The viability of cells in starting material and key in-process steps is a critical quality attribute for cell therapies. The functional integrity of cells can impact final product efficacy and potency. Methods to measure these attributes are mostly performed by flow cytometry. Lack of standardized methods and complexity introduced by traditional hands-on flow cytometry workflows can lead to variability and inconsistent results, leading some developers to omit cell health evaluation during their process.

Through this work, we showcase the development of an automated flow cytometry–based T Cell Health Annexin V (NL) Assay to reduce the complexity of at-line cell health monitoring.

This assay is designed to provide a standardized measure of T cell apoptosis and viability by incorporating key indicators in a six-marker panel (CD45, CD3, CD4, CD8, Annexin V, and viability dye). Implemented on the Accellix, a small footprint and fully automated flow cytometry platform, the assay requires minimal operator input, reducing hands-on time, training requirements, and user-dependent variability. The resulting assay supports reproducible automated at-line monitoring of T cell health and aims to provide cell therapy developers with a practical tool to improve in-process testing, reduce costs, and support robust quality control in early and late-stage development.

For nearly four decades, magnetic bead-based cell isolation has been the industry standard. To advance cell therapy workflows, the next generation of tools to support improvements in therapeutics require a more flexible, scalable, and gentle approach. Akadeum’s GMP-compliant microbubble technology offers superior outcomes for manufacturing workflows.  Akadeum’s microbubble kits provide flexibility to fit many workflows, enabling high-performance cell isolations on Day 0 as well as gentle cleanup steps at the terminal processing step.  Unlike conventional methods, microbubbles are compatible with existing lab instrumentation and offer a simple, easy-to-use negative selection workflow. 

These factors combined have been shown to improve cell purity, cell recovery, cell viability, cell editing efficiency, expansion, preservation of stemness – all while reducing the time of the workflow and reducing COGS.  Recent comparison from Dark Horse Consulting Group compared an Akadeum workflow to a approved autologous therapeutic workflow with leading magnetics and showed a 40% total cost savings, with 35% total time savings, and two times more potent cells – leading to greater accessibility with a smaller footprint, a faster path to clinical success in a cost-effective manner.

Microbubbles allow for a streamlined manufacturing process, reducing processing times and producing higher quality cells. This technology has demonstrated improvements in cell viability, potency, purity, and recovery compared to traditional magnetic separation methods. Workflows being pursued using Akadeum’s kits include instrument free negative T cell isolation in under an hour for truly untouched cells, bulk cleanup for stem cell isolation prior to positive selection to reduce workflow time by up to 80%, and depletion of TCRαβ-expressing cells after gene editing, ensuring a purer final product for allogeneic therapies. Enabling workflow flexibility and gentle isolations, Akadeum microbubble technology yields a higher quality cell product for therapeutics in a shorter timeframe, capable of scaling from process development to over 50 billion cells processed. This poster is an opportunity to share results from the cell therapy workflow improvements realized by incorporating microbubble-based cell separation kits.

Closed system immunotherapy manufacturing workflows minimize manual touchpoints and reduce the risk of error and contamination. One of the challenges faced with closed system workflows is the manual delivery of cytokines, which can be labor-intensive, requiring reconstitution and aliquoting in a biological safety cabinet. To minimize contamination and simplify the workflow, we packaged ready-to-use liquid GMP cytokines in single-use ProPak™ bags with weldable tubing for easy integration into closed system workflows.

This innovative approach allows for optimal dosing of GMP cytokines without the need for manual manipulation or cytokine spiking in a biosafety cabinet, enhancing both scalability and safety in T cell therapy production. Here we demonstrate that IL-2, IL-7 and IL-15 GMP cytokines packaged into ProPaks can be recovered into media bags and maintain bioactivity. Additionally, ProPak GMP cytokines support T cell growth and maintain T cell viability equivalently to lyophilized cytokine formats. The implementation of ready-to-use liquid GMP cytokines in single-use bags with weldable tubing enhances manufacturability and safety in T cell therapy production.

