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Defined Bioscience

Engineered T cells from induced pluripotent stem cells: from research towards clinical implementation

This review highlights the potential of using induced pluripotent stem cells (iPSCs) to generate engineered T cells for adoptive cell therapy. iPSC-derived T cells offer a scalable, off-the-shelf solution with precise genetic control. The article focuses on advancing manufacturing methods toward clinical-grade, GMP-compliant production, aiming to make engineered T cell therapies safer, more efficient, and broadly accessible.

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Defined Bioscience

A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs

Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell–derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this “hybrid” approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC.

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Defined Bioscience

Nilotinib-induced alterations in endothelial cell function recapitulate clinical vascular phenotypes independent of ABL1

Nilotinib is a highly effective treatment for chronic myeloid leukemia but has been consistently associated with the development of nilotinib-induced arterial disease (NAD) in a subset of patients. To date, which cell types mediate this effect and whether NAD results from on-target mechanisms is unknown. We utilized human induced pluripotent stem cells (hiPSCs) to generate endothelial cells and vascular smooth muscle cells for in vitro study of NAD. We found that nilotinib adversely affects endothelial proliferation and migration, in addition to increasing intracellular nitric oxide. Nilotinib did not alter endothelial barrier function or lipid uptake. No effect of nilotinib was observed in vascular smooth muscle cells, suggesting that NAD is primarily mediated through endothelial cells. To evaluate whether NAD results from enhanced inhibition of ABL1, we generated multiple ABL1 knockout lines. The effects of nilotinib remained unchanged in the absence of ABL1, suggesting that NAD results from off- rather than on-target signaling. The model established in the present study can be applied to future mechanistic and patient-specific pharmacogenomic studies.

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Defined Bioscience

Multi-objective Bayesian algorithm automatically discovers low-cost high-growth serum-free media for cellular agriculture application

In this work, we applied a multi-information source modeling technique to solve a multi-objective Bayesian optimization problem involving the simultaneous minimization of cost and maximization of growth for serum-free C2C12 cells using a hyper-volume improvement acquisition function. In sequential batches of custom media experiments designed using our Bayesian criteria, collected using multiple assays targeting different cellular growth dynamics, the algorithm learned to identify the trade-off relationship between long-term growth and cost. We were able to identify several media with
more growth of C2C12 cells than the control, as well as a medium with 23% more growth at only 62.5% of the cost of the control. These algorithmically generated media also maintained growth far past the study period, indicating the modeling approach approximates the cell growth well from an extremely limited data set.

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Defined Bioscience

Future protein alternative: recent progress and challenges in cellular agriculture

With arising concerns regarding traditional livestock-based proteins, cultivated meat is emerging as a potential alternative. Cultivated meat, often called cultured meat, is defined as a meat produced in the lab by growing animal-derived cells. Considering the cultivated meat is based on animal-origin, it may present several advantages over other types of meat analogue in terms of flavor and nutritional properties. To be commercially available, there are several technical limitations to overcome. This indicates the necessity to integrate information to comprehensively review the current progress. In this review, history and background about the development of cultivated meat is described. Also, the recent progress in cell culture media and scaffold, which are essential components to grow animal cells, is presented. Next, the aspects regarding antibiotics to minimize the risk of bacterial contamination is discussed. Finally, the consumer perception and governmental regulations concerning the consumption and production of cultivated meat are addressed.

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Defined Bioscience

Evaluating the Efficacy of Serum-Free Media Supplemented with Protein Isolates for Bovine Satellite Cell Proliferation: A Sustainable Approach for Cultivated Meat Production

This study investigates the short- and long-term efficacy of serum-free media supplemented with protein isolates from algae, alfalfa, silkworm pupae, and grasshoppers for the proliferation of bovine satellite cells (BSCs). Fresh and spent media were analyzed to monitor metabolites, while cell proliferation was assessed using the CyQUANT assay. The results of the short-term growth study indicated that a lower concentration of alfalfa protein isolate (0.05 mg/mL) significantly enhanced cell proliferation, achieving a 1.47-fold increase compared to basal media, thereby demonstrating its potential as a viable alternative to fetal bovine serum (FBS). The capability of the developed serum-free media for cell passaging was examined using different coating strategies, with vitronectin outperforming others; however, it was unable to support the long-term growth of cells compared to FBS-containing media. Given the critical role of glutamine, this study also evaluates the impact of L-glutamine supplementation at different concentrations on cell growth and metabolism in alfalfa-based media. However, while glutamine supplementation showed trends toward increased cell growth, the enhancements were not statistically significant. Based on the results, this study highlights the potential of alfalfa protein isolate as a promising component of serum-free media for BSC proliferation and underscores the need for continued research into alternative protein sources and media formulations to support the sustainable and ethical production of cultivated meat.

