For all studies, Lin- cells were enriched from total BM isolated from femurs and tibias of donor mice (6–12 weeks of age) using the EasySep Mouse Hematopoietic Progenitor Cell Isolation Kit (STEMCELL Technologies) in conjunction with the RoboSep-S (STEMCELL Technologies) automated cell-isolation machine and confirmed by flow analysis. Cells were stained for lineage markers with a PE-Cy5-conjugated lineage marker cocktail including B220, Ter119, Tcrb, Cd8a, Cd3ε, Cd4, Ly6g/Ly6c, and Cd11b and with hematopoietic stem cell markers APC-conjugated anti-Cd117 (c-Kit) and PE-conjugated Ly6a/e (Sca-1) (BD Biosciences). Lin- cells were transduced with a lentiviral vector in a cell incubator at 2 × 106 cells/mL in either StemSpan SFEM II (STEMCELL Technologies) or StemMACS HSC Expansion Media (Miltenyi Biotec) growth media freshly supplemented with TPO (10 ng/mL), SCF (100 ng/mL), and FLT3 (50 ng/mL). Approximately 16–22 h later, cells were collected, washed at least three times with DPBS (without Ca2+ and Mg2+), and resuspended in an appropriate volume for dosing. A small number of cells (3,000–4,000 cells) were set aside for use in the colony-forming unit assay using M3434 media (STEMCELL Technologies) and were analyzed 7–10 days later.
Robosep s
Manufactured by STEMCELL
Sourced in Canada
The RoboSep-S is an automated cell separation system that utilizes magnetic particles and a user-defined protocol to isolate target cell populations from heterogeneous samples. It is designed to provide consistent, reproducible cell separation results with minimal hands-on time.
Automatically generated - may contain errors
Lab products found in correlation
Easysep human t cell isolation kit, by STEMCELL (3 mentions)
Cts immune cell serum replacement, by Thermo Fisher Scientific (2 mentions)
Immunocult xf t cell expansion medium, by STEMCELL (2 mentions)
96 well shuttle system, by Lonza (2 mentions)
Dynabead, by Thermo Fisher Scientific (2 mentions)
Stemmacs hsc expansion media, by Miltenyi Biotec (1 mentions)
Easysep mouse hematopoietic progenitor cell isolation kit, by STEMCELL (1 mentions)
Stemspan sfem 2, by STEMCELL (1 mentions)
Complete rpmi media, by Thermo Fisher Scientific (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Sa3800 spectral cell analyzer, by Sony (1 mentions)
Anti cd3 cd28 dynabeads, by Thermo Fisher Scientific (1 mentions)
Macsquant device, by Miltenyi Biotec (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Rhil 7, by Miltenyi Biotec (1 mentions)
Cd4 and cd8 microbeads, by Miltenyi Biotec (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Anti biotin microbead, by Miltenyi Biotec (1 mentions)
Rhil 15, by Miltenyi Biotec (1 mentions)
8 protocols using robosep s
1
Lentiviral Transduction of Murine Hematopoietic Progenitors
2
Isolation and Characterization of IL-7 Responsive CD8+ T Cells
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PBMCs were isolated from whole blood using SepMate tubes (STEMCELL Technologies). T cells were isolated using the negative-selection, human T cell enrichment kit on the RoboSep-S automated cell separator (STEMCELL Technologies) and diluted in complete RPMI media (ThermoFisher) with 10% fetal bovine serum (FBS). A total of 50,000 cells were added per well in 96-well plates. EV samples or recombinant IL-7 were added to the wells in a final volume of 200 μL and incubated overnight at 37°C and 5% CO2. The next day, the cells were pelleted, washed, and stained to assess IL-7 receptor levels on CD8+ T cells by flow cytometry using a SA3800 Spectral Cell Analyzer (Sony). Data were normalized to compare data from multiple donors.
3
Engineered T Cell Generation for CB-011 Therapy
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CB-011 cells were generated from healthy donor–derived PBMCs. T cells were isolated from cryopreserved PBMCs (STEMCELL Technologies) using RoboSep-S (STEMCELL Technologies) and the EasySep Human T-cell Isolation Kit (STEMCELL Technologies) and activated for 3 days in the presence of anti–CD3/CD28 Dynabeads (Gibco) along with recombinant human interleukin-2 (rhIL-2; 100 U/mL). Beads were removed and cells were expanded for 24 hours with rhIL-2 before nucleofection and transduction. T cells were nucleofected using a 96-well Shuttle System (Lonza) with the prepared Cas12a chRDNA complexes and subsequently transduced with recombinant adeno-associated virus 6 (Signagen) engineered to deliver the CB-011 CAR transgene and the B2M–HLA-E fusion transgene. Cells were cultured for an additional 8 days in ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies) supplemented with 5% CTS immune cell serum replacement (Gibco) and rhIL-2.
4
Isolation and Purification of Blood Cell Subsets
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CB samples collected in heparin lithium vacutainers were processed to isolate peripheral blood mononuclear cells (PBMC) by gradient centrifugation with Histopaque 1077 (Sigma-Aldrich, MO, USA) and cell subsets were obtained by immuno-magnetic cell sorting (RoboSep™-S, STEMCELL™ Technologies, Canada). B cells, T cells, granulocytes and hematopoietic progenitor cells (HPC) were respectively sorted with EasySep® human whole blood CD19, CD3, CD66b positive selection kit and human cord blood CD34 positive selection kit following manufacturer’s recommendations.
