Rapidly dissolving biomaterials for high-efficiency viral transduction

Abstract

Cell and gene therapy represent the frontier of genetic medicine for treating devastating diseases, yet they continue to face a critical bottleneck: inefficient genetic cell modification. While viral vectors remain our most powerful genetic delivery tools, their application suffers from significant transport insufficiencies with most viral particles wasted before reaching their cell targets. This fundamental challenge undermines the efficiency, safety, and economic viability of these potentially transformative therapies. Current transduction enhancers provide only partial solutions with significant drawbacks. RetroNectin works only with retroviruses and hematopoietic cells, spinoculation is time consuming and laborious, while polycationic polymers like polybrene pose toxicity concerns that preclude clinical applications. In this report, we introduce DUCTS (Dissolving Ultrafast Cell Transduction Sponges), a transduction enhancer based on uncrosslinked alginate cryogels that works within five minutes and completely dissolves, enabling rapid and easy workflows. DUCTS demonstrates broad versatility, boosting cell transduction across a variety of viral vectors (gamma retrovirus, lentivirus, and adeno-associated virus) in both suspension and adherent cells. Notably, DUCTS achieves comparable transduction efficiency while reducing viral concentration requirements by an order of magnitude compared to standard protocols. This cell- and virus-agnostic platform streamlines gene transfer procedures, reduces viral consumption and associated costs, and enhances the accessibility and scalability of cell and gene therapies.

Statement of significance:

In this report, we present DUCTS (Dissolving Ultrafast Cell Transduction Sponges), a major advance to improve transduction technology, addressing critical bottlenecks in cell therapy manufacturing. Unlike existing methods that require lengthy procedures, specialized equipment, or toxic additives, DUCTS achieves comparable transduction efficiency in just five minutes using a simple, dissolving alginate sponge. This platform works across multiple virus types (lentivirus, retrovirus, AAV) and cell types, dramatically reducing viral vector requirements by 10-fold while maintaining cell viability. A modified DUCTs can also simultaneously activate and transduce T cells in a single step to revolutionize CAR-T cell manufacturing. With exceptional shelf stability and GMP-compatible materials, DUCTS offers a practical solution to reduce costs, accelerate production timelines, and enhance accessibility of life-saving cell therapies for patients worldwide.

1. Introduction

Cell and gene therapies are revolutionizing treatment in a wide range of diseases previously considered untreatable [1,2]. From Chimeric Antigen Receptor (CAR) T cell treatments for oncology and autoimmune diseases [3–5] to engineered hematopoietic stem cells (HSCs) for blood disorders [6–9], efficient gene delivery to target cells remains critical [10,11].

While viral vectors offer permanent and efficient reprogramming after infection, their clinical application faces significant challenges in achieving highly efficient transduction [12,13]. The fundamental challenge in viral transduction stems from the complex dynamics of viral-cell interactions and rapid viral degradation [14–16]. Viruses require close contact with cells, but can only travel short distances by Brownian motion [16], resulting in inefficient delivery that necessitates high viral doses and leads to viral wastage. This inefficiency creates both economic and practical barriers to widespread clinical adoption.

Current approaches to improve transduction efficiency have significant limitations. Cationic polymer additives such as polybrene and protamine enhance viral binding and entry [17,18], but have undesired cytotoxicity [19]. Microfluidic systems effectively address transport issues by constraining cells and viruses [20–22] but require specialized equipment and hardware, limiting scalability. These constraints have resulted in limited clinical translation, with only a few reagents – including fibronectin fragment FN CH-296 (RetroNectin) [23,24] and LentiBOOST – used clinically for viral transduction. However, RetroNectin only promotes transduction of hematopoietic cells with retroviruses and shows reduced efficiency with other viruses and cell types [25] and while LentiBOOST works well for lentivirus, it requires careful optimization for different applications. Some transduction enhancers such as Vectofusin-1 are approved for clinical manufacturing [26], but their effectiveness varies with different viral pseudotypes and they can aggregate in certain media [27]. These issues create a continued need for optimized, user-friendly, and biocompatible transduction systems that work consistently across a broad range of cells and virus types.

Our previous work addressed some of these challenges through the development of a biomaterial-based macroporous 3D-sponge (Drydux) for efficient transduction of T cells [28]. Drydux enhanced cell transduction, increasing the percentage of successfully transduced primary T cells from a baseline of ~10 % without Drydux to >80 % with Drydux. This enhancement rivals the “gold-standard” transduction methods of lengthy low-speed centrifugation on RetroNectin-coated plates (spinoculation). Drydux also improved ease of handling, leading to its commercialization by Takara Bio. In addition, Drydux enabled same-day CAR T cell manufacturing [29]. However, Drydux has important limitations: it requires incubating cells in the sponge, followed by dissolution with chelators (i.e. EDTA). This incubation causes processing delays and raises concerns regarding Drydux-cell mechanical interactions [30–32]. The dissolution may also cause undesired toxicity if chelation factors are not fully removed [33,34].

