Name: Evaluation of 3D Spheroids for AAV Transduction Studies (Video) | JoVE Journal
Uploaded: 2026-08-14T13:25:00.000+00:00
Duration: 8 min 45 s
Description: Learn a 3D spheroid platform for evaluating AAV vector potency and transgene expression kinetics across diverse cell lines. This method enables quantitative, real-time assessment for gene therapy development.

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The authors thank Morten Seirup and Trish Hoang for providing the plasmid for AAV nanoluciferase production and the live substrate reagents. This project was supported by an appointment to the ORISE Research Participation Program at the CBER, U.S. Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education under an interagency agreement between the U.S. Department of Energy and the FDA.

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	<li>Najera SS, et al. AAV2 delivery of the saCas9 gene results in presentation of an HLA-A*02:01-restricted T cell epitope potent to induce T cell cytotoxicity. Mol Ther Methods Clin Dev. 2025;33(3):101506.</li>
	<li>Alhakamy NA, Curiel DT, Berkland CJ. The era of gene therapy: From preclinical development to clinical application. Drug Discov Today. 2021;26(7):1602-19.</li>
	<li>Arutyunyan IV, et al. Gene Expression Profile of 3D Spheroids in Comparison with 2D Cell Cultures and Tissue Strains of Diffuse High-Grade Gliomas. Bull Exp Biol Med. 2023;175(4):576-84.</li>
	<li>Cashen P, Manser B. Quality by design (QbD) for adeno-associated virus (AAV): A framework for a QbD assessment for AAV products within the chemistry manufacturing and controls (CMC) documentation [Internet]. Port Washington (NY): Pall Corporation; 2021. Available from: https://www.pall.com/.</li>
	<li>Center for Biologics Evaluation and Research. Potency Assurance for Cellular and Gene Therapy Products: Draft Guidance for Industry [Internet]. Silver Spring (MD): U.S. Food and Drug Administration; 2023. Available from: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/potency-assurance-cellular-and-gene-therapy-products.</li>
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	<li>Ohguro H, et al. 3D Spheroid Configurations Are Possible Indicators for Evaluating the Pathophysiology of Melanoma Cell Lines. Cells. 2023;12(5):759.</li>
	<li>Casey G, et al. Self-complementarity in adeno-associated virus enhances transduction and gene expression in mouse cochlear tissues. PLoS One. 2020;15(11):e0242599.</li>
	<li>Hutt JA, et al. Scientific and Regulatory Policy Committee Points to Consider: Nonclinical Research and Development of In Vivo Gene Therapy Products, Emphasizing Adeno-Associated Virus Vectors. Toxicol Pathol. 2022;50(1):118-46.</li>
	<li>El Harane S, et al. Cancer Spheroids and Organoids as Novel Tools for Research and Therapy: State of the Art and Challenges to Guide Precision Medicine. Cells. 2023;12(7):1001.</li>
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</ol>

The authors have nothing to disclose.

