Manufacturing Cell Research Articles

Process-Driven Layout Optimization of a Portable Hybrid Manufacturing Robotic Cell Structure

Hybrid manufacturing combines manufacturing processes (typically additive manufacturing and machining) exploiting the benefits of each and enabling repair scenarios. Such an approach can be integrated with a robot, and if made portable, can form a flexible machine tool that can be easily transported anywhere to enable repairs in the field. However, the design of the load-bearing structure determines its transportability, and its stiffness plays a crucial functional role under dynamic loads and affects the product quality. Finding the right balance between weight and stiffness requires accurate boundary conditions and an effective design. In this work, a method is proposed towards process-driven optimization of a portable manufacturing cell structure. The dynamic cutting forces are determined through a machining process model and, via a simplified model of the robot arm, the forces at the base of the robot are estimated. Since the frame consists of beams, the layout optimization method is applied, using the estimated process forces as boundary conditions to optimize the arrangement of beams. The proposed method achieved 0.05 mm displacement in the load-bearing structure under milling and an acceptable accuracy of the position of a hole’s center during drilling, while the overall weight reduced by 17.6%.

Effects of design and manufacturing deviations on compressive properties of glass sponge lattice structures manufactured by selective laser melting

Bio-inspiration offers researchers innovative perspectives and methodologies, stimulating the advancement of high-performance, high-durability, and added-value materials, structures, and products. Inspired by the lightweight, high-strength, and highly stable glass sponges (GSs), five types of glass sponge lattice structures (GSLSs) were successfully designed and manufactured by selective laser melting (SLM) with Ti6Al4V. By conducting a comparative analysis of unit cell design and manufacturing deviations, we assess their impact on the compressive properties of GSLSs. Notably, the original GSLS with circle-/grid-like heterogeneous geometries exhibits outstanding overall compression performance, including stability, stress dispersion and unique cell-by-cell and layer-by-layer fracture mechanisms among GSLSs, which are attributable to their reinforced diagonal struts and heterogeneous unit cells. Hence, three strategies for unit cell design and optimization were proposed. These discoveries guide the future design and optimization of lattice structures (LSs) and offer potential applications for GSLSs in aerospace, biomedicine, automobiles, architecture, and wearable products.

Enrichment of T-lymphocytes from leukemic blood using inertial microfluidics toward improved chimeric antigen receptor-T cell manufacturing

Chimeric antigen receptor cell therapy is a successful immunotherapy for the treatment of blood cancers. However, hurdles in their manufacturing remain including efficient isolation and purification of the T-cell starting material. Herein, we describe a one-step separation based on inertial spiral microfluidics for efficient enrichment of T-cells in B-cell acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia patient's samples. In healthy donors used to optimize the process, the lymphocyte purity was enriched from 65% (SD ± 0.2) to 91% (SD ± 0.06) and T-cell purity was enriched from 45% (SD ± 0.1) to 73% (SD ± 0.02). Leukemic samples had higher starting B-cells compared to the healthy donor samples. Efficient enrichment and recovery of lymphocytes and T-cells were achieved in ALL samples with B-cells, monocytes and leukemic blasts depleted by 80% (SD ± 0.09), 89% (SD ± 0.1) and 74% (SD ± 0.09), respectively, and a 70% (SD ± 0.1) T-cell recovery. Chronic lymphocytic leukemia samples had lower T-cell numbers, and the separation process was less efficient compared to the ALL. This study demonstrates the use of inertial microfluidics for T-cell enrichment and depletion of B-cell blasts in ALL, suggesting its potential to address a key bottleneck of the chimeric antigen receptor-T manufacturing workflow.

Towards the fabrication of flexible thin film CdTe solar cells: The significance of substrate surfaces

Thin film CdTe solar cells have been recognized as reliable alternative for the manufacture of cheap photovoltaic solar cells of the future, due to its excellent absorber characteristics and simple, low-cost manufacturability. However, for the attainment of higher cell performances, additional studies are needed to increase cell efficiency through further development of better quality films and new fabrication processes. In the substrate-structured CdTe thin film solar cells, the CdTe absorber layer is deposited directly onto the substrate or through a back contact layer. But the quality of deposited film is believed to depend on the type and smoothness of the substrate. In this work CdTe was deposited on different substrates by RF sputtering and the effects on the deposited films were studied in terms of their structural and morphological forms. The substrates used were: pure molybdenum sheets (Mo), molybdenum-sputtered on molybdenum (Mo/Mo), molybdenum-sputtered polyimide (PI/Mo) and molybdenum-sputtered glass (glass/Mo). The characterization tools used included XRD, SEM and AFM. The results showed that all surfaces produced uniform, compact and pinhole-free films; however, those on smoother surfaces produced larger as-deposited grain sizes of up to1.7μm as against 1.3 for rougher surfaces. Non-uniformities such as overgrowth and voids were observed, but only films on PI showed evidence of cracking and peel-offs.

