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A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells

Nature Biomedical Engineering (2024)Cite this article

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Abstract

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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Fig. 1: Schematics of CAR T-cell process on the microbioreactor and gas-permeable well plate.
Fig. 2: Comparable levels of activation and transduction, and significantly higher levels of CAR T-cell expansion on the microbioreactor compared with gas-permeable well plate.
Fig. 3: Differentiation and exhaustion phenotypes of CAR T cells from the microbioreactor and gas-permeable well plate.
Fig. 4: CAR T cells produced on the microbioreactor were highly functional in cytokine secretion, short-term cytotoxic activity and long-term persistence and proliferation.
Fig. 5: Functional CAR T cells can be produced in the microfluidic bioreactor from patient-derived starting material.
Fig. 6: Patient-derived CAR T cells generated on the microbioreactor exert anti-leukaemic activity in vivo.
Fig. 7: Estimating growth rates and metabolic rates using computational modelling.

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Data availability

The RNA-seq and gene-expression data are available from the Gene Expression Omnibus (GEO) under accession number GSE261103. The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are available for research purposes from the corresponding authors on reasonable request. Source data are provided with this paper.

Code availability

The custom code for computational modelling is available via GitHub at https://github.com/narendrasuhas/perfReactorCarT.

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Acknowledgements

This research is supported by the National Research Foundation, Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) programme, through the Singapore-MIT Alliance for Research and Technology Centre (SMART) Critical Analytics for Manufacturing Personalized-Medicine (CAMP) Inter-Disciplinary Research Group, and in part by the Integrated Manufacturing Programme for Autologous Cell Therapy (IMPACT) (IAF-PP, H18AHa0001) from the Agency for Science, Technology and Research (A*STAR), Singapore. The mouse work is supported by BMRC Central Research Fund (ATR), A*STAR, Singapore to Q.C., and the patient runs are supported by Goh Foundation Limited, Singapore to M.S.-F.S. and S.Y.S. The authors thank N. Tan at SMART CAMP, Singapore, for the coordination of patient sample collections; S. Y. Tan at the Institute of Molecular and Cell Biology (IMCB), A*STAR, Singapore, for assistance with in vivo mouse experiments; V. Au at IMCB, A*STAR, Singapore, for assistance with Luminex multiplex assays; C. Perez at MIT for assistance in interpreting the RNA-seq data; and K. S. Lee of Erbi Biosystems, part of MilliporeSigma, for technical assistance with the microbioreactor.

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Authors and Affiliations

  1. Critical Analytics for Manufacturing Personalized-Medicine (CAMP), Singapore-MIT Alliance for Research and Technology Centre (SMART), Singapore, Singapore

    Wei-Xiang Sin, N. Suhas Jagannathan, Denise Bei Lin Teo, Faris Kairi, Dedy Sandikin, Ka-Wai Cheung, Yen Hoon Luah, Xiaolin Wu, Joshua Jebaraj Raymond, Yie Hou Lee, Rajeev J. Ram, Lisa Tucker-Kellogg & Michael E. Birnbaum

  2. Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore, Singapore

    N. Suhas Jagannathan & Lisa Tucker-Kellogg

  3. Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), Singapore, Singapore

    Shin Yie Fong, Joel Heng Loong Tan & Qingfeng Chen

  4. Advanced Cell Therapy and Research Institute, Singapore (ACTRIS), Consortium for Clinical Research and Innovation, Singapore (CRIS), Singapore, Singapore

    Francesca Lorraine Wei Inng Lim

  5. Department of Haematology, Singapore General Hospital, Singapore, Singapore

    Francesca Lorraine Wei Inng Lim

  6. SingHealth Duke-NUS Oncology Academic Clinical Programme, SingHealth Duke-NUS Academic Medical Centre, Singapore, Singapore

    Francesca Lorraine Wei Inng Lim, Michaela Su-Fern Seng & Shui Yen Soh

  7. SingHealth Duke-NUS Cell Therapy Centre, SingHealth Duke-NUS Academic Medical Centre, Singapore, Singapore

    Francesca Lorraine Wei Inng Lim, Yie Hou Lee, Michaela Su-Fern Seng & Shui Yen Soh