We present here a streamlined in vitro&#160;3D platform for AAV development and potency testing. This platform leverages enhanced transduction, high sensitivity, and better translational predictability using three-dimensional cell culture that more closely resembles human tissue architecture. This system successfully differentiates transduction efficacy across AAV serotypes and regulatory elements, including single-stranded and self-complementary vectors, in multiple cell lines. We note that, in contrast to conventional 2D assays, where transduction efficiency is often defined as the percentage of transgene-positive cells, the 3D spheroid system does not readily allow single-cell-resolution quantification. Therefore, transduction efficiency in this study is defined as the bulk reporter signal intensity (fluorescent or luminescent), which reflects the combined contributions of the proportion of transduced cells and the level of transgene expression per cell. The high sensitivity of the spheroids substantially reduces the need for high product quantities, making it cost-effective, versatile, and physiologically relevant for AAV research and development.
This approach addresses key challenges with traditional 2D cell culture assays, which rely on adhesion to artificial surfaces and lack cellular organization, possibly&#160;leading to poor predictions of vector performance in clinical settings. The 3D spheroid system better mimics tissue structure and drug distribution patterns, providing vector behavior data that more closely matches real biological conditions22,23. The protocol uses simple ultra-low attachment plates, making it easily reproducible in standard laboratory settings.
This adaptable system could be used for AAV neutralization assays, to study AAV penetration, and to develop more complex co-culture experiments to investigate the effects of different cellular environments on AAV transduction. In addition, high-throughput drug and vector screening platforms can easily integrate with the spheroid platform, enabling automation and cost reduction in vector discovery workflows for the development of more targeted products.
A major practical advantage of spheroids is their ability to grow continuously for weeks without cell passaging, enabling extended studies of AAV activity. This stable culture system maintains important cell-cell interactions and matrix components, avoiding the interruptions caused by repeated cell harvesting required in 2D cultures24. The preserved cellular environment supports natural protein organization and membrane structures that are essential for evaluating AAV binding and entry into cells25.
Although 3D spheroids show great potential for AAV transduction assessments, they also present some translational shortcomings compared to in vivo&#160;models, primarily due to their simplified structure relative to native tissues. Spheroids lack critical physiological components, including vasculature for vector delivery, immune cells for assessing immunogenicity, and tissue-specific barriers such as the blood-brain barrier, that could significantly impact AAV performance26. These limitations indicate that spheroids, despite offering an intermediate level of complexity between conventional 2D cultures and in vivo animal models, remain insufficient to fully recapitulate the physiological microenvironment required for comprehensive AAV assessment.
The spheroid protocol presented here describes a single cell type at a time. This protocol can be developed to include 2&#8211;3 additional cell types that aggregate into a basic three-dimensional structure. More complex 3D models, such as organoids or microfluidics, are multicellular systems that recapitulate the cellular diversity and organization of specific organs, containing 10 or more distinct cell types arranged in tissue-like patterns. Such advanced 3D organoids can include multiple cells27, hydrogel-embedded cultures, hanging drop techniques, and microfluidic platforms11,27,28. While these methods provide more complex tissue architecture that could help further our understanding of AAV biology, they tend to require more specialized reagents, equipment, and considerable technical expertise. On the other hand, 3D spheroids formed in ultra-low attachment plates are considerably simpler and are appropriate for many laboratories due to their cost-effectiveness and reproducibility.
Critical steps for successful implementation of this protocol include maintaining high cell viability, as compromised cells can impair spheroid formation and transduction efficiency. The use of ultra-low attachment plates and optimization of centrifugation are also critical for achieving spheroid compactness. Optimizing MOI for each cell type and AAV serotype is essential to avoid weak signals or saturation. Selection of the appropriate detection method is also critical, particularly for kinetic analyses, where compatibility with continuous live-cell imaging systems is required. Several technical considerations are important for accurate interpretation of AAV transduction in 3D spheroids.
A poor or absent transduction signal may result from low viral titer, suboptimal MOI, or insufficient incubation time; this can be addressed by verifying viral titers, optimizing MOI, and extending incubation time (typically 3&#8211;5 days). Weak fluorescence or luminescence signals may reflect low transgene expression or limited reagent penetration within the 3D structure, which can be improved by optimizing exposure settings or allowing additional time for substrate diffusion. A major challenge in certain cell lines, such as HeLa cells, is autofluorescence, which can lead to high background signal and reduced sensitivity in fluorescence-based assays. To address this limitation, we implemented a luminescence-based nanoluciferase reporter system, which provides an improved signal-to-noise ratio and enables more accurate detection of transduction in autofluorescent backgrounds. In addition, background signal may arise from improper thresholding or media-related fluorescence and should be controlled using untreated wells (MOI = 0) to define baseline levels. Finally, signal saturation at high MOIs may occur due to excessive viral input or detector limitations; this can be mitigated by reducing the MOI range and optimizing detector exposure settings to maintain measurements within the linear dynamic range.
Overall, this 3D spheroid methodology provides a streamlined, reproducible system for evaluating AAV transduction, potency, and kinetics. By bridging the gap between conventional 2D assays and in vivo studies, it enables more informed vector development and supports the advancement of clinically relevant gene therapy strategies.

