eBook 2 The cutting-edge techniques advancing the safety of cell therapies From developing safety switches to using secreted exosomes, researchers are exploring new methods to make cell therapies safer and more effective. BY KATE HARRISON, PHD C ell therapies are rapidly evolving, and their reach spans multiple areas, including cancer therapy, immunotherapy, and regenerative medicine. While transference of autologous or allogenic cells into a patient for clinical purposes is not a new concept, many modern cell therapies — both approved and in development — now involve an element of genetic engineering to manipulate or enhance the cells’ natural capabilities. There are several different types of cell therapy, including stem cell-based therapies and non-stem cellbased therapies such as immune cells. Since the approval of the first cell therapy — Hemacord, a hematopoietic stem cell therapy — in 2011, stem cell therapies and immune cell therapies have seen incredible success, with 28 now approved by the Food and Drug Administration as of 2025 (1,2). However, there are still challenges to contend with — most notably, ensuring the safety of these powerful therapies and preventing severe adverse effects in patients. From developing safety switches to using secreted exosomes rather than cells themselves, multiple methods are being used to improve the safety of these living drugs. Flipping the switch: immune cell therapies As the field of immune cell therapies continues to develop, novel applications are arising, including the treatment of autoimmune diseases and the prevention of allogenic transplant rejection (3). However, to date, the most advanced, successful immune cell therapies have been T cell therapies for cancers, with seven chimeric antigen receptor (CAR) T cell therapies approved for clinical use and the first tumor infiltrating lymphocyte therapy — lifileucel — approved in 2024 (2). “CAR T cell therapy involves collecting T cells from a patient and engineering them ex vivo to express a CAR that recognizes a tumor antigen,” says Melita Irving, group leader at the Ludwig Institute for Cancer Research, University of Lausanne. CAR-expressing cells are then expanded and transferred back into the patient to eliminate the cancerous cells. Irving’s research focuses on engineering T cells to effectively control solid tumors by enhancing T cell receptors and using small molecule “switches” to control T cell function. Despite the successes of CAR T cell therapies in treating leukemias and lymphomas, they are still hindered by safety risks and therapeutic challenges. One of the main safety concerns is the side effects of CAR T cell therapy, including cytokine release syndrome (CRS) and neurotoxicity. CRS is a systemic immunological response that can manifest severe symptoms in up to 46% of patients (4). “The infused CAR T cells can activate myeloid cells such as macrophages,” explains Irving. “This triggers the production of high levels of inflammatory cytokines such as interleukin 6.” CRS can be followed by immune effector cell-associated neurotoxicity syndrome (though it can also occur independently of CRS), in which the blood–brain barrier is disrupted by the inflammation induced by CRS, allowing cytokine infiltrates into the central nervous system. This causes symptoms such as cerebral edema, thrombosis, and neuropsychiatric effects (5). Solid tumors also pose additional challenges for successful CAR T cell therapy. In addition to the immunosuppressive environment of the tumor, the heterogeneity and complexity of the malignant cells must also be considered. “It’s difficult to select the best antigens to target in solid tumors,” says Irving. “In B cell malignancies we can target CD19, which is primarily found on B cells. However, 2 | Overcoming Roadblocks in Cell Therapy “An OFF-switch could be used to transiently turn off the T cells to modulate the immune system and prevent CRS.” most antigens targeted on solid tumors are also found on healthy tissues, leading to on-target, off-tumor toxicity. Additionally, not all cells in a solid tumor will express the target antigen, leading to escape and expansion of malignant cells.” Irving’s research team is looking to circumvent these challenges by using small molecules to control T cell function, thereby improving the safety and efficacy of CAR T cells (6). They designed split ON- or OFF-switch CARs, consisting of separate receptor and signaling chains that are either associated or disengaged respectively by the presence of a small molecule. They used a lentiviral vector to introduce an inducible ON-switch, controlled by the small molecule venetoclax, to CARs. When venetoclax was withdrawn, the CAR T cells lost the ability to destroy tumor cells. Subsequently, the team developed an ON/OFF switch controlled by lenalidomide, which disrupted the CAR T cell chains. Irving hopes that these switch mechanisms can help prevent CRS and other toxicities induced by CAR T cell therapy. “An OFF-switch could be used to transiently turn off the T cells to modulate the immune