  8. Department of Paediatric Haematology and Oncology, KK Women’s and Children’s Hospital, Singapore, Singapore

    Michaela Su-Fern Seng & Shui Yen Soh

  9. Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, Cambridge, MA, USA

    Rajeev J. Ram

  10. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA

    Michael E. Birnbaum

  11. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, USA

    Michael E. Birnbaum

  12. Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA

    Michael E. Birnbaum

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  1. Wei-Xiang Sin
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W.-X.S., R.J.R., L.T.-K. and M.E.B. conceived the project. W.-X.S., D.B.L.T., F.K., S.Y.F., J.H.L.T., D.S., K.-W.C., Y.H. Luah, X.W. and J.J.R. conducted experiments and collected data. W.-X.S., N.S.J., D.B.L.T., K.-W.C., Y.H. Luah and X.W. analysed data. F.L.W.I.L., M.S.-F.S. and S.Y.S. provided patient samples. Y.H. Lee, Q.C., R.J.R., L.T.-K. and M.E.B. supervised the work. W.-X.S., N.S.J., R.J.R., L.T.-K. and M.E.B. wrote the paper. All authors edited and approved the paper.

Corresponding authors

Correspondence to Rajeev J. Ram, Lisa Tucker-Kellogg or Michael E. Birnbaum.

Ethics declarations

Competing interests

M.E.B. is an equity holder in 3T Biosciences, is a cofounder, equity holder and consultant of Kelonia Therapeutics and Abata Therapeutics, and receives research funding from Pfizer unrelated to this work. The other authors declare no competing interests.

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Nature Biomedical Engineering thanks Manel Juan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Details of CAR T cell process on the microbioreactor and gas-permeable well plate.

a, Left: Image of the microbioreactor system, comprised of a base station controller and a CO2 controller supporting up to four ‘pods’, which allows highly parallelized synchronous or asynchronous running of up to four separate cultures per instrument (W: 41 cm × D: 43 cm × H: 36 cm). Right: Image of the sterile, single-use consumable, consisting of a microfluidic chip or ‘cassette’ (W: 5 cm × D: 8 cm × H: 1 cm) connected to a bottle rack assembly (W: 10 cm × D: 13 cm × H: 18 cm). b, In an optimization experiment, T cells were isolated and activated on Day 0, transduced on either Day 0 (concurrent with activation) or Day 2, and expanded up to Day 14 in the microbioreactor, with either a step-wise ramping up of perfusion flow rate from 1 v.v.d. to 4 v.v.d. (starting at 1 v.v.d. approximately 48 h after transduction, ramping up by 1 v.v.d. every other day) or a constant high perfusion flow rate of 4 v.v.d. (starting from approximately 48 h after transduction). Cells were sampled on Day 14, the percentage of total live cells expressing CAR were quantified by flow cytometry, the viable cell count (VCC) in number of cells were determined with acridine orange-propidium iodide (AO-PI) staining using an automated cell counter, and the percentage of total live cells that were CCR7+ CD45RA+ (naïve and stem cell memory, Tn/Tscm), CCR7+ CD45RA– (central memory, Tcm), CCR7– CD45RA– (effector memory, Tem), and CCR7– CD45RA+ (terminally differentiated effector, Temra) were quantified by flow cytometry. c, In an optimization experiment, T cells were isolated and activated on Day 0, transduced on either Day 0 (concurrent with activation) or Day 1, and expanded up to Day 14 in the microbioreactor or gas-permeable well, with a constant high perfusion flow rate of 4 v.v.d. (starting from Day 2). Cells were sampled on Day 14, the percentage of total live cells expressing CAR were quantified by flow cytometry, the viable cell count (VCC) in number of cells were determined with acridine orange-propidium iodide (AO-PI) staining using an automated cell counter, and the percentage of total live cells that were CCR7+ CD45RA+ (naïve and stem cell memory, Tn/Tscm), CCR7+ CD45RA– (central memory, Tcm), CCR7– CD45RA– (effector memory, Tem), and CCR7− CD45RA+ (terminally differentiated effector, Temra) were quantified by flow cytometry. d, Image of the gas-permeable well plate. Created with BioRender.come, Details of activation, transduction, and expansion culture conditions on the microbioreactor and gas-permeable wells. Starting cell numbers were 2 million T cells in 2 mL culture volumes for both culture vessels. f, Culture volumes (dashed lines) and total volumes of medium used (solid lines) over time for the microbioreactor and gas-permeable wells. g, The microbioreactor dissolved O2 setpoint was 80%. When dissolved O2 levels fall below 80%, O2 would be injected into the headspace of the cassette to maintain dissolved O2 levels at a minimum of 80%. The microbioreactor pH setpoint was 7.40 ± 0.05. When pH is above 7.45, more CO2 (above the minimum of 5%) would be injected into the headspace of the cassette to lower the pH, and when pH is below 7.35, a basic carbonate/bicarbonate solution would be injected into the growth chamber to raise the pH. Dissolved O2 and pH control were activated 2 h after transduction.

