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GMP T Cell Expansion Medium and Activators for T Cell Therapy Development and Manufacturing
T cell therapy is a rapidly progressing treatment option for cancer patients, however, many therapies fail during clinical trials due to issues with safety, efficacy, or regulatory roadblocks. These challenges make it difficult for cell therapy manufacturers to reach the commercialization stage, resulting in costly therapies that are unaffordable and inaccessible to many patients. By using products manufactured according to current Good Manufacturing Practices (cGMPs), scientists can avoid costly regulatory delays. Furthermore, by considering their full workflow and choosing products that are already optimized and highly compatible, cell therapy manufacturers can streamline their process development and save time while reducing costs.
Achieve robust and consistent T cell expansion and activation using GMP ܲԴǰܱ™-XF with GMP ܲԴǰܱ™ Human T Cell Activators CD3/CD28/CD2 and CD3/CD28. These reagents have been optimized for use in T cell therapy development and manufacturing, providing excellent T cell growth and viability without the use of magnetic beads, feeder cells, or antigens. The ܲԴǰܱ™ T cell workflow streamlines T cell therapy development by enabling process standardization and scale-up from discovery through to clinical and commercial manufacturing.
ܲԴǰܱ™-XF and ܲԴǰܱ™ Activators were designed for use as ancillary materials in cell and gene therapy manufacturing according to USP 1043 and ISO 20399 guidelines and manufactured under relevant cGMP regulations. Full quality and traceability documentation is available to support cell therapy developers' risk assessments and regulatory filings, including Certificates of Origin and Certificates of Analysis. US FDA Master Files are in progress for the ܲԴǰܱ™ product line and will be filed in 2024.
On this page, you will find tools and resources that support your T cell therapy development, our answers to some frequently asked questions, and a form where you can sign up to try GMP ܲԴǰܱ™-XF and ܲԴǰܱ™ T Cell Activators.
Why Use ܲԴǰܱ™ for T Cell Therapy Manufacturing?
- Expand T cells for use in cell therapy development with a medium produced under relevant cGMPs
- Reduce variability by expanding T cells in serum- and xeno-free culture conditions
- Achieve robust T cell expansion with high viability
- Obtain T cells able to produce cytokines upon restimulation
- Activate T cells bead-free by combining ܲԴǰܱ™-XF medium with ܲԴǰܱ™ Human T Cell Activators from preclinical development through to commercial manufacturing
Generate High Yields of Viable and Functional T Cells—See the Data
Enhance your cell therapy development by choosing the high-performance combination of GMP ܲԴǰܱ™-XF and GMP ܲԴǰܱ™ T Cell Activators. See the data for each activator below:
Figure 1. Culture Protocol for T Cell Activation and Expansion
(A) T cells were isolated from fresh leukopaks (Catalog #70500) from healthy donors using the EasySep™ Human T Cell Isolation Kit (Catalog #17951) and cryopreserved in CryoStor® CS10 (Catalog #07930). On Day 0, T cells were thawed, washed, and resuspended at 1 x 106 cells/mL in ܲԴǰܱ™-XF (Catalog #100-0956) supplemented with 180 IU/mL Human Recombinant IL-2, ACF [link] (rhIL-2; Catalog #78145). Cells were seeded into 24-well tissue culture plates (1 mL/well) and stimulated with 25 µL/mL ܲԴǰܱ™ Human CD3/CD28/CD2 T Cell Activator (Catalog #100-0785)*. On Days 3, 5, 7, and 10, cells were re-seeded in fresh ܲԴǰܱ™-XF + rhIL-2 at low or high cell densities as outlined in the table (B). Cells were harvested on Day 12.
*A similar protocol was performed using ܲԴǰܱ™ Human CD3/CD28 T Cell Activator (Catalog #100-0784). Please reach out to our Product and Scientific Support team for additional information.