In this report, we present DUCTS (Dissolving Ultrafast Cell Transduction Sponges), a “next generation” scaffold designed to overcome the limitations of previous approaches. DUCTS create transient microfluidic flow without requiring crosslinking reagents, facilitating both cell-virus binding and rapid dissolution [35] without any chemical agents. Our results establish that DUCTS promotes viral adhesion within five minutes, maintains stability for at least one year, and works across a wide variety of cells including both adherent and non-adherent cells and virus types such as lentivirus, gamma retrovirus and adeno-associated (AAV) virus. Most importantly, DUCTS achieve equivalent viral transduction to mixing methods at 10-fold lower viral amounts, potentially addressing both efficiency and cost barriers in cell and gene therapy manufacturing.

2. Methods

2.1. Preparation of DUCTS

A 1 % alginate solution was made by dissolving low molecular weight (<70 kDa) alginate (Pronova UP VLVG, Co: Novamatrix) in sterile-filtered deionized (DI) water. The solution was then added to 2 mL microcentrifuge tubes. The tubes were then frozen vertically at −20 °C overnight and lyophilized for 72 hrs. DUCTS was stored at 4 °C in the refrigerator and kept dry until further use.

2.2. Preparation of antibody-conjugated alginate

To prepare DBCO conjugated antibodies, a 10-fold molar excess of Dibenzocyclooctyne-PEG4-N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS, A134–10, Click Chemistry Tools, USA) was added to anti-CD3 (Bio X Cell BE0001–2, clone OKT-3, 2 μg/μL) or anti-CD28 antibodies (Bio X Cell BE0291, clone CD28.2, 2 μg/μL) and incubated at room temperature for 1 h. Then the solution was purified by Amicon centrifugation (MWCO 10 kDa) at 10,000 g for 10 mins until the flow through was free from DBCO-PEG4-NHS (measured by characteristic absorbance of DBCO moiety at 309 nm using Nanodrop). The degree of DBCO incorporation (i.e. the number of DBCO per antibody) was determined from the absorbance scan of the purified conjugate (235-400 nm) using the following equation.

$$\frac{\mathit{Molarity.DBCO}}{\mathit{Molarity.antibody}}=\frac{A_{309}\mathit{DBCO}\times {\epsilon }_{280}\mathit{Ab}}{{\epsilon }_{309}\mathit{DBCO}\times A_{280c}\mathit{Ab}}$$

• A₃₀₉ DBCO = DBCO-Ab conjugate’s absorbance at 309 nm

• ε₂₈₀ Ab = 210,000M⁻¹cm⁻¹

• A_280C Ab = conjugate’s corrected absorbance at 280 nm = A₂₈₀ - (A₃₀₉ x CF DBCO)

• ε₃₀₉ DBCO = 12,000M⁻¹cm⁻¹

• A₂₈₀Ab = DBCO-Ab conjugate’s absorbance at 280 nm

• CF DBCO = DBCO correction factor at 280 nm = 1.089

To synthesize azide-conjugated alginate [36,37], low molecular weight (<70 kDa) alginate (Pronova UP VLVG, Co: Novamatrix) (4 μmol, 1 eq.) was dissolved overnight in 200 mL MES buffer (100 mM MES, 300 mM NaCl, pH 6.5). Azide-PEG4-Amine (1.6 mmol, 400 eq., Lumiprobe-1868) was added to the solution and the mixture was stirred for an additional 1 hour at room temperature. A mixture of 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (1.6 mmol, 400 eq.) and sulfo-N-hydroxysuccinimide (sNHS) (0.8 mmol, 200 eq.) was added in three equal doses eight hours apart and the solution was stirred for an additional eight hours. The solution was dialyzed against 4 L of water with successively lower salt content, changing solution 2–3 times per day. Dialyzed solutions were frozen and lyophilized under high vacuum. To determine the degree of substitution, azide-alginate samples were analyzed using DOSY NMR to assess purity and ¹H NMR spectra was recorded at 600 MHz using a protocol published by our lab [38].

To prepare antibody conjugated scaffolds, a 2 % solution of azide-alginate in sterile-filtered deionized (DI) water was incubated with DBCO-modified anti-CD3 and anti-CD28 antibodies (1 μg Ab/mg alginate) at 4 °C overnight to allow for click conjugation. Recombinant human interleukin (IL)-2 (PeproTech), at a concentration of 0.2 μg/mg alginate was next added, and the solution was stirred for 15 mins. Finally, the resulting solution was diluted to 1 % solution with equal volume sterile-filtered deionized (DI) water for 15 mins, cast in 48 well plates (0.3–0.6 mL/well) or 2 mL microcentrifuge tubes (0.5 mL – 1 mL/tube), frozen at −20 °C overnight and lyophilized. Scaffolds were stored at 4 °C before use.