Source data

Extended Data Fig. 2 Significantly higher levels of CAR T cell expansion on the microbioreactor compared with gas-permeable well plate.

a–d, h, For CAR-transduced runs (n = 3 wells/cassettes per donor), cells were sampled after seeding on Day 0, and on Days 1, 6, and 12. Viable cell density (VCD) in cells per ml (a), percentage cell viability (b), and viable cell count (VCC) in number of cells (c) were determined with acridine orange-propidium iodide (AO-PI) staining using an automated cell counter. Data shown are mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values are 0.0009, <0.0001, 0.0002 for Donor A, 0.0017, 0.0004, 0.0001 for Donor B, and 0.0015, 0.0002, <0.0001 for Donor C in (a), 0.1631, 0.0785, 0.5983 for Donor A, 0.0022, 0.0163, 0.0956 for Donor B, and 0.0093, 0.6308, 0.3567 for Donor C in (b), 0.0008, 0.0378, 0.0013 for Donor A, 0.0017, 0.1184, 0.0008 for Donor B, and 0.0015, 0.0056, 0.0009 for Donor C in (c) for Microbioreactor CAR versus Gas-permeable well CAR, on Days 1, 6, 12, as determined using two-tailed unpaired t-tests (df=4). (d) Same data as (c), plotting AO-PI VCCs on Days 6 and 12 on different scales to visualize differences. Data shown are individual values and mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values were determined using two-tailed unpaired t-tests (df=4). e–f, For non-transduced runs (n = 1 well/cassette per donor), cells were sampled after seeding on Day 0, and on Days 1, 4, 6, 8, and 12. Viable cell count (VCC) in number of cells were determined with acridine orange-propidium iodide (AO-PI) staining using an automated cell counter. (f) Same data as (e), plotting AO-PI VCCs on Days 4, 6, 8, and 12 on different scales to visualize differences. Data shown are mean ± SD of n = 3 donors. P values were determined using paired t-tests (d.f.=2). g, Mean ± SD and coefficients of variation of final viable cell counts of 12 runs each on the microbioreactor and the gas-permeable well, showing similar variability between runs for the two culture systems. h, Fold expansion relative to VCC of cell sample taken after cell inoculation on Day 0 were determined with acridine orange-propidium iodide (AO-PI) staining using an automated cell counter. Data shown are mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values are 0.1857, 0.0173, 0.0045 for Donor A, 0.2318, 0.0158, 0.0055 for Donor B, and 0.9500, 0.0265, 0.0042 for Donor C, for Microbioreactor CAR versus Gas-permeable well CAR, on Days 1, 6, 12, as determined using two-tailed unpaired t-tests (df=4). i, Cells were sampled before harvest on Day 12, and number of CAR+ viable T cells were quantified by AO-PI cell count and flow cytometry. Data shown are individual values and mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values were determined using two-tailed unpaired t-tests (df=4). j, Number of transduction units (TU) of lentiviral vector (LVV) used to transduce the number of cells on Day 1 at multiplicity of infection (MOI) of 5 was divided by the final CAR+ viable cell count to obtain the number of TU of LVV used per million CAR+ viable T cells produced. Data shown are individual values and mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values were determined using two-tailed unpaired t-tests (df=4).