system and prevent CRS,” says Irving. “While an on switch could be used to target a new tumor antigen when the safety profile of that CAR isn’t fully known. If the target is toxic, you can withdraw the small molecule and the CAR T cells will stop functioning.” Irving’s next steps are to move her CAR designs and strategies into clinical testing, as well as examining their efficacy in difficult-to-treat cancers with unmet medical need, such as sarcomas. It’s in the microdetails: stem cell therapies Stem cell therapies use the self-renewal and differentiation properties of stem cells to regenerate damaged tissue or replace defective cells with new, fully functioning versions. There are several different types of stem cells used in stem cell therapies, including mesenchymal stem cells (MSCs) and human pluripotent stem cells (hPSCs). hPSCs have enormous therapeutic potential but also present safety issues, such as teratoma formation from undifferentiated cells and immunological rejection, as well as ethical concerns if sourced from human embryos. In comparison, MSCs can be collected from umbilical cords, placentas, and adult tissues such as adipose tissue and bone marrow. “MSCs can create an immune-privileged area around themselves and actually secrete immune-modulating cytokines, so there is no risk of rejection,” explains Ayman Al-Hendy, researcher and gynaecologic surgeon at the University of Chicago and Khalifa University. Al-Hendy’s work focuses on using MSCs to treat and even prevent female infertility caused by premature ovarian insufficiency (POI), whether idiopathic or chemotherapy-induced. “In POI, the ova are healthy, but the somatic cells in the ovary that help the egg mature are defective, often exhibiting mitochondrial dysfunction,” says Al-Hendy. “MSCs secrete a lot of important growth factors that can reactivate the mitochondria, decrease inflammation and oxidative stress, inhibit apoptosis, and increase cell proliferation. In effect, they rejuvenate the ovarian cells and allow the ova to mature.” Previous research by the Al-Hendy group has shown that intraovarian injection of MSCs is a safe and effective method to restore fertility in an animal model of POI (7). Now, they hope to take this a step further by using MSC exosomes to treat POI rather than the cells themselves. “MSC cells are not associated with tumor formation or abnormal cell proliferation anyway,” explains Al-Hendy. “But exosomes are even safer — they don’t contain any donor DNA and only persist in the body for a few days, long enough to exert their positive effects, without any potential long-term negative effects.” The group showed that MSC exosomes restored functional ovarian morphology, follicular cell numbers, and overall fertility in a chemotherapy-induced POI mouse model. Notably, miRNA profiling of the exosomes in this study provided insight into the mechanisms of the exosomes, showing the presence of three miRNAs essential in regulating apoptosis, promoting pathways for cell survival, and reducing inflammation. “Synthetic molecules either work or they don’t,” says Al-Hendy. “But stem cell therapies are so versatile. By continuously tweaking the cells, for example, overexpressing these specific miRNAs, we can keep improving the success rate.” The same can be said for immune cell therapies such as CAR T cells — continuous enhancement of T cell receptors and the use of methods such as inducible switches will keep improving on the safety of cell therapies, and push towards curative therapies across a wide range of diseases. REFERENCES 1. Traynor, K. FDA approves first stem-cell therapy. Am J Health-Syst Ph 68, 2316 (2011). 2. Approved cellular and gene therapy products. https:// www.fda.gov/vaccines-blood-biologics/cellular-genetherapy-products/approved-cellular-and-gene-therapyproducts (2025). 3. Weber, E.W., Maus, M.V. & Mackall, C.L. The emerging landscape of immune cell therapies. Cell 181, 46-62 (2020). 4. Xiao, X. et al. Mechanisms of cytokine release syndrome and neurotoxicity of CAR T-cell therapy and associated prevention and management strategies. J Exp Clin Cancer Res 40, 367 (2021). 5. Sterner, R.C. & Sterner, R.M. Immune effector cell associated neurotoxicity syndrome in chimeric antigen receptor-T cell therapy. Front Immunol 13 (2022). 6. Attianese, G.M.P.G. et al. Dual ON/OFF-switch chimeric antigen receptor controlled by two clinically approved drugs. PNAS 121, e2405085121(2024). 