2.3. Scanning electron microscopy

DUCTS was sectioned using a sharp razor and subsequently coated with a 70 nm layer of AuPd (60 % Au, 40 % Pd) over 10 min at a rate of 7 nm/min. Analysis of the scaffold’s surface morphology was performed using a Hitachi S-3200 N Variable Pressure Scanning Electron Microscope (SEM).

2.4. Cell lines

Primary human T cells were collected from the peripheral blood of a healthy donor and were isolated from a buffy coat (Gulf Coast Regional Blood Center) using Lymphoprep medium (Accurate Chemical and Scientific Corporation) and cryopreserved in a freeze medium consisting of 50 % HyClone fetal bovine serum (GE Healthcare), 40 % RPMI-1640, and 10 % dimethyl sulfoxide (DMSO) (Sigma). Prior to use, the cells were thawed, resuspended in 9 mL of complete media, and centrifuged at 400 g for 5 min to remove DMSO. Cells were maintained in complete T cell media (50 % Click’s Medium (Irvine Scientific) and 50 % RPMI-1640 supplemented with 10 % HyClone fetal bovine serum (GE Healthcare), 2 mmol/L GlutaMax (Gibco), penicillin (100 units/mL), and streptomycin (100 mg/mL; Gibco)) and were then activated on plates coated with 1 μg/mL of CD3 (Miltenyi Biotec, 130–093–387, clone OKT-3) and CD28 (BD Biosciences, 555,725, clone CD28.2) agonistic monoclonal antibodies for 72 h. IL-7 (10 ng/mL) and IL-15 (5 ng/mL) was added at 48 h.

Murine T cells were isolated from spleen of C57 BI/6 J mice following negative selection and activated using mouse Dynabeads (Gibco) for 24 h in presence of IL-2 (40 units/mL). Murine T Cells were maintained in murine T cell media (RPMI-1640 (Gibco), 10 % FBS (HyClone), 2 mM GlutaMAX, 100 μM β-mercaptoethanol, 100 unit/mL of Penicillin and 100 μg/mL of streptomycin).

Primary canine T cells were maintained in complete canine T cell media (cTCM) prepared with Advanced RMPI 1640 (Gibco) supplemented with 10 mmol/L HEPES (pH 7.3, ThermoScientific), 2 mmol/L L-Glutamine (Gibco), 100 units/mL Penicillin and 100 units/mL Streptomycin (Gibco), and 10 % Fetal Bovine Serum (Cytiva, HyClone Laboratories). Canine T cells were collected from the peripheral blood of a healthy donor by North Carolina State University’s Lab Animal Resources veterinary team and were isolated using Lymphoprep medium (Accurate Chemical) and cryopreserved in a freeze medium consisting of 50 % FBS, 40 % Complete canine T cell media, and 10 % DMSO (Sigma). Prior to use, the cells were thawed, suspended in 9 mL cTCM, and centrifuged at 400 g for 5 min to remove DMSO. Cells were resuspended in warm cTCM and allowed to rest overnight in a humidified incubator, 38.8 °C, 5 % CO₂. Cells were then activated on plates coated with anti-canine CD3 antibodies (clone CA17.6F9, gifted by Peter Moore, UC Davis) and anti-canine CD28 antibodies (clone 5B8, purified into PBS from hybridoma gifted by Brian Hayes, Fred Hutchinson Cancer Research Center) for 72 h in the presence of IL-2 (200 units/mL) (Peprotech).

Daudi (B-lymphoblast), A20 (Mouse B cell lymphoma) and Raji (B-lymphoblast) cells were maintained in RPMI 1640 (Gibco) supplemented with 10 % fetal bovine serum (Gibco), 2 mmol L – 1 GlutaMax (Gibco), penicillin (100 units mL⁻¹) and streptomycin (100 mg mL⁻¹; Gibco).

HEK293T (human embryonic kidney cells), C28/I2 (human chondrocytes), B16-OVA (mouse melanoma), PANC-1 (human pancreatic carcinoma), GL261 (mouse glioma) and CT2A (mouse astrocytoma/glioma) cells were maintained in DMEM (GIBCO) supplemented with 10 % fetal bovine serum (Gibco), penicillin (100 units mL⁻¹) and streptomycin (100 mg mL⁻¹; Gibco).

Retroviral producer cell lines were maintained in IMDM supplemented with 10 % fetal bovine serum (Gibco), penicillin (100 units mL⁻¹), and streptomycin (100 mg mL⁻¹; Gibco).

All cell lines were maintained at 37 °C with 5 % CO₂ except for canine cells which were maintained at 38.8 °C with 5 % CO₂.