Innovative cell therapies encounter practical challenges in achieving effective clinical implementation, largely due to labor-intensive cell manufacturing processes that lack flexibility, consistency and scalability. Isolation of target cells from other contaminants is essential to streamline the cell therapy workflow. Currently, standard methods for T cell activation in bioprocessing rely on superparamagnetic beads or polymer-based reagents. However, these approaches  face limitations in scalability, cell recovery/viability, cost and complexity of beads removal. Lipid-shelled microbubbles (MB) have been widely used in clinical setting as ultrasound contrast agents for various indications over the past two decades. Their surface can now be functionalized to address challenges in T cell selection and activation.  Herein, we introduce a solution to select and activate cells of interest using streptavidin (STV) conjugated microbubble (MB) reagent that can be combined with any biotinylated ligand, so-called Modular MB Reagent. By leveraging the natural buoyancy of MB, targeted cells can be gently and efficiently separated and, concomitantly, activated. Experiments were conducted by preincubating 1 x 107 human Peripheral Blood Mononuclear Cells (PBMC) with biotinylated ligands, followed by incubation with STV-conjugated MB. Target cells were gently isolated via centrifugation, and both the isolated cells and PBMC were analyzed using flow cytometry.

By employing specific biotinylated anti-human CD3 and CD28 antibodies (Abs), the MB enabled both selection and activation of T cells.  This process led to significant enrichment of target cells with high recovery and preserved cell viability (Figure 1). Using Dynabeads™ Human T-Activator CD3/CD28 (Gibco™) as benchmark, the proportion of activated (CD25+) T cells was similar 3-day after activation, while T cell expansion was donor-dependent and slightly lower for cells activated with MB (Figure 2). The ongoing process optimization emphasized the key role of the antibodies for efficient selection, activation and expansion as same concentrations of Abs from different suppliers showed diverse fold of expansion (Figure 3).  In conclusion, our innovative, cost-effective and versatile lipid-based MB offers similar results to existing T cell activation reagent. It results in purified and activated T cell population, with similar expansion to current standard. The process is bead-free, scale independent and easily amenable to automated cell therapy manufacturing purposes. Our next steps focus on developing a Modular MB Reagent kit, designed for clinical translation.  

Background: Cell therapy manufacturers require robust analytical methods that deliver consistent results across geographically distributed manufacturing sites. Flow cytometry quality control faces significant challenges with biological controls, which exhibit donor-to-donor variability that complicates site-to-site method transfer and comparability studies. As companies scale from single-site to multi-site manufacturing networks, establishing standardized controls is critical for successful technology transfer. Cell mimics represent an advanced solution for achieving standardization and analytical comparability across future manufacturing sites. Objective: To establish a framework for cross-site analytical comparability using hydrogel-based cell mimics (TruCytes™, Slingshot Biosciences) as standardized flow cytometry controls on the Cellares Cell Q™, a high-throughput automated QC testing platform. This proof-of-concept study evaluated compatibility metrics across multiple laboratories within Cellares' California facility to validate readiness for future technology transfer to our planned New Jersey manufacturing site, assessing whether cell mimic controls could eliminate the variability inherent to donor materials and monitor instrument performance.

Methods: We conducted a multi-laboratory comparability study testing TruCytes Lymphocyte Subset panels and negative controls across multiple Cytek Northern Lights flow cytometers in Quality Control and Analytical Development laboratories at our California site. Different operators evaluated two TruCyte reagent lots using a standardized fluorescent antibody panel to assess T cell (CD3+CD4+, CD3+CD8+), B cell (CD19+), NK cell (CD16+CD56+), and monocyte (CD14+) populations. Inter-laboratory reproducibility was compared against manufacturer specifications and parallel donor PBMC to simulate site-to-site variability scenarios.

Compatibility with Cellares' automated Cell Q platform was verified, including automated reconstitution protocols to support future closed-system manufacturing. Results: Cell mimics demonstrated measurable improvements over biological controls. Lot-to-lot comparability showed remarkable consistency: <6% difference between TruCyte Lymphocyte Subset reagent lots across all cell populations. In contrast, donor PBMC exhibited significant variability compared to the cell mimic specifications, with T cytotoxic cells (93%) and NK cells (72%) showing the most donor-dependent variation. Inter-laboratory precision with cell mimics showed 0-10% coefficient of variation (CV) for each parameter across instruments, compared to 3-9% CV for donor materials. Both Quality Control and Analytical Development laboratories achieved <8.2% difference from manufacturer specifications for T cell, B cell, monocyte, and NK cell populations, confirming lot-to-lot and operator-independent accuracy.

The tight, reproducible population clustering of cell mimic controls enabled standardized gating strategies applicable across both laboratories, while donor cell populations required case-by-case manual adjustments and longer analysis times.

