A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells
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ABSTRACT:
PROVIDER: PRJNA1085289 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28260645_1.fastq.gz | Fastqsanger.gz | |||
| SRR28260645_2.fastq.gz | Fastqsanger.gz | |||
| SRR28260646_1.fastq.gz | Fastqsanger.gz | |||
| SRR28260646_2.fastq.gz | Fastqsanger.gz | |||
| SRR28260647_1.fastq.gz | Fastqsanger.gz |
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Project description:The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale-out of cell-therapy manufacturing.
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Project description:Adoptive T cell therapy (ACT) using chimeric antigen receptor (CAR) engineered T cells is currently being explored in multiple cancer types beyond leukemia/lymphoma. A key step in CAR-T cell manufacturing is the activation and expansion of T cells, which facilitates viral transduction, however, may hamper T cell fitness and reduce in vivo persistence. We developed “T-Expand” for T cell activation and expansion, comprising dextran-based nanoparticles (NPs) conjugated with anti-CD3 and anti-CD28 antibodies. The NPs triggered robust polyclonal expansion of human T cells with efficiency in the range of commercial microbeads (Dynabeads™). Engineered in the presence of T-Expand, CD19 CAR T cells exhibited enhanced proliferative capacity, cytotoxicity and persistence in vitro, and furthermore, showed superior anti-lymphoma activity in mouse models, resulting in complete tumor clearance at one fourth of the CAR T cell dose. Importantly, T-Expand is biocompatible with no observed toxicity, circumventing removal steps after T cell expansion compared to DynabeadsTM. As a biocompatible T cell expansion platform, T-Expand simplifies the manufacturing process while enhancing T cell persistence and functionality, and thereby holds promise for increasing clinical efficacy of CAR T cell therapy.
2025-11-19 | GSE308384 | GEO
Project description:We developed the microfluidic-oscillatory-washing-based ChIP-Seq (MOWChIP-Seq) protocol. We achieved genome-wide mapping of histone modifications (H3K4me3 and H3K27ac) with as few as 100 cells. Moreover, the automated microfluidic platform dramatically reduced assay time and has a potential for future scale-up.
2015-07-21 | GSE65516 | GEO
Project description:Cell culture conditions impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products, but the optimal approach remains unknown. Separate CD4+ and CD8+ cultures offer a potential advantage but complicate manufacturing and may affect cell expansion and function. We evaluated the co-culture of CD4+ and CD8+ cells at a defined ratio at culture initiation. We observed that the presence of CD4+ cells markedly improves expansion of CD8+ CAR T cells, and CD8+ cells cultured in isolation exhibit a hypofunctional phenotype and transcriptional signature compared with those co-cultured with CD4+ cells. Mixed-culture CAR T cells also confer superior anti-tumor activity in vivo compared with separately expanded, co-infused cells. CD4+ cell effects on CD8+ cells are mediated through both cytokines and direct cell contact, including CD40L-CD40 and CD70-CD27 interactions.
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Project description:In this work, we address the impact of CAR density on the in vitro and in vivo functionality of BCMA-CAR T cells. Phenotypic, functional, transcriptomic and epigenomic studies at bulk and single cell level revealed different profiles between CAR T cells with high and low expression of the CAR molecule (CARHigh and CARLow T cells). We analyzed the phenotype of CARHigh and CARLow T cell populations before and after stimulation with tumor cells. Sorted CD4+ and CD8+ CARHigh and CARLow T cell populations from six different CAR T cell productions were profiled using high-throughput RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin (ATAC-seq). To better understand the heterogeneity of CAR T cells and the influence of CAR level on their transcriptomic profile, we performed single cell transcriptomic analysis on 43,981 CAR T cells from three independent productions.
2022-10-05 | GSE197849 | GEO
Project description:In this work, we address the impact of CAR density on the in vitro and in vivo functionality of BCMA-CAR T cells. Phenotypic, functional, transcriptomic and epigenomic studies at bulk and single cell level revealed different profiles between CAR T cells with high and low expression of the CAR molecule (CARHigh and CARLow T cells). We analyzed the phenotype of CARHigh and CARLow T cell populations before and after stimulation with tumor cells. Sorted CD4+ and CD8+ CARHigh and CARLow T cell populations from six different CAR T cell productions were profiled using high-throughput RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin (ATAC-seq). To better understand the heterogeneity of CAR T cells and the influence of CAR level on their transcriptomic profile, we performed single cell transcriptomic analysis on 43,981 CAR T cells from three independent productions.
