vendors.

Abstract

Adeno-associated viral vectors (AAVs) are a remarkable tool for investigating the central nervous system (CNS). Innovative capsids, such as AAV.PHP.eB, demonstrate extensive transduction of the CNS by intravenous injection in mice. To achieve comparable transduction, a 100-fold higher titer (minimally 1 x 10¹¹ genome copies/mouse) is needed compared to direct injection in the CNS parenchyma. In our group, AAV production, including AAV.PHP.eB relies on adherent HEK293T cells and the triple transfection method. Achieving high yields of AAV with adherent cells entails a labor- and material-intensive process. This constraint prompted the development of a protocol for suspension-based cell culture in conical tubes. AAVs generated in adherent cells were compared to the suspension production method. Culture in suspension using transfection reagents Polyethylenimine or TransIt were compared. AAV vectors were purified by iodixanol gradient ultracentrifugation followed by buffer exchange and concentration using a centrifugal filter. With the adherent method, we achieved an average of 2.6 x 10¹² genome copies (GC) total, whereas the suspension method and Polyethylenimine yielded 7.7 x 10¹² GC in total, and TransIt yielded 2.4 x 10¹³ GC in total. There is no difference in in vivo transduction efficiency between vectors produced with adherent compared to the suspension cell system. In summary, a suspension HEK293 cell based AAV production protocol is introduced, resulting in a reduced amount of time and labor needed for vector production while achieving 3 to 9 times higher yields using components available from commercial vendors for research purposes.

Introduction

Adeno-associated virus (AAV) was discovered in 1965 and has since been used in a myriad of applications¹. AAVs have been applied in neuroscience research to study gene and neuronal function, map neurocircuits, or produce animal models for disease². Traditionally, this is done by injecting directly at the site of interest, as most natural serotypes do not cross the blood-brain barrier or need a high dose to do so¹^,²^,³.

With the discovery of AAV.PHP.B⁴ and next-generation capsids such as AAV.PHP.eB⁵ and AAV.CAP-B10⁶, it is possible to target the central nervous system (CNS) using a simple systemic injection. Spatial mapping reveals the cells targeted by AAV.PHP.eB at cellular level⁶^,⁷. In combination with specific promoters/enhancers, these capsids offer extensive opportunities for neuroscientists to study genes and brain function by non-invasive AAV delivery⁴^,⁸.

While a lower dose is needed for AAV.PHP.eB (typically 1 to 5 x 10¹¹ Genome Copies (GC)/mouse) compared to AAV9 (4 x 10¹² GC/mouse)⁷, still more vector needs to be produced compared to direct injection strategies (typically 1 x 10⁹ GC/µL injection). Most natural serotypes can be produced using the classical adherent cell culture system in combination with iodixanol purification⁹^,¹⁰^,¹¹^,¹². For AAV.PHP.eB this entails a labor-intensive process to culture and transfect cells to obtain sufficient vectors for one experiment⁸. Therefore, the production of AAV in suspension cell culture in conical tubes was developed. Conical tubes, with a capacity of up to 300 mL, are compact, saving both incubator space and plastics. Suspension cells are much easier to culture and handle in large amounts than adherent cells on 15 cm plates. The transfection components of the protocol remain the same. Therefore, plasmids previously used with the adherent system can easily be used in this protocol based on production in suspension cells. The protocol was successfully transferred to other researchers in the laboratory and successfully used for various capsids and constructs.

Protocol

All experimental procedures were approved by the institutional animal care and use committee of the Royal Netherlands Academy of Sciences (KNAW) and were in accordance with the Dutch Law on Animal Experimentation under project number AVD8010020199126. In Figure 1, a schematic overview of the complete protocol is provided. From seeding cells to AAV purification, the protocol takes 6 days to complete.