AAV vectors have become one of the preferred gene delivery systems for in vivo applications due to their safety profile and ability to achieve persistent gene expression1. AAV gene therapy is showing success in the treatment of multiple rare disease indications, with hundreds of products in the development pipeline2. Furthermore, emerging applications include in vivo delivery of genome editing components such as Cas9, with great potential to aid in treating hard-to-treat indications3. Key development challenges include high manufacturing costs and the limited predictive capacity of current in vitro assays for determining product quality and clinical efficacy4.
The advancement of AAV therapeutics is fundamentally limited by the lack of predictive, standardized activity assays that can accurately forecast clinical performance4,5. These assays are a critical component of the development pipeline, as they are used for multiple purposes, including screening libraries of engineered vectors, optimizing capsid modifications for enhanced targeting, and evaluating tissue-specific tropism. They also serve to characterize the influence of regulatory sequences on transgene expression6. Beyond candidate selection, AAV transduction assays also support critical manufacturing quality control through potency assays used for lot release, lot comparability studies, and stability assessments6,7.
The scientific challenge lies in developing assays that capture the complexity of vector-host interactions while maintaining practical feasibility for routine use7,8. Current approaches include utilizing immortalized cell lines that represent the target organ in traditional 2D monolayer cultures. These culture conditions lack physiological relevance and require high drug doses. This creates a significant gap between laboratory predictions and clinical translatability. Novel engineered cell lines, such as AAVR-overexpressing or CRISPR-activated systems, represent important advances but fail to recapitulate the three-dimensional architecture and extracellular matrix interactions characteristic of native tissues9,10,11. Next-generation models that better simulate in vivo microenvironments could bridge this translational divide12,13,14.
We recently developed a 3D spheroid system optimized for AAV transduction to improve the translatability of AAV transduction assays. This system significantly enhances AAV transduction efficiency across multiple cell lines and AAV serotypes compared to the gold-standard 2D assays. In addition to the improved sensitivity and conservation of the product used in the assay, these models improve upon 2D monolayers by providing realistic cell-cell interactions that vary with cellular location within the spheroid. They also enable meaningful investigation of drug penetration that simulates actual organ conditions11,15,16. Furthermore, this spheroid approach offers practical advantages over more complex organoid or co-culture systems, as it uses standard laboratory equipment and avoids specialized medium or scaffold requirements. This simplicity ensures reproducibility and scalability, making it well-suited for routine gene therapy testing and vector development workflows17.
This 3D spheroid platform is particularly well-suited for applications requiring sensitive detection of AAV transduction at low doses and comparative evaluation of vector genome designs. It also supports assessment of serotype-dependent tropism. It is especially advantageous for cell lines with relatively low transduction efficiency that require high AAV doses in conventional 2D cultures. In these contexts, the enhanced sensitivity and improved physiological relevance of the 3D platform are critical. However, this system may be less appropriate for studies requiring immune components, vascular delivery, or full tissue complexity. This is because spheroids do not fully recapitulate the in vivo&#160;environment.
Thus, we present a straightforward 3D spheroid protocol optimized for AAV transduction assays, with two critical and complementary readouts: transduction efficiency (defined here as total reporter signal per spheroid) and transduction kinetics. This method provides a cost-effective, physiologically relevant, informative platform for AAV potency evaluation, vector design, and neutralization assays.

<table><tbody><tr><td>37C incubator</td><td>ThermoFisher Scientific</td><td>#51025983</td><td>Standard culture conditions</td></tr><tr><td>A375 cells</td><td>ATCC</td><td>CRL-1619</td><td>Cell line</td></tr><tr><td>AAV1 plasmid</td><td>Addgene</td><td>112862</td><td>AAV plasmid</td></tr><tr><td>AAV2 plasmid</td><td>Addgene</td><td>104963</td><td>AAV plasmid</td></tr><tr><td>AAV5 plasmid</td><td>Addgene</td><td>104964</td><td>AAV plasmid</td></tr><tr><td>AAV8 plasmid</td><td>Addgene</td><td>112864</td><td>AAV plasmid</td></tr><tr><td>AAV9 plasmid</td><td>Addgene</td><td>112865</td><td>AAV plasmid</td></tr><tr><td>BioTek GEN5 plate reader</td><td>Agilent Technologies</td><td>NA</td><td>End-point readout for fluorescence or luminescence</td></tr><tr><td>Centrifuge</td><td>ThermoFisher Scientific</td><td>#TSO-MX1R</td><td>Maintain spheroid suspension</td></tr><tr><td>Corning&amp;trade; Cell Culture Phosphate Buffered Saline (1X)</td><td>Fisher Scientific</td><td>#MT21040CMX</td><td>PBS to rinse cells</td></tr><tr><td>DMEM high glucose, GlutaMAX supplement, pyruvate</td><td>ThermoFisher Scientific</td><td>#10569010</td><td>Complete growth medium</td></tr><tr><td>Extracellular NanoLuc Inhibitor&amp;nbsp;</td><td>Promega</td><td>N235B</td><td>Quenches any Nanoluc&amp;reg; signal coming from the extracellular environment&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Sigma-Aldrich</td><td>#F4135-500mL</td><td>Provide growth factors</td></tr><tr><td>HEK293T Cells</td><td>ATCC</td><td>CRL-3216</td><td>Cell line</td></tr><tr><td>HeLa Cells</td><td>ATCC</td><td>CRM-CCL-2</td><td>Cell line</td></tr><tr><td>HEP3B Cells</td><td>ATCC</td><td>HB-8064</td><td>Cell line</td></tr><tr><td>HEPG2 Cells</td><td>ATCC</td><td>HB-8065</td><td>Cell line</td></tr><tr><td>Huh7 Cells</td><td>ABM</td><td>T8973</td><td>Cell line</td></tr><tr><td>IncuCyte S3 Inverted microscope</td><td>Sartorius</td><td>NA</td><td>Monitor spheroid formation/growth</td></tr><tr><td>IVIS Spectrum Imager</td><td>Spectralinvivo</td><td>NA</td><td>End-point readout for luminescence</td></tr><tr><td>Nano-Glo&amp;reg; Live Cell Assay&amp;nbsp;</td><td>Promega</td><td>N2013</td><td>Luminescent substrate reagent for live cell detection</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>ThermoFisher Scientific</td><td># 15140122</td><td>Prevent bacterial contamination</td></tr><tr><td>T75 Tissue Culture flasks with ventilation</td><td>Fisher Scientific</td><td>#NC0509296&amp;nbsp;</td><td>Flasks to grow cells</td></tr><tr><td>Trypsin-EDTA (0.05%)</td><td>ThermoFisher Scientific</td><td>#25300120</td><td>Enzymatic detachment of cells</td></tr><tr><td>Ultra-low attachment plates</td><td>Corning</td><td>#7007</td><td>Prevent cell adhesion to promote spheroid formation</td></tr></tbody></table>

