Name: Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices (Video) | JoVE Journal
Uploaded: 2025-12-05T12:50:00.000+00:00
Duration: 9 min 30 s
Description: Learn a G-Rex protocol for scalable CAR-T and CIK cell expansion using GMP-compatible devices. This method enables high-density cultures with minimal intervention and robust clinical viability for adoptive cell therapy applications.

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The CAR construct was provided under MTA by St. Jude Children&#39;s Research Hospital. This work was supported by ScaleReady G-Rex Grant 2025; University of Padova - SOMMAGGIO_BIRD24_01; AIRC Foundation under 5x1000 2019 - ID. 22759 program; Alleanza Contro il Cancro, Progetto CAR-T_2 RCR-2023-23684268. This research received &#34;Ricerca Corrente&#34; funding from the Italian Ministry of Health to cover publication costs. Figures were created with GraphPad Prism 9 Software and BioRender.com.

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This protocol outlines the implementation of an efficient and scalable platform for the generation of two different ACT products, namely CAR-T and CIK cells, from PBMCs using G-Rex culture systems. We implemented proof-of-concept functional and phenotypic assays to validate the scalability of the approach. We showed that both phenotype and activity of the expanded cells are maintained during scale-up, ensuring the system suitability for research applications and direct clinical translation. Indeed, we successfully demonstrated that both CAR-T and CIK cells can be efficiently expanded with high cell yield, while maintaining a functional effector phenotype and potent antigen-specific cytotoxicity across different culture devices.
Combined with the gas-permeable membrane technology of the culture devices, this expansion protocol leads to faster expansion kinetics, reduces manual handling, and allows consistent high yields across donors. These features make the protocol highly reproducible and less labor-intensive compared to standard culture systems20,21, which often involves multiple media exchanges and increased risk of cell loss and contamination.
Moreover, this protocol enables seamless transition across manufacturing scales: from small-scale open systems (G-24, suitable for preclinical research and screening, for example, of CAR constructs, cytokine combinations, or donor variability) to larger-scale open systems (G-6M, for pre-GMP validation), and ultimately to larger-scale closed systems (for GMP production). As described in this study, the progression from small-scale to larger-scale open systems requires only proportional adjustments of volumes and cell input relative to device size, which represents a significant practical advantage. Likewise, the only implementation needed for the transition from open to closed systems for GMP manufacturing, and thus to adapt the G-6M protocol to large-scale GMP production, is utilizing appropriate GMP-grade reagents and closed system devices (G-10M-CS with a surface area of 10 cm2, corresponding to a single G-6M well, or G-100M-CS with a surface area of 100 cm&#178;, corresponding to the G-100M open counterpart) without any further modification of the protocol itself. These closed devices differ from their open counterparts in the integration of sterile tubing connections for automated media handling, which work in conjunction with a liquid handling pump (GatheRex) to create a fully closed environment. This closed system approach eliminates manual media exchanges and reduces contamination risks, making it ideally suited for GMP-compliant manufacturing environments where sterility and reproducibility are paramount. Notably, scalability represents a critical feature for bridging the gap between early-stage discovery and clinical translation, particularly in academic or early biotech settings with limited resources.
Further implementation of this protocol toward full GMP compliance should consider transitioning from FBS-containing media to serum-free/xeno-free formulations, aligning with current regulatory trends from the FDA and EMA, in order to eliminate batch-to-batch variability, remove potential xenogeneic contamination risks, and enhance regulatory compliance27.
Moreover, the minimal intervention required by the proposed expansion system provides an ideal platform for implementing multi-dimensional phenotypic profiling28 by reducing procedure-induced artifacts that might confound phenotypic analysis during manufacturing procedures. Additionally, beyond CAR-T and CIK cells, the system could be highly adaptable to other lymphocyte populations and engineered immune cells, broadening its utility in immunotherapy development29,30.
Although the protocol is robust and does not require modifications, a few steps are particularly critical for success. For CIK cells expansion, the timing of cytokine addition is essential to ensure optimal differentiation and expansion. For CAR-T cells, the transduction should occur at 24 h after activation to maximize efficiency, and lentiviral aliquots should be thawed only once, as repeated freeze-thaw cycles significantly reduce transduction efficiency31.
Biological variability among primary cell preparations naturally contributes to differences in expansion rates and functional activity, reflecting donors&#39; diversity. This inevitable and intrinsic variability can be effectively managed by performing parallel expansions from multiple donors, ensuring robust and reproducible outcomes across experiments.
In conclusion, this protocol enables rapid, scalable, and reliable expansion of functional effector lymphocytes directly from PBMCs, offering flexibility for both early-phase research and large-scale clinical translation. This system can be readily adapted to various cell types and genetic modifications, providing a versatile platform for immunotherapy development.