1. Reagent preparation

Plasmid purification
1. Perform plasmid extraction as described by the manufacturer with a few adjustments to ensure sterility of the plasmids.
2. Prepare 70% ethanol using sterile distilled water and absolute ethanol.
3. After the isopropanol centrifugation step, dry plasmids in the flow cabinet hood and add sterile 70% ethanol to the tubes. After ethanol precipitation, dry the plasmids in the flow cabinet hood and resuspend the plasmid in sterile distilled water. Take care to use sterilized microcentrifuge tubes.
4. Take an aliquot for quantification, restriction analysis, and, if needed, sequencing. It is advised to check the integrity of the inverted terminal repeats (ITRs) as mutations can have a negative impact on yield. Adjust the sample concentration to 1 µg/µL.
Preparation of 10x Tween lysis buffer
1. Dissolve 30.3 g Tris buffer and 2.033 g MgCl₂.6H₂O in 400 mL of sterile distilled water, set pH to 8.0, and increase total volume to 450 mL.
2. Keep Tween 20 sterile, add 50 mL of Tween 20 to buffer solution in the hood, and filter sterilize using a 0.2 µM vacuum filter.
Iodixanol dilutions
1. Iodixanol solution is provided at 60% concentration. Use the starting solution to make 15%, 25%, and 40% solutions.
2. For 15% solution, dilute 60 mL of 60% solution with 48 mL of 5 M NaCl and 48 mL of PBS-MK (5x PBS with 5 mM MgCl₂ and 12.5 mM KCl), and add distilled water up to 240 mL.
3. For 25% solution, dilute 67 mL of 60% solution and 32 mL of PBS-MK. Add distilled water up to 160 mL. Mix well and add 1.6 mL of phenol red solution.
4. For 40% solution, dilute 160 mL of 60% solution with 48 mL of PBS-MK and add distilled water up to 240 mL. For 60% solution, add 1 mL of phenol red to 100 mL of 60% solution.
PBS 5% sucrose solution
1. Add 25 g of sucrose to a 500 mL bottle of DPBS without calcium or magnesium. Shake well and filter using a 0.2 µM bottle vacuum filter.
Polyethylene glycol (PEG) 8000 solution
1. Dissolve 400 g of PEG 8000 and 24 g of NaCl into sterile distilled water and adjust to a final volume of 1000 mL. Stir with heating until fully dissolved. Adjust pH to 7.4 using pH paper.
2. Filter sterilize using a 0.2 µM bottle vacuum filter to obtain a 40% PEG 8000 solution. Note that filtration will take a while due to the viscosity of the solution-store at 4 ˚C.

2. Culture of HEK293 suspension cells

Use wipes soaked in 70% ethanol for cleaning. Clean biosafety hood, prepare and clean all materials needed for culturing cells.
Prewarm 30 mL of suspension cell medium to 37 °C in a 50 mL conical culturing tube. Retrieve viral production cells from liquid nitrogen. Quickly thaw cells in a 37 °C water bath. Just before the vial is completely thawed, clean it with a wipe soaked in 70% ethanol and transfer the vial to the biosafety hood.
NOTE: The details of the viral production cells used here are provided in the Table of Materials.
Quickly transfer cells to the prewarmed 50 mL conical culturing tube. Culture cells in a shaking incubator at 37 °C, 80% humidity, 8% CO₂, 200 revolutions per min (RPM), and a shaking diameter of 50 mm. Passage cells, once viability is above 90% and cell density is above 1-3 x 10⁶ cells/mL.
NOTE: The incubator used has a shaking diameter of 50 mm; culture conditions should be adjusted for a different shaking diameter.
Passage cells 3-4 days after thawing. Check culture tube(s) to make sure there are no signs of contamination; for example, fungus will appear as a discolored (black or green) ring.
Prepare 400 µL of 0.4% trypan blue solution per sample using a 1 mL stripette.
Quickly retrieve suspension cells from the incubator. Immediately extract 500 µL from the tube using a 1 mL stripette. Gently pipet up and down.
Add 100 µL of cell suspension to the prepared trypan blue tube. Gently invert the tube to mix; do not pipet to mix, as this will lead to cell death. Return cells to incubator.
Take 50 µL of trypan blue-treated cell suspension and apply it to the hemocytometer slide. Count total viable cells and calculate the amount of cell suspension and medium needed for passage or transfection the following day.
Warm medium in the incubator for 10-15 min. Add cell suspension to warmed medium and return to incubator. For passaging cells for 3 days (i.e., Monday-Thursday), set to 0.5 x 10⁶ cells/mL; for passaging cells for 4 days (i.e., Thursday-Monday), set to 0.3 x 10⁶ cells/mL.
NOTE: According to the manufacturer, viral production cells double every 26 h and should be between 3.5 x 10⁶ and 5.5 x 10⁶ before passaging. Cells can be kept for up to passage 20.