Conclusions: Cell mimics minimize the biological variability that is inherent to traditional biological controls, directly addressing FDA guidance on analytical similarity and comparability protocols for multi-site manufacturing. The ability to apply standardized gating strategies across laboratories, which is challenging with unique donor materials, dramatically reduces method transfer complexity, operator training burden, and validation timelines. These results de-risk our planned expansion to future manufacturing facilities by demonstrating that cell mimic controls can deliver site-independent and operator-independent results, which are essential for rapid technology transfer and comparability studies. Impact: This work validates a paradigm shift from variable biological controls to standardized synthetic alternatives for quality control testing in cell therapy. Furthermore, Slingshot Biosciences’ cell mimics have shown to be superior reference controls that are made to be precise, highly reproducible, and scalable.

The demonstrated advantages enable: (1) seamless technology transfer from California to New Jersey and future global facilities with pre-validated analytical flow cytometry methods, (2) elimination of donor-dependent analytical variability that confounds cross-site comparisons, (3) regulatory confidence through manufacturer-certified, traceable standards versus uncharacterized donor samples, and (4) accelerated commercial scale-up by removing biological variability as a barrier to multi-site manufacturing. For an industry pursuing geographically distributed manufacturing to meet global patient demand, replacing inherent donor and instrument performance variability with predictable experimental accuracy represents a critical advancement in analytical standardization. Regulatory Significance: Demonstrates quantifiable improvements in analytical procedure standardization in accordance with ICH Q14 guidelines and provides a validated framework for FDA comparability protocols in multi-site cell therapy manufacturing networks, replacing highly variable biological materials with manufacturer-certified, traceable synthetic standards.

Machine learning analysis of induced pluripotent stem cell metabolism predicts optimal culture conditions for improved cell yield and functionality.   ShaNelle Yelity, Despina Pleitez, and Alex Sargent.   Induced pluripotent stem cells (iPSCs) hold tremendous promise in regenerative medicine to treat injury and disease. Culturing and expanding iPSCs can be challenging and labor intensive, as iPSCs are acutely sensitive to culture conditions and often require frequent media exchanges.

Here, we developed a novel culture protocol for iPSCs using gas permeable culture devices, the G-REX® bioreactors, eliminating the need for media exchanges for up to 10 days.

Metabolic analysis of extended iPSC culture was performed using machine learning (ML) to evaluate and optimize culture conditions for iPSC expansion and functionality. Using gas permeable culture devices and ML designed culture conditions, we increased iPSC expansion up to 3-fold and reduced labor by 80% by eliminating the need for multiple media exchanges and passaging. iPSCs had high viability, consistently defined colonies, high expression of pluripotency markers, and were capable of functional differentiation into all primary germ layers. This novel and scalable approach for iPSC expansion for cell and tissue therapies provides an enhanced application for regenerative medicine.

CREATe Tx employs its proprietary chemogenetic technology to develop a small molecule-controlled CNS gene therapy platform for the treatment of a broad spectrum of neurological disorders. Our approach inverts the traditional pharmaceutical R&D paradigm: instead of discovering new ligands for existing targets, we engineer new targets for existing ligands.  This platform enables precise pharmacological control of neuronal activity within spatially and neurochemically defined populations.

Our lead indication is drug-resistant focal epilepsy: after having our receptor expressed in the seizure focus, we succeeded in preventing the generation of seizures with oral pharmacotherapy in an in vivo mouse model.

Stable gene insertion using non-viral delivery is essential to unlocking the next generationof safe and accessible cell and gene therapies. Currently, viral vectors carry highmanufacturing costs and insertional mutagenesis concerns, while electroporation (EP)affects cell yield and viability. Lipid nanoparticles (LNPs) can be a promising alternativeoption for engineering cells for persistent expression of target genes, as they offer afavorable safety profile and are both cost-effective and scalable. In this work, we mappedout the critical process parameters (CPPs) governing homology-directed repair (HDR) inprimary T cells using LNP-mediated cargo delivery. Cas9 mRNA, a chemically synthesizedguide RNA (sgRNA), and a ~100 nt single-stranded donor oligonucleotide (ssODN) wereproduced using a novel LNP composition and scalable production platform. Ahaemagglutinin (HA) epitope tag was knocked in at the CD5 locus as an easily quantifiableread-out for CPP optimization. CD3+ primary T cells from healthy donors were cultured inwell-plates and the CRISPR LNPs added to the media in a one-step process, without furthercell manipulation. Various parameters were identified and systematically varied, including(and not limited to) the length of cell activation, cell density, nucleic acid dose and theRNA/DNA molar ratios. Through multiple rounds of optimizations, LNPs achieved onaverage 31 ± 7% HDR in n=5 T cell donors, detected through dual CD5/HA flow cytometry 4days post-LNP administration.

