Microfluidic alginate sponge activates and transduces T cells
The Lenti‑X T‑Cell Transduction Sponge (T‑cell sponge) is a modified version of our Lenti‑X Transduction Sponge that is optimized specifically for human T cells. The T-cell sponge expedites and streamlines T‑cell lentiviral transduction for research applications, including CAR T‑cell therapy (Agarwalla et al. 2022).
Benefits of the T‑cell sponge supported by the data below:
Expedited CAR T‑cell development by combining transduction and activation into one step
Reduction of CAR T-cell production time by days
Consistent activation across a range of multiplicity of infection (MOI) values
Increased or comparable transduction efficiency and cell viability compared to other transduction methods
Effective scaling (from 2 x 106–10 x 106 cells)
Minimal phenotypic and transcriptomic impact to the resulting cells
Reduction in the amount of virus required
Omission of T‑cell isolation by using PBMCs directly as T‑cell source in the T-cell sponge
Applications
Lentiviral transduction for:
T‑cell engineering
Gene editing
Gene and protein expression
Therapeutic model generation
Whole-genome screening
CAR T‑cell therapy research using lentivirus for gene modification
The T‑cell sponge is a dissolvable macroporous alginate matrix (Figure 1, Panel A) that allows users to activate and transduce T cells, achieving lentiviral transduction efficiency comparable or superior to traditional methods, with only one hands-on step. The alginate microfluidics system promotes transduction by facilitating colocalization of cells and lentiviral particles (Figure 1, Panel B) without the use of expensive instrumentation.
Figure 1. Structure and format of the T‑cell sponge. Panel A. The Lenti‑X T‑Cell Transduction Sponge is a lyophilized alginate-based cake with a structure of ~1 cm in diameter and ~0.5 cm in depth to which a mixture of cells and viruses are added, resulting in high lentiviral transduction efficiency. Every sponge features a complex microfluidic pore structure with pore sizes ranging from 20–300 µm. The scanning electron microscopy image shown is at 150X magnification. Panel B. Schematic showing the macroporous structure, which enables microfluidics-based colocalization of the cells and virus.
The T‑cell sponge is embedded with an optimized blend of T‑cell activation reagents (rhIL‑2 and anti-human CD3 and CD28 antibodies), allowing T‑cell activation and transduction in a single hands-on step. By incorporating the T‑cell sponge into workflows, users can avoid time-consuming spinoculation and harsh chemical transduction enhancers while minimizing cell handling, which can affect viability. Additionally, the effective colocalization of cells and lentiviral particles allows users to reduce the total amount of virus required, which saves users money by decreasing the cost per reaction.
Results
T‑cell activation remains consistent and comparable to other activation methods
The T‑cell sponge was evaluated for T‑cell activation while being transduced at multiple MOIs. This activation was compared against a CD3/CD8 conjugate method and an unstimulated control (Figure 2). Activation was quantified as the percentage of cells expressing CD69+.
Figure 2. T‑cell activation analysis at different MOIs. Activation, determined by CD69+ expression at 48 hr, was compared across an unstimulated control and two methods: CD3/CD28 conjugate and the Lenti‑X T‑cell Transduction Sponge (T‑cell sponge). The CD3/CD28 conjugate was evaluated at MOI 5; the T‑cell sponge was evaluated at MOI of 0, 5, and 10. An average across the samples for the T‑cell sponge was calculated (final column).
These data support the use of the T‑cell sponge for T‑cell activation with comparable or superior activation compared to the CD3/CD28 conjugate method.
T‑cell sponge shows improved transduction efficiency compared to spinoculation, which requires an additional activation step
Lentiviral transduction of T cells can be done via spinoculation, a centrifugation method that increases transduction efficiency by modulating cytoskeletal dynamics in response to centrifugal stress (Guo et al. 2011). However, spinoculation is time-consuming and can result in decreased cell viability.
The T‑cell sponge increases transduction efficiency by gently bringing viral particles and T cells closer together—without the need to expose cells to centrifugal force that can affect T-cell viability—while also activating T cells with its embedded CD3 and CD28 antibodies.