3. Transfection

Day 1
1. At 24 h before transfection, culture 1 x 10⁶ cells per/mL in 300 mL of media.
Day 2
1. Warm medium, plasmids, and transfection reagent to room temperature. For the amounts to prepare, see Table 1.
2. Briefly vortex plasmids and reagents. Add plasmids to the prepared medium, vortex. Add transfection reagent and vortex briefly. Do not agitate the mixture after this step. Incubate at room temperature for 30 min.
3. In the meantime, count cells prepared on day 1. Cells should be between 2 and 2.5 x 10⁶ cells per mL and > 95% viable.
4. After incubation, dropwise, add the transfection mixture to cells while gently swirling the tube. Incubate for 72-96 h.

4. Harvesting the cells

Day 5
1. Add 33 mL of 10x cell lysis buffer (500 mM Tris pH 8, 10% Tween 20, 20 mM MgCl₂), mix by gently shaking, and incubate at 37 °C for 1.5 h with shaking.
2. Centrifuge at 3428 x g at 4 °C for 60 min. Filter through a 0.45 µM PES vacuum filter to clarify the cell lysate, leaving the clarified lysate in the container of the filter.
  NOTE: Optional after filtration: take 50 µL of sample for quantitative-PCR (Q-PCR).
3. Add 90 mL of 40% PEG 8000 solution to 360 mL of filtered cell lysate.
4. Clean the stir bar with 70% ethanol and add to the sample. Stir on ice or in the cold room at 300 RPM for 1 h. Incubate at 4 °C without stirring overnight.
  NOTE: Incubation times of 1 h to 72 h have been tested. Longer incubation times have a negative effect on overall protein precipitation.

5. Iodixanol purification

Day 6
1. Transfer the entire PEG precipitated culture volume sample to a clean, large conical tube and centrifuge at 2820 x g and 4°C for 15 min.
2. Discard the supernatant and resuspend the PEG pellet in 15 mL of DPBS with calcium and magnesium. The pellet is difficult to resuspend; take the time to do this carefully.
3. Transfer the resuspended part to a clean 50 mL tube. PEG will remain on the sides of the bioreactor tube. Add an extra 10 mL, taking care to gather all the PEG precipitate. Transfer all to a 50 mL container for a total volume of 25-30 mL.
4. Add 40 µL of DNaseI (10 U/µL). Place in the incubator for 1 h 37°C.
  NOTE: Optional after incubation of DNase: take 50 µL of sample for Q-PCR.
5. Clean stir bars that were used for PEG precipitation well with chloride solution followed by 70% ethanol. Leave stir bars in 70% ethanol for the next experiment.
6. Fill a 25 mm x 89 mm polyallomer tube using a glass Pasteur pipette with 15.5 mL of concentrated cell lysate in each tube. After adding cell lysate, exchange the glass Pasteur pipette for a new one.
7. Add iodixanol solutions from 15%-60%: infuse 9 mL of the 15% iodixanol solution gently beneath the cell lysate. Next, add 5 mL of the 25% and 40% iodixanol solutions, and lastly, add 5 mL of the 60% iodixanol solution.
8. Top off the tube using a syringe with DPBS +/+ to remove most of the air bubbles, taking care not to disturb iodixanol layers. Seal the tube using an electrical tube topper.
9. Centrifuge in a non-swing rotor for 1 h 10 min at 490,000 x g at 16 °C in an ultracentrifuge. Remove from ultracentrifuge, assemble the tubes in a metal clasp, and prepare collection tubes. Open the rotor in the hood in case of spills during spinning.
10. Prepare one 50 mL tube (for discarding the rotor tube), one 15 mL tube (for collecting virus), a 5 mL syringe, and a 30G and 19G needle per gradient.
11. Puncture a hole in the top of the tube with a 30G needle. Leave the needle in place. Place a 19G needle on the syringe.
12. Carefully puncture the tube just below the 40%/60% interface, which can be seen by the phenol red indicator. Make sure the needle bevel is facing the 40% layer.
13. Remove the 30G needle from the top of the tube using the non-dominant hand. With the needle beveled up, slowly extract the virus/iodixanol. The aim is to extract between 3 mL and 4.5 mL. About halfway and well into the 40%/clear layer, rotate the needle for the bevel to face down and continue to extract to avoid collection from the protein layer.
14. Prepare the 50 mL tube with the non-dominant hand, carefully extract the needle while placing the tube in the 50 mL tube and discard. Dilute the AAV/iodixanol suspension 5x in DPBS by filling up to 15 mL.
15. Transfer to a centrifugal filter tube and centrifuge at 3428 x g, 4 °C for 10 min. Discard flow-through; gently remove the holder of the filter and tip the bottom container into a waste bottle. Add 15 mL of PBS-5% sucrose to filtrate and resuspend.
16. Centrifuge at 3428 x g at 4 ˚C for 20 min. Repeat buffer exchange at least 3x. Store virus (~150-250 µL) at 4 °C (PHP.B variants a maximum of 3 months) or aliquot into 50 µL aliquots and store at -80 °C for long term.
  NOTE: PHP.B capsid variants are sensitive to freeze/thaw, so this should be avoided.
17. Perform titration as described in a previous protocol¹⁰.