Viability of the cells remained high at 96 ± 5% at the time ofHDR detection, relative to untreated controls. The aforementioned results reflect no addedenhancers; however, when we tested various small molecules, such as NHEJ inhibitors,HDR rates further improved to over 50% in primary T cells. Finally, we compared theoptimized LNP protocol to EP which resulted in similar HA+ frequencies in the cellpopulation. However, most notably, the yield of viable edited cells by LNP was an order ofmagnitude higher than EP owing to improved cell viability and proliferation. All together,this data demonstrates how LNPs can achieve clinically relevant knock-in frequences andshowcases the benefits of LNPs as a non-viral alternative for gene insertion. The CPPevaluation offers a ready-to-implement framework applicable to a diverse set oftherapeutic loci, providing for a foundational dataset for the rapid application of LNPs to enable the next generation of T cell therapies.

The manufacturing of cell therapies (including autologous and allogeneic CAR‑T, TCR, HSC, NK, TIL, Treg, and γδ T cells) remains complex, manual, and costly, limiting scalability and patient access. ElevateBio provides access to a breadth of technologies and deep expertise in manufacturing processes, services, and regulatory path and has partnered with Sartorius to develop a novel, automated end‑to‑end cell therapy manufacturing system (NE2ES) to address these industry challenges. Here, we present engineered T‑cell process development data generated on a novel bioreactor system (NBS) within the NE2ES and characterized using ElevateBio’ s advanced analytics. Within NE2ES’s digitally connected, closed‑system workflow, robotics orchestrate standard unit operations—cell selection, activation and transduction, expansion, harvest, and washing—using a single‑use disposable flow path.

Configurable software manages recipes, production orders, electronic batch records, and alarms while integrating in‑line sensors for real‑time monitoring. Across multiple donors automated cultures achieved cell yields, viability, phenotype, and potency comparable to or exceeding standard manual processes. The platform’s flexibility and closed, single‑use design reduces development time, improves reproducibility, reduces operator hands‑on time, and enables rapid scale‑out to more than 10X batches within the footprint typically required for a single manual GMP process, with operations feasible in lower grade cleanroom classifications. By increasing process consistency, reducing cost of goods, and enabling effective scale‑out, this NE2ES offers an accelerated path to successful commercial production and greater accessibility for patients.

ElevateBio integrates end-to-end GMP manufacturing capabilities, including process and analytical development, with gene editing technologies and R&D expertise to power transformative cell and gene therapies. Here, we have utilized advanced analytics including single-cell RNA sequencing and flow cytometry to profile engineered T cells.  T cells from multiple donors were engineered using a lentiviral vector and subsequently exposed to antigen-positive target cells at varying concentrations. Analytical profiling was performed on 20 samples consisting of over 50,000 T cells from which we identified donor- and dose-dependent shifts in T cell phenotypes across Helper, Cytotoxic, Naïve, Memory, and Effector populations; exhaustion and proliferation states; and cytokine secretion profiles.

By analytically isolating key T cell populations, we further resolved drug-response trajectories and functional treatment outcomes.  Overall, application of advanced analytics can be applied to manufactured edited cells on two levels, cell surface protein and single-cell gene expression, to provide an innovative and adaptable technique that advances cell and gene therapies.

LigronBio, is a groundbreaking biotechnology company, harnessing the power of its innovative platform, AI-TriMatrix AnalyzerTM. Integrating computational chemistry, bioinformatics, Artificial Intelligence (AI), and Machine Learning (ML), LigronBio is at the forefront of discovering rational molecular glues for challenging undruggable targets in human cancer and CNS therapy, addressing unmet medical needs. Through this platform, we employ computational tools to design compounds that modulate protein-protein interactions, leading to the formation of ternary complexes for targeted protein degradation - a notable strategy includes the introduction of the L-tag assay, an innovative method for spatial distribution analysis within ternary complexes and assessing effects on protein ubiquitination and facilitating the optimization of drug development processes.  

