Large-scale manufacturing is a bottleneck for industrializing gene therapies. Here we outline a HEK293T adeno-associated virus (AAV) production workflow comparing adherent, suspension, and microcarrier processes. Microcarriers delivered high total titers and translated from spinner flask to a 1 L stirred-tank bioreactor with consistent parameters.
Key takeaways
- Microcarrier culture enables adherent-like growth in a suspension system and reduces labor and contamination risk compared to multilayer vessels.
- In this study, adherent and microcarrier processes achieved higher total AAV titers than a suspension culture.
- The microcarrier process scaled to a 1 L bioreactor, maintaining process parameters and titer trends.
Introduction
Gene therapies are increasingly progressing toward late-stage clinical trials and market approval to treat genetic diseases, cancers, and other diseases. However, large-scale production of viruses is still a limiting factor in the industrialization of gene therapy.
Most AAV and other viruses are produced using static systems through transfection of HEK293T cells. These systems, such as multi-layer vessels, are labor-intensive, show inconsistent gas exchange, and hinder cell monitoring, which results in high production costs and high contamination risks (1). On the other hand, suspension cultures, which are a convenient way for industrialization, can alter cell morphology and metabolism, affecting the quality and yield of the viruses produced (2, 3).
Microcarrier cultures offer a hybrid solution by enabling the suspension culture of adherent cells. Microcarriers are 60 to 2500 µm support matrices that provide a high surface area for the growth of adherent cells in scalable single-use bioreactors. For example, 5 mg Cytodex™ at 3 g/L in a 50 L bioreactor provides an equivalent surface area to 94 ten-layer vessels with cells grown in monolayers. Additionally, because it can produce large quantities of cells without the need for large-capacity equipment it reduces labor demand and the risk of contamination (4).
Scientists at Obio Technology have successfully adapted an adherent HEK293T cell line to grow in animal component-free and serum-free suspension conditions in shaker flasks. After comparing adherent, suspension, and microcarrier culture for AAV production, they believe that microcarrier culture is suitable for large-scale production of viral vectors to support future gene therapy manufacturing processes.
Materials and methods
Microcarrier preparation
Cytodex 1 microcarriers were hydrated and sterilized according to the manufacturer’s instructions. Microcarriers were hydrated in Dulbeccos-PBS at 50 mL/g Cytodex 1, washed once using the same volume of D-PBS, and then autoclaved at 121°C for 60 min. Before use, the microcarriers were incubated at 37°C for 4 h, in a 50 mL/g Dulbecco's modified eagle medium (DMEM) solution with 10% Fetal bovine serum.
Cell preparation
Adherent HEK293T cells were thawed in a 37°C water bath, resuspended with medium, and transferred to a T-75 flask. The cell culture was split when confluent, after 2 d of growth, using a recombinant enzyme for cell dissociation. Each passage was performed at a ratio of 1:4 to 1:6 in the T-flask expansion process.
Suspension HEK293T cells were adapted to serum-free, animal component-free medium in shake flasks.
Cell cultures
Adherent HEK293T cells were cultured on Cytodex 1 microcarriers at 3 g/L in a spinner flask with a final working volume of 125 mL. Cells were inoculated at a density of 4 × 105 cells/mL. AAV transfection was performed once the cell density on the microcarriers reached 1.0 to 1.5 × 106 cells/mL.
For adherent T-flask cultures, HEK293T cells were inoculated into T-25 flasks at a density of 4 × 105 cells/mL in 5 mL of medium. AAV transfection was performed 24 h post-inoculation, once cell confluence exceeded 70%.
For suspension cultures, suspension-adapted HEK293T cells were inoculated at 2 × 106 cells/mL into shake flasks with 20 mL of medium. AAV transfection was carried out 1 to 4 h after inoculation.
Table 1 lists the parameters for the microcarrier, adherent, and suspension culture processes.
Table 1. Parameters of the three culture processes
| Stage of culture | Parameters | Culture processes | ||
| Microcarrier | Adherent | Suspension | ||
| Cell culture | Culture vessel | Spinner 125 | T-25 | Flask 125 |
| Culture volume (mL) | 125 | 5 | 20 | |
| Inoculation cell density (× 106 cells/mL) | 0.4 | 0.4 | 2 | |
| Microcarrier concentration (g/L) | 3 | NA1 | NA1 | |
| Culture time (h) | 24 | 24 | 1–4 | |
| Transfection | Transfection medium | DMEM | DMEM | Opti-MEM |
| Backbone:envelope:target:PEI2 (µg/mL) | 1:0.5:1:2 | 1:0.5:1:2 | 1:0.5:1:2 | |
| Time to exchange medium after transfection (h) | 6–10 | 6–10 | NA1 | |
| Harvest | Time to harvest (h) | 96 | 96 | 96 |
| Harvest for supernatants or cells | Both | Both | Both | |
1 NA is not applicable.
2 PEI is polyethylenimine.
AAV transfection
Cells in all three processes were transfected with an adeno-associated viral vector that expresses green fluorescent protein (AAV2-GFP).
For microcarrier cultures, the microcarriers were settled by stopping agitation. Afterwards, 80% of the supernatant was carefully removed, and fresh complete medium was added to 60% initial culture volume. The transfection mix was prepared in 10 mL serum-free medium with plasmid ratios for target, envelope, and backbone of 1:0.5:1 (µg/mL). The transfection reagent PEI was added at a 1:1 (µg/mL) ratio of total plasmid:PEI. An 80% medium exchange was performed 6 to 8 h post-transfection.
For the adherent process, an 80% medium exchange was performed with the supernatant, and a 20% volume of DMEM was used to prepare the transfection solution, following the same steps as for transfection of microcarrier cultures. The medium exchange was performed 6 to 8 h after transfection.