Representative Results

Most academic labs use adherent HEK293T cells for AAV production⁸^,⁹. While this works relatively well when small amounts of AAV are needed for direct injection, a 100-fold higher titer (minimally 1 x 10¹¹ GC/mouse) is needed to achieve similar transduction with systemic capsids such as AAV.PHP.eB.

In this protocol, the production of AAV using suspension HEK293 cells cultured in conical tubes was established. Small-scale cultures can be done in 50 mL tubes and large-scale in 600 mL tubes. For specific shakers (see Table of Materials), a rack can be used in which up to 16 large tubes can be cultured simultaneously. This rack is used in combination with special inserts for 50 mL tubes (Figure 2A). This system offers a significant gain in time for this protocol's culturing and transfection segment. The initial setup of production was done with reporter constructs for luciferase and green fluorescent protein (GFP)¹⁰^,¹¹. In parallel, the GFP construct was produced in 12, 15 cm² plates as described in a previously published protocol¹². On average, a yield of 7.7 x 10¹² GC total was achieved using polyethylenimine (PEI) and 2.4 x 10¹³ GC using TransIt (Figure 2B). For PEI, this is an almost 3-fold improvement; for TransIt, it is a 9.2-fold improvement over titers obtained with adherent culture (2.6 x 10¹²). When used to transfect suspension cells, TransIt gives approximately a 3-fold improvement in yield compared to PEI (Figure 2B).

Subsequently, the performance of the AAV vectors was tested in vivo. GFP vectors were produced with PEI and TransIt and compared to GFP vectors produced with the classical adherent system. At 4 weeks after injection, mice (6 weeks C57BL/6 female mice weighing 20-25 g) were sacrificed, and GFP expression in mouse's brains was evaluated by histology (Figure 3A). Comparative analysis was done on sagittal brain sections. No difference was observed in the transduction pattern of the virus produced using these production methods (Figure 3B). The luciferase activity of the vectors produced using either method was also assessed. Transduction was measured in vivo for 3 weeks; the expression pattern over time is similar no matter which transfection reagent is used (Figure 4).

Next, the protocol was tested and implemented by other members of the team (Table 2). The production of other capsids with PEI was successful, yielding an average yield of 3.5 x 10¹² GC vectors. For capsid B10 production, a 3-fold improvement can be achieved by using TransIt versus PEI. For another project, several constructs packaged in AAv.PHP.eB gave an average yield of 3.7 x 10¹², which is enough to inject 7 mice intravenously at a dose of 5 x 10¹¹ GC per mouse. These results illustrate that the protocol can be successfully used by several researchers for various capsids and construct combinations.