Committed to addressing unmet medical needs, LigronBio recently achieved a significant milestone by identifying a lead compound with direct activity on RAF1. This breakthrough is a testament to the success of our rationally designed approach holding tremendous promise for the treatment of multiple cancers,  neurological and infectious diseases.

The field of Cell and Gene Therapy (C&GT) has generated a variety of novel solutions to the treatment of certain diseases such as cancer and genetic disorders. In this space, viral vectors are employed as the drug product itself, or a critical reagent to generate the therapeutic cell. These viral vectors require a variety of analytics to be performed for the evaluation of their critical quality attributes. Among these attributes are genomic titer, particle titer, and vector potency. While the analytics for genomic and particle titer, often PCR and ELISA-based, respectively, vary modestly among viral vectors, potency analytics are more varied. As each viral vector is unique in its intended bioactivity, no one-size-fits all potency assay is in place and therefore each program must develop product specific potency assays. Despite the commonality of the physical and biochemical techniques applied to evaluate genomic and particle titer, there are some differences that necessitate one or more augments to these methods. For instance, while qPCR and ddPCR can be utilized to titer DNA-carrying vectors, RNA-containing ones require the addition of a reverse transcriptase step. Moreover, to evaluate particle titer, due to the variation in particle size and composition of the base vector, ELISA, HPLC, microscopy, and light scattering techniques are employed. Each of these methods require some variation in their execution to accommodate for these vector differences. Potency assays, however, require the greatest variation among vectors to characterize their bioactivity.

Vector potency refers to the construct’s ability to exert its intended therapeutic effect upon the target cell type. As such, the potency assay must be developed to directly evaluate the therapeutic effect of the vector whether this effect is oncolytic for the treatment of cancer cells, or transducing, resulting in short, or long, term expression of a specific gene product. Incompatible, or inefficient, methods to determine potency can lead to program complications and regulatory delays. At Matica, we have developed a variety of potency assays to support our clients’ programs. These include one of multiple different viral vectors generated using Lentiviruses, AAV’s, Adenoviruses, and others. Each vector exhibits its own characteristics which determine the analytical techniques that are applicable. For instance, Adenoviruses, often employed as oncolytic vectors, allow for a more direct characterization through traditional plaque assays, as these vectors are cytopathic. Whereas AAV’s, due to their nonpathogenic nature, traditional infectivity assays are not applicable. Instead, to evaluate an AAV vector’s infectivity, a TCID50 can be employed, where the readout is by qPCR or ddPCR after a short infection period. We show here multiple methods developed and executed at Matica, on different representative vectors systems, to demonstrate the variety of techniques available to interrogate viral vector potency along with data showing characterization of genomic and particle titer.

Progressing manufacturing of Bacterial-Derived Extracellular Vesicles to clinical stage   Introduction   Extracellular vesicles (EVs) have lately raised as an attractive new therapeutic modality for both immunotherapy and as vectors for various cargos such as nucleic acids and proteins. This new class of therapies is emerging as a transformative area of biopharmaceutical innovation, with potential applications in therapeutics, drug delivery, and vaccines. As for all new modalities the translation from research to clinical and commercial use is not without challenges, particularly in developing effective manufacturing processes and qualified analytical methods meeting the CMC requirements of Good Manufacturing Practices (GMP). This talk aims to put the light on some of the challenges that need to be addressed for progressing the scientific novelty of bacterial EVs and the practicalities of modern GMP-compliant manufacturing.   Methods   Drawing on the extensive GMP expertise of a NorthX Biologics, a leading CDMO with several EV projects in development, this presentation will outline some of the principles of GMP as applied to the production of bacterial EVs, emphasizing the distinct challenges and considerations unique to this field. Topics will include the development of robust and reproducible upstream processes for vesicle production, scalable purification strategies tailored to the specific properties of EVs, and strategies to characterize the complex products to verify consistency, safety, and efficacy.   Results   Special focus will be given to:   • Describing the road map for the translation from bench to bedside.   • Critical quality attributes (CQAs) for EVs and their alignment with regulatory expectations.   • Analytical challenges in characterizing EVs for purity, potency, and sterility.   • Risk management and contamination control strategies tailored to bacterial systems.   Summary/Conclusion   By addressing these key topics, this presentation seeks to give inventors working in the EV field some useful practical insights into the GMP landscape for bacterial EVs. As the field advances, leveraging GMP expertise and collaborative partnerships early in the product development journey will be essential to translating the promise of bacterial EVs into real-world therapeutic breakthroughs.            Event banner   Thank you for completing your ISEV2025 abstract submission.     Do not hesitate to contact our office (contact@isev.org) if you have any questions.   ISEV2025 Abstract Submissions: ISEV 2025 Annual Meeting   You can access your Abstract at any time by clicking here.    Presentation Type   Late Breaking Oral   Abstract Status:   Complete   Abstract ID:   2031487   Abstract Title:   Time for GMP? Progressing manufacturing of Bacterial-Derived Extracellular Vesicles to clinical stage.   Author(s)       Ola Tuvesson (he/him/his) (Role: Presenting Author)   Abstract Information      Topic/Keyword   Disease and therapy   Subtopic:   EV therapeutics development   Regulatory compliance   Is the presenting author a Junior or Senior Researcher?   Senior Researcher   Is this abstract submitted by a member of industry?   Yes   Is the research in your abstract funded by industry?   Yes   Introduction   Extracellular vesicles (EVs) have lately raised as an attractive new therapeutic modality for both immunotherapy and as vectors for various cargos such as nucleic acids and proteins. This new class of therapies is emerging as a transformative area of biopharmaceutical innovation, with potential applications in therapeutics, drug delivery, and vaccines. As for all new modalities the translation from research to clinical and commercial use is not without challenges, particularly in developing effective manufacturing processes and qualified analytical methods meeting the CMC requirements of Good Manufacturing Practices (GMP). This talk aims to put the light on some of the challenges that need to be addressed for progressing the scientific novelty of bacterial EVs and the practicalities of modern GMP-compliant manufacturing.  