Source data

Extended Data Fig. 3 Significantly higher levels of CAR T cell expansion on the microbioreactor compared with gas-permeable well plate.

a–d, Cells were sampled after seeding on Day 0, and on Days 1, 6, and 12, and viable cell density (VCD) in cells/mL (a), percentage cell viability (b), viable cell count (VCC) in number of cells (c), and fold expansion relative to VCC of cell sample taken after cell inoculation on Day 0 (d) were determined with trypan blue (TB) staining using an automated cell counter. Data shown are mean ± SD of n = 3 technical replicate wells/cassettes per donor. e, Cells were sampled before harvest on Day 12, and number of CAR+ viable T cells were quantified by TB cell count and flow cytometry. Data shown are individual values and mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values were determined using two-tailed unpaired t-tests (df=4). f, Volume of medium used for the entire 12-day CAR T cell production process was divided by the final CAR+ viable cell count to obtain the volume of medium used per million CAR+ viable T cells produced. Data shown are individual values and mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values were determined using two-tailed unpaired t-tests (df=4).

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Extended Data Fig. 4 T-cell differentiation and exhaustion phenotypes from the microbioreactor and the gas-permeable well plate.

a–e, Cells were sampled on Days 6 and 12, and the percentage of CCR7+ CD45RA+ (naïve and stem cell memory, Tn/Tscm), CCR7+ CD45RA– (central memory, Tcm), CCR7– CD45RA– (effector memory, Tem), and CCR7– CD45RA+ (terminally differentiated effector, Temra) cells gated on CAR+ live cells (a), on CD4+ cells (b), on CAR+ CD4+ cells (c), on CD8+ cells (d), and on CAR+ CD8+ cells (e) were quantified by flow cytometry. Data shown are mean ± SD of n = 3 technical replicate wells/cassettes per donor. f–g, Cells were sampled on Days 6 and 12, and the percentage of total live cells expressing CD127 (f) and CD57 (g) were quantified by flow cytometry. Data shown are individual values and mean ± SD of n = 3 technical replicate wells/cassettes per donor. P values were determined using two-tailed unpaired t-tests (df=4).

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Extended Data Fig. 5 Bulk RNA-seq analysis of cell products from the microbioreactor and gas-permeable wells.

Day 12 cell products (n = 3 healthy donors from the microbioreactor and n = 3 healthy donors from gas-permeable wells) were analysed by bulk RNA-seq. a, Principal component analysis (PCA) of the gene expression profiles from the bulk RNA-seq samples (microbioreactor, MBR and gas-permeable well plate, GWP). b, Volcano plot of differentially expressed genes. The x-axis shows the fold change in gene expression between the microbioreactor (MBR) and gas-permeable well plate (GWP), and the y-axis shows the statistical significance of the differences. Genes with a |log2(FoldChange)| ≥ 1 and Benjamini-Hochberg false discovery rate (FDR) adjusted P-value ≤ 0.05 were defined as differentially expressed. Upregulated genes in the microbioreactor (752 genes) are shown in red and downregulated genes in the microbioreactor (452 genes) are shown in green. c, Top ten most significantly enriched Gene Ontology (GO) biological processes for all differentially expressed genes (top), top ten most significantly enriched GO biological processes for upregulated genes (middle), and the four significantly enriched GO molecular functions for downregulated genes (bottom). The x-axis is the ratio of the number of differentially expressed genes linked with the GO term to the total number of differentially expressed genes, and the y-axis is the GO terms. The size of a point represents the number of genes annotated to the specific GO term, and the colour represents the statistical significance of the enrichment. d, Heat map of all 1,204 differentially expressed genes. e–i, Gene signatures of T cell states were obtained from Anderson et al., Nat Med, ref. 64. Each column represents a different sample, and the columns are always ordered as microbioreactor (MBR) Donors A, B, C and gas-permeable well plate (GWP) Donors A, B, C. Differentially expressed genes are annotated with asterisks, and non-differentially expressed genes are not annotated. e, Heat map of naïve gene signature. f, Heat map of central memory (CM) gene signature. g, Heat map of effector memory (EM) gene signature. h, Heat map of effector gene signature. i, Heat map of exhaustion gene signature. PDCD1 (PD-1), LAG3, and HAVCR2 (TIM-3) genes are boxed. j, Gene expression levels (FPKM) in the cell products harvested from the microbioreactor (MBR) and gas-permeable well plate (GWP) (before stimulation with NALM6 tumour cells) for proteins that were differentially secreted after stimulation with NALM6 tumour cells. IL4, IL5, IL13, IL3, CSF2 (GM-CSF), and IFNG genes were upregulated in the microbioreactor, CD274 (PD-L1) was upregulated in the gas-permeable well plate, whereas CSF3 (G-CSF), LTA (TNF-β), and TNF (TNF-α) were not differentially expressed. Benjamini-Hochberg false discovery rate (FDR) adjusted P-values are shown.