Figure 1: Schematic overview of production. On day 1 (D1), cells are cultured in 300 mL tubes. On day 2 (D2), cells are transfected with the relevant plasmids. On day 5 (D5), cells are harvested and lysed using a 10x tween lysis buffer. After lysis, cell debris is removed by centrifugation. Subsequently, the cell lysate is filtered to remove large proteins, and the AAV virus is precipitated using Polyethylene glycol (PEG) 8000. On day 6 (D6), the iodixanol purification and concentration are performed. The PEG concentrate from the previous day is treated with DNase and afterward placed through an iodixanol gradient. The purified vectors are subsequently desalted and concentrated using a centrifugal filter. Please click here to view a larger version of this figure.

Figure 2: Incubator set-up and AAV yields. (A) Viral production cells are cultured in a shaking incubator with a 50 cm shaking diameter. This incubator is equipped with an adapter plate for 600 mL tubes and special inserts for 50 mL tubes produced by the mechatronics department. (B) Following the initial setup, reporter constructs using either polyethyleneimine (PEI; 7.7 x 10¹² GC total, n=5) or TransIt (2.4 x 10¹³ GC total, n=5) as transfection method and compared to the yields achieved with the classical adherent method (2.6 x 10¹² GC total, n=5) as described in Verhaagen et al.¹². Data are presented as mean ± Standard deviation. Please click here to view a larger version of this figure.

Figure 3: GFP expression pattern after in vivo evaluation. (A) Schematic depiction of a typical AAV vector construct containing the ubiquitous promoter CMV immediate-early enhancer, chicken β-actin promoter, and rabbit β-Globin splice acceptor site (CAG) followed by Green fluorescent protein (GFP), the Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) and a polyadenylation tail (pA)¹³. Here, 6-week-old female mice (n=3) received a tail vein injection containing 5 x 10¹¹ total genomic copies (gc)/mouse. At 4 weeks after injection, mice were sacrificed, and brains were analyzed by histology. (B) Representative depiction of the transduction pattern in sagittal sections, using adherent HEK293T cells and suspension cells with polyethyleneimine (PEI) or TransIt transfection reagent, a similar transduction pattern is observed irrespective of production method used. Scale bar = 1000 µm. Please click here to view a larger version of this figure.

Figure 4: Similar luciferase activity irrespective of transfection method. (A) Schematic depiction of the construct used containing the ubiquitous promoter CMV immediate-early enhancer, chicken β-actin promoter, and rabbit β-Globin splice acceptor site (CAG) luciferase (LUC) with a V5 tag and a bovine growth hormone polyadenylation tail (BGHpA), 5 x 10¹¹ GC total/mouse (n=3) was injected via the tail vein, luciferase activity in the cranial area (circled in red, in example image) as bioluminescent image was measured weekly. (B) Bioluminescent activity was measured as relative luminescence units (RLU) depicted as a blue bar for PEI production and a brown bar for TransIt. No significant difference in luciferase activity in the selected brain area was observed between groups. Data are presented as mean ± Standard deviation. Please click here to view a larger version of this figure.

Table 1: Transfection parameters. Please click here to download this Table.

Table 2: Yields of the implemented protocol. Suspension protocol used by other researchers to produce AAV. Please click here to download this Table.

Discussion

Systemic administration of AAV is a powerful tool for gene transfer to the CNS; however, the production of AAV is an expensive and laborious process. By using suspension cells, labor and plastics are reduced compared to the adherent culture of HEK293T on 15 cm² plates. Furthermore, the conical tubes implemented here are easy to handle and maximize the use of laboratory space. The protocol was set up by two researchers and subsequently applied by others in the lab. A series of productions by three independent researchers yielded, on average, enough vectors per batch to inject 6-7 mice.