Methods   Drawing on the extensive GMP expertise of a NorthX Biologics, a leading CDMO with several EV projects in development, this presentation will outline some of the principles of GMP as applied to the production of bacterial EVs, emphasizing the distinct challenges and considerations unique to this field. Topics will include the development of robust and reproducible upstream processes for vesicle production, scalable purification strategies tailored to the specific properties of EVs, and strategies to characterize the complex products to verify consistency, safety, and efficacy.   Results   Special focus will be given to:   • Describing the road map for the translation from bench to bedside.   • Critical quality attributes (CQAs) for EVs and their alignment with regulatory expectations.   • Analytical challenges in characterizing EVs for purity, potency, and sterility.   • Risk management and contamination control strategies tailored to bacterial systems.   Summary/Conclusion   By addressing these key topics, this presentation seeks to give inventors working in the EV field some useful practical insights into the GMP landscape for bacterial EVs. As the field advances, leveraging GMP expertise and collaborative partnerships early in the product development journey will be essential to translating the promise of bacterial EVs into real-world therapeutic breakthroughs.   Funding   MISEV      The MISEV 2023 guidelines are:   Not relevant to this work       Don't forget, the submission process closes Friday, January 31, 2025 11:59 PM      Technical Support   Email: Help@ConferenceAbstracts.com   Phone: (410) 638-9239   About the Abstract ScoreCard®   The Abstract ScoreCard® enables meeting planners to collect submissions and manage educational data online. It has been designed to fulfill the promise of our mission statement: Meeting Education Made Easy. To learn more about CadmiumCD's services, please contact us at info@CadmiumCD.com or call (410) 638 9239.      Organizer   For content related questions, please contact Ally McGuire at contact@isev.org or (856) 423-1621    Technical Support   Should you need technical support, please email support@gocadmium.com or call (410) 638-9239 between the hours of 9am - 9pm ET, Monday - Friday to reach a support specialist.    About the Abstract Scorecard   The Abstract Scorecard® enables meeting planners to collect submissions and manage educational data online. It has been designed to fulfill the promise of our mission statement: Bring Your Event Together. To learn more about Cadmium's services, please contact us at info@gocadmium.com or call (410) 638 9239 or visit our website at www.gocadmium.com.

Genome editing is rapidly gaining traction in many clinical applications. However, the evaluation of editing outcomes remains time consuming and impedes R&D timelines, especially when multiple iterations are needed to optimize workflows.    Amplicon sequencing (amp-seq) is considered the gold standard for quantification of editing efficiency. However, amp-seq is time consuming (~1 week turnaround time), and can be costly. Faster methods have been developed, but these often pose a trade-off of accuracy. The introduction of new workflows into research pipelines also requires additional equipment and/or extensive assay optimization. Hence, there is a critical need for fast, accurate, and cost-effective quantification of gene editing efficiency.  To address this need, we have developed an assay which can be easily run on standard lab equipment for quantification of indels, knock-ins, and off-target activity. The assay is called QUiCKR (Quantification Using initial CRISPR Kinetic Rates) and offers results in 20 min with minimal manual steps.