Adoptive cell therapy (ACT) has become an important pillar of advanced therapies for cancer treatment. This approach is based on administering ex vivo expanded cellular products to achieve anti-tumor efficacy1. To date, many cell types have been used in ACT, ranging from antigen-aspecific approaches, e.g., natural killer (NK)2, cytokine-induced killer (CIK)3,4, natural killer T (NKT)5, or γδ T cells6, to highly specific and personalized ones, such as tumor-infiltrating lymphocytes (TILs)7 and engineered cell-based therapies, e.g., chimeric antigen receptor (CAR)1,8 and T cell receptor (TCR) cells9,10.
CAR-T cells are genetically modified T cells generated by ex vivo viral transduction to express a synthetic immune receptor designed to recognize a specific molecular target. The CAR structure consists of an antigen-specific single-chain variable Fragment (scFv) antibody combined with intracellular signaling domains, including the TCRζ chain and costimulatory molecules. Upon antigen engagement, the intracellular domain delivers activation signals that promote T cell proliferation and antitumor activity11.
CIK cells are a heterogeneous population of activated T lymphocytes that acquire expression of CD56 during ex vivo expansion from peripheral blood mononuclear cells (PBMCs) or umbilical cord blood (UCB)12. This expansion involves stimulation with Interferon-γ (IFN-γ), anti-CD3 monoclonal antibody, and Interleukin-2 (IL-2), followed by culture in IL-2 for 14 days. CIK cells functionally resemble both NK and T cells, exerting antitumor activity through both major histocompatibility complex (MHC)-independent and dependent mechanisms13,14,15,16. These properties allow CIK cells to be used in both allogeneic and autologous therapeutic settings with minimal graft-versus-host disease (GvHD) risk17.
In clinical practice, both CAR-T and CIK cell therapies require the administration of large numbers of cells. CAR-T cells are infused at 50-200 million doses per patient18. Similarly, CIK cells are typically given at doses of up to 10 billion cells per infusion, with patients often receiving multiple infusions depending on the treatment protocol19. Traditional cell culture methods using flasks or plates are inefficient, time-consuming, and require frequent passaging, making the large-scale expansion unfeasible and increasing the risk of contamination. Automated or semi-automated systems using bag cultures or bioreactors allow for large-scale expansion but have limitations, i.e., shear stress or the need for density adjustments and frequent medium perfusion20,21, and the handling of one batch at a time, leading to high costs in both academic and industrial contexts. In this challenging situation, an alternative is represented by the gas-permeable rapid expansion (G-Rex) device, a static culture method featuring a silicon membrane at the bottom of the well, which ensures efficient oxygen exchange and increased height of the vessels, allowing for high-volume medium capacity. Thus, the system supports cell growth without medium replacement or density adjustment22,23. G-Rex-M systems have demonstrated scalability up to 5000 mL and are compatible with automated harvest systems, providing a contamination-free and closed culture platform that is easily translatable from academic to clinical settings for the development of advanced therapy medicinal products (ATMPs)24.
In the outlined procedure, we leveraged the advantages and characteristics of G-Rex devices to optimize the expansion protocol of both CAR-T and CIK cells. We demonstrated that both cell types can be efficiently expanded and scaled up, maintaining a consistent phenotype and functional profile across different culture systems. A schematic overview of the expansion protocol for both CIK and CAR-T cells is shown in Figure 1.
<img alt="CAR-T and CIK cell protocols diagram showing PBMCs activation, lentivirus engineering, cell expansion." class="xfigimg" src="/files/ftp_upload/69369/69369fig1v2.jpg" data-altupdatedat="1784001604000" data-alt="Figure 1"> 