The use of HEK293 suspension cells has been described earlier¹³^,¹⁴. However, the suspension cell line described is not freely available for research purposes. This is a bottleneck for academic researchers when applying the described methods. The suspension cell line described in this protocol is freely available for research purposes. The culture of suspension cells was done in conical tubes instead of Erlenmeyer's to save space and time. With Erlenmeyer's, up to 4 productions can be cultured simultaneously, versus 12 productions using conical tubes. Conical tubes can be directly transferred to the centrifuge for harvest.

One alternative possibility for a suspension cell culture system is the baculoviral system. An advantage of the baculo system is that these cells and media are publicly available and can easily be applied. For academics, repositories such as Addgene are available, containing a large library of ready-to-use AAV transfer plasmids¹². While these can also have caveats, such as faulty ITRs, they serve as a good source for plug-and-play experiments. The choice was made to keep a HEK293-based production system as available plasmids can directly be used for production without the transfer of the construct to baculovirus.

For transfection, polyethyleneimine is relatively cheap and is, therefore, the transfection reagent of choice. As an alternative, a novel transfection method (TransIt) was evaluated to increase the yield of vectors. A three-fold improvement was observed using half the amount of plasmids. This makes TransIt an interesting alternative transfection reagent when larger stocks are needed. A limitation of this is the cost, making it less interesting for small batches.

In summary, here, a protocol is presented that can be used to produce high-yield AAV in suspension cells in an academic laboratory setting. Tools are described that can be used in an academic laboratory setting to produce AAV at a high yield.

Divulgations

The authors have nothing to disclose.

Acknowledgements

This work was supported by a grant from the Royal Netherlands Academy of Arts and Sciences (KNAW) research fund and a grant from Start2Cure (0-TI-01). We thank Leisha Kopp for her input and advice in the setup of the protocol. Figures were created using Biorender.

Materials

39 mL, Quick-Seal Round-Top Polypropylene Tube, 25 x 89 mm – 50Pk	Beckman Coulter	342414
Adapter 600 mL conical tubes, for rotor S-4×1000,	eppendorf	5920701002
Adapter Plate fits 16 bioreactors of 600 ml	Infors HT/ TPP	587633
Aerosol-tight caps, for 750 mL round buckets	eppendorf	5820747005
Centrifuge 5920 R G, 230 V, 50-60 Hz, incl. rotor S-4×1000, round buckets and adapter 15 mL/50 mL conical tubes	eppendorf	5948000315
Distilled Water	Gibco	15230147
DNase I recombinant, RNase-free	Roche	4716728001
DNase I recombinant, RNase-free	Roche	4716728001
DPBS, calcium, magnesium	Gibco	14040091
DPBS, no calcium, no magnesium	Gibco	14190144
Fisherbrand Disposable PES Filter Units 0,2	Fisher	FB12566504
Fisherbrand Disposable PES Filter Units 0,45	Fisher	FB12566505
Holder for 50 ml culture tubes also fits falcon tube	Infors HT/ TPP	31362
Holder for 600 ml cell culture tube	Infors HT/ TPP	66129
Incubator Minitron 50 mm	Infors HT	500043
LV-MAX Production Medium	Gibco	A3583401
N-Tray Universal	Infors HT/ TPP	31321
OptiPrep – Iodixanol	Serumwerk bernburg	1893
PEI MAX – Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40,000)	Poly-sciences	24765-100
Phenol red solution	Sigma-Aldrich	72420100
Poly(ethylene glycol) 8000	Sigma-Aldrich	89510
TransIT-VirusGEN	Mirus	Mir 6706
Trypan Blue Solution, 0.4%	Gibco	5250061
TubeSpin Bioreactors-50ml	TTP	87050
TubeSpin Bioreactors-600ml	TTP	87600
Viral Production Cells	Gibco	A35347
Vivaspin 20 MWCO 100 000	Cytvia	28932363

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Pietersz, K. L., Nijhuis, P. J., Klunder, M. H., van den Herik, J., Hobo, B., de Winter, F., Verhaagen, J. Production of High-Yield Adeno Associated Vector Batches Using HEK293 Suspension Cells. J. Vis. Exp. (206), e66532, doi:10.3791/66532 (2024).