7. Park, H.S. et al. Safety of intraovarian injection of human mesenchymal stem cells in a premature ovarian insufficiency mouse model. Cell Transplant 30 (2021) 3 | Overcoming Roadblocks in Cell Therapy CREDIT: MELITA IRVING AND AYMAN AL-HENDY Melita Irving (left) leads a group developing inducible safety switches for CAR T cells, while Ayman Al-Hendy (right) is exploring the use of MSC exosomes to treat POI. Now you can effortlessly transfer your user-defined assays between instruments with our innovative assay transfer feature – designed to support standardization and boost reproducibility across your entire manufacturing site network. Experience exceptional resolution and improved separation, making dim and rare populations easier to resolve with the outstanding performance of our BD FACSLyric™ Flow Cytometer. Confidently transfer your cell therapy assays Simplify your assay transfer with our BD FACSLyric™ Flow Cytometer Scan here to learn more > worldwide. bdbiosciences.com/celltherapy The BD FACSLyric™ Flow Cytometer is a Class 1 Laser Product. In the U.S., the BD FACSLyric™ Flow Cytometer is for In Vitro Diagnostic Use with BD FACSuite™ Clinical Application for up to six colors. In the U.S., the BD FACSLyric™ Flow Cytometer is for Research Use Only with BD FACSuite™ Application for up to 12 colors. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, BD FACSLyric and FACS are trademarks of Becton, Dickinson and Company or its affiliates. All other trademarks are the property of their respective owners. © 2025 BD. All rights reserved. BD-149392 (v1.0) 0525 Engineering Solutions for Cell Therapy Roadblocks Written by Steven Gibney, PhD Designed by AnnaMaria Vasco Tumor entry barrier One of the first challenges cell therapies must overcome are the dense extracellular matrix (ECM) and abnormal vasculature that prevent cell therapies from penetrating into solid tumors. A challenging environment Maintaining persistence The tumor microenvironment actively suppresses immune function through inhibitory molecules, metabolic restrictions, and recruitment of immunosuppressive cells. Therapeutic cells often exhibit limited durability, with rapid exhaustion creating a narrow window of therapeutic action. Advanced delivery systems enhance precision, reduce off-target effects, and improve overall therapeutic efficiency. Immunomodulatory enhancements convert immunosuppressive environments into productive therapeutic landscapes. Enhancing cell persistence can convert a single treatment into long-term disease control using: These barriers to entry can be broken down using: Safety switches can be engineered to control toxicity and enable precise management of therapeutic activity, minimizing adverse events while preserving efficacy. This includes the use of: Converting immunosuppressive signals to stimulatory ones allows therapeutic cells to thrive in previously hostile environments. A variety of tools can be used to transform the tumor landscape in favor of anti-cancer activity: • Chemokine receptor engineering to enhance migration toward tumor signals • Heparanase expression to break down extracellular matrix • Vascular normalization factors to repair blood vessel and enhances access • TGF-β receptors neutralize suppressive cytokines • Metabolic engineering ensures cellular fitness in nutrient-poor environments • Hypoxia-resistant designs maintain function in low-oxygen conditions • Memory stem cell features that sustain therapeutic populations over time • Metabolic reprogramming to ensure optimal energy utilization • Epigenetic modifications which maintain functional state despite challenges Toxicity complications Using immune cells against cancer can trigger severe systemic reactions caused by cytokine release, creating potentially life-threatening complications. • Inducible control systems that provide tunable cell activation • Therapeutic designs that require multiple signals for full activation • Logic-gated activation to ensure precision targeting Maintaining function in a hostile microenvironment Develop an effective toolkit E F Cytokine release and neurotoxity Cytokines Cell-based therapies are a revolutionary approach to treating cancer and other diseases. However, on the path from laboratory development to clinical efficacy, researchers face numerous biological and manufacturing obstacles. Using cutting-edge engineering solutions, it is possible to dismantle and overcome these barriers, unlocking the full therapeutic potential of immune and stem cell therapeutics. Genetic modifications rewrite cellular capabilities, creating cells with enhanced targeting, persistence, and therapeutic potential. These solutions represent more than individual technologies, they are interconnected strategies. Combining these tools makes it possible to create a new generation of cell therapies capable of overcoming the most challenging therapeutic landscapes. FINE FS·Т SCIENCE ToOLS YEARS FINE Enhancing CAR T cell metabolism to overcome hypoxia in brain tumors Metabolic preconditioning with metformin and rapamycin improves CAR T cell function in the hypoxic brain tumor environment. BY STEVEN GIBNEY, PHD Glioblastoma multiforme (GBM) remains a highly aggressive and deadly brain tumor despite current treatment strategies. While chimeric antigen receptor (CAR) T cell therapy has been effective against blood cancers, its efficacy against solid tumors like GBM has been limited. A major obstacle in the treatment of solid tumors is the hypoxic, immunosuppressive tumor microenvironment (TME) that diminishes T cell function and leads to exhaustion (1). Researchers investigating this challenge discovered that CAR T cells lose their oxidative