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This paper deals with an overview of flowshop group scheduling problems in the manufacturing environment. The aim of this paper is twofold: (i) making a comprehensive survey of research on flowshop group scheduling problems in manufacturing systems, and (ii) presenting a bibliometric analysis. We address the general definition of flowshop group scheduling problems and provide a taxonomy of methodologies used in previous literature. The papers are presented from several perspectives, including the utilised objective functions, a transformation of problem structure, benchmarks in existing literature, and solution approaches. Additionally, bibliometric analysis, including keyword and journal analyses, is conducted for articles published between 1986 and 2022. Finally, suggestions for future developments are listed to further consolidate this area. [ABSTRACT FROM AUTHOR]

As globalisation and competition grow rapidly, adopting a flexible manufacturing philosophy becomes more critical for manufacturers. Cellular Manufacturing System (CMS) is among one of the most favourable ones which has been deeply studied in academia since this philosophy was coined in 1925. To design the manufacturing system efficiently and effectively, the management team needs to have a comprehensive representation of the definitions, classifications, solution approach to cellular manufacturing design problems. In this study, exact methods, heuristic approaches, metaheuristic techniques, and artificial intelligence strategies for cellular manufacturing design are reviewed. A comparison of solution approaches is discussed and based on this analysis, some suggestions for future research are proposed. Moreover, we tried to highlight the importance of investigating sustainability decisions related to CMS problems, and integration of supply chain decisions with CMS decisions which have not been sufficiently studied to identify a research direction for researchers to model and analyse those issues. Finally, we briefly discuss the placement of CMS as one of the major production systems in the industry 4.0 evolution paradigm. Reviewing the adoption of industry 4.0 techniques by CMS sheds light on the contribution of methodological approaches in transition to intelligent CMS. [ABSTRACT FROM AUTHOR]

Biomarkers are valuable tools in clinical research where they allow to predict susceptibility to diseases, or response to specific treatments. Likewise, biomarkers can be extremely useful in the biomanufacturing of therapeutic proteins. Indeed, constraints such as short timelines and the need to find hyper-productive cells could benefit from a data-driven approach during cell line and process development. Many companies still rely on large screening capacities to develop productive cell lines, but as they reach a limit of production, there is a need to go from empirical to rationale procedures. Similarly, during bioprocessing runs, substrate consumption and metabolism wastes are commonly monitored. None of them possess the ability to predict the culture behavior in the bioreactor. Big data driven approaches are being adapted to the study of industrial mammalian cell lines, enabled by the publication of Chinese hamster and CHO genome assemblies which allowed the use of next-generation sequencing with these cells, as well as continuous proteome and metabolome annotation. However, if these different -omics technologies contributed to the characterization of CHO cells, there is a significant effort remaining to apply this knowledge to biomanufacturing methods. The correlation of a complex phenotype such as high productivity or rapid growth to the presence or expression level of a specific biomarker could save time and effort in the screening of manufacturing cell lines or culture conditions. In this review we will first discuss the different biological molecules that can be identified and quantified in cells, their detection techniques, and associated challenges. We will then review how these markers are used during the different steps of cell line and bioprocess development, and the inherent limitations of this strategy. • Biomarkers characterize the physiological state of cells and help predict their behavior. • A biomarker can consist of any macromolecule present in the cell that can be detected and quantified. • Discovering and identifying biomarkers requires specific and customized methods. • In an industrial context, biomarkers enable a more rational and efficient approach to bioprocessing. • Biomarkers are often specific to environmental conditions and, as such, have limitations. [ABSTRACT FROM AUTHOR]

Biomedical research has witnessed significant strides in manufacturing chimeric antigen receptor T cell (CAR-T) therapies, marking a transformative era in cellular immunotherapy. Nevertheless, existing manufacturing methods for autologous cell therapies still pose several challenges related to cost, immune cell source, safety risks, and scalability. These challenges have motivated recent efforts to optimize process development and manufacturing for cell therapies using automated closed-system bioreactors and models created using artificial intelligence. Simultaneously, non-viral gene transfer methods like mRNA, CRISPR genome editing, and transposons are being applied to engineer T cells and other immune cells like macrophages and natural killer cells. Alternative sources of primary immune cells and stem cells are being developed to generate universal, allogeneic therapies, signaling a shift away from the current autologous paradigm. These multifaceted innovations in manufacturing underscore a collective effort to propel this therapeutic approach toward broader clinical adoption and improved patient outcomes in the evolving landscape of cancer treatment. Here, we review current CAR immune cell manufacturing strategies and highlight recent advancements in cell therapy scale-up, automation, process development, and engineering. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) represent a new axis of intercellular communication that can be harnessed for therapeutic purposes, as cell-free therapies. The clinical application of mesenchymal stromal cell (MSC)-derived EVs, however, is still in its infancy and faces many challenges. The heterogeneity inherent to MSCs, differences among donors, tissue sources, and variations in manufacturing conditions may influence the release of EVs and their cargo, thus potentially affecting the quality and consistency of the final product. We investigated the influence of cell culture and conditioned medium harvesting conditions on the physicochemical and proteomic profile of human umbilical cord MSC-derived EVs (hUCMSC-EVs) produced under current good manufacturing practice (cGMP) standards. We also evaluated the efficiency of the protocol in terms of yield, purity, productivity, and expression of surface markers, and assessed the biodistribution, toxicity and potential efficacy of hUCMSC-EVs in pre-clinical studies using the LPS-induced acute lung injury model. hUCMSCs were isolated from a cord tissue, cultured, cryopreserved, and characterized at a cGMP facility. The conditioned medium was harvested at 24, 48, and 72 h after the addition of EV collection medium. Three conventional methods (nanoparticle tracking analysis, transmission electron microscopy, and nanoflow cytometry) and mass spectrometry were used to characterize hUCMSC-EVs. Safety (toxicity of single and repeated doses) and biodistribution were evaluated in naive mice after intravenous administration of the product. Efficacy was evaluated in an LPS-induced acute lung injury model. hUCMSC-EVs were successfully isolated using a cGMP-compliant protocol. Comparison of hUCMSC-EVs purified from multiple harvests revealed progressive EV productivity and slight changes in the proteomic profile, presenting higher homogeneity at later timepoints of conditioned medium harvesting. Pooled hUCMSC-EVs showed a non-toxic profile after single and repeated intravenous administration to naive mice. Biodistribution studies demonstrated a major concentration in liver, spleen and lungs. HUCMSC-EVs reduced lung damage and inflammation in a model of LPS-induced acute lung injury. hUCMSC-EVs were successfully obtained following a cGMP-compliant protocol, with consistent characteristics and pre-clinical safety profile, supporting their future clinical development as cell-free therapies. [ABSTRACT FROM AUTHOR]