A numerical simulation and analysis of chalcogenide BaZrS3-based perovskite solar cells utilizing different hole transport materials

Recently, barium zirconium sulfide (BaZrS3) chalcogenide perovskite (CP) material has attracted much attention in the photovoltaic community because of its excellent light harvesting ability, stability, and nontoxicity. BaZrS3 has shown great potential in becoming the best alternative to hybrid halide perovskites. Herein, we have numerically simulated and optimized the performance of a device with the general architecture FTO/WS2/BaZrS3/HTL/Au, using SCAPS-1D (version 3.3.07) software. In this general device, inorganic CuSCN, Cu2O and organic poly(3-hexylthiophene) (P3HT) were inserted, and performance was evaluated as hole transport layer (HTL) materials. Moreover, the influence of various parameters on the device, such as the effect of changing the absorber defect density, varying the operation temperature, and using different metal back contacts, were examined. The best HTL material was organic P3HT, and it exhibited a power conversion efficiency (PCE) of 13.86 %, with a Fill factor (FF) of 11.97 %, open circuit voltage (Voc) of 6.58 V, and current density (Jsc) of 17.59 mA cm−2. The other tested inorganic CuSCN and Cu2O HTL showed a PCE of 13.83 and 13.70 %, respectively. For all the devices, the optimum density of defects was kept at 1.0 × 1015 cm−2. It was established that the P3HT-based device can operate and be stable between 240–––340 K temperature window, while devices with CuSCN and Cu2O can work between 280 – 400 K temperature range. Thus, the CuSCN- and Cu2O-based devices were more stable because they could form a more compact structure that can withstand relatively higher operation temperatures than organic-based devices with P3HT as HTL. Finally, it was established that relatively cheaper metals such as Pt, Ni, and Pd can be suitable alternatives to expensive gold metal back contact. It is expected that this work would shed additional light on the utilisation of BaZrS3 as a promising absorber material in the actual manufacturing of solar cells for clean energy production.

Lithium Plating at the Cell Edge Induced by Anode Overhang during Cycling in Lithium-Ion Batteries: Part I. Modeling and Mechanism

The anode overhang is usually cited to prevent lithium plating at the cell edges of lithium-ion batteries. Still, numerous reports in the literature show lithium plating at the cell edge, which is typically referred to as edge plating. Edge plating is often attributed to inhomogeneous lithium distribution, thermal gradients, or pressure-dependent effects. This work presents an easy-to-implement two-dimensional electrochemical model demonstrating inhomogeneous lithiation induced by the anode overhang, which can explain experimentally observed edge plating. First, the mechanism of inhomogeneous lithiation due to the anode overhang is explained in detail. Then, a parameter study on charge protocol and geometric cell properties is presented, and the implications for cell applications are analyzed. Finally, the findings are discussed and put into a broader perspective of cell design, manufacturing, and fast charging application. In Part II of this work, the simulation is validated experimentally using multi-reference electrode single-layer pouch cells.

Advantages of high cell concentration prior to cryopreservation of initial leukapheresis in CAR-T cell therapy.

Chimeric antigen receptor (CAR) T-cell therapy is increasingly used in patients affected by B-cell lymphoma and acute lymphoblastic leukemia. For logistical reasons, initial apheresis products may be cryopreserved for shipment to manufacturing centers. Due to the characteristics of these patients, cells are often collected in large volumes, meaning more bags must be cryopreserved. This requires increased storage, time and monetary costs. In this context, we aimed to evaluate a high cell concentration cryopreservation protocol by centrifugation to standardize the initial CAR-T manufacturing procedure. Sixty-eight processes of leukapheresis of 57 patients affected by refractory/relapsed B cell lymphoma and 9 patients affected by acute lymphoblastic leukemia who were eligible for anti-CD19 CAR-T cell treatment performed between June 2019 and October 2022 were analyzed. Whole blood count, percentage and number of T cells were assessed on the apheresis final product. The apheresis product, which was alternatively stored overnight at 4°C, was centrifuged, adjusting the volume to approximately 40 mL. The product was immediately cryopreserved to achieve a final cell concentration of 50-200×106 cells/ml for cryopreservation. Leukapheresis volume was reduced by almost fivefold (median: 185 to 40 mL), resulting in a higher product concentration in one bag. In addition, the number of non-target cells (monocytes, platelets and erythrocytes) was also reduced during the development of CAR-T cell therapy, thereby maintaining T lymphocyte levels and providing a purer starting material. The advantages of the protocol include reducing economic costs, saving storage space, simplifying the manufacturing process, and facilitating shipping logistics. In conclusion, we present a validated, simple, and cost-effective cell enrichment processing protocol that provides high-quality cryopreserved products as starting material for the CAR-T cell manufacturing process.