Figure 1: Overview of CAR-T and CIK cell expansion protocols using G-24 or G-6M culture devices. CAR-T and CIK cells are expanded from isolated PBMCs starting on Day 0. For the expansion of CAR-T cells, PBMCs are stimulated on Day 1 with anti-CD3 and anti-CD28 antibodies, genetically engineered on Day 2, and subsequently cultured with hIL-7 and hIL-15 supplementation every 3-4 days, with final harvest for functional assays on Day 9. In the G-6M format, cytokine supplementation is limited to Day 2, with no additional cytokine feeding required throughout the culture period. For the expansion of CIK cells, PBMCs are stimulated on Day 1 with rhIFN-γ and on Day 2 with anti-CD3 antibody and rhIL-2, which is then provided every 3-4 days. In G-24, a medium change is required concurrently with the cytokine supplement. CIK cells are harvested on Day 14 for downstream functional assays. <a href="https://www.jove.com/files/ftp_upload/69369/69369fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Brilliant Stain Buffer</td><td>BD Biosciences</td><td>566349</td><td></td></tr><tr><td>CD28 Antibody, anti-human</td><td>Miltenyi Biotech</td><td>130-093-375</td><td>Clone 15E8</td></tr><tr><td>CD3 Antibody, anti-human</td><td>Miltenyi Biotech</td><td>130-093-387</td><td>Clone OKT3</td></tr><tr><td>CD3 BV510</td><td>BD Biosciences</td><td>740147</td><td>Clone UCHT1</td></tr><tr><td>CD314 APC (NKG2D)</td><td>Biolegend</td><td>320808</td><td>Clone 1D11</td></tr><tr><td>CD4 BV650</td><td>BD Biosciences</td><td>563875</td><td>Clone SK3</td></tr><tr><td>CD45RA PerCP-Cy5.5</td><td>BD Biosciences</td><td>563429</td><td>Clone HI100</td></tr><tr><td>CD56 PE</td><td>Biolegend</td><td>318306</td><td>Clone HCD56</td></tr><tr><td>CD62L FITC</td><td>BD Biosciences</td><td>555543</td><td>Clone FREG-56</td></tr><tr><td>CD8 BV450</td><td>BD Biosciences</td><td>562428</td><td>Clone RPA-T8</td></tr><tr><td>CellTrace Violet Cell Proliferation Kit</td><td>ThermoFisher scientific</td><td>C34557</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>ThermoFisher scientific</td><td>D2650</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Phosphate Buffered Saline (D-PBS)</td><td>Sigma Aldrich</td><td>D5652</td><td></td></tr><tr><td>E-plate 16</td><td>Agilent</td><td>5469813001</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS), Value</td><td>ThermoFisher Scientific</td><td>A5256701</td><td></td></tr><tr><td>Fixable Viability Stain 780 (FVS780)</td><td>BD Biosciences</td><td>565388</td><td></td></tr><tr><td>G-Rex 24 Well Plate&amp;nbsp;</td><td>Wilson Wolf</td><td>80192M</td><td></td></tr><tr><td>G-Rex 6M Well Plate&amp;nbsp;</td><td>Wilson Wolf</td><td>80660M</td><td></td></tr><tr><td>HEPES Buffer Solution</td><td>Lonza</td><td>ECM0180D</td><td></td></tr><tr><td>His Antibody</td><td>Miltenyi Biotech</td><td>130-120-718</td><td></td></tr><tr><td>Human Albumin 20%</td><td>CSL Behring</td><td></td><td></td></tr><tr><td>Human IL-15 Recombinant Protein</td><td>PeproTech</td><td>200-15</td><td></td></tr><tr><td>Human IL-7 Recombinant Protein</td><td>PeproTech</td><td>200-07</td><td></td></tr><tr><td>LSRFortessa X-20</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Lymphoprep</td><td>STEMCELL Technologies</td><td>18060</td><td></td></tr><tr><td>MCF-7</td><td>ATCC</td><td>HTB-22</td><td>Breast cancer cell line&amp;nbsp;</td></tr><tr><td>Penicillin and Streptomycin</td><td>Lonza</td><td>ECB3001D</td><td></td></tr><tr><td>Physiological solution (0.9% Sodium Chloride)</td><td>Monico</td><td></td><td></td></tr><tr><td>Protamine Sulfate</td><td>Sigma Aldrich</td><td>P3369</td><td></td></tr><tr><td>Raji</td><td>ATCC</td><td>CCL-86</td><td>Burkitt&#039;s lymphoma cell line&amp;nbsp;</td></tr><tr><td>Recombinant Human IFN-gamma</td><td>R&amp;amp;D Systems</td><td>285-IF-100/CF</td><td></td></tr><tr><td>Recombinant IL-2 (Proleukin)</td><td>Novartis</td><td></td><td></td></tr><tr><td>rhCD19-His</td><td>Invitrogen</td><td>A42607</td><td></td></tr><tr><td>Roswell Park Memorial Institute 1640 (RPMI-1640)</td><td>Euroclone</td><td>ECB9006L</td><td></td></tr><tr><td>Stable L-Glutamine</td><td>Lonza</td><td>ECB3004D</td><td></td></tr><tr><td>Trypan-Blue solution</td><td>Sigma Aldrich</td><td>T8154</td><td></td></tr><tr><td>xCELLigence Real-Time Cell Analysis</td><td>Agilent</td><td></td><td></td></tr><tr><td>X-VIVO 15 Serum-free Hematopoietic Cell Medium</td><td>Lonza</td><td>02-053Q</td><td></td></tr></tbody></table>