2.5. Retrovirus production

Green Fluorescent Protein (GFP)-encoding gamma retrovirus was prepared using a stably transfected FLYRD118 packaging cell line (gift from Dotti lab, UNC–CH) [39]. For retroviral production, cells were plated in T75 or T175 flasks and incubated until ~90 % confluency in complete IMDM media. After 24 h, the virus-containing supernatant was collected, filtered (0.45 μm), aliquoted, and snap-frozen at −80 °C. A second collection was performed after 48 h. CD19-chimeric antigen receptor (CAR)-encoding retrovirus was prepared using transient transfection described previously. Briefly, 2.5 × 10⁶ 293T cells were seeded in 10 cm cell culture dish and transfected with the plasmid mixture containing CD19 CAR transfer plasmid, the Peg-Pam-e packaging plasmid encoding MoMLV gag-pol, and the RDF plasmid encoding the RD114 envelope, using the GeneJuice transfection reagent (Merck Millipore). To generate murine retrovirus 293T cells were transfected with retroviral vector and the pCL-Eco plasmid encoding gag/pol/env. Supernatants containing the retroviruses were collected 48 and 72 h after transfection and filtered with 0.45 μm filters.

2.6. Viral titer and multiplicity of infection (MOI)

The viral titer was determined using a standard flow cytometry assay. HEK293T cells were exposed to serial dilutions of the viral stocks (1X, 10X, 50X, 100X, 1,000X dilutions), and after 72 h, GFP expression was evaluated by flow cytometry. The viral titer (transducing units (TU) per mL) was calculated for populations with 5–20 % GFP+ cells using the equation: Titer (TU/mL) = (initial cell count * %GFP+)/(volume of virus * dilution factor). MOI was calculated as the ratio of the number of transducing viral particles used to the actual number of cells.

2.7. Transduction by conventional methods

Gold-standard transduction with gamma retrovirus was done by spinoculation on RetroNectin coated plates following standard procedure [29]. Transduction efficiency for retroviral experiments was assessed by flow cytometry after 72 h.

To transduce cells with lentivirus, cells were mixed with lentivirus in presence of 8 μg/ml polybrene (Sigma) or without any transduction enhancer. For spinoculation with polybrene, cell-virus mixture was plated, and plate was centrifuged at 1000 g for 90 min. Next day, cells were washed to remove polybrene and cultured for additional 3 days and transduction efficiency was determined using flow cytometry.

Transduction with AAVs (adeno-associated virus), serotype 6, was done by mixing 1 × 10⁶ cells and virus (MOI 1 × 10⁴) in 50 μL of media and pipetting on a 24 well plate. Seeded plates were incubated for 20 min in a 5 % CO2 incubator at 37 °C, and then supplemented with additional 2 mL media. Transduction efficiency was assessed by flow cytometry at 24 h.

For no enhancer groups, viral supernatant was concentrated ten-fold by Amicon centrifugation (MWCO 100 kDa, Millipore) at 4 °C, 2500 g, 15–20 min, where applicable. Concentrated virus was mixed with 1 million cells at MOI 4 for lentiviruses and retroviruses or MOI 1 × 10⁴ for AAV in 50 μL media and pipetted onto a 24 well plate. Seeded plates were incubated for 20 min in a 5 % CO2 incubator at 37 °C. Then the wells were supplemented with additional 2 mL media.

2.8. DUCTS mediated transduction

Viral supernatant was concentrated ten-fold by Amicon centrifugation (MWCO 100 kDa, Millipore) at 4 °C, 2500 g, 15–20 min, where applicable. Concentrated virus was mixed with 1 million cells at MOI 2–4 for lentiviruses and retroviruses or MOI 1 × 10⁴ for AAV in 50 μL media and pipetted onto the top of DUCTS. Control scaffolds i.e. scaffolds with no virus were seeded with 1 million cells suspended in cell culture medium. Seeded scaffolds were incubated at room temperature for 5 min. 1 mL of media was added to each scaffold and incubated for 2 min at room temperature. The solution was then mixed with a pipette until the entire scaffold was fully dissolved. The solution was transferred to a cell culture plate and an additional 1 mL of media was added.

All samples were then kept in a 5 % CO2 incubator at 37 °C. After 1–3 days of culture, cells were analyzed for GFP/CAR expression by flow cytometry. >95 % of cells were viable.

2.9. Characterization of CAR T cells

To determine transduction stability, CAR T cell phenotype and their functionality against tumor cells, CAR T cells generated using DUCTS or RetroNectin and spinoculation method were cultured in complete T cell media. Cells were assessed for their immunophenotypic composition to determine the percentages of stem cell memory (SCM) (CCR7+, CD45RA+), central memory (CM) (CCR7+, CD45RA−), effector memory (EM) (CCR7−CD45RA−), and effector (Eff) (CCR7−CD45RA+) phenotypes. To assess the ability of CAR T cells to kill CD19 positive tumor cells, Daudi cells were co-cultured with CAR T cells generated using either method. Briefly, 50,000 tumor cells were cocultured with CAR T cells at 1:1 and 1:5 effector to target (E:T) ratios with the number of CAR T cells normalized based on transduction efficiency. No normalization was done in “no virus” controls, which used T cell numbers without any adjustments. On day 5 of co-culture, cells were collected, T cells were stained for CD3, and Daudi cells were stained for CD20. Dead cells were gated out using live/dead stains, and percentages of residual tumor cells were assessed.