Fractions were checked for purity by flow cytometry with the MACSQuant® device (Miltenyi Biotec, Germany) using CD20-VioBlue®; CD4-(VIT4)-FITC; CD8-PE and CD66abce-APC fluorescent antibodies, following manufacturer’s recommendations. Cell fractions with purity higher than 95% were kept for microchimerism analysis.
Fractions were checked for purity by flow cytometry with the MACSQuant® device (Miltenyi Biotec, Germany) using CD20-VioBlue®; CD4-(VIT4)-FITC; CD8-PE and CD66abce-APC fluorescent antibodies, following manufacturer’s recommendations. Cell fractions with purity higher than 95% were kept for microchimerism analysis.
5
Generating CAR T Cells from PBMCs
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For CAR T production, CD8+ and CD4+ T cells were isolated from PBMCs by either a custom sequential positive selection program using a RoboSep-S and EasySep CD8 and CD4 Positive Selection Kits (Stemcell Technologies) or manually by sequential positive selection using CD4 and CD8 MicroBeads (Miltenyi Biotec). The negative cell fraction was cryopreserved in FBS/10% DMSO for subsequent CD14+ isolation. T cells were stimulated with Dynabeads and cultured in RPMI-1640 (Gibco) with 10% FBS (VWR Seradigm), 2 mM l-glutamine (Gibco), 0.5 ng/mL rhIL-15 (Miltenyi Biotec) and either 50 U/mL rhIL-2 (CD8+ T cells; Chiron Corporation) or 5 ng/mL rhIL-7 (CD4+ T cells; Miltenyi Biotec). T cells were transduced with CD19 CAR-HER2tG lentivirus via spinoculation with protamine sulfate (APP Pharma), expanded, enriched for the HER2tG+ subset using biotinylated Trastuzumab antibody (online supplemental table 1 ) and anti-Biotin Microbeads (Miltenyi Biotec), and further expanded prior to cryopreservation or use in luciferase killing assays. Briefly, CAR T cells were added to eGFP:ffluc GEMs or eGFP:ffluc Raji cells and killing was measured by luminescence 10 min after luciferin addition, using lysis in 1% SDS as a positive control.
6
Magnetic-Bead Enrichment of CD19+ Cells
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CD19 magnetic‐bead‐selection and enrichment of cells of interest was performed with the RoboSep‐S instrument (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's protocol. Briefly, the sample was diluted 5‐fold using RoboSep dilution buffer (STEMCELL Technologies, Vancouver, Canada), mixed gently, and spun at 300 g for 10 minutes, followed by the aspiration of the supernatant. The sample was then diluted to the original volume with RoboSep buffer and mixed well. If required, the sample was filtered through a pre‐wetted 70 µm strainer (Fisher Scientific, Waltham, MA) to remove bone fragments and cell aggregates or debris. The sample was diluted 1:1 in EasySep RBC buffer (STEMCELL Technologies, Vancouver, Canada), transferred to a 14 ml centrifuge tube, and processed on the RoboSep‐S instrument using the RoboSep HLA Chim WB CD19 Positive Selection Kit (STEMCELL Technologies, Vancouver, Canada). In‐house magnetic‐bead selection achieved CD19+ cell purities within the stated range of the manufacturer (94.3%–99.6%).
7
Automated T-cell Isolation from PBMCs
8
Generation of CB-010 CAR-T Cells
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CB-010 CAR-T cells were generated from healthy donorÀderived peripheral blood mononuclear cells. To summarize, T cells were isolated from cryopreserved peripheral blood mononuclear cells (STEM-CELL Technologies, Vancouver, Canada) using RoboSep-S (STEMCELL Technologies) and the EasySep Human T cell Isolation Kit (STEMCELL Technologies) and activated for 3 days in the presence of anti-CD3/ CD28 beads (Dynabeads; Gibco, Billings, MT, USA) along with recombinant human interleukin 2 (rhIL-2; 100 units/mL). Beads were removed and cells were expanded for 24 h with rhIL-2 before nucleofection and transduction. Cas9 chRDNA complexes to edit the TRAC and PDCD1 loci were generated by combining purified Cas9 enzyme, 1£ CCE buffer (20 mmol/L HEPES:KOH, pH 7.4; 10 mmol/L MgCl 2 ; 150 mmol/L KCl; 5% glycerol) and annealed chRDNA-chacr complexes at a molar ratio of 1:3 Cas9:chRDNA. T cells were nucleofected using a 96-well Shuttle System (Lonza, Basel, Switzerland) with Cas9 chRDNA complexes and subsequently transduced with rAAV6 (Signagen, Frederick, MD, USA) engineered to deliver the non-targeting CAR transgene or the CB-010 CAR transgene. Cells were cultured for an additional 8 days in ImmunoCult-XF T Cell Expansion Medium (STEMCELL Technologies) supplemented with 5% CTS Immune Cell Serum Replacement (Gibco) and rhIL-2.
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