clinimacs cd8 reagent  (Miltenyi Biotec)

Journal: Nucleic Acids Research

Article Title: Engineering of BZ transposase and transposon donor vector for enhanced efficiency and safety in gene delivery applications

doi: 10.1093/nar/gkaf935

Figure Lengend Snippet: CAR modification efficiency using the MDP and STDP. ( A ) CAR positive rates were measured by flow cytometry analysis of T cells after co-transfection with 5 μg STDP or MDP along with BZ327 mRNA (10 or 20 μg) ( n = 4 independent healthy donors). ( B ) Fold change in CAR + cell expansion was measured following the cell co-transfection as described in panel (A). ( C ) Cell viability was measured by flow cytometry analysis after cell co-transfection as depicted in panel (A). ( D ) CD3 + CD4 + and CD3 + CD8 + T cells were detected co-transfected as described in panel (A) via flow cytometry (top), and CD4+/CD8 + T cell ratios were calculated in all groups (bottom). ( E ) T cell phenotypes, including Tscm, Tcm, Tem, and Teff were detected using CD45RA and CCR7 antibodies via flow cytometry (top). Percents of T cell subpopulations were measured on the bottom. ( F , G ) The specific cytotoxicity (%) of CAR-T cells generated using MDP or STDP plasmids was assessed against H929 (left) and Raji (right) cells at different ratios (E:T ratio: effector to target ratio). Data are shown as the mean ± SEM. P values (* P < 0.05) were calculated by one-way ANOVA with Dunnett's multiple comparisons test (four independent replicates). ( H – K ) The inflammatory secretion levels of IL-2, TNF-α, IFN-γ, and IL-6 were detected in the supernatant of H929 cell assay (E:T = 1:2) via CBA.

Article Snippet: T cells were isolated from human PBMCs by using CliniMACS® CD8 Reagent (Miltenyi Biotec, Germany) and CliniMACS® CD4 Reagent (Miltenyi Biotec, Germany).

Techniques: Modification, Flow Cytometry, Cotransfection, Transfection, Generated

Journal: Biomolecules

Article Title: Introducing CAR-T Therapy in Kazakhstan: Establishing Academic-Scale Lentiviral Vector and CAR-T Cell Production

doi: 10.3390/biom15081166

Figure Lengend Snippet: Miltenyi Biotec technology for automated clinical-grade CAR-T cell manufacturing using the CliniMACS Prodigy platform. ( a ) CAR-T Cell manufacturing timeline. Key production steps: (i) T-cell isolation and activation (Day 0)—CD4 + /CD8 + T-cells are immunomagnetically selected from leukapheresis product and activated using GMP-compliant T-Cell TransAct reagent; (ii) lentiviral transduction (Day 1); and (iii) automated expansion culture (Days 3–12) with continuous monitoring. ( b ) Instrument control interface showing the activity matrix that directs the automated manufacturing process. The closed-system design ensures minimal manual handling while maintaining GMP compliance throughout cultivation, media exchange, and harvest procedures.

Article Snippet: In the next step, peripheral blood mononuclear cells (PBMCs) were incubated with magnetic beads conjugated to anti-CD4 and anti-CD8 antibodies (CliniMACS CD4 Reagent, Cat. 200-070-213; CD8 Reagent, Cat. 200-070-215; Miltenyi Biotec).

Techniques: Cell Isolation, Activation Assay, Transduction, Control, Activity Assay

Journal: Biomolecules

Article Title: Introducing CAR-T Therapy in Kazakhstan: Establishing Academic-Scale Lentiviral Vector and CAR-T Cell Production