2.10. Flow cytometry

All samples were analyzed using a flow cytometer, and a minimum of 10,000 events were acquired per sample. Viability Dye (Sytox) was added before acquisition. Transduced cells were analyzed by gating on FSC singlets, viable cells and GFP/CAR positive cells (Supplementary Fig. 9). Results were analyzed using Flow Jo 9 (FlowJo LLC).

2.11. Statistical analysis

All statistical analysis was done using two-tailed Student’s t-test, or multiple comparisons one way ANOVA test using GraphPad prism and noted in figures with p-values.

2.12. Figure preparation

Schematic figures were created using Biorender under the following publication licenses: https://BioRender.com/gxsc071; https://BioRender.com/lt72iug; https://BioRender.com/8re3o1r; https://BioRender.com/a2m832k.

3. Results

3.1. Development of DUCTS – non-crosslinked dissolving sponges for cell transduction

Although a limited number of other materials enhance transduction [39], our prior work on Drydux scaffolds focused mainly on calcium-crosslinked alginate cryogels as the primary scaffold material [28,35,40,41]. In that system, cells and viruses are combined and then seeded onto dry, macroporous sponges. We selected alginate for its Good Manufacturing Production (GMP)-compliant, FDA-approved, and Generally Recognized as Safe (GRAS) status. Alginate materials offer broad biocompatibility and low toxicity, enabling their use as both in vitro reagents and in vivo implantable materials.

Through systematic investigation of the material properties underlying the Drydux phenomenon, we identified three critical factors that govern transduction enhancement: sponge hygroscopy, macroporosity and rapid liquid absorption into the sponge [28,35]. These insights led us to explore alternative approaches to both increase imbibition rate and reduce the required incubation times of cells within the scaffold.

We reasoned that lower calcium concentration would reduce scaffold integrity, thereby increasing absorption speeds. Our previous studies demonstrated that between 0.1 and 0.3 % w/v, calcium concentration had minimal impact on performance [35], leading us to explore sponges entirely lacking crosslinker. In addition, we reasoned that uncrosslinked porous alginate would also dissolve quickly, enabling rapid cell recovery and obviating the need for de-crosslinking agents such as EDTA.

Based on these insights, we developed non-crosslinked DUCTS through a streamlined manufacturing process. A 1 % low molecular weight alginate (VLVG) solution was added to 2 mL centrifuge tubes, frozen at −20 °C, and then lyophilized (Fig. 1A) to produce DUCTS. Scanning electron microscopy (SEM) of the dry DUCTS revealed a highly interconnected network with 100–200 μm pores (Fig. 1B), providing ideal architecture for rapid fluid imbibition and cell-virus collisions. This architecture of DUCTS provides a simple workflow for cell transduction with minimal hands-on time. The cell/virus solution is added onto DUCTS, where it is rapidly absorbed, followed by gentle resuspension to ensure complete dissolution and effective cell transduction (Fig. 1C, Supplementary video 1).

Fig. 1.

Dissolving ultrafast cell transduction sponges (DUCTS). (A) schematic for synthesis of DUCTS through freezing and lyophilization of low molecular weight alginate. (B) SEM image of DUCTS surface showing a loose matrix of pores. C) Transduction workflow using DUCTS. The simple workflow allows for the transduction of cells by simply loading cells and virus, absorption of the cell/virus solution, and resuspending in media to fully dissolve. D) Cell transduction of primary human T cells using CD19 CAR-encoding gamma retrovirus with (purple) and without (red) DUCTS. Gold-standard low-speed centrifugation (spinoculation) on RetroNectin-coated plates (green) shown for comparison. Non-transduced cells served as negative controls (black). P values calculated by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Data represent mean ± S.E.M.

We benchmarked DUCTS performance against the current gold standard for retroviral transduction: spinoculation with RetroNectin [17,42], which requires multiple-hour centrifugations on RetroNectin-coated plates. Negative controls included cells and virus mixed in a droplet equivalent in volume to the loading volume of DUCTS, as well as cells added to DUCTS without virus. As shown in Fig. 1D, at equivalent retrovirus doses (MOI = 2), DUCTS achieved transduction efficiencies comparable to gold-standard spinoculation. Importantly, cells incubated with DUCTS showed >90 % viability (Supplementary Fig. 1) and this transduction was stable over 15 days (Supplementary Fig. 2).

DUCTS also significantly enhanced viral transduction in G-Rex flasks, specialized cell culture vessels with gas-permeable silicone membranes that enable high-density cell expansion and previously required off-column spinoculation [43] (Supplementary Fig. 3). We observed reduced cell proliferation compared to samples spinoculated outside the G-Rex column. We attribute this reduction to incomplete DUCTS dissolution, as the G-Rex protocol employed gentle addition of media to DUCTS, without the rigorous pipetting and mechanical agitation used in the tube-based method. Optimizing the dissolution procedure in G-Rex flasks may improve cell expansion outcomes.