efficient and rapid generation of car-t and cytokine-induced killer cells in gmp-scalable devices

Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices

Adoptive cell therapies (ACT) have progressed rapidly in recent years, playing a pivotal role in cancer immunotherapy and significantly influencing treatment outcomes. A critical step in cell therapy development, at both preclinical and clinical stages, is the manufacturing process. Conventional static culture methods require frequent cell manipulation and are often limited by reduced gas exchange and nutrient supply, which can compromise cell yield and phenotype. Here, we present a step-by-step, reproducible, and scalable protocol for the ex vivo expansion of two ACT products, namely Chimeric Antigen Receptor (CAR)-T and Cytokine-Induced Killer (CIK) cells, using Gas-permeable Rapid expansion (G-Rex) devices. G-Rex devices are specifically designed to enhance nutrient exchange and support high-density cultures with minimal user intervention. Among various ACT approaches, CAR-T cells have demonstrated remarkable success in treating hematological malignancies, facilitating a rapid advance from experimental research into clinical applications. In addition to CAR-T cells, CIK cells have also been widely applied in&#160;clinical trials due to their characteristic phenotype and the absence of treatment-related adverse events and Graft-versus-Host Disease (GvHD). This protocol outlines the key technical steps for both cell types, starting with the isolation and seeding of peripheral blood mononuclear cells (PBMCs). Briefly, PBMCs are stimulated with anti-CD3/CD28 antibodies, followed by lentiviral transduction to expand CAR-T cells, or with Interferon-&#947; (IFN-&#947;), anti-CD3 antibody, and Interleukin 2 (IL-2) to obtain CIK cells. Flow cytometry is employed to monitor the viability and phenotype of the cells, and functional assays are performed to confirm the therapeutic potential of the cell product and to validate the scalability of the approach. Overall, this protocol provides a practical solution for producing large numbers of effector cells in a preclinical setting, facilitating clinical application with several immune cell populations. Finally, it offers a scalable solution for high-yield effector cell manufacturing.