doi: 10.3390/biom15081166

Figure Lengend Snippet: Analysis of CD4+ and CD8+ subsets among CAR+ T cells. ( a ) Distribution of CD4 + and CD8 + cells in CAR + T-cell populations. No significant differences between V1 and V2 groups were found; ( b ) CD4/CD8 ratio in CAR + T-cells (V1: n = 6; V2: n = 6) compared to untransduced (CAR − ) T-cells (n = 12); ( c ) subset analysis of CD4 + CAR + T-cells, proportions of naïve (Tn), central memory (Tcm), effector memory (Tem), and terminally differentiated effector (Temra) cells in different CAR-T products are shown as violin plots. There are no significant differences between V1 and V2 groups of CD4 + CAR + T-cells; ( d ) Pie charts depict the distribution among CD4 + CAR + T-cells with immunophenotypes of naïve (Tn), central memory (Tcm), effector memory (Tem), and terminally differentiated effector (Temra) cells; ( e ) Dot plot depicts cross-hair gating used to separate T-cell subsets (Temra, Tn, Tem, Tcm) in leukapheresis material from a healthy donor, based on CD45RA and CCR7 expression; ( f ) Representative data for a CAR-T cell product generated with the V1 vector. The cell population gated on CD4 + cells is displayed, showing the distribution of T-cell subsets (CD45RA vs. CCR7); ( g ) Representative data for a CAR-T cell product generated with the V2 vector. The cell population gated on CD8 + cells is displayed, showing the distribution of T-cell subsets (CD45RA vs. CCR7); ( h ) Analysis of CD8 + CAR + T-cells revealed vector-dependent subset distribution, with V1 favoring Tcm and V2 favoring Tem. Significant differences were observed between vectors for these subsets ( p < 0.01); ( i ) Pie charts depict the distribution among CD8 + CAR + T-cells of Tn, Tcm, Tem, Temra, stratified by vector. Panels ( a , b ) display medians with 95% confidence intervals; ( c , h ) show violin plots with medians and interquartile ranges; ( d , i ) show mean shares from total; The red color represents group V1, the blue color represents group V2, and the black line on panel ( b ) shows untransduced cells in the combined groups V1 and V2; ns = not significant, ** p ≤ 0.01.

Article Snippet: In the next step, peripheral blood mononuclear cells (PBMCs) were incubated with magnetic beads conjugated to anti-CD4 and anti-CD8 antibodies (CliniMACS CD4 Reagent, Cat. 200-070-213; CD8 Reagent, Cat. 200-070-215; Miltenyi Biotec).

Techniques: Expressing, Generated, Plasmid Preparation

Journal: Scientific Reports

Article Title: Cryopreserved leukapheresis enables scalable and distributed CAR-T manufacturing: a multi-platform comparative study

doi: 10.1038/s41598-025-14865-5

Figure Lengend Snippet: Comparison of quality between fresh leukapheresis, cryopreserved leukapheresis, and cryopreserved PBMCs. ( A ) Flow chart of direct cryopreservation of leukapheresis versus cryopreservation of leukapheresis after isolation of PBMCs. ( B ) WBC Classification by Sysmex XS-1000i. NEUT = Neutrophil; LYMPH = Lymphocyte; MONO = Monocyte, Cryo = Cryopreserved. ( C ) T-, B-, and NK- cell subsets in Lymphocytes were analyzed by flow cytometry. ( D ) A flow cytometer analyzed CD4 and CD8 subsets in T-cells. ( E ) Different phenotypes of differentiated subpopulations were analyzed by flow cytometry. Tn = Naïve T cell; TCM = Central Memory T cell; TEM = Effector Memory T cell; Teff = Effector T cell. ( F ) Cell viability in different stages of the process. Pre-separation, Before T cell separation; Post-separation, After T cell separation; Pre-transduction, Before CAR transduction. The p -values for panels B-F were derived from two-way ANOVA (Fresh-LEUK n = 5, Cryo-LEUK n = 8, Cryo-PBMC n = 3). ( G ) Cell recovery from cryopreserved leukapheresis and cryopreserved PBMCs. Cell recovery = concentration of thawed cells/ concentration of frozen cells ( n = 3). ( H ) Cell cryopreservation recovery from leukapheresis and PBMCs, Cell cryopreservation recovery = cell number of frozen cells/ cell number of leukapheresis. ( n = 5). ( I ) Cell sorting yield from cryopreserved leukapheresis and cryopreserved PBMCs. ( n = 5). Scatter dot plots with bars show the mean and SEM. The p -values for panels G-I were calculated using t-tests. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Specifically, 2 μL of CliniMACS CD4 (Miltenyi, 200-070-213, DE) and CD8 (Miltenyi, 200-070-215, DE) microbeads were added per 1 × 10 7 cells for positive selection, with purity validated by flow cytometry.