Exploiting green energy potential via FinTech: The role of DLT-based crowdfunding in PV and ESS investments

This article explores the interplay of green energy potential and innovative financial technology (FinTech). To achieve this, an in-depth study on the technological, economic, and environmental aspects of residential-scale solar energy and storage investments was performed. As the first part of the study, residential-scale solar energy and associated energy storage investments were modeled in the assessed countries. As the next step, the effects of Distributed Ledger Technology (DLT) enabled crowdfunding on the modeled investments were highlighted with a Proof-of-Concept (PoC) DLT-enabled investment tool. As the last step, the Life Cycle Assessments (LCAs) of the investments were carried out for each of the assessed countries in order to provide better investment decisions to various investors. The results of this research indicate that with the integration of novel Fintech tools, the Levelized Cost of Storage (LCOS) values of the assessed investments may be reduced by up to 58%. Meanwhile, the LCA results highlighted that solar silicon production and battery cell manufacturing have the highest LCA results, accounting for 19.1 and 63.8 kgCO2eq/kWh respectively. Meanwhile, the transportation sector had an emission of 9 kgCO2eq/kWh. Thus, with cleaner manufacturing and transportation, the emissions from PV and ESS investments may be reduced drastically.

Residual protein analysis by SDS-PAGE in clinically manufactured BM-MSC products.

Residual substances that are considered hazardous to the recipient must be removed from final cellular therapeutic products manufactured for clinical purposes. In doing so, quality rules determined by competent authorities (CAs) for the clinical use of tissue- and cell-based products can be met. In our study, we carried out residual substance analyses, and purity determination studies of trypsin and trypsin inhibitor in clinically manufactured bone marrow-derived mesenchymal stromal/stem cell products, using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method. Despite being a semiquantitative method, SDS-PAGE has several benefits over other methods for protein analysis, such as simplicity, convenience of use, and affordability. Due to its convenience and adaptability, SDS-PAGE is still a commonly used method in many laboratories, despite its limits in dynamic range and quantitative precision. Our goal in this work was to show that SDS-PAGE may be used effectively for protein measurement, especially where practicality and affordability are the major factors. The results of our study suggest a validated method to guide tissue and cell manufacturing sites for making use of an agreeable, accessible, and cost-effective method for residual substance analyses in clinically manufactured cellular therapies.

Analogical environmental cost assessment of silicon flows used in solar panels by the US and China

Achieving carbon neutrality requires deployment of large-scale renewable energy technologies like solar photovoltaic (PV) panels. Nevertheless, methods to ascertain the overall environmental impacts PVs and further improve their sustainability are under-investigated. In an effort to provide more understanding of this crucial topic, this research focuses on silicon flows—a key element for manufacturing crystalline silicon PVs. Using system dynamics modeling, we conduct a comprehensive environmental cost assessment of the silicon flows used in PVs based on a comparative analysis between the United States and China as the leading global PV manufacturers. Despite the advancement in wafer quality, material usage reductions and overall price decreases achieved in recent decades, our results project a substantial increase in energy and water consumption in China related to Metallurgical Grade Si (MG-Si), Solar Grade Si (SoG-Si) and cell manufacturing by 2030. An approximate 6.5 times increase of energy and water consumption is observed for c-Si cell manufacturing in China between 2010 and 2020. In 2030, increases of 70% in energy consumption and 69% in water use are estimated for Chinese MG-Si and SoG-Si production. The most significant environmental impact is observed in silicon cell and module manufacturing in both countries, particularly concerning GHG, SOx and NOx emissions. This study provides valuable insights into the environmental impacts of these two major solar panel manufacturing countries by examining the silicon life cycle, from production to end-of-life.