Although several cell-based therapies have received FDA approval, and others are showing promising results, scalable, and quality-driven reproducible manufacturing of therapeutic cells at a lower cost remains challenging. Challenges include starting material and patient variability, limited understanding of manufacturing process parameter effects on quality, complex supply chain logistics, and lack of predictive, well-understood product quality attributes. These issues can manifest as increased production costs, longer production times, greater batch-to-batch variability, and lower overall yield of viable, high-quality cells. The lack of data-driven insights and decision-making in cell manufacturing and delivery is an underlying commonality behind all these problems. Data collection and analytics from discovery, preclinical and clinical research, process development, and product manufacturing have not been sufficiently utilized to develop a "systems" understanding and identify actionable controls. Experience from other industries shows that data science and analytics can drive technological innovations and manufacturing optimization, leading to improved consistency, reduced risk, and lower cost. The cell therapy manufacturing industry will benefit from implementing data science tools, such as data-driven modeling, data management and mining, AI, and machine learning. The integration of data-driven predictive capabilities into cell therapy manufacturing, such as predicting product quality and clinical outcomes based on manufacturing data, or ensuring robustness and reliability using data-driven supply-chain modeling could enable more precise and efficient production processes and lead to better patient access and outcomes. In this review, we introduce some of the relevant computational and data science tools and how they are being or can be implemented in the cell therapy manufacturing workflow. We also identify areas where innovative approaches are required to address challenges and opportunities specific to the cell therapy industry. We conclude that interfacing data science throughout a cell therapy product lifecycle, developing data-driven manufacturing workflow, designing better data collection tools and algorithms, using data analytics and AI-based methods to better understand critical quality attributes and critical-process parameters, and training the appropriate workforce will be critical for overcoming current industry and regulatory barriers and accelerating clinical translation. [ABSTRACT FROM AUTHOR]

The production of commercial autologous cell therapies such as chimeric antigen receptor T cells requires complex manual manufacturing processes. Skilled labor costs and challenges in manufacturing scale-out have contributed to high prices for these products. We present a robotic system that uses industry-standard cell therapy manufacturing equipment to automate the steps involved in cell therapy manufacturing. The robotic cluster consists of a robotic arm and customized modules, allowing the robot to manipulate a variety of standard cell therapy instruments and materials such as incubators, bioreactors, and reagent bags. This system enables existing manual manufacturing processes to be rapidly adapted to robotic manufacturing, without having to adopt a completely new technology platform. Proof-of-concept for the robotic cluster's expansion module was demonstrated by expanding human CD8+ T cells. The robotic cultures showed comparable cell yields, viability, and identity to those manually performed. In addition, the robotic system was able to maintain culture sterility. Such modular robotic solutions may support scale-up and scale-out of cell therapies that are developed using classical manual methods in academic laboratories and biotechnology companies. This approach offers a pathway for overcoming manufacturing challenges associated with manual processes, ultimately contributing to the broader accessibility and affordability for personalized immunotherapies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article presents the possibilities of automating production pre-processing and post-processing operations for the Material Extrusion - MEX process based on Fused Filament Fabrication technology. Automation is based on hardware and software solutions. For this purpose, a special research station was developed, equipped with a warehouse of working platforms, a 3D printer and a collaborative robot that integrates individual elements of the manufacturing process. The developed solution allows for increasing the efficiency of the manufacturing cell and reducing the operator's involvement in manual operations at the pre-processing and post-processing stages. [ABSTRACT FROM AUTHOR]

CD19-targeted chimeric antigen receptors (CAR) T cells are one of the most remarkable cellular therapies for managing B cell malignancies. However, long-term disease-free survival is still a challenge to overcome. Here, we evaluated the influence of different hinge, transmembrane (TM), and costimulatory CAR domains, as well as manufacturing conditions, cellular product type, doses, patient's age, and tumor types on the clinical outcomes of patients with B cell cancers treated with CD19 CAR T cells. The primary outcome was defined as the best complete response (BCR), and the secondary outcomes were the best objective response (BOR) and 12-month overall survival (OS). The covariates considered were the type of hinge, TM, and costimulatory domains in the CAR, CAR T cell manufacturing conditions, cell population transduced with the CAR, the number of CAR T cell infusions, amount of CAR T cells injected/Kg, CD19 CAR type (name), tumor type, and age. Fifty-six studies (3493 patients) were included in the systematic review and 46 (3421 patients) in the meta-analysis. The overall BCR rate was 56%, with 60% OS and 75% BOR. Younger patients displayed remarkably higher BCR prevalence without differences in OS. The presence of CD28 in the CAR's hinge, TM, and costimulatory domains improved all outcomes evaluated. Doses from one to 4.9 million cells/kg resulted in better clinical outcomes. Our data also suggest that regardless of whether patients have had high objective responses, they might have survival benefits from CD19 CAR T therapy. This meta-analysis is a critical hypothesis-generating instrument, capturing effects in the CD19 CAR T cells literature lacking randomized clinical trials and large observational studies. [ABSTRACT FROM AUTHOR]

The present work assesses the manufacturing method for producing ceramic-based solid oxide fuel cells (SOFC) using compaction and sintering methods, which are combined by adding pore former to improve the porosity of the produced anode. The ratio of pore former varies between 5%, 10% and 15%. The base cermet material in this work is NiOYSZ (Nickle Oxide/Yttria Stabilized Zirconia), which is suitable for high-temperature SOFC. The base material is mixed with pore former at the given ratio, followed by ultrasonic cleaning. This work uses polyethylene glycol (PEG, 10%) as binder material. The produced slurry is then dried in electric oven, continued with the compaction process at a pressure of 135MPa and sintered for 4 hours at 1200°C. The addition of pore former leads to significant improvement in the porosity level, with the highest value of 16.67%. It contributes to the excellent performance of the produced anode, particularly for the permeability value. The NiO-YSZ with 15% pore former has the highest permeability value with average of 0.37 m². Moreover, the detailed permeability profile shows the baseline ratio for adding pore former is between 5% and 10%, which experiences significant variation in the porosity level and permeability value. [ABSTRACT FROM AUTHOR]