Techniques: Comparison, Isolation, Flow Cytometry, Transduction, Derivative Assay, Cell Recovery, Concentration Assay, FACS

Journal: Scientific Reports

Article Title: Cryopreserved leukapheresis enables scalable and distributed CAR-T manufacturing: a multi-platform comparative study

doi: 10.1038/s41598-025-14865-5

Figure Lengend Snippet: Generation of CAR-T cells by non-viral vector and lentiviral transduction. ( A ) Cell viability during the preparation of non-viral CAR-T cells from day 0 to day 9. ( n = 3). ( B ) Fold expansion during the generation of non-viral CAR-T cells from day 0 to day 9. ( n = 3). ( C ) Different phenotypes of differentiated subpopulations and CAR + within T cells from fresh and cryopreserved leukapheresis were analyzed by flow cytometry. ( n = 3). ( D ) Exhaustion markers PD-1, TIM-3, and LAG-3 in non-viral CAR-T cells were analyzed by Flow cytometry. ( n = 3). ( E ) Cytotoxicity of non-viral CAR T cells co-cultured with tumor cells in a 2:1 and 4:1 E: T ratio for 24 h. ( n = 3). ( F ) Cell viability in the process of LV CAR-T preparation through lentiviral technology. MOI = 1.5. ( n = 3). ( G ) Fold expansion during the generation of LV CAR-T cell cultures. ( n = 3). ( H ) Different phenotypes of differentiated subpopulations and CAR + within T cells from fresh and cryopreserved leukapheresis were analyzed by flow cytometry. ( n = 3). ( I ) Exhaustion markers PD-1, TIM-3, and LAG-3 in LV CAR-T cells were analyzed by Flow cytometry. ( n = 3). ( J ) Cell viability of the Fast CAR-T cell process. ( n = 2). ( K ) Recovery of Fast CAR-T cells from thawing. ( n = 2). ( L ) CD4 + and CD8 + T cell subsets of fast CAR-T cells 3 days post-thaw from fresh and cryopreserved leukapheresis. ( n = 2). ( M ) Different phenotypes of differentiated subpopulations and CAR + within T cells on 3 days post-thaw from fresh and cryopreserved leukapheresis. ( n = 2). ( N ) Cytotoxicity of fast CAR T cells co-cultured with tumor cells in a 1:2, 1:1, 2:1, and 4:1 E: T ratio for 24 h. ( n = 2). Scatter dot plots with bars show the mean and SEM. P -values were from t-tests.

Article Snippet: Specifically, 2 μL of CliniMACS CD4 (Miltenyi, 200-070-213, DE) and CD8 (Miltenyi, 200-070-215, DE) microbeads were added per 1 × 10 7 cells for positive selection, with purity validated by flow cytometry.

Techniques: Plasmid Preparation, Transduction, Flow Cytometry, Cell Culture

Journal: Scientific Reports

Article Title: Cryopreserved leukapheresis enables scalable and distributed CAR-T manufacturing: a multi-platform comparative study

doi: 10.1038/s41598-025-14865-5

Figure Lengend Snippet: Comparability study of immune cell culture in cryopreserved leukapheresis with cryopreserved PBMCs. ( A ) CD4 and CD8 subsets in Lymphocytes were analyzed by flow cytometry. ( n = 3). ( B ) Different phenotypes of differentiated subpopulations within T cells were analyzed by flow cytometry. ( n = 3). ( C ) Cell viability during the T cells from day 0 to day 9. ( n = 3). ( D ) Fold expansion of the T cells from day 0 to day 9. ( n = 3). ( E ) Cell viability during the NK and NKT cells from day 0 to day 15. ( n = 3). ( F ) Total fold expansion during the NK and NKT cells from day 0 to day 15. ( n = 3). ( G ) NK and NKT cell subsets in Lymphocytes were analyzed by flow cytometry on day 11 of the cell cultures. ( n = 3). ( H ) Specific fold expansion of NK cells and NKT cells at day 11 of cell cultures. ( n = 3). Scatter dot plots with bars show the mean and SEM. P -values were from t-tests.

Article Snippet: Specifically, 2 μL of CliniMACS CD4 (Miltenyi, 200-070-213, DE) and CD8 (Miltenyi, 200-070-215, DE) microbeads were added per 1 × 10 7 cells for positive selection, with purity validated by flow cytometry.

Techniques: Cell Culture, Flow Cytometry