The protocol described here provides an efficient and robust workflow for the ex vivo expansion and engineering of effector cells from isolated PBMCs, namely CAR-T and CIK cells. This method requires minimal cell manipulation and takes advantage of the gas-exchange capacity of G-Rex devices, providing superior nutrient availability and oxygenation compared to conventional static culture systems23,24. A key advantage of this protocol is its inherent scalability from small-scale 24-well devices (G-24), suitable for preclinical studies, to larger 6M devices (G-6M), compatible with translational or clinical manufacturing needs, without requiring any changes to the protocol other than proportional scaling.
Using this protocol, both CAR-T and CIK cells were successfully expanded in parallel from healthy donor PBMCs in G-24 and G-6M formats, as described in Sections 2-4. At the end of the culture period, both cell types exhibited high fold expansion and viability, with comparable proliferation between the two device formats (Figure 2, Supplementary Table 1, Supplementary Table 2), highlighting the reproducibility of the protocol across different culture scales.
To validate the scalability of the culture devices, effector cells were further characterized phenotypically and functionally. For the CD19 CAR-T cells, we quantified CAR expression and evaluated the CD4+ and CD8+ subsets, noting a predominance of CD19 CAR+ CD8+ T cells (Figure 3A-B, Supplementary Table 1). For CIK cells, we assessed the frequency of T (CD3+CD56-), NK (CD3-CD56+), and CIK (CD3+CD56+) populations (Figure 3D), as well as the expression of the activating receptor NKG2D (Figure 3E, Supplementary Table 2). Moreover, we observed overall comparable phenotypes and expression of specific population markers between the G-24 and G-6M culture formats for both CAR-T and CIK cells. The differentiation status of both CD19 CAR-T and CIK cell products was analyzed by CD62L and CD45RA staining (Figure 3C-F, Supplementary Table 1, Supplementary Table 2), revealing similar memory and effector phenotypes between cells cultured in G-24 and G-6M formats. These findings demonstrate that scaling up the process does not compromise the phenotypic integrity or differentiation status of the expanded effector cell populations.
Lastly, to evaluate functional potency, we employed target-specific cytotoxicity assays. Both CD19 CAR-T and CIK cells expanded in either device exhibited potent cytolytic activity, confirming the maintenance of functional properties across scale (Figure 4). Overall, these results support the suitability of the expanded products for downstream applications in preclinical models and potentially clinical testing.
<img alt="CD19 CAR-T and CIK cells bar graph; fold increase, viability analysis in immunotherapy research." class="xfigimg" src="/files/ftp_upload/69369/69369fig2.jpg" data-altupdatedat="1784001604000" data-alt="Figure 2"> 
Figure 2: Proliferation and viability ofCD19 CAR-T and CIK cells. CAR-T cells (n = 5) and CIK cells (n = 6) expanded from PBMCs in G-24 and G-6M plates were collected on Day 9 and Day 14 of expansion, respectively, to perform cell count and evaluation of the (A-C) fold increase and (B-D) percentage of viable cells. Histograms represent the mean expression ± standard deviation (SD), and dots indicate single donors (see Supplementary Table 1 and Supplementary Table 2 for individual values). Data was analyzed by two-tailed multiple paired t-test (A-C) or multiple two-tailed Wilcoxon matched-pairs rank test (B-D), with Holm-Sidak correction for multiple comparisons (*p &lt; 0.05). <a href="https://www.jove.com/files/ftp_upload/69369/69369fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="CD19 CAR-T cells analysis; bar charts on cell subpopulations and phenotypes; data comparison G-24/G-6M." class="xfigimg" src="/files/ftp_upload/69369/69369fig3v2.jpg" data-altupdatedat="1784001604000" data-alt="Figure 3"> 