Cell therapies such as genetically modified T cells have emerged as a promising and viable treatment for hematologic cancers and are being aggressively pursued for a wide range of diseases and conditions that were previously difficult to treat or had no cure. The process development requires genetic modifications to T cells to express a receptor (engineered T cell receptor (eTCR)) of specific binding qualities to the desired target. Protein reagents utilized during the cell therapy manufacturing process, to facilitate these genetic modifications, are often present as process-related impurities at residual levels in the final drug product and can represent a potential immunogenicity risk upon infusion. This manuscript presents a framework for the qualification of an assay for assessing the immunogenicity risk of AA6 and Cas9 residuals. The same framework applies for other residuals; however, AAV6 and Cas9 were selected as they were residuals from the manufacturing of an engineered T cell receptor cellular product in development. The manuscript: 1) elucidates theoretical risks, 2) summarizes analytical data collected during process development, 3) describes the qualification of an in vitro human PBMC cytokine release assay to assess immunogenicity risk from cellular product associated process residuals; 4) identifies a multiplexed inflammatory innate and adaptive cytokine panel with pre-defined criteria using relevant positive controls; and 5) discusses qualification challenges and potential solutions for establishing meaningful thresholds. The assessment is not only relevant to establishing safe exposure levels of these residuals but also in guiding risk assessment and CMC strategy during the conduct of clinical trials. [Display omitted] • Process-related impurities/residuals in cell and gene therapies production must be characterized for their potential to induce innate and adaptive immune responses prior to patient administration of the final drug product. • AAV empty capsids and Cas9 protein have the potential to induce both innate and adaptive responses. • A positive response was first detected at 7.2 × 1011 AAV6 capsids/mL for adaptive cytokines at 7 days, and 7.2 × 1012 capsids/mL for innate cytokines at 48 hours. For both innate cytokine release at 48 hours as well as adaptive cytokines at 7 days, it required 143 nM of Cas9 protein to see significant cytokine release. • Overall, for both AAV6 and Cas9, theoretical post-infusion maximum concentrations were approximately 30- and 100-fold below levels associated with immune activation in healthy donors as determined preliminarily in the in vitro human Peripheral Blood Mononuclear Cells (PBMC) assay. • Future studies for cell therapy including residual levels and association or lack of association with safety adverse events, including immunogenicity, are needed to validate prediction assessments such as the one presented here. [ABSTRACT FROM AUTHOR]

With the success of chimeric antigen receptor T-cell (CAR-T) and similar cellular-based therapies, the demand for collection of autologous mononuclear cells by apheresis (MNC(A)) from blood by apheresis has increased. From an apheresis technical standpoint, the collection of MNC(A) is relatively straightforward, especially when compared with collection of hematopoietic progenitor cells (HPC(A)). Most of the collection for MNC(A) are performed for the commercial entities, who use the product for manufacturing cellular therapeutics. We have noticed discrepancies in the handling and apheresis processes required by different companies in obtaining essentially the same product (all companies in the study manufacture CAR-T-based products). We have analyzed the MNC collection requirements from all FDA-approved CAR-T cellular products and some investigational products collected at University of Nebraska Medical Center. We identified discrepancies in the process and suggested mitigation strategies. Step-by-step analysis of the collection requirements. Review of the current guidelines and recommendations on this issue. Multiple discrepancies in the collection process have been identified, even in the products collected for the same company. Practical approach of satisfying all the requirements based on University of Nebraska Medical Center experience has been suggested. The current recommendations from multiple sources were reviewed in discussion. [ABSTRACT FROM AUTHOR]

Vγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. Specifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. We found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity. This study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement. [ABSTRACT FROM AUTHOR]

Mammalian cell line stability is an important consideration when establishing a biologics manufacturing process in the biopharmaceutical and in vitro diagnostics (IVD) industries. Traditional Chinese hamster ovary (CHO) cell line development methods use a random integration approach that requires transfection, selection, optional amplification, screenings, and single-cell cloning to select clones with acceptable productivity, product quality, and genetic stability. Site-specific integration reduces these disadvantages, and new technologies have been developed to mitigate risks associated with genetic instability. In this study, we applied the Leap-In® transposase-mediated expression system from ATUM to generate stable CHOK1 pools for the production of four recombinant antibody reagents for IVD immunoassays. CHO cell line stability is defined by consistent antibody production over time. Three of the CHOK1 pools maintained productivity suitable for manufacturing, with high antibody yields. The productivity of the remaining CHOK1 pool decreased over time; however, derivative clones showed acceptable stability. l-glutamine had variable effects on CHOK1 cell line or stable pool stability and significantly affected antibody product titer. Compared with traditional random integration methods, the ATUM Leap-In system can reduce the time needed to develop new immunoassays by using semi site-specific integration to generate high-yield stable pools that meet manufacturing stability requirements. [ABSTRACT FROM AUTHOR]

Flexible manufacturing cells provide significant advantages in low-volume mass-customization production but also induce added complexity and technical challenges in terms of integration, control, and extensibility. The variety of closed-source industrial protocols, the heterogeneous equipment, and the product's manufacturing specifications are main points of consideration in the development of such a system. This study aims to describe the approach, from concept to implementation, for the development of the controller for a flexible manufacturing cell consisting of heterogeneous equipment in terms of functions and communication interfaces. Emphasis is put on the considerations and challenges for effective integration, extensibility, and interoperability. Scheduling and monitoring performed by the developed controller are demonstrated for a manufacturing cell producing microfluidic devices (bioMEMS) that consists of six workstations and a robot-based handling system. Communication between the system controller and the workstations was based on open-source technologies instead of proprietary software and protocols, to support interoperability and, to a considerable extent, code reusability. [ABSTRACT FROM AUTHOR]

The Cell Formation Problem (CFP) is a widely studied issue that aims to group machines effectively based on criteria such as productivity, lower costs, and greater efficiency. In recent years, more characteristics were summarized relating to this problem. This paper provides a bibliographic examination of methodologies addressing the CFP in cellular manufacturing, focusing on novel approaches such as alternative routes and machine reliability. The articles were obtained from Scopus and Web of Science and filtered using the PRISMA methodology. Classification based on objective functions, constraints, and methodologies facilitated informative visualizations for analysis. Findings indicate a focus on capital utilization optimization, with cost reduction via intercellular moves minimization as the primary objective. Common constraints include limits on the number of machines per cell, restricting machines to a single cell and singular production routes per part. The genetic algorithm predominates as a non-exact solution approach, while the " ε -constraint" method is commonly used. This study offers insights into contemporary trends in solving the CFP with alternative routings and machine reliability, aiding researchers and professionals in the field to improve the quality of their investigations. [ABSTRACT FROM AUTHOR]