evaluation of 3d spheroids for aav transduction studies

Evaluation of 3D Spheroids for AAV Transduction Studies

Adeno-associated viruses (AAVs) are potent vectors used for gene delivery in gene therapy products. Their development requires in vitro&#160;systems that can reliably detect differences in vector design, serotype performance, regulatory element strength, and expression kinetics. These systems must also support applications such as potency assessment and vector optimization. Here, we describe a streamlined three-dimensional spheroid platform optimized for evaluating AAV potency, transgene expression kinetics, and serotype-specific transduction efficacy across diverse cell lines. Uniform spheroids are generated using ultra-low attachment plates and maintained under conditions that support stable architecture and long-term imaging. Following AAV transduction, fluorescent or luminescent readouts are monitored in real time using live-cell imaging systems. This enables quantitative assessment of reporter signal, dose responsiveness, regulatory element activity, and onset time through continuous kinetic imaging. The platform effectively discriminates between potent and weak vector genome designs and among multiple AAV serotypes. This method demonstrates robust performance across both slowly and rapidly dividing cell lines. These results establish its utility as a scalable and physiologically relevant system for preclinical gene therapy evaluation and development.

Three-dimensional spheroid culture systems improve upon conventional 2D models for AAV gene therapy development. This methodology enables sensitive detection of AAV transduction at low doses, enhanced physiological relevance, and robust long-term monitoring. The platform supports potency assessment, high-throughput screening, and vector optimization across diverse cell lines and AAV serotypes.

Adeno-associated viruses (AAVs) are potent vectors used for gene delivery in gene therapy products. Their development requires in vitro&#160;systems that ...