Figure 3: Assessment of the phenotype of CD19 CAR-T and CIK cells expanded in G-24 and G-6M devices. The expression of CAR molecules on CD19 CAR-T cells (n = 5) was analyzed by flow cytometry to assess (A) the transduction efficiency in G-24 and G-6M expansion protocols, as well as the expression of (B) CD4+/CD8+ and (C) naïve/memory subpopulations in the CD3+CAR+ subset. The phenotype of bulk CIK cells (n = 6) was analyzed by flow cytometry to investigate (D) the percentage of CIK (CD3+CD56+), NK (CD3-CD56+) and T (CD3+CD56-), as well as the expression of (E) NKG2D and (F) naïve/memory subpopulation in the CD3+CD56+ subset (G-24 n=5, G-6M n=6). Naïve (CD45RA+CD62L+), Central Memory (CM, CD45RA-CD62L+), Effector Memory (EM, CD45RA-CD62L-), and Effector Memory CD45RA-positive (EMRA, CD45RA+CD62L-). Histograms represent the mean expression ± SD, and dots indicate single donors (see Supplementary Table 1 and Supplementary Table 2 for individual values). Data was analyzed by two-tailed multiple paired t-test (B-C-D-F) or two-tailed Wilcoxon matched rank test (A-E), with Holm-Sidak correction for multiple comparisons (*p &lt; 0.05). <a href="https://www.jove.com/files/ftp_upload/69369/69369fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="CAR-T cell therapy efficacy; bar and line graphs; specific lysis in Burkitt's lymphoma, breast cancer." class="xfigimg" src="/files/ftp_upload/69369/69369fig4.jpg" data-altupdatedat="1784001604000" data-alt="Figure 4"> 
Figure 4: Evaluation of CD19 CAR-T and CIK cell cytotoxicity. (A) Specific lysis of CD19 CAR-T cells (orange, n = 5) or untransduced (UTD) cells (gray, n = 5) against the Burkitt's lymphoma Raji cell line (as a suspension cell line model) was performed by a 6 h flow cytometry-based killing assay. Results are shown as the mean specific lysis ± SD. (B) Cytotoxicity of CIK cells cultured in either G-24 (light blue, n = 7) or G-6M (dark blue, n=6) against the breast cancer MCF-7 cell line (as an adherent cell line model) was monitored every 15 min over 24 h using a real-time cell analyzer. Results are shown as the mean specific lysis ± SEM. Data was analyzed by Mann-Whitney test, Holm-Sidak correction for multiple comparisons (*p &lt; 0.05). <a href="https://www.jove.com/files/ftp_upload/69369/69369fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Gating strategy used for flow cytometry analyses. Representative plots illustrating the gating strategies for (A) CAR-T cell and (B) CIK cell phenotypic characterization, and (C) fluorescent cell dye-based cytotoxicity assay. <a href="https://www.jove.com/files/ftp_upload/69369/Suppl_Figure%201_RE.pdf" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Growth, viability, and phenotype of CD19 CAR-T cell cultures from individual donors. Total cell number, fold increase, viability, and phenotypic characterization of CD19-CAR T cells harvested on Day 9 of culture in G-24 and G-6M devices are shown for each donor. <a href="https://www.jove.com/files/ftp_upload/69369/Suppl_table_1_RE.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Table 2: Growth, viability, and phenotype of CIK cell cultures from individual donors. Total cell number, fold increase, viability, and phenotypic characterization of CIK cells harvested at Day 14 of culture in G-24 and G-6M devices are shown for each donor. <a href="https://www.jove.com/files/ftp_upload/69369/Suppl_table_2_RE.xlsx" target="_blank">Please click here to download this File.</a>