Technological parameters of thin-film pulsed laser scribing for perovskite photovoltaics

Abstract Over the past decade, the power conversion efficiency of halide perovskite solar cells has shown a rapid increase to 26.1%. The significant efficiency growth and the relative simplification of the technology for obtaining thin-film solar cells due to liquid printing methods determine the high potential for the low-cost perovskite solar cells manufacturing. However, efficient use of cell geometry is comparable to the size of standard crystalline-Si wafers (156:156 mm and more). Therefore, modular geometry similar to amorphous-Si solar cell approaches is used to scale perovskite solar cells. Serial electrical connection of thin-film cells requires precise processing of the conductive layers that form the device p-i-n structure. The subject of research is the development of a full pulsed laser scribing cycle for inverted perovskite solar cells. In this work, we propose a study of a laser-patterning technology In2O3:SnO2 (ITO) conductive layer and a photoactive perovskite layer Cs0,2(CH(NH2)2)0,8PbI3. Process regimes of transparent conducting electrodes based on ITO and halide perovskite layer Cs0,2(CH(NH2)2)0,8PbI3 laser patterning were obtained. The optimal parameters for the multipass mode processing of ITO and perovskite layer were determined. The cell was electrically isolated at a scribe line width of 30 μm.

Tutorial on laser locking techniques and the manufacturing of vapor cells for spectroscopy

Abstract This tutorial provides a hands-on entry point about laser locking for atomic vapor research and related research such as laser cooling. We furthermore introduce common materials and methods for the fabrication of vapor cells as a tool for this research. Its aim is not to be exhaustive, but rather to provide an overview about the possible techniques that are actively employed in labs today. Some critical parameters of locked laser system for use with thermal atomic vapors are introduced and discussed. To exemplify this, we describe a versatile locking system that caters for many of the needs we found during our research with thermal atomic vapors. We also emphasize the compromises we took during our decision-making process.

High-temperature degradation of tetragonal zirconia in solid oxide fuel and electrolysis cells: A critical challenge for long-term durability and a solution

Solid oxide fuel and electrolysis cells (SOCs) are promising devices for efficiently generating electricity and hydrogen/syngas, respectively. The preferred cell configuration by many industrial developers consists of a Ni(O)-3YSZ fuel electrode support or a 3YSZ electrolyte support, which allows the manufacturing of large-area cells. 3YSZ, like many other transformable tetragonal zirconia ceramics, is well-known to be susceptible to hydrothermal degradation at temperatures below 400 °C (LTD) but is widely believed to withstand this degradation at higher temperatures. In this work, we present our recent finding that 3YSZ is, in fact, susceptible to high-temperature degradation (HTD) after operation over 8500–10,000 h. The degradation leads to substantial embrittlement of the cells. In addition, we report on the excellent resistance to LTD and HTD of a newly developed fine-grained Ni-3YSZ support. This study highlights the importance of using HTD-resistant tetragonal zirconia ceramics in developing the next generation of strong and durable ceramic-supported SOCs.

Peptoid-Cross-Linked Hydrogel Stiffness Modulates Human Mesenchymal Stromal Cell Immunoregulatory Potential in the Presence of Interferon-Gamma.

Human mesenchymal stromal cell (hMSC) manufacturing requires the production of large numbers of therapeutically potent cells. Licensing with soluble cytokines improves hMSC therapeutic potency by enhancing secretion of immunoactive factors but typically decreases proliferative ability. Soft hydrogels, however, have shown promise for boosting immunomodulatory potential, which may compensate for decreased proliferation. Here, hydrogels are cross-linked with peptoids of different secondary structures to generate substrates of various bulk stiffnesses but fixed network connectivity. Secretions of interleukin 6, monocyte chemoattractive protein-1, macrophage colony-stimulating factor, and vascular endothelial growth factor are shown to depend on hydrogel stiffness in the presence of interferon gamma (IFN-γ) supplementation, with soft substrates further improving secretion. The immunological function of these secreted cytokines is then investigated via coculture of hMSCs seeded on hydrogels with primary peripheral blood mononuclear cells (PBMCs) in the presence and absence of IFN-γ. Cocultures with hMSCs seeded on softer hydrogels show decreased PBMC proliferation with IFN-γ. To probe possible signaling pathways, immunofluorescent studies probe the nuclear factor kappa B pathway and demonstrate that IFN-γ supplementation and softer hydrogel mechanics lead to higher activation of this pathway. Overall, these studies may allow for production of more efficacious therapeutic hMSCs in the presence of IFN-γ.