Figure 2. ܲԴǰܱ™ Reagents Result in Robust T Cell Expansion Under Low Cell Density Culture Conditions
T cells were cultured with ܲԴǰܱ™-XF, Human Recombinant IL-2, ACF, and ܲԴǰܱ™ Human CD3/CD28/CD2 T Cell Activator for 12 days following the low cell density protocol described in Figure 1. Cells were harvested and analyzed at different time points. Cell count and viability were assessed using the NucleoCounter® NC-250™ automated cell analyzer, and cell surface marker expression was analyzed by flow cytometry. (A) Points and bars in the graph represent T cell viability and average cumulative fold expansion, respectively, for each assessed culture time point. An average viability of 92.8 ± 1.8% and a total fold expansion of 1497 ± 143 were achieved by Day 12 in the low cell density cultures. Data represent mean ± SEM (n = 11). (B) The ratio of CD4+ T cells to CD8+ T cells (CD4:CD8) was comparable between Day 0 and Day 12 in 10 out of 11 donors. (C) The frequency of T cells expressing PD-1, TIM-3, and LAG-3 was upregulated upon stimulation and subsequently decreased to basal levels (n = 4). (D) The frequency and number of T cell subtypes in CD4+ and CD8+ populations during the 12-day culture period are shown. Cell numbers were calculated based on 106 starting T cells. The number of central memory T cells remained constant from day 7 to 12. T cell subsets: naive and T memory stem cell (TN/SCM) CCR7+CD45RO-; central memory (TCM) CCR7+CD45RO+; effector memory (TEM) CCR7-CD45RO+; effector cells (TEFF) CCR7-CD45RO- (n = 4 - 11).
Figure 3. ܲԴǰܱ™ Reagents Support T Cell Activation and Expansion Following High Cell Density Protocol
T cells were cultured with ܲԴǰܱ™-XF, Human Recombinant IL-2, ACF, and ܲԴǰܱ™ Human CD3/CD28/CD2 T Cell Activator for 12 days following the high cell density protocol described in Figure 1. Cells were harvested at different time points and analyzed. Cell count and viability were assessed using the NucleoCounter® NC-250™ automated cell analyzer, and cell surface marker expression was analyzed by flow cytometry. (A) Points and bars in the graph represent T cell viability and average cumulative fold expansion, respectively, for each assessed culture time point. An average viability of 89.3 ± 2.3% and a total fold expansion of 67 ± 7 were achieved by Day 12 in the high cell density cultures (mean ± SEM, n = 4). (B) The ratio of CD4+ T cells to CD8+ T cells (CD4:CD8) was measured at the beginning and end of culture. The CD4:CD8 ratios increased in two out of four donors tested. (C) The frequency of T cells expressing PD-1, TIM-3, and LAG-3 increased upon stimulation and subsequently decreased to basal levels by Day 12 of culture (n = 4), with the exception of LAG-3 expression in the CD8⁺ population. (D) Data shows the frequency and number of T cell subtypes in CD4+ and CD8+ populations at the indicated time points. Cell numbers were calculated based on 106 starting T cells. The number of central memory T cells continued to increase from Day 7 to Day 12 of culture. T cell subsets: naive and T memory stem cells (TN/SCM) CCR7+CD45RO-; central memory (TCM) CCR7+CD45RO+; effector memory (TEM) CCR7-CD45RO+; effector cells (TEFF) CCR7-CD45RO- (n = 4).
Figure 4. Bioreactor-Based Expansion with ܲԴǰܱ™ Reagents Yields High Numbers of Viable, Functional T Cells
(A) T cell expansion workflow in Xuri™ Cell Expansion System W25: T cells were isolated from fresh leukopaks using the EasySep™ Human T Cell Isolation Kit and cryopreserved in CryoStor® CS10. On Day 0, cryopreserved cells were thawed and resuspended at 1 x 106 cells/mL in ܲԴǰܱ™-XF supplemented with 180 IU/mL Human Recombinant IL-2, ACF. 5 x 107 T cells were seeded in a T175 tissue culture flask and activated with ܲԴǰܱ™ Human CD3/CD28/CD2 T Cell Activator at 25 μL/mL. On Day 3, cell density was adjusted to 1 - 2.5 x 105 cells/mL by adding fresh ܲԴǰܱ™-XF with IL-2. On Day 5, the cell suspension containing 3 x 108 cells/L of culture volume was inoculated into a Xuri™ 2L Cellbag™ Bioreactor and cultured until Day 10. Once the cell density reached 1.5 x 106 cells/mL, perfusion was initiated at 0.5 L/day on Day 7 and increased to 1 L/day on Day 9. The culture was harvested on Day 10. (B) Points and bars in the graph represent T cell viability and average cumulative fold expansion, respectively, for each assessed culture time point. On average, 92.7 ± 0.7% viability and a total fold expansion of 503 ± 51 were achieved by Day 10 (mean ± SEM, n=4). (C) Changes in the ratio of CD4+ T cells to CD8+ T cells (CD4:CD8) were measured at the beginning and end of culture. Some variability among donors was observed (n = 4). (D) The frequency of T cells expressing PD-1, TIM-3, and LAG-3 increased by Day 3 and subsequently decreased from Day 3 to Day 10 (n = 4). (E) The frequency and number of T cell subtypes in CD4+ and CD8+ populations are shown. Cell numbers were calculated based on 106 starting T cells. The number of central memory T cells peaked on Day 7 of the culture. T cell subsets: naive and T memory stem cells (TN/SCM) CCR7+CD45RO-; central memory (TCM) CCR7+CD45RO+; effector memory (TEM) CCR7-CD45RO+; effector cells (TEFF) CCR7-CD45RO- (n = 4). (F) The frequencies of expanded T cells expressing IFN-ɣ and TNF-ɑ upon stimulation with PMA and ionomycin were 66.6 ± 2.1% and 96.3 ± 1.1%, respectively, in the CD4+ population, and 73.2 ± 11.4% and 93.8 ± 1.3%, in the CD8⁺ population. Additionally, the frequencies of expanded CD8+ T cells expressing perforin and granzyme B without stimulation were 23.3 ± 9.1% and 37.7 ± 6.8%, respectively (n = 4).