The article focuses on the concepts of Cell Image Segmentation (CIS) and the gradual introduction of cell counting. Motivated by the rapid development of Machine learning (ML) methods, which is carried out in this investigation. ML is evolving from theory to practical applications, with deep neural network models extensively used in academia and business for various applications, including image counting and natural language processing. These advancements can greatly influence medical imaging technologies, data processing, diagnostics, and healthcare in general. Main objectives of the research are to provide an overview of biological cell counting methods in microscopic images and to explore deep learning (DL)-based image segmentation approaches. The study expertly describes current trends, cutting-edge learning technologies, and platforms utilized for DL approaches. Cell counting is one of the most researched and challenging subjects in computer vision systems. Academics are increasingly interested in this area due to its real-time applications in biology, biochemistry, medical diagnostics, computer vision-based cell tracking systems for large populations, and stem cell manufacturing. Counting cells in the biological field is beneficial. For instance, the ratio of white blood cells to cancer cells in the blood can help determine the origin of a disease. Biologists also need to count cells within cell cultures to monitor the time-dependent growth of cells during bacterial experiments. Numerous methods for cell counting have been developed, after addressing the challenges with Cell Counting algorithms; the article explores promising future directions in CIS and cell counting research fields. [ABSTRACT FROM AUTHOR]

Abstract\nKEY POINTSHigh-purity quartz (HPQ) is the only naturally occurring and economically viable source for the production of silicon. Silicon is a critical mineral, and a key component in modern technologies such as semiconductors and photovoltaic cells. Critical minerals support the move towards a greater reliance on electrification, renewable energy sources and economic security. The global transition to net zero carbon emissions means there is a growing need for new discoveries of HPQ to supply the silicon production chain. HPQ deposits are identified in a multitude of geological settings, including pegmatites, hydrothermal veins, sedimentary accumulations and quartzite; however, deposits of sufficient volume and quality are rare. Quartz is abundant throughout Australia, but the exploration and discovery of HPQ occurrences are notably under-reported, making assessment of the HPQ potential in Australia extremely difficult. This paper presents a much-needed summary of the state of the HPQ industry, exploration and deposit styles in Australia.High-purity quartz (HPQ) is a key material for the manufacture of photovoltaic cells, semiconductors and other high-technology applications.HPQ can be recovered from a variety of different source rocks in a range of geological settings.Currently, the HPQ industry in Australia is under-utilised for high-technology applications, and historical exploration and mining records are under-reported and opaque.This review presents an outline of the characteristics, processing requirements and end uses of HPQ, and a summary of the operations, deposits, exploration targets and known occurrences of HPQ in Australia. [ABSTRACT FROM AUTHOR]

With the long persistence of complex, chronic diseases in society, there is increasing motivation to develop cells as living medicine to treat diseases ranging from cancer to wounds. While cell therapies can significantly impact healthcare, the shortage of starter cells meant that considerable raw materials must be channeled solely for cell expansion, leading to expensive products with long manufacturing time which can prevent accessibility by patients who either cannot afford the treatment or have highly aggressive diseases and cannot wait that long. Over the last three decades, there has been increasing knowledge on the effects of electrical modulation on proliferation, but to the best of the knowledge, none of these studies went beyond how electro‐control of cell proliferation may be extended to enhance industrial scale cell manufacturing. Here, this review is started by discussing the importance of maximizing cell yield during manufacturing before comparing strategies spanning biomolecular/chemical/physical to modulate cell proliferation. Next, the authors describe how factors governing invasive and non‐invasive electrical stimulation (ES) including capacitive coupling electric field may be modified to boost cell manufacturing. This review concludes by describing what needs to be urgently performed to bridge the gap between academic investigation of ES to industrial applications. [ABSTRACT FROM AUTHOR]

Mesenchymal stem and stromal cells (MSCs) hold potential to treat a broad range of clinical indications, but clinical translation has been limited to date due in part to challenges with batch-to-batch reproducibility of potential critical quality attributes (pCQAs) that can predict potency/efficacy. Here, we designed and implemented a microcarrier-microbioreactor approach to cell therapy manufacturing, specific to anchorage-dependent cells such as MSCs. We sought to assess whether increased control of the biochemical and biophysical environment had the potential to create product with consistent presentation and elevated expression of pCQAs relative to established manufacturing approaches in tissue culture polystyrene (TCPS) flasks. First, we evaluated total cell yield harvested from dissolvable, gelatin microcarriers within a microbioreactor cassette (Mobius Breez) or a flask control with matched initial cell seeding density and culture duration. Next, we identified 24 genes implicated in a therapeutic role for a specific motivating indication, acute respiratory distress syndrome (ARDS); expression of these genes served as our pCQAs for initial in vitro evaluation of product potency. We evaluated mRNA expression for three distinct donors to assess inter-donor repeatability, as well as for one donor in three distinct batches to assess within-donor, inter-batch variability. Finally, we assessed gene expression at the protein level for a subset of the panel to confirm successful translation. Our results indicated that MSCs expanded with this microcarrier-microbioreactor approach exhibited reasonable donor-to-donor repeatability and reliable batch-to-batch reproducibility of pCQAs. Interestingly, the baseline conditions of this microcarrier-microbioreactor approach also significantly improved expression of several key pCQAs at the gene and protein expression levels and reduced total media consumption relative to TCPS culture. This proof-of-concept study illustrates key benefits of this approach to therapeutic cell process development for MSCs and other anchorage-dependent cells that are candidates for cell therapies. [ABSTRACT FROM AUTHOR]

Cumulative evidence from several pre-clinical studies suggests that restoration of plasma DNase activity in a thrombo-inflammatory state may improve clinical outcomes. Following injury, hyperactivated immune cells release large amounts of granular proteins together with DNA, which often accumulate in the surrounding environment in so-called neutrophil extracellular traps (NETs). Degradation of excess NETs by systemic DNase administration offers a promising therapeutic approach to ameliorate inflammation and dissolve intravascular clots. In order to expand the therapeutic utility of human DNase I, a variant of the enzyme was developed that has both a prolonged systemic half-life and a higher catalytic activity compared to Dornase alfa (Pulmozyme®), the recombinant form of DNase I approved for inhaled therapy of cystic fibrosis. The hyperactive enzyme was "PASylated" by genetic fusion with a strongly hydrophilic and biodegradable PAS-polypeptide to increase its hydrodynamic volume and retard kidney filtration. A stable TurboCell™ CHO-K1-based cell line was generated which is suitable for the future production of PASylated DNase I according to good manufacturing practice (GMP). Furthermore, a robust bioprocess strategy was devised and an effective downstream process was developed. The final protein product is characterized by excellent purity, favorable physicochemical properties, a 14-fold higher DNA-degrading activity than Dornase alfa and a sustained pharmacokinetic profile, with a 22-fold slower clearance in rats. [ABSTRACT FROM AUTHOR]