To compare the two methods (spinoculation vs. T‑cell sponge), transduction efficiency was evaluated at MOIs of 0, 5, and 10 over 11 days using GFP+ expression (Figure 3).
Figure 3. Transduction efficiency comparison between the T‑cell sponge and spinoculation with polybrene (Pb). Transduction efficiency was measured by GFP+ expression at MOIs of 0 (control), 5, and 10 at Days 2, 4, 7, 9, and 11. Day 7 data are listed above the 7‑day bar for both methods. The use of the T‑cell sponge consistently results in higher transduction efficiency compared to spinoculation and Pb at Days 7, 9, and 11.
The T‑cell sponge consistently results in higher transduction efficiency at Days 7, 9, and 11 compared to spinoculation, supporting the use of the T‑cell sponge as a gentle method to increase transduction efficiency, while also activating T cells with the embedded anti-human CD3 and CD28 antibodies.
T‑cell transduction efficiency remains consistent and high across a range of cell numbers
To ensure the efficacy of transduction for a wide range of cell number inputs, the T‑cell sponge was tested across a five-fold range (2 x 106–10 x 106 cells, Figure 4). GFP-expression data were collected at 2, 4, 7, and 9 days for the T‑cell sponge and for the cells transduced via spinoculation. For both transduction methods, the percentage of GFP-expressing cells increases with time and then plateaus (Day 2 shows the lowest percentage of cells expressing GFP, and Days 7 and 9 show the highest).
Figure 4. Transduction efficiency using the Lenti‑X T‑Cell Transduction Sponge during scaling. The T‑cell sponge was tested with input amounts from 2 x 106–1 x 107 cells. Spinoculation was tested at 4 x 106 and 1 x 107 cells. GFP-expression data were collected at 2, 4, 7, and 9 days for the T‑cell sponge and for the cells transduced via spinoculation. All reactions occurred at MOI 5.
The percentage of GFP-expressing cells following transduction remains consistent for 4 x 106–1 x 107 cell ranges using the T‑cell sponge. However, T cells transduced via spinoculation show considerable variation between the 4 x 106 –1 x 107 cell ranges. These data support the use of the T‑cell sponge for transduction across a wide range of inputs, which can all be accomplished in a single well. Less efficiently, transduction via plated spinoculation requires multiple wells of a 24‑well plate to maintain cell density requirements.
The advantages of the T‑cell sponge give researchers the flexibility to scale up or down and activate their T cells at the same time.
Sponge-transduced T cells exhibit a normal phenotype
Post-transduced cells were analyzed after nine days to determine if phenotype or cellular exhaustion markers differed between cells transduced via spinoculation and those transduced using the T‑cell sponge (Figure 5). The ratio of CD4+ to CD8+ cells was determined, and LAG‑3 and PD‑1 expression were used to evaluate cell exhaustion.
Figure 5. T cells transduced with the transduction sponge are phenotypically the same as spinoculated cells. 4 x 106 human primary T cells were mixed with a lentivirus expressing ZsGreen1 at the indicated MOIs and added to the Lenti‑X T‑Cell Transduction Sponge. Spinoculated samples were activated with a CD3/CD28 reagent for two days, then exposed to the lentivirus with polybrene (8 µg/ml), centrifuged at 1,500g for 90 min, and incubated overnight. Panel A. Activated and transduced cells were expanded out for nine days using a G‑Rex24 Well Plate and then analyzed for CD4 and CD8 expression using FACS analysis. Panel B. Activated, transduced, and expanded cells were also analyzed for the presence of exhaustion markers PD‑1 and LAG‑3 using FACS analysis.
The ratio of CD4+ and CD8+ T cells nine days post-transduction remains around 1:1 for T cells transduced with the T‑cell sponge. Spinoculation results in slightly more CD4+ cells than CD8+ cells. Nine days poststimulation exhaustion markers remain relatively low for cells transduced with either method.
These data indicate that the cells activated and transduced by the T‑cell sponge exhibit a normal phenotype and similar exhaustion markers compared to the cells transduced via spinoculation, which requires a separate activation step. While producing T cells with normal phenotypes and similar exhaustion markers to those prepared via spinoculation, the T cells prepared with the T‑cell sponge are ready in substantially less time.