In vivo manufacture and manipulation of CAR-T cells for better druggability.

The current CAR-T cell therapy products have been hampered in their druggability due to the personalized preparation required, unclear pharmacokinetic characteristics, and unpredictable adverse reactions. Enabling standardized manufacturing and having clear efficacy and pharmacokinetic characteristics are prerequisites for ensuring the effective practicality of CAR-T cell therapy drugs. This review provides a broad overview of the different approaches for controlling behaviors of CAR-T cells in vivo. The utilization of genetically modified vectors enables in vivo production of CAR-T cells, thereby abbreviating or skipping the lengthy in vitro expansion process. By equipping CAR-T cells with intricately designed control elements, using molecule switches or small-molecule inhibitors, the control of CAR-T cell activity can be achieved. Moreover, the on-off control of CAR-T cell activity would yield potential gains in phenotypic remodeling. These methods provide beneficial references for the future development of safe, controllable, convenient, and suitable for standardized production of CAR-T cell therapy products.

Sustainable Manufacturing of Perovskite-CIGS Tandem Solar Cells through Lamination with Metal-Free Transparent Conductive Adhesives

Sustainable Manufacturing of Perovskite-CIGS Tandem Solar Cells through Lamination with Metal-Free Transparent Conductive Adhesives

Abstract CT081: A phase 1/2 study investigating the safety and efficacy of autologous TAC T cells in subjects with unresectable, locally advanced, or metastatic claudin 18.2+ solid tumors

Abstract Background: CLDN18.2 is a tight junction protein found in differentiated gastric epithelial cells, which can become abnormally or ectopically expressed in a number of solid tumor indications. This exposure profile illustrates the potential of CLDN18.2 as a candidate for targeted therapy. To date, there are no approved therapeutic agents directed against this target. The T cell antigen coupler (TAC) technology is an approach to modifying T cells ex vivo, which allows recognition and cytotoxicity of tumor cells by co-opting the natural T cell receptor. TAC T cells demonstrate a safer profile than chimeric antigen receptor T cells. TAC01-CLDN18.2 is an autologous T-cell product comprising T cells expressing CLDN18.2 TAC. Methods: This is a first-in-human study (NCT05862324) to investigate the safety and preliminary anti-tumor activity of TAC01-CLDN18.2 in CLDN18.2+/HER2- solid tumors. Subjects will undergo leukapheresis and may receive bridging anticancer therapy, if deemed necessary by the Investigator, during cell manufacturing. Prior to TAC01-CLDN18.2 infusion, subjects will undergo low-intensity lymphodepletion chemotherapy. In Phase I dose escalation, TAC01-CLDN18.2 will be administered at increasing doses (Cohorts 1-3) in adult subjects after ≥2 lines of therapy using the classic 3+3 dose escalation study design. Subjects with pancreatic ductal adenocarcinoma (PDAC) may have been treated with 1 line of prior therapy. CLDN18.2 expression levels will be determined centrally using a clinical trial assay validated across relevant indications. Dose limiting toxicities will be assessed up to 28 days from TAC01-CLDN18.2 infusion. A second dose may be administered, according to preidentified clinical and safety criteria. In Phase II, dose expansion groups will further evaluate the efficacy, safety, and pharmacokinetics of the optimal TAC01-CLDN18.2 dose, with the option of redosing. Indications will include gastric and esophageal adenocarcinoma (group A), PDAC (group B) and ovarian and non-small cell lung cancer (group C) in subjects after ≥2 and <4 lines of prior therapy (subjects with PDAC may have been exposed to 1 therapy). Definitions of eligible CLDN18.2 expression levels will be based on retrospective analysis of data from Phase 1 in association with clinical efficacy. A Simon 2-stage design will be used to enroll ≤57 subjects in Group A and ≤22 subjects in Group C. Group B will be exploratory and will enroll ≤10 subjects with an opportunity of cohort enrichment based on clinical efficacy data. The first patient was enrolled in December and continues to participate in the trial. Citation Format: Ecaterina E. Dumbrava, Daniel Olson, Samuel Saibil, Davendra P. Sohal, Syma Iqbal, Alejandro Urgelles, Maria Apostolopoulou, Amy Mueller, Kara Moss, Deyaa Adib, Ben Schlecter. A phase 1/2 study investigating the safety and efficacy of autologous TAC T cells in subjects with unresectable, locally advanced, or metastatic claudin 18.2+ solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT081.