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (T n/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O 2 from day 5 onward, we could generate >60% T n/scm -like cells, with longer time in hypoxia correlating with a higher percentage of T n/scm -like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) monocyte and macrophage therapies are promising solid tumor immunotherapies that can overcome the challenges facing conventional CAR T cell therapy. mRNA lipid nanoparticles (mRNA‐LNPs) offer a viable platform for in situ engineering of CAR monocytes with transient and tunable CAR expression to reduce off‐tumor toxicity and streamline cell manufacturing. However, identifying LNPs with monocyte tropism and intracellular delivery potency is difficult using traditional screening techniques. Here, ionizable lipid design and high‐throughput in vivo screening are utilized to identify a new class of oxidized LNPs with innate tropism and mRNA delivery to monocytes. A library of oxidized (oLNPs) and unoxidized LNPs (uLNPs) is synthesized to evaluate mRNA delivery to immune cells. oLNPs demonstrate notable differences in morphology, ionization energy, and pKa, thereby enhancing delivery to human macrophages, but not T cells. Subsequently, in vivo library screening with DNA barcodes identifies an oLNP formulation, C14‐O2, with innate tropism to monocytes. In a proof‐of‐concept study, the C14‐O2 LNP is used to engineer functional CD19‐CAR monocytes in situ for robust B cell aplasia (45%) in healthy mice. This work highlights the utility of oxidized LNPs as a promising platform for engineering CAR macrophages/monocytes for solid tumor CAR monocyte therapy. [ABSTRACT FROM AUTHOR]

In this study we demonstrate detection of redox peaks in Li‐ion pouch cells during cyclic voltammetry (CV), via the optical signal of plasmonic based fibre optic sensors placed inside Li‐ion pouch cells. The sensors are placed inside the pouch cells during manufacture, allowing diagnostic data to be obtained from directly inside the cells in real time. We further present data showing the battery cell remains agnostic to the presence of fibre optic sensors over repeated cycling through analysis of the formation cycles, electrochemical impedance spectroscopy (EIS) analysis, coulombic efficiency data, capacity data and post‐mortem materials analysis. This study provides evidence of the utility of fibre optic based diagnostic sensors, and in particular the novel use of plasmonic based fibre optic sensors, as an in situ battery diagnostic technique and potential research tool to investigate battery cell phenomena. [ABSTRACT FROM AUTHOR]

This paper presents a collaborative manufacturing cell implemented in a laboratory setting, focusing on developing learning-based interaction abilities to enhance versatility and ease of use. The key components of the system include 3D real-time volumetric monitoring for safety, visual recognition of hand gestures for human-to-robot communication, classification of physical-contact-based interaction primitives during handover operations, and detection of hand–object interactions to anticipate human intentions. Due to the nature and complexity of perception, deep-learning-based techniques were used to enhance robustness and adaptability. The main components are integrated in a system containing multiple functionalities, coordinated through a dedicated state machine. This ensures appropriate actions and reactions based on events, enabling the execution of specific modules to complete a given multi-step task. An ROS-based architecture supports the software infrastructure among sensor interfacing, data processing, and robot and gripper controllers nodes. The result is demonstrated by a functional use case that involves multiple tasks and behaviors, paving the way for the deployment of more advanced collaborative cells in manufacturing contexts. [ABSTRACT FROM AUTHOR]

Industrial automation in the manufacturing environment has revolutionized production and manufacturing in many industries, generating significant improvements in efficiency, quality, and process effectiveness. However, it has also posed challenges related to feedback in manufacturing environment monitoring systems, and increasing the effectiveness, productivity, and quality in industrial production. Feedback systems in the manufacturing environment are fundamental to industrial automation, which is why an application has been developed for the detection of elements in a printed circuit board manufacturing cell. The solution presented in this article proposes implementing a continuous feedback system with the ability to provide real-time information to identify the location of elements in a manufacturing cell and potentially detect anomalies, with the goal of improving the manufacturing process appropriately. [ABSTRACT FROM AUTHOR]

This work presents an image‐based hybrid model incorporating the initial spatial distribution for mesenchymal stem cell (MSC) cultivation process design. First, three levels of seeding bias in static MSC cultivation were experimentally investigated, and the resulting initial distribution was quantified using phase contrast microscopy image analysis. Second, the observed spatial heterogeneity was defined as a new parameter by calculating the standard deviation of the seeded cell number fraction among numerical 8 × 8 tiles on two‐dimensional plates. Third, the parameter was incorporated into kinetic models to consider spatial growth limitation. The model was then applied to simulate MSC cultivation processes with experimental data. Using dynamic and stochastic simulation outputs, feasible ranges of cell‐harvesting time could be determined to satisfy a given requirement for the minimum cell number and acceptable confluency level. The developed hybrid model could serve as a basis for quantitative decision‐making in the design of MSC cultivation processes. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this article is to conduct a bibliometric analysis of the cellular manufacturing system (CMS) literature published from 1982–2021 to identify key issues and trends for the future. Design/methodology/approach: A six-stage methodology is used to conduct a literature review, which includes: (1) article collection; (2) inclusion/exclusion criteria; (3) reviewing the articles; (4) analyzing the articles; (5) framework development; and (6) future research directions. A total of 936 CMS-specific articles are reviewed. This paper made use of three software tools: the R package, VOSviewer and SciMAT. Findings: According to the findings, the majority of CM researchers focused on cell formation and design. The USA, Iran and India are the top three leading publishers. Additionally, the gap and future direction of CM are discussed. Originality/value: To the best of the authors' knowledge, the current study is the first attempt to investigate CMS evaluation through bibliometric and thematic analysis and provides a decisional framework as well as steps for CMS adoption. For individuals who are interested in understanding more about CMS and its evolution, this paper offers a starting point. [ABSTRACT FROM AUTHOR]

The safety and efficacy of both cell and gene therapies have been demonstrated in numerous preclinical and clinical trials. Chimeric antigen receptor T (CAR-T) cell therapy, which leverages the technologies of both cell and gene therapies, has also shown great promise for treating various cancers. Advancements in pertinent fields have also highlighted challenges faced while manufacturing cell and gene therapy products. Potential problems and obstacles must be addressed to ease the clinical translation of individual therapies. Literature reviews of representative cell-based, gene-based, and cell-based gene therapies with regard to their general manufacturing processes, the challenges faced during manufacturing, and QC specifications are limited. We review the general manufacturing processes of cell and gene therapies, including those involving mesenchymal stem cells, viral vectors, and CAR-T cells. The complexities associated with the manufacturing processes and subsequent QC/validation processes may present challenges that could impede the clinical progression of the products. This article addresses these potential challenges. Further, we discuss the use of the manufacturing model and its impact on cell and gene therapy. [ABSTRACT FROM AUTHOR]