The assay protocol consists of generating a DNA sample dilution series, which is added to a pre-plated reagent mix containing Cas12, crRNA and fluorescent reporters, followed by fluorescence readout (Fig 1a). The editing efficiency is then quantified using a machine-learning-based software.   Current CRISPR based detection assays typically have a binary output, and are ill-suited for precise quantification. In contrast, the QUiCKR assay extracts the kinetic rates of Cas12 trans-cleavage to quantify gene editing efficiency, with amp-seq level accuracy. Pre-plated reagents remain stable during long-term storage, which provides users with ready-to-use reagents, a quick workflow, and accurate results. With minor adjustments to the sample input, the dynamic range can be shifted for accurate detection of single percent editing efficiencies.

Gene-modified cell therapies, such as engineered T cells or Natural Killer (NK) cells expressing chimeric antigen receptors (CARs), are among the most promising treatments for cancer and autoimmune diseases. Traditionally, these therapies rely on lentiviral vectors to transfer the gene of interest; however, this approach is associated with lengthy manufacturing timelines, high costs, and potential safety risks. To overcome these limitations, our research focuses on lipid-based solutions as a promising alternative for producing CAR-T and CAR-NK cells. In this study, innovative lipid-based solution LipidBrick® Cell Ready was employed first for transient protein expression with the production of functional ex vivo mRNA-based CD19 CAR-T cells and then for stable cell modification through the delivery of genetic engineering tools such as CRISPR using Cas9 mRNA and sgRNA targeting the TRAC gene. Optimization of T cell transfection was initially performed in 96-well plates before scaling up to a bioreactor.

To demonstrate the versatility of this solution, diverse nucleic acid payloads (mRNA and nanoplasmid) and various cell types (T cells, monocyte-derived dendritic cells, macrophages, and hematopoietic stem cells) were successfully transfected with our LipidBrick® Cell Ready reagent. Finally, we’ve shown that our ready-to-use solution LipidBrick® Cell Ready could be efficient to generate functional CD19 CAR-NK cells.

Background and Aim: The advancement of cell therapy manufacturing demands robust, flexible, and closed-system solutions that maintain cell quality while improving workflow consistency and scalability. This study evaluates the performance of two automation-enabled instruments designed to enhance distinct unit functions in a cell therapy manufacturing process. The Gibco™ CTS™ Rotea™ Counterflow Centrifugation System was assessed for its ability to concentrate and wash T cells while preserving recovery, viability, and phenotype integrity. Complementary studies were conducted with the soon to be released Gibco™ CTS™ Compleo™ Fill and Finish System, developed for automated formulation and fill of cell therapy products. Performance metrics include fill-volume precision, cell concentration accuracy, and volume accuracy and reproducibility across multiple dispense outputs (1–10 fills, 0.5–750 mL range). Together, these evaluations characterize how modular automation can improve process control, reduce operator variability, and support scalable manufacturing workflows in cell therapy production. Methods, Results & Conclusion: To assess the performance and consistency of automation-enabled systems in cell therapy manufacturing, a series of workflow experiments were performed targeting both cell processing and fill-finish operations. Primary human T cells were concentrated and washed using the CTS Rotea Counterflow Centrifugation System, while formulation and dispensing studies were conducted with the CTS Compleo Fill and Finish System using cryopreservation media. Performance indicators included cell recovery, viability, and phenotype stability for the processing phase, and volumetric accuracy and cell concentration uniformity across dispensed samples for fill-finish. Methods were also developed to evaluate connectivity between the two systems, including approaches for sterile transfer and connection between concentration and fill-finish operations.

During concentration and wash steps, the CTS Rotea system achieved greater than 80% recovery of primary T cells, with no significant loss in viability with phenotypic profiles remaining consistent before and after processing. In the fill-finish evaluations, the CTS Compleo system delivered high precision across volumes ranging from 750 mL to 0.5 mL, with percent deviation from target volumes under 5% and cell concentration variability below 10% among seven dispensed outputs at a starting concentration of 1×10⁶ cells/mL. Cell recovery and viability were maintained throughout, indicating minimal impact from the dispensing process in the presence of cryoprotective media, supporting the suitability of this automated workflow for manufacturing-relevant cryopreservation processes.