phosphorylation (OXPHOS) activity when infiltrating brain tumors, despite maintaining glycolytic function (2). Flow cytometry analysis showed that ATP synthase (ATP5a), a key OXPHOS marker, steadily decreased in tumor-infiltrating CAR T cells over time, compared to those originally derived from spleen CAR T cells. Further investigation confirmed that the brain TME is significantly more hypoxic than subcutaneous tumors, directly impacting CAR T cell metabolism (2). To overcome the negative impact of the hypoxic TME, the researchers conducted screenings to identify promising metabolic regulators, finding that a combination of metformin and rapamycin (Met+Rap) enhanced CAR T cell function under hypoxic conditions. This pretreatment activated AMPKα and inhibited mammalian target of rapamycin pathways, leading to upregulation of PGC-1α, the master regulator of mitochondrial biogenesis. Seahorse assays demonstrated that Met+Rap significantly increased spare respiratory capacity in CAR T cells, enabling better survival in oxygen-deprived environments. The Met+Rap pretreatment also promoted the central memory T cell phenotype and protected CAR T cells from exhaustion. In vitro studies showed that while untreated CAR T cells rapidly lost cytotoxic function under hypoxia, Met+Rap-treated cells maintained their tumor-killing abilities even after chronic antigen stimulation. These cells preserved both glycolytic markers (Glut1) and OXPHOS markers (ATP5a) under hypoxic conditions, unlike their untreated counterparts. In vivo studies also provided compelling evidence of clinical potential. A single intravenous infusion of Met+Rap-pretreated CAR T cells improved survival in mice with intracerebral gliomas. All mice receiving these enhanced CAR T cells survived to 80 days post-tumor inoculation, while mice receiving untreated CAR T cells succumbed by day 41. Mass cytometry analysis revealed that Met+Rap-pretreated CAR T cells not only infiltrated tumors in greater numbers but also reduced immunosuppressive myeloid-derived suppressor cells through an IFN-γ-dependent mechanism. The Met+Rap pretreatment strategy also proved effective in human CAR T cells across multiple donors. Treatment enhanced their mitochondrial function and maintained tumor-killing capabilities under hypoxic conditions. This metabolic conditioning approach offers several advantages over previous strategies, including the elimination of potential in vivo side effects, since treatment occurs only during the in vitro expansion phase. The findings suggest that metabolic preconditioning could significantly advance CAR T therapy for GBM and potentially for other solid tumors characterized by hypoxic, immunosuppressive microenvironments. REFERENCES 1. Watchmaker, P.B., Colton, M., Pineo-Cavanaugh, P.L. et al. Future development of chimeric antigen receptor T cell therapies for patients suffering from malignant glioma. Curr Opin Oncol 34, 661-669 (2022). 2. Hatae, R., Kyewalabye, K., Yamamichi, A. et al. Enhancing CAR-T cell metabolism to overcome hypoxic conditions in the brain tumor microenvironment. JCI Insight 9(7), e177141 (2024). 7 | Overcoming Roadblocks in Cell Therapy An easy yet efficient approach to lentiviral transduction An innovative, non-toxic, and dissolvable microfluidic sponge enhances lentiviral transduction across diverse cell types without complex equipment or harsh chemicals. Transduction — the final, sometimes challenging step in a gene delivery workflow Lentiviruses are a popular tool for delivering genes into target cells because they infect a wide range of host cells — including both dividing and nondividing cells — and integrate their genetic material into the host genome for stable expression. Lentiviral vectors are used in cell and gene therapy for many applications, such as gene and protein expression, cell line development, therapeutic model generation, gene therapy, and chimeric antigen receptor expression in T cells. Transduction is the final step in the lentiviral gene delivery workflow, transporting the gene of interest (GOI) into target cells, but inefficient transduction presents challenging roadblocks. A low transduction efficiency means researchers may not have enough cells expressing their GOI to use in downstream applications, causing them to backtrack and expend even more time and resources. When target cells fail to express the GOI, it can be hard to determine what went wrong, either in the transduction or another upstream process. A significant amount of time is spent troubleshooting and optimizing each step in the process — delaying the advancement of research projects. Drawbacks of current transduction methods Common transduction methods have significant challenges, requiring methods that aim to improve efficiency. The standard lentiviral transduction method involves placing the target cells and lentivirus — containing the GOI — into a well. The cells ready to be transduced are on the bottom of the well, with the lentivirus in solution on top. This method relies on diffusion, with much of the virus remaining