Research progress on optimization of in vitro isolation, cultivation and preservation methods of dental pulp stem cells for clinical application.

Due to high proliferative capacity, multipotent differentiation, immunomodulatory abilities, and lack of ethical concerns, dental pulp stem cells (DPSCs) are promising candidates for clinical application. Currently, clinical research on DPSCs is in its early stages. The reason for the failure to obtain clinically effective results may be problems with the production process of DPSCs. Due to the different preparation methods and reagent formulations of DPSCs, cell characteristics may be affected and lead to inconsistent experimental results. Preparation of clinical-grade DPSCs is far from ready. To achieve clinical application, it is essential to transit the manufacturing of stem cells from laboratory grade to clinical grade. This review compares and analyzes experimental data on optimizing the preparation methods of DPSCs from extraction to resuscitation, including research articles, invention patents and clinical trials. The advantages and disadvantages of various methods and potential clinical applications are discussed, and factors that could improve the quality of DPSCs for clinical application are proposed. The aim is to summarize the current manufacture of DPSCs in the establishment of a standardized, reliable, safe, and economic method for future preparation of clinical-grade cell products.

Magnetic CAR T cell purification using an anti-G4S linker antibody

Chimeric antigen receptor (CAR) redirected T cells are successfully employed in the combat against several hematological malignancies, however, are often compromised by low transduction rates making refinement of the CAR T cell products necessary. Here, we report a broadly applicable enrichment protocol relying on marking CAR T cells with an anti-glycine4-serine (G4S) linker antibody followed by magnetic activated cell sorting (MACS). The protocol is broadly applicable since the G4S peptide is an integral part of the vast majority of CARs as it links the VH and VL recognition domains. We demonstrate the feasibility by using the canonical second generation CARs specific for CEA and Her2, respectively, obtaining highly purified CAR T cell products in a one-step procedure without impairing cell viability. The protocol is also applicable to a dual specific CAR (tandem CAR). Except for CD39, T cell activation/exhaustion markers were not upregulated after separation. Purified CAR T cells retained their functionality with respect to antigen-specific cytokine secretion, cytotoxicity, and the capacity to proliferate and eliminate cognate tumor cells upon repetitive stimulation. Collectively, the one-step protocol for purifying CAR T cells extends the toolbox for preclinical research and specifically for clinical CAR T cell manufacturing.

Optimizing lentiviral vector formulation conditions for efficient ex vivo transduction of primary human T cells in chimeric antigen receptor T-cell manufacturing

Background aimsChimeric antigen receptor (CAR) T-cell products are commonly generated using lentiviral vector (LV) transduction. Optimal final formulation buffer (FFB) supporting LV stability during cryostorage is crucial for cost-effective manufacturing. MethodsTo identify the ideal LV FFB composition for ex vivo CAR-T production, primary human T cells were transduced with vesicular stomatitis virus G-protein (VSV-G) -pseudotyped LVs (encoding a reporter gene or an anti-CD19-CAR). The formulations included phosphate-buffered saline (PBS), HEPES, or X-VIVOTM 15, and stabilizing excipients. The functional and viral particle titers and vector copy number were measured after LV cryopreservation and up to 24 h post-thaw incubation. CAR-Ts were produced with LVs in selected FFBs, and the resulting cells were characterized. ResultsPost-cryopreservation, HEPES-based FFBs provided higher LV functional titers than PBS and X-VIVOTM 15, and 10% trehalose-20 mM MgCl2 improved LV transduction efficiency in PBS and 50 mM HEPES. Thawed vectors remained stable at +4°C, while a ≤ 25% median decrease in the functional titer occurred during 24 h at room temperature. Tested excipients did not enhance LV post-thaw stability. CAR-Ts produced using LVs cryopreserved in 10% trehalose- or sucrose-20 mM MgCl2 in 50 mM HEPES showed comparable transduction rates, cell yield, viability, phenotype, and in vitro functionality. ConclusionA buffer consisting of 10% trehalose-20 mM MgCl2 in 50 mM HEPES provided a feasible FFB to cryopreserve a VSV-G -pseudotyped LV for CAR-T-cell production. The LVs remained relatively stable for at least 24 h post-thaw, even at ambient temperatures. This study provides insights into process development, showing LV formulation data generated using the relevant target cell type for CAR-T-cell manufacturing.