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Defined Bioscience

Stress-free cell aggregation by using the CEPT cocktail enhances embryoid body and organoid fitness

Embryoid bodies (EBs) and self-organizing organoids derived from human pluripotent stem cells (hPSCs) recapitulate tissue development in a dish and hold great promise for disease modeling and drug development. However, current protocols are hampered by cellular stress and apoptosis during cell aggregation, resulting in variability and impaired cell differentiation. Here, we demonstrate that EBs and various organoid models (e.g., brain, gut, kidney) can be optimized by using the small molecule cocktail named CEPT (chroman 1, emricasan, polyamines, trans-ISRIB), a polypharmacological approach that ensures cytoprotection and cell survival. Application of CEPT for just 24 h during cell aggregation has long-lasting consequences affecting morphogenesis, gene expression, cellular differentiation, and organoid function. Various qualification methods confirmed that CEPT treatment enhanced experimental reproducibility and consistently improved EB and organoid fitness as compared to the widely used ROCK inhibitor Y-27632. Collectively, we discovered that stress-free cell aggregation and superior cell survival in the presence of CEPT are critical quality control determinants that establish a robust foundation for bioengineering complex tissue and organ models.

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Defined Bioscience

A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells

Human pluripotent stem cells (hPSCs) are capable of extensive self-renewal yet remain highly sensitive to environmental perturbations in vitro, posing challenges to their therapeutic use. There is an urgent need to advance strategies that ensure safe and robust long-term growth and functional differentiation of these cells. Here, we deployed high-throughput screening strategies to identify a small-molecule cocktail that improves viability of hPSCs and their differentiated progeny. The combination of chroman 1, emricasan, polyamines, and trans-ISRIB (CEPT) enhanced cell survival of genetically stable hPSCs by simultaneously blocking several stress mechanisms that otherwise compromise cell structure and function. CEPT provided strong improvements for several key applications in stem-cell research, including routine cell passaging, cryopreservation of pluripotent and differentiated cells, embryoid body (EB) and organoid formation, single-cell cloning, and genome editing. Thus, CEPT represents a unique poly-pharmacological strategy for comprehensive cytoprotection, providing a rationale for efficient and safe utilization of hPSCs.

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Defined Bioscience

Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail

Human pluripotent stem cells (hPSCs) are inherently sensitive cells. Single-cell dissociation and the establishment of clonal cell lines have been long-standing challenges. This inefficiency of cell cloning represents a major obstacle for the standardization and streamlining of gene editing in induced pluripotent stem cells for basic and translational research. Here we describe a chemically defined protocol for robust single-cell cloning using microfluidics-based cell sorting in combination with the CEPT small-molecule cocktail. This advanced strategy promotes the viability and cell fitness of self-renewing stem cells. The use of low-pressure microfluidic cell dispensing ensures gentle and rapid dispensing of single cells into 96- and 384-well plates, while the fast-acting CEPT cocktail minimizes cellular stress and maintains cell structure and function immediately after cell dissociation. The protocol also facilitates clone picking and produces genetically stable clonal cell lines from hPSCs in a safe and cost-efficient fashion. Depending on the proliferation rate of the clone derived from a single cell, this protocol can be completed in 7–14 d and requires experience with aseptic cell culture techniques. Altogether, the relative ease, scalability and robustness of this workflow should boost gene editing in hPSCs and leverage a wide range of applications, including cell line development (e.g., reporter and isogenic cell lines), disease modeling and applications in regenerative medicine.

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Defined Bioscience

Microbial lysates as low-cost serum replacements in cellular agriculture media formulation

Cultivated meat, the process of generating meat in vitro without sacrificing animals, is a promising alternative to the traditional practice of livestock agriculture. However, the success of this field depends on finding sustainable and economical replacements for animal-derived and expensive fetal bovine serum (FBS) that is typically used in cell culture processes. Here, we outline an effective screening process to vet the suitability of microbial lysates to support the growth of immortalized bovine satellite cells (iBSCs) and mackerel (Mack1) cells. We show that easily producible, low-cost whole-cell lysates from Vibrio natriegens can be used to create serum-free media for the long-term growth of iBSCs. The optimized medium, named “VN40” (basal B8 media containing Vibrio natriegens lysate proteins at 40 µg/mL), outperforms previously established serum-free media while maintaining cell phenotype and myogenicity. Overall, this study shows a novel approach to producing serum-free media for cultivated meat production using microbially-derived lysates.