Collectively, these results demonstrate that the two instruments provide complementary capabilities essential to advancing automated cell therapy manufacturing. The CTS Rotea system supports efficient cell handling while preserving product quality, and the CTS Compleo platform achieves accurate, repeatable formulation and dispensing at clinically relevant scales. Together, they illustrate how modular automation can improve yield, reproducibility, and process control across the manufacturing continuum. Future work will investigate integration of these platforms into a unified, closed workflow to further streamline autologous and small-scale allogeneic cell therapy production.  For Research Use or Manufacturing of Cell, Gene, or Tissue-Based Products. Caution: Not intended for direct administration into humans or animals.  © 2025 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.

SCN2A variants are one of the most common genetic causes of intractable epilepsy in children, particularly in developmental and epileptic encephalopathies (DEEs) which can present with uncontrolled seizures at birth, accounting for 1-2% of all epileptic encephalopathies. There is significant genotype-phenotype heterogeneity in SCN2A-related disorders (SRD) which include neurologic symptoms of seizures, developmental delay, choreoathetosis, and autism spectrum disorder (ASD). A substantial fraction of causal variants is gain-of-functon(GOF) or mixed function, functionally associated with increased open-probability or greater sodium current flux. Individualized allele-selective antisense oligonucleotides (ASOs) were designed to target heterozygous intronic SNPs for decreased expression of mutant SCN2A transcript while preserving the wild-type copy.

Efficacy measures were also individualized to phenotype. Improvements in seizure control, development, and quality-of-life were seen with no ASO-related adverse events. Haplotype phasing in a separate cohort of infants with SRD diagnosed by rapid whole genome sequencing identified 16% of patients with compatible SNPs. Allele-selective ASOs demonstrate potential to decrease seizures and impact neurodevelopment, providing a pathway for potential benefit in n-of-1 to n-of-more SRD patients.

Background: Frontotemporal Dementia (FTD) is a debilitating neurodegenerative disease characterized by rapid cognitive and behavioral decline. Currently, there are no effective treatments. The aggregation and dysfunction of TDP-43, an RNA/DNA-binding protein, are hallmark pathological changes in FTD. One of the major cellular consequences of TDP-43 mis-localization is disrupted mitochondrial dynamics and function, which has been shown to directly contribute to neuronal degeneration and induce cognitive decline in the TDP-43 mouse model of FTD. Our group has shown that SynCav1 (Synapsin driven Caveolin-1 overexpression) preserves mitochondrial homeostasis in multiple neurodegenerative diseases, including AD and ALS. Thus, the current study aims to evaluate the effect of SynCav1 on mitochondrial health and cognitive function in the TDP-43 mouse model of FTD.       Methods: AAV-PhP.eB-SynCav1 or control AAV-PhP.eB-SynNull (5 × 10¹¹ v.g/mouse) was systemically delivered via retro-orbital injection into TDP-43 mice at 10 weeks old. Open-field and fear-conditioning tests were performed at 22 weeks to evaluate locomotion, learning, and memory, respectively. Hippocampal ultrastructure was examined by transmission electron microscopy (TEM). Mitochondrial dynamics were assessed by immunoblotting (IB) of fusion/fission markers.     Results: Systemic delivery of AAV-PhP.eB-SynCav1 achieved a significant pro-cognitive effect in the TDP-43 mouse model of FTD.

Compared to transgenic negative (TGN) mice, TDP-43 SynNull untreated group exhibited significant memory deficits, whereas SynCav1 treated TDP-43 mice demonstrated improved contextual recall and recovered extinction learning. Transmission electron microscopy analysis of the hippocampus revealed that TDP43-SynNull mice exhibited reduced excitatory synapse counts, vesicle density, and excessive mitochondrial fragmentation. In contrast, TDP43-SynCav1 group showed preserved synapse numbers, vesicle counts, and similar mitochondrial morphology as TGN group. Furthermore, IB analysis of hippocampal tissue revealed that the TDP43-SynNull group exhibited increased expression of p-DRP and p-MFF, while TDP43-SynCav1 group showed similar expression levels as TGN group.     Conclusion: Together, these findings suggest that SynCav1 preserved cognitive function by inhibiting excessive mitochondrial fission activity in the hippocampus.  Future studies will focus on optimizing and identifying specific cortical/hippocampal promoters to avoid off-target effects.