This protocol describes a scalable method for the expansion of Adoptive Cell Therapies (ACT) products, namely Chimeric Antigen Receptor (CAR)-T cells and Cytokine-Induced Killer (CIK) cells, using Gas-permeable Rapid expansion (G-Rex) devices, demonstrating efficient and robust growth in a clinically relevant setting.

Adoptive cell therapies (ACT) have progressed rapidly in recent years, playing a pivotal role in cancer immunotherapy and significantly influencing ...

We describe a G-Rex protocol for the efficient and scalable expansion of adoptive cell therapy products, including CAR-T and CIK cells. The core challenge lies in setting up a protocol to support high-density cultures all while ensuring minimal user intervention and robust clinical scalability. To begin, resuspend 2 times 10 to the power of 6 freshly isolated peripheral blood mononuclear cells in 2 milliliters of complete culture medium, supplemented with 5%FBS referred to as CCMF.Transfer the suspension into the wells of a 24-well G-Rex culture vessel. Add 4 microliters of anti-CD3 antibody and 20 microliters of anti-CD28 antibody directly into each well to activate the T cells and incubate at 37 degrees Celsius with 5%carbon dioxide. After overnight stimulation, harvest and count the activated T cells using the Trypan Blue exclusion assay to assess viability, calculate 1 times 10 to the power of 6 viable activated cells to seed for each well of the G-Rex culture vessel.Transfer the cells into a tube and centrifuge at 300 g for 5 minutes. Now, prepare the transduction medium for each well by combining the required lentiviral vector volume with 5 microliters of an 8 milligram per milliliter stock solution of protamine sulfate. Use CM medium supplemented with 5%FBS and without penicillin or streptomycin to bring the total volume to 0.8 milliliters per well.Then, resuspend the pelleted cells in 0.8 milliliters of the prepared transduction medium and transfer the suspension into a new well of the G-Rex culture vessel. Perform the same procedure for the untransduced control condition, avoiding the addition of the lentiviral vector. Incubate the cells overnight at 37 degrees Celsius with 5%carbon dioxide.On day 2, increase the culture volume to a total of 8 milliliters per well by adding 7.2 milliliters of fresh CCMF supplemented with human interleukin-7 and interleukin-15. Next, on day 5, refresh the culture medium by removing 6 milliliters of conditioned medium from each well without disturbing the cells at the bottom. Replace it with 6 milliliters of fresh CCMF containing human interleukin-7 and interleukin-15.On day 9, gently remove and discard 5 milliliters of conditioned medium from each well. Using a 1000-microliter pipette, resuspend the CAR-T cells in the remaining 3 milliliters of medium and transfer them to a 15-milliliter tube. Count the CAR-T cells and check their viability using the Trypan Blue exclusion assay.Seed 1 times 10 to the power of 6 freshly isolated peripheral blood mononuclear cells for each well of the G-24 culture vessel in 8 milliliters of CCM supplemented with recombinant human interferon-gamma. Incubate the culture vessel at 37 degrees Celsius with 5%carbon dioxide. On day 1, after 22 to 24 hours, add 4 microliters of anti-CD3 antibody directly into each well and 4 microliters of recombinant human interleukin-2.Incubate the cells again at 37 degrees Celsius with 5%carbon dioxide. Then, on days 5, 9, and 13, remove 6 milliliters of conditioned medium from each well without disturbing the settled cells. Replace it with 6 milliliters of fresh CCM supplemented with recombinant human interleukin-2.On day 14, gently remove and discard 5 milliliters of conditioned medium from each well. Resuspend the cytokine-induced killer cells in the remaining 3 milliliters of medium and transfer them into a 15-milliliter tube. Assess the total cell number and viability using the Trypan Blue exclusion assay.For CAR-T cell expansion, perform activation of peripheral blood mononuclear cells on day 0 and lentiviral transduction on day 1 as demonstrated earlier. Maintain all concentrations and seeding densities but scale up all volumes to match the G-6M device format. On day 2, bring the total culture volume in each well to 100 milliliters using CCMF medium, calculate and add human interleukin-7 and human interleukin-15 to reach final concentrations of 10 nanograms per milliliter each.On day 9, remove approximately 90 milliliters of medium from each well using a serological pipette. Resuspend the CAR-T cells, the remaining 10 milliliters of medium, and transfer the cell suspension to a 15-milliliter tube for subsequent assays. For CIK cell expansion, perform seeding of peripheral blood mononuclear cells on day 0 following the CIK cell expansion protocol described previously.On day 1, bring the total culture volume to 100 milliliters using CCM and add recombinant human interleukin-2 and anti-CD3 antibody. Every 3 to 4 days, supplement each well with recombinant human interleukin-2 at a final concentration of 500 international units per milliliter without changing the medium. On day 14, remove approximately 90 milliliters of medium from each well using a serological pipette.Finally, resuspend the cytokine-induced killer cells in the remaining 10 milliliters of medium and transfer the cells into a 15-milliliter tube for further assays. Both CD19 CAR-T and CIK cells expanded in parallel from healthy donor peripheral blood mononuclear cells in G-24 and G-6M devices exhibited high fold expansion and viability with comparable proliferation between the two device formats. For CD19 CAR-T cells, CAR expression and the CD4 and CD8 positive subsets were comparable in G-24 and G-6M devices.For the bulk CIK cell population, the frequency of T, NK, and CIK populations as well as the expression of the activating receptor NKG2D were equal across scales. The differentiation profile of CD19 CAR-T and CIK cells, including naive, central memory, effector memory, and effector memory CD45RA-positive subpopulations, was similar between G-24 and G-6M expansion formats. CD19 CAR-T cells expanded in both G-24 and G-6M devices displayed potent cytolytic activity against Burkitt's lymphoma target cells, confirming functional preservation across scale.CIK cells cultured in both G-24 and G-6M devices demonstrated strong cytotoxicity against MCF-7 breast cancer target cells with overlapping lysis curves across 24 hours. Our G-Rex protocol overcomes complex scaling hurdles, enabling a seamless and proportional transition of cell product manufacturing from preclinical to clinical. Our protocol is highly reproducible and less labor-intensive than standard preclinical cell culture systems, leading to consistent high yields across donors.We advance adoptive cell therapy to clinical application by standardizing the protocol and enabling an easier translational use for patients.