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Extended Data Fig. 6 CAR T cells produced on the microbioreactor were highly functional in terms of cytokine release and cytolytic activity against tumour cells.

a–c, Harvested T cells and NALM6 cells were co-cultured overnight at a 1:1 CAR+ T cell:NALM6 cell ratio, and cytokine secretion in cell culture supernatants were analysed by Luminex multiplex assays. Cells from one run of each donor were used for this assay. Data shown are mean ± SEM of n = 3 donors (except for NTC unstim, for which only Donor A was measured), each assayed in technical triplicates. NTC: non-transduced control. CAR: CAR T cells (unsorted). Unstim: T cells alone. Stim: Co-cultured with NALM6 cells. P values were determined using two-tailed unpaired t-tests (df=4). d, Harvested T cells and NALM6 cells were co-cultured overnight at various CAR+ T cell:NALM6 cell ratios, and the number of remaining NALM6 cells were analysed by flow cytometry. Percentage specific lysis were calculated relative to wells containing only NALM6 cells (0% lysis) and wells containing only T cells (100% lysis). Cryopreserved cells from one run of each donor were thawed for this assay. Data shown are mean ± SD of n = 3 technical replicate measurements. Adjusted P values are 0.0031, 0.0007 for Donor A, 0.0004, 0.0002, 0.0030 for Donor B, and 0.0005, <0.0001 for Donor C, for Microbioreactor CAR versus Gas-permeable well CAR, in increasing order of E:T ratio, as determined using two-tailed unpaired t-tests (df=4) with Holm-Sidak’s multiple comparison correction.

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Extended Data Fig. 7 Healthy donor CAR T cells generated on the microbioreactor exert anti-leukemic activity in vivo.

a, Experimental design: NSG mice were intravenously injected with 0.5 million NALM6-luciferase cells, and then treated with 0.5 million CAR+ viable T cells or corresponding numbers of non-transduced T cells six days later, followed by monitoring of tumour burden via bioluminescent imaging (BLI) (n = 5 mice for each group). Created with BioRender.comb, Kaplan-Meier curve for overall survival. P values are 0.0019 for Microbioreactor CAR versus Microbioreactor NTC, 0.0016 for Gas-permeable well CAR versus Gas-permeable well NTC, 0.3173 for Microbioreactor CAR versus Gas-permeable well CAR, and 0.6015 for Microbioreactor NTC versus Gas-permeable well NTC, as determined using log-rank test (df=1). c–e, Tumour bioluminescent signal was monitored every 2–7 days by imaging for luciferase activity, and total photon counts were quantified. c, Data shown are mean ± SD of n = 5 mice. P values between groups are 0.0038 for Microbioreactor CAR versus Microbioreactor NTC, 0.0009 for Gas-permeable well CAR versus Gas-permeable well NTC, 0.1424 for Microbioreactor CAR versus Gas-permeable well CAR, and 0.3643 for Microbioreactor NTC versus Gas-permeable well NTC, as determined by two-way ANOVA or main effects model with Geisser-Greenhouse correction. Adjusted P values are 0.0049 for Microbioreactor CAR versus Gas-permeable well CAR (Day 14), as determined using two-tailed unpaired t-tests (df=8) with Holm-Sidak’s multiple comparison correction. d, Data shown are individual values from individual mouse. e, Images of bioluminescent signal after injection of luciferin substrate. Radiance scale was the same within each timepoint and different across timepoints in order to show NALM6 tumour engraftment on Day 0.

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Sin, WX., Jagannathan, N.S., Teo, D.B.L. et al. A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01219-1

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