2022-10-05 | GSE197848 | GEO
Project description:In this work, we address the impact of CAR density on the in vitro and in vivo functionality of BCMA-CAR T cells. Phenotypic, functional, transcriptomic and epigenomic studies at bulk and single cell level revealed different profiles between CAR T cells with high and low expression of the CAR molecule (CARHigh and CARLow T cells). We analyzed the phenotype of CARHigh and CARLow T cell populations before and after stimulation with tumor cells. Sorted CD4+ and CD8+ CARHigh and CARLow T cell populations from six different CAR T cell productions were profiled using high-throughput RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin (ATAC-seq). To better understand the heterogeneity of CAR T cells and the influence of CAR level on their transcriptomic profile, we performed single cell transcriptomic analysis on 43,981 CAR T cells from three independent productions.
2022-10-05 | GSE197843 | GEO
Project description:Genome-wide epigenetic changes such as histone modifications form a critical layer of gene regulations and have been implicated in a number of different disorders such as cancer and inflammation. Progress has made to decrease the input required for gold-standard genome-wide profiling tools like chromatin immunoprecipitation followed by next generation sequencing (i.e. ChIP-seq) to allow scarce primary tissues of specific type from patients and lab animals to be tested. However, there has been very little effort to rapidly increase the throughput of these low-input tools. In this report, we demonstrate LIFE-ChIP-seq (Low-Input Fluidized-bed Enabled Chromatin Immunoprecipitation combined with sequencing), an automated and high-throughput microfluidic platform capable of running multiple sets of ChIP assays in as little as 1 h with as few as 50 cells per assay. Our technology will enable testing of a large number of samples and replicates with low-abundance primary samples in the context of precision medicine.
2018-06-01 | GSE102932 | GEO
Project description:Background: Autologous CD19 CAR T cell therapy leads to durable responses and improved survival in patients with relapsed or refractory large B cell lymphoma (R/R LBCL). Among approved CAR T cell products, axicabtagene ciloleucel (axi-cel; CD19/CD28) has greater real-world efficacy and cytokine-associated toxicity than tisagenlecleucel (tisa-cel; CD19/4-1BB), for reasons that are poorly understood. Methods: Here we report single cell RNA sequencing (scRNA-seq) of 57 pre-infusion CAR T cell products from axi-cel (n=39) and tisa-cel (n=18) patients treated as standard-of-care for R/R LBCL, and their biological associations with clinical outcomes. In vitro CAR manufacturing conditions mimicking those known for axi-cel and tisa-cel were performed using CD19/CD28z or CD19/4-1BBz constructs. Results: ScRNA-seq revealed that axi-cel and tisa-cel are markedly different products. Axi-cel is comprised of more CD4 central memory, CD8 central memory, and CD8 effectors, whereas tisa-cel is comprised of more proliferative CD4 and CD8 cells. Across multiple T cell subsets, axi-cel had greater expression of immune response pathways and protein synthesis and trafficking pathways vs. tisa-cel. On comparison of infusion product CAR transgene-positive (CAR+) cells to CAR transgene-negative (CAR-) T cells, axi-cel CAR+ cells had vastly different gene expression than axi-cel CAR- cells. Unexpectedly, tisa-cel CAR+ cells were highly similar to tisa-cel CAR- cells. Under recapitulated CAR-T manufacturing conditions known to be utilized for axi-cel and tisa-cel, we found that CAR+ cells differed from CAR- cells early after manufacturing yet became more similar to CAR- cells after prolonged expansion. Prolonged time in expansion culture, as utilized during tisa-cel manufacturing, greatly decreased naïve and central memory T cell subsets. Conclusions: Following manufacture, axi-cel is less differentiated and has greater immune activation compared to tisa-cel, potentially accounting for its greater efficacy and toxicity in patients. Our data support the conclusion that tisa-cel is adversely affected by its manufacturing rather than by the CAR construct.
2025-07-30 | GSE297676 | GEO