For the suspension process, the transfection solution was prepared in a 10% culture volume of Opti-MEM medium, following the same steps as for transfection of microcarrier cultures. No medium exchanged was performed after transfection.
The transfection parameters for the three culture processes are shown in Table 1.
AAV harvest
All cells were harvested 96 h after transfection. The parameters for the three culture processes are shown in Table 1.
For the microcarrier process, the harvested media was centrifuged to separate the supernatant from the cells (on microcarriers). The cells were resuspended in the same volume of D-PBS and subjected to three freeze-thaw cycles. After the last thawing step, the cells were digested with benzonase nuclease (Mg2+) at 37°C for 2 h. Similar steps were followed for adherent and suspension processes. The titer was determined on the cells and supernatant by quantitative PCR (qPCR).
Bioreactor scale-up process
The parameters for producing AAV were confirmed in a 1 L bioreactor (Table 2), and samples were quantitated at 48, 72, 96, and 120 h post-transfection.
Table 2. Parameters for bioreactor culture
| Agitation (rpm) | Aeration rate | Temp (°C) | Dissolved oxygen (%) | pH |
| 62.5 ± 2.5 | Compressed air: 2% (12 mL/min), Oxygen: automated |
37 | 50 | 7.2 ± 0.15 |
Results
The growth rates of HEK293T cells in microcarrier and adherent cultures were similar, with a doubling time of about 24 h. The cell density was higher in the microcarrier culture as compared with T-flasks. The morphologies of HEK293T cells after transfection in the three culture processes are shown in Figure 1.
Fig 1. Cell morphology and AAV2-GFP levels of the three culture processes
The total titers for the microcarrier and adherent cultures were approximately 2.9- and 4.4-fold higher, respectively, than the total titer for the suspension culture (Fig 2, Table 3).
Fig 2. AAV production titers for the different cell culture types. The titer was determined through the presence of viral genomes (v.g.) by qPCR.
Table 3. AAV production titers
| Culture processes | qPCR titers (v.g./mL) | ||
| Cells | Supernatants | Total | |
| Adherent | 5.87 × 1011 | 1.08 × 1011 | 6.95 × 1011 |
| Suspension | 1.40 × 1011 | 1.67 × 1010 | 1.57 × 1011 |
| Microcarrier | 3.95 × 1011 | 6.44 × 1010 | 4.59 × 1011 |
The microcarrier culture process was confirmed by a 1 L bioreactor culture test (Table 4). As the culture time increased, the titer of the supernatant and cells also increased (Fig 3). The trend was more obvious in the curve for the cells, which was up to 3.3 × 1011 v.g./mL at 96 h and 4.5 × 1011 v.g./mL at 120 h (determined via qPCR).
Table 4. The microcarrier process in a 1 L bioreactor
| Stage of culture | Conditions | Parameters |
| Cell culture | Inoculum density (× 106 cells/mL) | 0.4 |
| Agitation (rpm) | 45 | |
| Culture volume (mL) | 400 | |
| Microcarrier concentration (g/L) | 3 | |
| Cell culture time (h) | 24 | |
| Transfection | Backbone:envelope:target:PEI (µg/mL) | 1:0.5:1:2 |
| Time to exchange medium after transfection (h) | 6–8 | |
| Harvest | Time to harvest (h) | 48, 72, 96, 120 |
| Harvest for supernatants or cells | Both |
Fig 3. The AAV titer for a microcarrier process in a 1 L bioreactor.
Conclusions
Microcarriers enabled high AAV titers with HEK293T cells in both spinner flask and in a 1 L stirred-tank bioreactor. The data suggest that the developed microcarrier process is suitable for large-scale AAV production.
FAQ
Q1. Why choose microcarriers over suspension for HEK293T AAV production?
Microcarriers allow adherent HEK293T cells to retain their morphology and metabolism closer to static culture while running in stirred systems, which can improve total titer versus the same cells adapted to suspension.
Q2. What Cytodex 1 concentration and inoculation density worked here?
3 g/L Cytodex 1 with 0.4 × 10⁶ cells/mL inoculation, followed by transfection and medium exchange at 6 to 8 h post-transfection.
Q3. When should I harvest?
This workflow harvested at 96 h for the three process modes; the 1 L time course showed continued titer increases through 120 h, so optimal timing should be analyzed case by case.
Q4. Does the process translate to a stirred-tank bioreactor?
Yes. A 1 L run with dissolved oxygen (DO) 50%, pH ~ 7.2, and ~ 62.5 rpm agitation reproduced the microcarrier process and showed the expected titer trend over time.
Q5. Any practical tips for transfection on microcarriers?
Complex plasmids and PEI to 1:1 (µg/mL) after a short incubation; perform a medium exchange at 6 to 8 h to improve transfection environment; avoid over agitation during early attachment.
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ACKNOWLEDGEMENTS
We gratefully acknowledge Yongfa Lan, HaiRui Yang, and QingRui You from Obio Technology for permission to share their work.
REFERENCES
- Yang J, Guertin P, Jia G, Lv Z, Yang H, Ju D. Large-scale microcarrier culture of HEK293T cells and Vero cells in single-use bioreactors. AMB Express. 2019;9(1):70. doi: 10.1186/s13568-019-0794-5.
- Wang D, Tai PW, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18:358-378. doi: 10.1038/s41573-019-0012-9.
- Naso MF, Tomkowicz B, Perry WL, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;31(4):317-334. doi: 10.1007/s40259-017-0234-5.
- Chen XY, Chen JY, Tong XM, Mei JG, Chen YF, Mou XZ. Recent advances in the use of microcarriers for cell cultures and their ex vivo and in vivo application. Biotechnol Lett. 2020;42:1-10. doi: 10.1007/s10529-019-02738-7.
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