CAR-T cell therapy has shown remarkable promise in treating B-cell malignancies, which has sparked optimism about its potential to treat other types of cancer as well. Nevertheless, the Expectations of CAR-T cell therapy in solid tumors and non-B cell hematologic malignancies have not been met. Furthermore, safety concerns regarding the use of viral vectors and the current personalized production process are other bottlenecks that limit its widespread use. In recent years the use of gene editing technology in CAR-T cell therapy has opened a new way to unleash the latent potentials of CAR-T cell therapy and lessen its associated challenges. Moreover, gene editing tools have paved the way to manufacturing CAR-T cells in a fully non-viral approach as well as providing a universal, off-the-shelf product. Despite all the advantages of gene editing strategies, the off-target activity of classical gene editing tools (ZFNs, TALENs, and CRISPR/Cas9) remains a major concern. Accordingly, several efforts have been made in recent years to reduce their off-target activity and genotoxicity, leading to the introduction of advanced gene editing tools with an improved safety profile. In this review, we begin by examining advanced gene editing tools, providing an overview of how these technologies are currently being applied in clinical trials of CAR-T cell therapies. Following this, we explore various gene editing strategies aimed at enhancing the safety and efficacy of CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

This study focuses on reducing the weight of the stack applied to the unit cell during the structuring process of a solid oxide fuel cell (SOFC) stack. By using a porous Ag coating layer as a current collector, separator plates and connectors are eliminated and the manufacturing cost of the stack can thereby be reduced. Additionally, the performance and stability of the stack are significantly improved by reducing the contact resistance between cells. The Ag coating layer used in this study can be optimized by adjusting the coating thickness and the ratio of graphene and Ag paste, which affects dispersibility and porosity. The optimized Ag porous coating layer makes it possible to manufacture a stack cell with a simple structure without requiring a separator, and excellent performance is confirmed in a long-term test of 4000 h. [Display omitted] • Highlights (for review). • Reduced stack weight and manufacturing costs through porous Ag coating as a current collector. • Eliminated separator plates and connectors, enhancing stack performance and stability. • Optimized Ag porous coating layer for simplified stack cell structure and improved performance. • Demonstrated excellent long-term performance in a 4000-h test. [ABSTRACT FROM AUTHOR]

Background: There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac). Methods: We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks. Results: The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone. Conclusions: The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications. [ABSTRACT FROM AUTHOR]

Exploiting the ability to directly deposit larger‐sized, flexible, alloying‐type sodium‐ion battery electrodes without the need of additional inactive components and processing steps is a strategic way to achieve higher gravimetric capacities which will be suitable for commercial‐level sodium‐ion (SIB) pouch cell manufacturing. In this regard, a simple, template‐free electroplating protocol is reported for designing phase‐pure tin antimonide (TA)‐based negatrode. An optimum control over the potentiostatic electroplating parameters allows the preferential formation of dendritic nanostructures over an electroplated foam‐based current collector. The prepared negatrode and Mn‐based Prussian blue analog positrode in full coin cell format deliver an average nominal working voltage of 3.0 V with a discharge energy density of 342 Wh kg−1. In single‐layer full pouch cell format, an 8.01 mAh capacity (89.89 mAh g−1, 593.3 µAh cm−2) is achieved at an undeformed state and 97.12% capacity is retained at 180° bend condition. In multi‐layer full pouch cell format, a 35.86 mAh capacity (80.58 mAh g−1, 531.2 µAh cm−2) is achieved with 62.77% capacity retention after 200 cycles (0.125 C). The results of this work showcase how a simple, template free, aqueous electrolyte based electroplating protocol can be developed for the direct fabrication of large‐sized, flexible, alloying‐type negatrodes for commercial SIB pouch cells. [ABSTRACT FROM AUTHOR]

The intriguing biomineralization process in nature endows the mineralized biological materials with intricate microarchitected structures in a facile and orderly way, which provides an inspiration for processing ceramics. Here, we propose a simple and efficient manufacturing process to fabricate cellular ceramics in programmed cell-based 3D configurations, inspired by the biomineralization process of the diatom frustule. Our approach separates the ingredient synthesis from architecture building, enabling the programmable manufacturing of cellular ceramics with various cell sizes, geometries, densities, metastructures, and constituent elements. Our approach exploits surface tension to capture precursor solutions in the architected cellular lattices, allowing us to control the liquid geometry and manufacture cellular ceramics with high precision. We investigate the geometry parameters for the architected lattices assembled by unit cells and unit columns, both theoretically and experimentally, to guide the 3D fluid interface creation in arranged configurations. We manufacture a series of globally cellular and locally compact piezoceramics, obtaining an enhanced piezoelectric constant and a designed piezoelectric anisotropy. This bioinspired, surface tension-assisted approach has the potential to revolutionize the design and processing of multifarious ceramic materials for structural and functional applications in energy, electronics and biomedicine. Three-dimensional architecture engineering allows for higher performance ceramics with more diverse applications. Here, the authors report a simple and efficient manufacturing process for cellular ceramics in programmed cell-based 3D configurations. [ABSTRACT FROM AUTHOR]

In exploring the challenges of bone repair and regeneration, this review evaluates the potential of bone tissue engineering (BTE) as a viable alternative to traditional methods, such as autografts and allografts. Key developments in biomaterials and scaffold fabrication techniques, such as additive manufacturing and cell and bioactive molecule-laden scaffolds, are discussed, along with the integration of bio-responsive scaffolds, which can respond to physical and chemical stimuli. These advancements collectively aim to mimic the natural microenvironment of bone, thereby enhancing osteogenesis and facilitating the formation of new tissue. Through a comprehensive combination of in vitro and in vivo studies, we scrutinize the biocompatibility, osteoinductivity, and osteoconductivity of these engineered scaffolds, as well as their interactions with critical cellular players in bone healing processes. Findings from scaffold fabrication techniques and bio-responsive scaffolds indicate that incorporating nanostructured materials and bioactive compounds is particularly effective in promoting the recruitment and differentiation of osteoprogenitor cells. The therapeutic potential of these advanced biomaterials in clinical settings is widely recognized and the paper advocates continued research into multi-responsive scaffold systems. [ABSTRACT FROM AUTHOR]