We also assessed the proliferation, T cell subsets, phenotype and cytotoxicity of CAR T cells generated using DUCTS and compared these to conventional methods (RetroNectin and spinoculation) used in the clinics. Over 15 days of culture, DUCTS generated CAR T cells showed similar proliferation profile compared to conventional methods (Supplementary Fig. 4). Looking at CAR T cell composition, less expanded cells showed nearly equal %CD4 and %CD8 population and cells expanded for longer duration showed higher (~60 %) CD8 percentages compared to CD4 percentages across groups (Supplementary Fig. 4A–B) as previously reported [40,41]. Further looking at the phenotypes of unexpanded and expanded cells, we found similar percentages of naive/stem cell memory, central memory, effector memory and effector populations across DUCTS and conventionally generated cells (Supplementary Fig. 4C–D). Additionally, CAR T cells produced using either method demonstrated similar tumor cell killing against CD19+ Daudi cells when co-cultured at 1:1 and 1:5 E:T ratios (Supplementary Fig. 4F).

3.2. DUCTS works across volumes, seeding times, alginate types and is stable for over one year

We evaluated the effect of DUCTS volume and incubation times for transducing activated primary human T cells with gamma retrovirus. The testing protocol involved adding a suspension of cells and virus particles to different DUCTS volumes, allowing for complete absorption, incubating for specified time periods, and finally diluting with cell culture media to fully dissolve DUCTS and isolate cells. Systematic time course analysis revealed that DUCTS-mediated transduction was suboptimal without any incubation, but a brief five-minute incubation proved sufficient for highly efficient cell transduction (Fig. 2A). Extended incubation times beyond five minutes yielded only marginal improvements in transduction efficiency. Notably, although the largest DUCTS volume (1.5 mL) appeared to enhance transduction even without deliberate incubation, we observed that these larger scaffolds required more time to fully absorb the liquid, effectively creating an incubation period even at the nominal “zero” timepoint.

Fig. 2.

DUCTS mediated transduction is dependent on incubation time but independent of alginate type. A) Transduction of activated human T cells seeded onto DUCTS with GFP-encoding retrovirus. Different DUCTS volumes and incubation times were tested. B) Transduction of activated human T cells with GFP-encoding retrovirus on DUCTS made from different alginate varieties. C) Shelf life of DUCTS at 4 °C as assessed by transduction efficiency after one year of storage. Activated human T cells were transduced on DUCTS with GFP expressing gamma retrovirus. P values calculated by one-way ANOVA with Tukey’s correction. Data represent mean ± S.E.M.

To determine the optimal composition for DUCTS, we next assessed a variety of alginate formulations with different guluronic acid to mannuronic acid (G/M) ratios and viscosities (Supplementary Table 1) for their ability to enhance viral transduction. As shown in Fig. 2B, DUCTS-mediated transduction enhancement was largely independent of alginate composition, and only some loss of enhancement was observed with the smallest molecular weight (MW) material and lower guluronic ratio (G/M ratio ≤ 1). This compositional flexibility represents a significant advantage for manufacturing scale-up and quality control.

Furthermore, DUCTS demonstrated exceptional shelf stability, maintaining equivalent transduction enhancement after storage at 4 °C for one year with no loss in transduction efficiency (Fig. 2C), highlighting its potential for commercial applications.

3.3. DUCTS works through facilitating cell-virus adhesion

Having established that DUCTS enhances transduction within just five minutes and fully dissolves after this brief incubation, we sought to better understand its mechanism of action. Given the very short incubation times, we hypothesized that DUCTS primarily enhances cell-virus adhesion, rather than affecting downstream processes like viral fusion, internalization, or genome integration. To test this hypothesis, we conducted parallel retroviral transduction experiments on DUCTS using a 5-minute incubation at either 4 °C or at room temperature (25 °C) [44].

To isolate the specific impact of DUCTS on cell-virus adhesion, we centrifuged and washed the 4 °C samples after incubation to remove soluble polymer and unbound virus (Fig. 3A). As positive controls, cells incubated with DUCTS at room temperature were not washed, but were directly diluted with 1 mL media. Both sample groups were subsequently cultured for 72 h to allow time for viral internalization, genome integration and protein expression. DUCTS rapidly absorbed the cell/virus solutions and dissolved at both temperatures.

Fig. 3.

DUCTS enhances transduction through facilitating viral binding. A) Schematic of experiment. At 4 °C, viruses can bind to cells, but fusion and cell entry is restricted. B) Transduction with DUCTS at 4 °C and removing unbound virus only slightly reduced cell transduction, suggesting DUCTS enhances viral binding to cells, rather than fusion or downstream activities such as reverse transcription, nuclear transport or integration.