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Research

JoVE Journal

Cancer Research

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

The lymphodepletive and immunomodulatory effects of chemotherapy and radiation standard of care can be leveraged to enhance the antitumor efficacy of T cell immunotherapy. We outline a method for generating EGFRvIII-specific chimeric antigen receptor (CAR) T cells and administering them in the context of glioblastoma standard of care.

Generation of CAR T Cells for Adoptive Therapy in the Context of Glioblastoma Standard of Care

The goal of this protocol is to manufacture pathogen-specific clinical-grade T cells using a bench-top, automated, second generation cell enrichment device that incorporates a closed cytokine capture system and does not require dedicated staff or use of a GMP facility. The cytomegalovirus pp65-specific-T cells generated can be directly administered to patients.

Automated Cell Enrichment of Cytomegalovirus-specific T cells for Clinical Applications using the Cytokine-capture System

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Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells

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High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Presented is a protocol for the expansion of Gamma-Delta (γδ) T cell drug product. Lymphocytes are isolated by elutriation and γδ-enriched with zoledronic acid and interleukin-2. Alpha-beta T cells are depleted using a clinical-grade magnetic separation device. The γδ cells are co-cultured with K562-derived, artificial antigen-presenting cells and expanded.

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

This protocol outlines the process of transducing primary human T cells with a gene of interest, ensuring compatibility with Good Manufacturing Practice (GMP) standards.

A GMP-Compliant Procedure for the Generation of Gene-Modified T cells

This article details the manufacturing process for chimeric antigen receptor T cells for clinical use, specifically using an automated cell processor capable of performing viral transduction and cultivation of T cells. We provide recommendations and describe pitfalls that should be considered during the process development and implementation of an early-phase clinical trial.

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

This protocol streamlines retroviral vector production and murine T cell transduction, facilitating the efficient generation of mouse CAR-T cells.

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells

Here, we describe a method for creating double-emulsion droplets encapsulating one T cell and a cancer target cell to examine cell killing at the single-cell level. This method allows for dual quantification of both cytotoxic molecules and target-cell apoptosis within a large population of T cells.

Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

A detailed protocol is provided for using CRISPR/Cas9 technology to achieve highly efficient targeted knock-in of large, multicistronic constructs in primary human T cells via the homology-mediated end joining (HMEJ) DNA repair pathway. T cells engineered with this cGMP-adaptable protocol maintain excellent cell expansion, cytotoxicity, and cytokine production.

Non-Viral Engineering of Primary Human T Cells via Homology-Mediated End-Joining Targeted Integration of Large DNA Templates

Learn a G-Rex protocol for scalable CAR-T and CIK cell expansion using GMP-compatible devices. This method enables high-density cultures with minimal intervention and robust clinical viability for adoptive cell therapy applications.

Efficient and Rapid Generation of CAR-T and Cytokine-Induced Killer Cells in GMP-scalable Devices

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