Figure 5. High-Efficiency TRAC Knockout of T Cells
(A) The protocol for non-viral genetic modification and expansion of T cells: On Day 0, T cells were isolated from fresh leukopaks using the EasySep™ Human T Cell Isolation Kit. Cells were resuspended at 1 x 106 cells/mL in ܲԴǰܱ™-XF supplemented with 180 IU/mL Human Recombinant IL-2, ACF (rhIL-2). Cells were seeded into 24-well tissue culture plates at 1 mL/well and stimulated with ܲԴǰܱ™ Human CD3/CD28/CD2 T Cell Activator at 25 μL/mL. Following 3 days of activation, RNP complexes containing ArciTect™ Cas9 Nuclease (Catalog #76004) and sgRNA targeting the TRAC gene were delivered to T cells by electroporation (Neon® Transfection System). Cells were then returned to culture in ܲԴǰܱ™-XF supplemented with rhIL-2 for another 9 days of expansion following the low cell density protocol. (B) Representative flow cytometry plots of TCRαβ and CD3 expression analysis on Days 2 and 9 post-T cell editing. (C) The gene editing efficiency was assessed by measuring the frequency of the remaining TCRαβ+CD3+ cells in the mock (91.8 ± 1.1%) and edited T cells (16.1 ± 2.6%) 2 days post-editing. The frequency of TRAC-edited cells was maintained throughout the culture duration. (D) The viability of T cells was measured using the NucleoCounter® NC-250™ automated cell analyzer across culture periods. On Day 3, the viability of T cells further decreased immediately following transfection. (E) Cumulative cell expansion post-electroporation was comparable between mock-treated and TRAC-edited T cells, suggesting that the edited T cells are functional. Data represent mean ± SEM (n = 3).
Try GMP ܲԴǰܱ™ Now
Are you interested in testing how your T cells perform in GMP ܲԴǰܱ™-XF with GMP ܲԴǰܱ™ Human T Cell Activators? Sign up below to have a sales representative contact you about an introductory offer to try ܲԴǰܱ™-XF Medium and Human T Cell Activators in your lab.
Advancing T Cell Therapies: Insights from the Translational T Cell Talks
On June 11, 2024, ƽ Technologies hosted the half-day virtual event “Translational T Cell Talks: Scaling for the Future” in collaboration with Scientist.com. The presentations from distinguished scientists and industry leaders, provided insights into the latest developments in translational T cell research and CAR T cell innovations, and concluded with a discussion on improving collaboration between industry and academia.
Frequently Asked Questions
What is GMP and why is it important for my research?
Products manufactured according to current Good Manufacturing Practices (cGMPs) helps facilitate ancillary material qualification and risk assessment by cell therapy developers streamlining regulatory reviews of regulatory filings with global authorities, such as the US Food and Drug Administration (FDA) or the European Medicines Agency in Europe (EMA).
All of ƽ Technologies’ GMP products have or will have Master Files available, which contain detailed information regarding product components and manufacturing processes. To learn more about our approach to quality and compliance, including GMP, explore the resources on our Quality Page. There you’ll also find information on the regulatory support we can provide to help you move your development from the bench to the clinic.
What does serum- and xeno-free mean for cell culture media?
ܲԴǰܱ™-XF is a serum- and xeno-free medium, which means that the finished product was not manufactured with any primary raw materials derived directly from non-human animals. Recombinant materials are generated with non-human animal DNA sequences, resulting in a product that does not contain any raw materials (serum, plasma, or hemolymph), but may contain other biological materials.
Interested in other solutions for your T cell therapy workflow? View more products here.
Additional Resources for T Cell Therapy Research and Development