Notably, the five-minute incubations at both 4 °C and 37 °C resulted in robust transduction enhancement (Fig. 3B). Although a small difference in transduction enhancement was noted and was statistically significant, the substantial level of transduction achieved after just five minutes at 4 °C – conditions that support viral adhesion, but minimize entry or other downstream processes [26] – strongly suggests that DUCTS primarily enhances viral adhesion to target cells. This rapid enhancement of viral binding at low temperatures represents a significant advantage for transduction of highly sensitive cell types that may be compromised by extended processing times or temperature fluctuations.

3.4. DUCTS transduces cells across a range of cell numbers and concentrations

We next assessed the effects of loading volume as well as cell/virus concentrations on transduction efficiency. With calcium-crosslinked materials (Drydux) we previously reported that transduction efficiency is reduced with increasing liquid volumes [35]. Activated human T cells and retrovirus particles were suspended in 25–500 μL of media (1 million cells, MOI=4) and added on to 1 mL DUCTS. At volumes below 100 μL, transduction efficiency did not decrease, in contrast to what was seen with Drydux. However, higher loading volumes (200 μL, 500 μL) decreased transduction efficiency (Supplementary Fig. 5). We also explored the number of cells that could efficiently be transduced in a single, 1 mL DUCTS. DUCTS efficiently transduced 0.5–10 million T cells with retrovirus (MOI=4) by scaling the loading volume but keeping cell/virus concentration the same (1 million cells and 4 million TUs of virus per 50 μL). We suspect that at high volumes, scaffold collapse and dissolution becomes too fast, abrogating the effects of the pores. Having said that, even the 500 μL volume (10 million cells and 40 million TUs of virus) showed some transduction enhancement (Supplementary Fig. 6).

3.5. DUCTS enhances transduction across multiple virus and cell types

Having established DUCTS enhances retroviral transduction of human T cells (Fig. 1D, 4A), we next assessed the versatility of DUCTS-enhanced transduction by testing its performance with additional viruses and cell types.

Fig. 4.

DUCTS can transduce cells with a wide variety of viral vectors. A-C) Human T cells were transduced by mixing on plates or on DUCTS with gamma retrovirus (A), lentivirus (B) or AAVs (C) encoding for GFP. Cells were analyzed for gene expression by flow at 72 h (A and B) or 24 h (C). P values calculated by one-way ANOVA with Tukey’s correction. Data represent mean ± S.E.M.

We first tested whether DUCTS could enhance transduction of murine and canine T cells using CAR-encoding retrovirus (Supplementary Figure 7). Interestingly, we observed species-specific enhancement effects, with DUCTS outperforming spinoculation on RetroNectin coated plates in murine T cells but underperforming in canine T cells.

Next, we tested two alternative viral vectors: lentivirus and adeno-associated viruses (AAVs). With VSVG-pseudotyped lentiviruses, DUCTS significantly enhanced T cell transduction compared to simple mixing, the most common clinical transduction protocol (Fig. 4B). DUCTS also enhanced lentiviral transduction of a wide variety of adherent and non-adherent cell lines (Supplementary Figure 8).

When tested with AAVs, DUCTS enhanced T cell transduction over simple mixing with serotype 6 AAVs. Simple mixing with AAVs is reported to have poor transduction with primary T cells, especially at low MOIs (1 × 10⁴ vg/cell) [45], so this enhancement is important and the results were striking: transduction efficiency on DUCTS was 7-fold higher than with simple mixing (Fig. 4C), demonstrating substantial enhancement for this clinically relevant vector.

Collectively these results demonstrate that DUCTS consistently enhances cell transduction processes across multiple viral vectors and cells, highlighting its versatility as a transduction enhancement platform.

3.6. DUCTS substantially reduces viral doses needed for cell transduction

A critical factor limiting widespread implementation of adoptive cellular therapies remains the high cost viral vectors [46,47]. Having established that DUCTS significantly increases the transduction efficiency at equal viral doses, we next investigated whether DUCTS could enable comparative transduction at reduced viral concentrations. We transduced human T cells with a serial dilution of lentivirus using either DUCTS or simple mixing, and quantified transduction efficiency across the dose range. We aimed at a transduction efficiency of 30 %, in line with many clinical trials [48–50]. The results were compelling: DUCTS achieved transduction efficiencies similar to those of simple mixing at a 10-fold lower viral dose (Fig. 5), presenting a compelling economic case for DUCTS as an enabling technology that could substantially reduce manufacturing costs for adoptive cell therapies.

Fig. 5.

DUCTS transduces cells at 10 fold lower virus doses and demonstrates consistent transduction across varying cell numbers. A) Transduction of activated human T cells with different dilutions of virus with and without DUCTS analyzed at 72 h. Data represent mean ± S.E.M. B) Transduction of different numbers of T cells on a 1 mL DUCTS sample.