in the supernatant, which is wasted, as it never encounters the target cells. In an attempt to boost transduction efficiency, virus and cell contact is increased by adding a centrifugation step (spinoculation) with harsh chemical enhancers such as polybrene, which is toxic to some cells. This can have unknown downstream effects on cells. An alternative and possibly better way to increase contact between the virus and the cells is a microfluidic approach, which places the cells and virus close together in small spaces. While this does increase transduction efficiency, it requires expensive instruments and expertise. Sponge microfluidics: an effective, gentle, and yet easy-to-use transduction method The Lenti-X™ Transduction Sponge provides a microfluidic environment for lentivirus transduction in a small, easy-to-use sponge. The sponge is made of innocuous sodium alginate which is dried to produce a pore size of 20–300 µm. Within the pores of the sponge, the cells and viruses are gently colocalized, mimicking a microfluidic device — but without the large capital investment, making the sponge a cost-effective alternative. This close contact between virus particles and cells increases the rate of transduction. The sponge has a transduction efficiency up to five times higher than static transductions, and facilitates transduction equivalent to or better than spincoculation with an uncomplicated workflow. Researchers simply mix the target cells and lentivirus, then add the mixture to the sponge. After a short 24-hour incubation, the alginate sponge is depolymerized with non-toxic buffer, releasing successfully transduced, healthy cells ready for any downstream application. The sponge protocol does not require spinoculation, nor harsh chemical enhancers that are common in standard transduction methods yet achieves equivalent or higher transduction efficiencies across varying multiplicities of infection (MOI) and cell lines than spinoculation. The sponge has proven to be gentle on cells, resulting in high cell viability. Compatibility of sponge microfluidic transduction with diverse cells and particles In addition to being easier, gentler on cells, and more cost-effective than other transduction methods, the sponge is cell- and particle-type agnostic. Unlike some transduction methods, a wide variety of cell types can be used, even difficult-to-transduce primary cells, including CD34+ hematopoietic stem cells, natural killer cells, and T cells. CD34+ cells demonstrated a tenfold reduction in virus requirement while maintaining the same transduction efficiency. T cells transduced with the sponge showed high transduction efficiency, while maintaining equivalent phenotypes and similar gene expression patterns as cells transduced with other methods. The sponge is not just for lentivirus transduction — it also works with particles such as retrovirus, adeno-associated virus, and virus-like particle-like vesicles. Simplifying viral transduction workflow while increasing transduction efficiency The simple, groundbreaking transduction sponge protocol effortlessly enables successful transductions. Faster than other transduction methods, it also preserves cell viability through a protocol that requires less cell handling. Whether a lentivirus novice or an expert, researchers can easily achieve successful transductions using the sponge’s microfluidics without the need for complicated protocols, such as spinoculation, harsh chemical enhancers, or expensive specialized equipment. By adding the Lenti-X Transduction Sponge to their workflow to enable successful downstream use of the target cells, researchers can achieve reliable and effective transductions the first time. This article is contributed by Thomas Quinn, R&D Group Leader at Takara Bio USA, Inc 8 | Overcoming Roadblocks in Cell Therapy BY THOMAS QUINN IMAGINE... Maximizing reproducible results with low oxygen physioxic cell culture Tel: (858) 535-0545 Toll-Free: (877) 755-3309 Email: info@embrient.com Meet the low-cost standard for hypoxic environment cell culture that enabled HIF-1a Nobel Prize-winning research The Modular Incubator Chamber (MIC-101) is a compact and versatile tool designed as a cost-effective system for maintaining cell cultures under stable hypoxic (physioxic) or hyperoxic conditions. It offers a straightforward approach to controling oxygen levels using its closed system, preventing multi-well plate edge evaporation, and tailoring gas concentrations to your specific requirements. Learn more: embrient.com Benefits • Simple to use • Trustworthy, air-tight sealing • Eliminates the edge effect • Minimizes consumption of expensive tri-gas mix • Stable environment up to 72 hours • Easy to clean • Versatile internal trays accommodate common labware such as petri dishes, multi-well plates and cell culture flasks How it Works Simply place your tissue culture plates, flasks, or petri dishes in the chamber, attach a flow meter to the unit, flush for several minutes with the desired gas mixture, then seal the chamber and place in your 37˚C incubator.