Sponge-transduced T cells exhibit minimal transcriptomic impact
In addition to analyzing T‑cell phenotype, a transcriptomic analysis was also done to determine how transduction with the T‑cell sponge affected gene expression (Figure 6).
Figure 6. Cells transduced with the T‑cell sponge display similar gene expression changes to those transduced by standard methods. 4 x 106 human primary T cells were activated using one of the following methods for two days: T-cell sponge, a CD3/CD28 reagent (“Conjugate”), RetroNectin reagent + anti‑CD3 (“RN + anti‑CD3”), or anti‑CD3/CD28-coated beads (“Beads”). Cells were then transduced with a lentivirus expressing ZsGreen1 at an MOI of 5 in the presence of polybrene (8 µg/ml), followed by centrifugation at 1,500g for 90 min. For the T‑cell sponge transduction, 4 x 106 T cells were mixed with the same lentivirus (MOI = 5), incubated within the sponge for two days, and then released. Data were collected 48 hr post-transduction. RNA was isolated using the SMART‑Seq mRNA LP kit and sequenced on an Illumina® NextSeq® instrument. Heat map and clustering are based on the top 200 most-differentially expressed genes for each activation/transduction condition.
The heat map and hierarchical clustering show differentially expressed genes in activated and transduced cells at 48 hours post-transduction for four activation/transduction methods (Figure 6). The data indicate no significant difference in the expression pattern of the top 200 differentially expressed genes, indicating that the T‑cell sponge is both an effective and transcriptionally inert tool for transducing T cells.
PBMCs can be used as the T‑cell source, saving days during a workflow
Peripheral blood mononuclear cells (PBMCs), obtained from a standard blood draw, contain T cells. The process for isolating T cells from PBMCs for transduction is labor intensive and expensive. The T‑cell sponge is able to remove that isolation step by selectively activating and transducing T cells directly from a PBMC sample.
The efficacy of the T‑cell sponge from PBMC samples was evaluated by comparing cell subtypes pre‑ and post-transduction (Figure 7).
Figure 7. T cells can be specifically activated and transduced when starting with PBMCs with the T‑cell sponge. 1 x 107 unstimulated PBMCs were combined with lentivirus encoding ZsGreen1 at an MOI of 3 and 12, and then directly applied to a T‑cell transduction sponge. After 48 hr, the cells were released from the sponge for further culture and analysis. Panel A. The composition of the PBMC population was assessed by FACS prior to lentiviral transduction with the sponge. Panel B. Transduction efficiency and cell phenotypes were analyzed at 48 hr post-transduction by FACS for ZsGreen1 expression and cell phenotypes.
The data show that the sponge effectively and selectively activates and transduces T cells specifically from PBMC samples, eliminating the need for T‑cell isolation and potentially saving days in the T‑cell engineering and validation workflow.
Conclusions
The data presented in this technote support the Lenti‑X T‑Cell Transduction Sponge as an effective tool for T‑cell activation and transduction, allowing users to achieve transduction efficiency gains without the use of additional activation steps or chemical enhancers, which can affect cell viability. The T‑cell sponge also minimizes hands-on time and allows users to start from PMBC samples.
T‑cell transduction efficiency is better or comparable to difficult and time-consuming spinoculation and does not require an additional activation step
Sponge-transduced T cells do not require an additional activation step
PBMCs can be used as the T‑cell source, saving days during a workflow
T‑cell activation remains consistent and is comparable to other activation methods
T‑cell transduction efficiency remains consistent and high across a range of cell numbers
Sponge-transduced T cells exhibit a normal phenotype
Sponge-transduced T cells exhibit minimal transcriptomic impact
References
Agarwalla, P. et al. Bioinstructive implantable scaffolds for rapid in vivo manufacture and release of CAR‑T cells. Nat. Biotechnol.40, 1250–1258 (2022).
Guo, J., Wang, W., Yu, D. & Wu, Y. Spinoculation triggers dynamic actin and cofilin activity that facilitates HIV-1 infection of transformed and resting CD4 T cells. J. Virol.85, 9824–9833 (2011).
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