We developed a sensitive, physiologically relevant 3D spheroid platform to evaluate AAV transduction activity and improve the prediction of vector performance for gene therapy. This protocol is applicable for evaluating AAV activity in vector potency testing, serotype screening, transgene optimization, neutralization studies, and in vitro preclinical gene therapy.To start, grow the cells in a T75 cell culture flask until they reach 70% confluency under standard conditions. After trypsinizing the cells, prepare a 1 to 10 dilution for routine cell passaging. Incubate the cells again until they reach 70% confluency, then harvest them. After assessing cell viability, thoroughly pipette the cell suspension to ensure it is well mixed. Perform two-fold serial dilutions of the cells in an ultra-low attachment 96-well plate, starting with 1 times 10 to the power of 4 cells per well in 200 microliters. Following the experimental layout shown, add 1 volume of complete medium to all rows except the top row. Then add two volumes of 1 times 10 to the power of 4 cells per well in the top row. Serially dilute row 1A by adding one volume of row 1A into row 1 B, and continue in the same manner. Centrifuge the ultra-low attachment plate at 350 x g for five minutes to pull the cells to the center and promote cell-to-cell contact. Incubate at 37 degrees Celsius, undisturbed for 24 to 48 hours for spheroid formation. Allow three days for the diameter to reach about 400 micrometers. Check spheroid size uniformity, ensuring cells are resuspended evenly and plates are flat. Monitor spheroid morphology. Select mature spheroids that exhibit tight cell-to-cell adhesion rather than loose cell aggregates.Once the cell suspensions are spun down, transfer the plate to a 37 degrees Celsius incubator equipped with a live-cell imaging instrument. Log in to the acquisition software. Select Schedule to acquire and Scan on schedule, then select Create vessel to New. Then choose scan type and select spheroid. Pick the channels of interest. Select phase with bright field. Then choose green or red. Set the desired magnification to 10x. Pick the plate model and its position in the drawer. Select the wells to image and enter the experiment name. For the analysis setup, select Differ analysis until later. Set scans to be added every six hours and scan indefinitely. Check daily for spheroid growth by logging into the imaging software. Select View recent scans and double-click on the desired experiment. After selecting Brightfield in the image channels panel, use the Measure image feature to measure the diameters of the spheroids. Remove the plate on the day of optimized spheroid formation and use it for adeno-associated virus, or AAV, transduction.Prepare a 96-well round bottom plate containing 297 microliters of 2% FBS-supplemented DMEM without antibiotics. Add 33 microliters of AAV stock to the medium to obtain a starting multiplicity of infection, or MOI, of 1 times 10 to the power of 6. Then prepare 10-fold serial dilutions by transferring 33 microliters of each dilution into 297 microliters of medium. Aspirate all medium from the previously prepared spheroid plate using a multichannel pipette. Transfer 100 microliters of the desired MOI of AAV from the dilution plate to the treatment plate using a multichannel pipette. Place the plates back into the imaging instrument. Start monitoring transduction by tracking green fluorescent intensity over 72 hours. Add 100 microliters of complete DMEM per well every three days to replenish nutrients. Place the plate back in the imaging instrument and start recording transduction efficiency every six hours. Analyze the data by creating an analysis definition specific to the cells and reporter genes of interest. Perform endpoint analysis using fluorescence readouts. Similarly, perform AAV transduction in 3D spheroids with luminescent reporters, followed by transduction analysis using bioluminescent imaging.Each cell line exhibited distinct morphological features and varying levels of compaction after three days of seeding. Hepatic cell lines formed more compact, denser spheroids than other cell lines. HEK293T formed a very round spherical appearance with a distinct necrotic core. A375 formed an irregularly shaped cell aggregate. Spheroids were highly susceptible to AAV transduction, with strong transduction detected at very low AAV titers across different cell lines for self-complementary GFP. Spheroid transduction of single-stranded GFP was observed at lower levels than self-complementary GFP. A 100-fold difference in relative fluorescence was observed between the self-complementary and single-stranded transgenes at a MOI of 10 to the power of 6 in HEK293T cells. A three-fold difference in relative fluorescence was observed between the self-complementary and single-stranded transgenes in HEP3B cells. All four AAV serotypes were successfully detected using both the fluorescent plate reader and the live-cell imaging system. AAV2 showed the highest transduction efficacy, followed by AAV1 in both cell lines. All AAV serotypes showed efficient transduction using the luminescent system, enabling precise differentiation of transduction efficacy. AAV2 was the most effective serotype in HeLa spheroids, while AAV9 had the lowest transduction efficacy. AAV2 self-complementary GFP transduction was detectable at infection rates as low as 10 to the power of 1, with a noticeable increase in signal after about 34 hours compared to untreated cells. The AAV2 single-stranded GFP signal was weaker and slower than the self-complementary GFP signal.The key challenge when performing this protocol is maintaining high cell viability, producing uniform, compact spheroids, and optimizing AAV dosing for each cell type and serotype. This protocol can assess transgene expression and functionality using RT-qPCR, Western blotting, ELISAs, and somatic activity assays, and onset time kinetic analysis. Future studies can incorporate co-cultures, organoids, microfluidics, immune cells, or vasculature to better mimic the in vivo microenvironment.

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Learn a 3D spheroid platform for evaluating AAV vector potency and transgene expression kinetics across diverse cell lines. This method enables quantitative, real-time assessment for gene therapy development.

Evaluation of 3D Spheroids for AAV Transduction Studies

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