The CliniMACS Prodigy closed system is widely used for the manufacturing of chimeric antigen receptor T cells (CAR-T cells). Our study presents an extensive immunophenotypic and functional characterization and comparison of the properties of anti-CD19 CAR-T cell products obtained during long (11 days) and short (7 days) manufacturing cycles using the CliniMACS Prodigy system, as well as cell products manufactured from different donor sources of T lymphocytes: from patients, from patients who underwent HSCT, and from haploidentical donors. We also present the possibility of assessing the efficiency of transduction by an indirect method. Seventy-six CD19 CAR-T cell products were manufactured using the CliniMACS Prodigy automated system. Immunophenotypic properties, markers of cell activation and exhaustion, antitumor, anti-CD19 specific activity in vitro of the manufactured cell products were evaluated. As an indirect method for assessing the efficiency of transduction, we used the method of functional assessment of cytokine secretion and expression of the CD107a marker after incubation of CAR-T cells with tumor targets. The CliniMACS Prodigy platform can produce a product of CD19 CAR-T cells with sufficient cell expansion (4.6 × 109 cells—median for long process [LP] and 1.6 × 109—for short process [SP]), transduction efficiency (43.5%—median for LP and 41.0%—for SP), represented mainly by T central memory cell population, with low expression of exhaustion markers, and with high specific antitumor activity in vitro. We did not find significant differences in the properties of the products obtained during the 7- and 11-day manufacturing cycles, which is in favor of reducing the duration of production to 7 days, which may accelerate CAR-T therapy. We have shown that donor sources for CAR-T manufacturing do not significantly affect the composition and functional properties of the cell product. This study demonstrates the possibility of using the CliniMACS Prodigy system with a shortened 7-day production cycle to produce sufficient amount of functional CAR-T cells. CAR transduction efficiency can be measured indirectly via functional assays. [ABSTRACT FROM AUTHOR]

The printing company's process performance depends on the possibility of providing requested products and managing the existing constraints of fixed machine layouts and high setup times between different products. Process inefficiencies caused by these factors reflect on throughput, production times, and resource utilization. The changes that improve one part of the production system usually affect other parts, needing additional optimization, and it is very useful to test the feasibility of proposed solutions with simulation before implementation. This paper presents a new algorithm for continuous improvement of enterprises performance, combining the lean approach with cellular manufacturing, and simulation. The performance is observed in a way that a certain setup influences the system in its entirety, rather than on a specific part of that system. The results are presented through models developed within the production optimization phase, representing various ways in which the continuous improvement algorithm can unfold. Each of them comes with its advantages and disadvantages, all intending to create more efficient production processes that generate less production waste. [ABSTRACT FROM AUTHOR]

The mercury cell manufacturing process, which has been extensively used in chlor-alkali plants to produce chlorine and caustic soda by electrolysis, represents a major source of Hg environmental pollution. At Saline di Volterra (Tuscany, Italy), solution mining by pumping water into halite deposits was applied to produce brines for a mercury-cell chlor-alkali plant. The Hg-contaminated, exhausted brines were pumped back at depth into the rock salt field in order to renew the available resources. Activities ceased in 1994, following the leakage dispersion of highly contaminated Hg(0)-bearing brines into the environment. The mercury content in the soil, measured during a survey conducted in 2000, reached 334 mg/kg, highlighting diffuse contamination in the floodplain. By 2009, the Hg concentration had generally decreased and was mostly confined to the topsoil layer. In order to evaluate the present Hg soil pollution, a geochemical survey was carried out in 2023, almost thirty years after the contamination event. The obtained data indicated the occurrence of legacy Hg, which reached 25.5 mg/kg in some soil samples. Speciation analysis for the most contaminated soil revealed that Hg(0) represented about 17.3% of the total Hg and that water-soluble and organic Hg fractions were negligible. These results suggest that the originally released metallic mercury has volatilized and likely oxidized, becoming practically immobile in the soil. A risk assessment, performed by applying Hg speciation analysis, indicated that the mercury in the soil does not carry a risk of non-cancerous effects for different exposure routes in case of subsequent use of the site and that the formerly contaminated area can now be converted into a leisure area. [ABSTRACT FROM AUTHOR]

This article experimentally examines methods for implementing the philosophies of Lean Six Sigma (LSS) in a High-Mix Low-Volume (HMLV) manufacturing environment. HMLV environments present unique challenges to LSS paradigms because of the need for extraordinary operational flexibility and customer responsiveness. The subject HMLV manufacturer for this experimentation manufactures (among 8500 others) an example component for which 3 machines work independently to perform the necessary operations to manufacture this component. The experiment that is the subject of this research seeks to adapt LSS philosophies to develop treatments to improve the performance of the manufacturing of this component. These LSS-inspired treatments included 1) using cellular manufacturing methods, and the 3 machines as a single work cell to manufacture the component, and 2) using a single multipurpose machine to perform all operations required to manufacture the component. The results of this experiment demonstrate that the cellular manufacturing method was the most effective to reduce costs, to standardize operations at a process level, and to increase throughput. The single machine processing method improved production rates and on-time delivery relative to the baseline, but greatly increased lead time, thereby increasing total cost per part. These results highlight the importance of critically assessing the application of LSS within HMLV environments compared to the Low-Mix High-Volume (LMHV) environments where LSS is traditionally successful. HMLV manufacturers and researchers can use these findings to identify the most effective methods for their specific needs and to design interventions that will improve system-level manufacturing performance in high mix environments. [ABSTRACT FROM AUTHOR]

NK cell therapeutics have gained significant attention as a potential cancer treatment. Towards therapeutic use, NK cells need to be activated and expanded to attain high potency and large quantities for an effective dosage. This is typically done by ex vivo stimulation with cytokines to enhance functionality or expansion for 10-14 days to increase both their activity and quantity. Attaining a robust methodology to produce large doses of potent NK cells for an off-the-shelf product is highly desirable. Notably, past reports have shown that stimulating NK cells with IL-12, IL-15, and IL-18 endows them with memory-like properties, better anti-tumor activity, and persistence. While this approach produces NK cells with clinically favorable characteristics supported by encouraging early results for the treatment of hematological malignancies, its limited scalability, variability in initial doses, and the necessity for patient-specific production hinder its broader application. In this study, stimulation of NK cells with PM21-particles derived from K562-41BBL-mbIL21 cells was combined with memory-like induction using cytokines IL-12, IL-15, and IL-18 to produce NK cells with enhanced anti-tumor function. The use of cytokines combined with PM21-particles (cytokine and particle, CAP) significantly enhanced NK cell expansion, achieving a remarkable 8,200-fold in 14 days. Mechanistically, this significant improvement over expansion with PM21-particles alone was due to the upregulation of receptors for key stimulating ligands (4-1BBL and IL-2), resulting in a synergy that drives substantial NK cell growth, showcasing the potential for more effective therapeutic applications. The therapeutic potential of CAP-NK cells was demonstrated by the enhanced metabolic fitness, persistence, and anti-tumor function both in vitro and in vivo. Finally, CAP-NK cells were amenable to current technologies used in developing therapeutic NK cell products, including CRISPR/Cas9-based techniques to generate a triple-gene knockout or a gene knock-in. Taken together, these data demonstrate that the addition of cytokines enhanced the already effective method of ex vivo generation of therapeutic NK cells with PM21-particles, yielding a superior NK cell product for manufacturing efficiency and potential therapeutic applications. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) are promising cell therapy candidates, but their debated efficacy in clinical trials still limits successful adoption. Here, we discuss proceedings from a roundtable session titled "Failure to Launch Mesenchymal Stromal Cells 10 Years Later: What's on the Horizon?" held at the International Society for Cell & Gene Therapy 2023 Annual Meeting. Panelists discussed recent progress toward developing patient-stratification approaches for MSC treatments, highlighting the role of baseline levels of inflammation in mediating MSC treatment efficacy. In addition, MSC critical quality attributes (CQAs) are beginning to be elucidated and applied to investigational MSC products, including immunomodulatory functional assays and other potency markers that will help to ensure product consistency and quality. Lastly, next-generation MSC products, such as culture-priming strategies, were discussed as a promising strategy to augment MSC basal fitness and therapeutic potency. Key variables that will need to be considered alongside investigations of patient stratification approaches, CQAs and next-generation MSC products include the specific disease target being evaluated, route of administration of the cells and cell manufacturing parameters; these factors will have to be matched with postulated mechanisms of action towards treatment efficacy. Taken together, patient stratification metrics paired with the selection of therapeutically potent MSCs (using rigorous CQAs and/or engineered MSC products) represent a path forward to improve clinical successes and regulatory endorsements. [Display omitted] [ABSTRACT FROM AUTHOR]