3.7. Simultaneous T cell activation and transduction by modified T cell specific DUCTS

Current manufacturing protocols for adoptive cell therapies require separate T cell activation prior to viral transduction, introducing significant time and complexity to the manufacturing process [51]. Conventional transduction methods are particularly ineffective for non-activated peripheral blood mononuclear cells (PBMCs), which remain largely refractory to viral transduction [52,53]. Developing streamlined methods to directly transduce non-activated PBMCs would eliminate separate steps like activation and spinoculation-mediated transduction, reducing overall processing time.

To address this challenge, we tested whether DUCTS could be functionalized to simultaneously activate and transduce T cells in a single step. We developed immunologically active DUCTS by conjugating anti-CD3 and anti-CD28 antibodies directly to the alginate polymers through click chemistry [29], creating T cell-specific activation scaffolds (T-DUCTS). These modified polymers were supplemented with IL-2 (0.2 μg/mg alginate) and formulated into DUCTS. As demonstrated in Fig. 6 and Supp. Fig. 10 functionalized DUCTS enabled activation and direct transduction of unstimulated PBMCs with lentiviral vectors. Notably, the T cell-specific DUCTS exhibited substantially higher transduction efficiency compared to previously published calcium crosslinked scaffolds [29]. These innovations represent a significant advance in T cell engineering technology by consolidating activation and transduction into a single step while maintaining or improving gene delivery efficiency. The resulting streamlined manufacturing process could substantially reduce production costs, processing times, and complexity for adoptive cell therapies.

Fig. 6.

DUCTS can be modified to simultaneously activate and transduce T cells. A) Schematic of modified DUCTS that can simultaneously activate and transduce T cells. B) T cell specific DUCTS facilitate simultaneous activation and transduction of unstimulated T cells. Unstimulated human PBMCs were combined with CAR-encoding lentivirus and seeded onto DUCTS. CAR expression in T cells was analyzed at 72 h. Controls groups include no virus and no scaffold, no virus on a T-DUCTS scaffold, antibody in solution with no scaffold, DUCTS (no conjugated antibody) and a calcium crosslinked, antibody-conjugated material. P values calculated by one-way ANOVA with Tukey’s correction. Data represent mean ± S.E.M.

4. Discussion

Viral vectors constitute the cornerstone of gene therapy, with AAV, lentiviral and retroviral vectors leading clinical applications due to their distinct advantages in different therapeutic contexts [10]. Yet despite its promise, the rapidly expanding cell and gene therapy field faces critical vulnerabilities: manufacturing bottlenecks, high costs, limited viral vector supply, and inconsistent transgene integration. These aren’t merely theoretical challenges – they’ve triggered actual treatment delays for cancer patients awaiting CAR T cell therapies [46,47].

The development of DUCTS emerged from a key observation: brief cell-virus contact on a calcium-crosslinked (DryDux) scaffold could achieve significant transduction [28,35,39,41]. By strategically eliminating the calcium crosslinks used in our previous approach, DUCTS leverages rapid liquid absorption and promotes viral-cell binding, facilitating robust gene delivery. This simple yet effective approach achieves clinical-level transduction efficiencies of large numbers of cells (10 million) while using 10-fold less virus.

DUCTS offers several distinctive advantages that address current industry challenges. First, ~10-fold reduction in viral vector requirements directly addresses manufacturing bottlenecks and cost concerns. Second, DUCTS versatility across vector platforms and cell types enables standardization of manufacturing protocols to reduce regulatory complexity and accurate clinical translation. Third, antibody-functionalized DUCTS eliminates entire manufacturing steps by combining T cell activation and transduction into a single operation, reconceptualizing the manufacturing workflow for T cell therapies. Finally, the rapid absorption and dissolution steps in DUCTS workflows make them fully compatible with automated, closed-system bioreactor platforms, such as the Prodigy (Miltenyi Biotec), Cocoon (Lonza) devices, and G-Rex (Wilson Wolf) flasks, which are increasingly adopted in clinical cell therapy production. DUCTS’ minimal handling requirements could facilitate rapid, scalable, and GMP-compliant manufacturing processes.

Beyond simplified workflow, the superior performance of antibody-modified DUCTS compared to previous calcium-crosslinked scaffolds [29] reflects a fundamental theoretical advance. Unlike static scaffolds that constrain cells within rigid structures, DUCTS creates a dynamic microenvironment that transitions from solid to liquid. This transformation liberates antibody-conjugated polymers to engage with T cells in their natural suspended state, enhancing activation and subsequent viral entry.

In summary, DUCTS is a versatile, vector-agnostic platform offering a simplified, scalable, and cost-effective approach to gene delivery. Its year-long stability and independence from alginate composition and cell number ensure robust performance across numerous applications. Furthermore, building upon the established commercial success of Drydux, DUCTS presents a practical solution for streamlining cell therapy manufacturing. As the field continues to grow, DUCTS’ ability to reduce viral vector consumption while maintaining high transduction efficiency positions it as a promising platform to enhance the accessibility and scalability of cell and gene therapies.

Supplementary Material

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.actbio.2025.12.047.