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Defined Bioscience

Sustainable Alternatives to Fetal Bovine Serum: Evaluating the Role of Plant and Insect Protein Isolates in Serum-Free Media for Bovine Satellite Cell Proliferation in Cultivated Meat Production

This study explores the development and efficacy of serum-free media supplemented with various plant and insect protein isolates as a sustainable alternative to fetal bovine serum (FBS) for cultivated meat production. Focusing on the proliferation and differentiation of bovine satellite cells (BSCs), we tested proteins derived from various plants, insects, and legume byproducts. Our results demonstrated that alfalfa, chickpea cooking liquid, tofu whey, and mung bean proteins at lower concentrations (0.05–0.25 mg/mL) significantly enhanced cell proliferation, with mung bean media outperforming the others. Differentiation assays showed that cells cultured in serum-free media supplemented with mung bean protein maintained their myogenic potential, forming well-differentiated myotubes similar to those observed in FBS-containing media. Live/dead imaging and metabolic profiling further supported these findings, showing healthy cell morphology and active metabolism in the optimized protein media. However, higher protein concentrations resulted in cytotoxic effects. This research highlights the potential of plant protein isolates as key components in serum-free media, providing an ethical, cost-effective, and sustainable media for cultivated meat production. While insect-derived proteins were not effective in supporting cell growth, optimizing their extraction and concentration may enhance their future applicability. Further refinement and long-term studies are needed to improve media composition and scalability for commercial use.

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Defined Bioscience

Reactivation of the G1 enhancer landscape underlies core circuitry addiction to SWI/SNF

Several cancer core regulatory circuitries (CRCs) depend on the sustained generation of DNA accessibility by SWI/SNF chromatin remodelers. However, the window when SWI/SNF is acutely essential in these settings has not been identified. Here we used neuroblastoma (NB) cells to model and dissect the relationship between cell-cycle progression and SWI/SNF ATPase activity. We find that SWI/SNF inactivation impairs coordinated occupancy of non-pioneer CRC members at enhancers within 1 hour, rapidly breaking their autoregulation. By precisely timing inhibitor treatment following synchronization, we show that SWI/SNF is dispensable for survival in S and G2/M, but becomes acutely essential only during G1 phase. We furthermore developed a new approach to analyze the oscillating patterns of genome-wide DNA accessibility across the cell cycle, which revealed that SWI/SNF-dependent CRC binding sites are enriched at enhancers with peak accessibility during G1 phase, where they activate genes involved in cell-cycle progression. SWI/SNF inhibition strongly impairs G1-S transition and potentiates the ability of retinoids used clinically to induce cell-cycle exit. Similar cell-cycle effects in diverse SWI/SNF-addicted settings highlight G1-S transition as a common cause of SWI/SNF dependency. Our results illustrate that deeper knowledge of the temporal patterns of enhancer-related dependencies may aid the rational targeting of addicted cancers.

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Defined Bioscience

Simple and effective serum-free medium for sustained expansion of bovine satellite cells for cell cultured meat.

Cell-cultured meat could create a more sustainable and ethical food system, but progress has been limited by the lack of affordable, serum-free media. Building on the low-cost B8 medium for iPSCs, we adapted it for bovine satellite cells by adding recombinant albumin, creating “Beefy-9.” This formulation supports long-term cell growth over seven passages (39-hour doubling time) without losing myogenicity. Beefy-9 offers a foundation for serum-free media for other meat-relevant species, advancing cultured meat research.

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Defined Bioscience

Negligible-Cost and Weekend-Free Chemically Defined Human iPSC Culture.

Human iPSC culture is routine but limited by high media costs, frequent changes, and variable differentiation. We developed B8, an optimized, low-cost medium using in-lab–produced recombinant FGF2, TGFβ3, and NRG1, reducing costs to 3% of commercial media. B8 supports derivation and long-term culture of 34 hiPSC lines (100+ passages) while enabling a weekend-free feeding schedule without compromising pluripotency or differentiation potential.

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Defined Bioscience

Large-scale cultured meat production: Trends, challenges and promising biomanufacturing technologies.

Future food systems need protein-rich diets, but traditional meat cannot sustainably meet global demand. This paper reviews bioprocess technologies for scaling cultured meat, covering cell line development, media, scaffolding, and bioreactors. It highlights current challenges, research opportunities, and tradeoffs between scale, cost, quality, and footprint. Broader issues—social acceptance, regulations, and environmental impacts—are discussed, emphasizing that the shift from livestock to alternative proteins is already underway and will accelerate in the coming decade.

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Defined Bioscience

Food safety considerations and research priorities for the cultured meat and seafood industry.

Cell-cultured meat and seafood could sustainably meet rising protein demand, but safety validation is key for consumer trust and commercialization. This review maps potential hazards in the manufacturing process through workshops with 87 experts, using a process diagram to identify chemical and biological risks. Most hazards mirror those in conventional foods, so existing safety methods largely apply, though novel inputs may require extra evaluation. Further research on inputs, contamination risks, and standardized (animal-free) safety assessments is recommended to ensure safe, scalable production.

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