The successful development of CD19-targeted chimeric antigen receptor (CAR) T-cell therapies has led to an exponential increase in the number of patients recieving treatment and the advancement of novel CAR T products. Therefore, there is a strong need to develop streamlined platforms that allow rapid, cost-effective, and accurate measurement of the key characteristics of CAR T cells during manufacturing (i.e., cell number, cell size, viability, and basic phenotype). In this study, we compared the novel benchtop cell analyzer Moxi GO II (ORFLO Technologies), which enables simultaneous evaluation of all the aforementioned parameters, with current gold standards in the field: the Multisizer Coulter Counter (cell counter) and the BD LSRFortessa (flow cytometer). Our results demonstrated that the Moxi GO II can accurately measure cell number and cell size (i.e., cell volume) while simultaneously assessing simple two-color flow cytometry parameters, such as CAR T-cell viability and CD4 or CAR expression. These measurements are comparable with those of gold standard instruments, demonstrating that the Moxi GO II is a promising platform for quickly monitoring CAR T-cell growth and phenotype in research-grade and clinical samples. [ABSTRACT FROM AUTHOR]

Objectives: Autologous chimeric antigen receptor (CAR) T‐cell therapy of B‐cell malignancies achieves long‐term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR‐T manufacturing, the compromised fitness of patient‐derived T cells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR‐NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. Methods: To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in‐depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR‐NK manufacturing pipeline. Results: We identified adult peripheral blood‐derived NK cells to be the superior source for generating a CAR‐NK cell product because of a higher maximum yield of CAR‐expressing NK cells combined with potent natural, as well as CAR‐mediated anti‐tumor effector functions. Conclusions: Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre‐ and post‐transduction and high on‐target cytotoxicity make peripheral blood‐derived NK cells a feasible and attractive CAR‐NK cell product for clinical utility. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell therapy has transformed cancer immunotherapy. However, significant challenges limit its application beyond B cell-driven malignancies, including limited clinical efficacy, high toxicity, and complex autologous cell product manufacturing. Despite efforts to improve CAR T cell therapy outcomes, there is a growing interest in utilizing alternative immune cells to develop CAR cells. These immune cells offer several advantages, such as major histocompatibility complex (MHC)-independent function, tumor microenvironment (TME) modulation, and increased tissue infiltration capabilities. Currently, CAR products from various T cell subtypes, innate immune cells, hematopoietic progenitor cells, and even exosomes are being explored. These CAR products often show enhanced antitumor efficacy, diminished toxicity, and superior tumor penetration. With these benefits in mind, numerous clinical trials are underway to access the potential of these innovative CAR cells. This review aims to thoroughly examine the advantages, challenges, and existing insights on these new CAR products in cancer treatment. [ABSTRACT FROM AUTHOR]

Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cellbased therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/antiinflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics. [ABSTRACT FROM AUTHOR]

Introduction: The clinical success of chimeric antigen receptor-modified T cells (CAR-T cells) for hematological malignancies has not been reproduced for solid tumors, partly due to the lack of cancer-type specific antigens. In this work, we used a novel combinatorial approach consisting of a versatile anti-FITC CAR-T effector cells plus an FITC-conjugated neuroblastoma (NB)-targeting linker, an FITC-conjugated monoclonal antibody (Dinutuximab) that recognizes GD2. Methods: We compared cord blood (CB), and CD45RA-enriched peripheral blood leukapheresis product (45RA) as allogeneic sources of T cells, using peripheral blood (PB) as a control to choose the best condition for anti-FITC CAR-T production. Cells were manufactured under two cytokine conditions (IL-2 versus IL-7+IL-15+IL-21) with or without CD3/CD28 stimulation. Immune phenotype, vector copy number, and genomic integrity of the final products were determined for cell characterization and quality control assessment. Functionality and antitumor capacity of CB/45RA-derived anti-FITC CAR-T cells were analyzed in co-culture with different anti-GD2-FITC labeled NB cell lines. Results: The IL-7+IL-15+IL-21 cocktail, in addition to co-stimulation signals, resulted in a favorable cell proliferation rate and maintained less differentiated immune phenotypes in both CB and 45RA T cells. Therefore, it was used for CART cell manufacturing and further characterization. CB and CD45RA-derived anti-FITC CAR-T cells cultured with IL-7+IL-15+IL-21 retained a predominantly naïve phenotype compared with controls. In the presence of the NB-FITC targeting, CD4+ CB-derived anti-FITC CAR-T cells showed the highest values of costimulatory receptors OX40 and 4-1BB, and CD8+ CAR-T cells exhibited high levels of PD-1 and 4-1BB and low levels of TIM3 and OX40, compared with CART cells form the other sources studied. CB-derived anti-FITC CAR-T cells released the highest amounts of cytokines (IFN-γ and TNF-α) into co-culture supernatants. The viability of NB target cells decreased to 30% when co-cultured with CB-derived CAR-T cells during 48h. Conclusion: CB and 45RA-derived T cells may be used as allogeneic sources of T cells to produce CAR-T cells. Moreover, ex vivo culture with IL-7+IL-15+IL-21 could favor CAR-T products with a longer persistence in the host. Our strategy may complement the current use of Dinutuximab in treating NB through its combination with a targeted CAR-T cell approach. [ABSTRACT FROM AUTHOR]