Cryopreserved PBMC was thawed at a 37 ℃-water bath and washed and resuspended in CAR-T culturing medium composed of 1% GlutaMAX (ThermoFisher Scientific, Waltham, MA), 1% HEPES (ThermoFisher Scientific, Waltham, MA), 0.2% N-Acetyl-l -cysteine (CHENG YI, Wenzhou, China) and 5% human plasma in X-VIVO15 (LONZA, Valais, Switzerland). Then, PBMC was rested for 20–24 h at 37 ℃ with 5% CO2 in a cell culture flask. Finally, PBMC has been washed and resuspended in an isolation buffer for CD3+ T cell selection. This study tested three isolation media: DPBS (Corning Incorporated, Corning, NY), X-VIVO15 (LONZA, Valais, Switzerland) basic medium, and CAR-T culturing medium. X-VIVO15 basic medium consists of X-VIVO15 (LONZA, Valais, Switzerland) with 0.2% NAC (CHENG YI, Wenzhou, China), 1% HEPES (ThermoFisher Scientific, Waltham, MA), and 1% GlutaMAX (ThermoFisher Scientific, Waltham, MA). Positive selection of CD3+ T cells was made by co-incubating CTS™ Dynabeads™ CD3/CD28 (ThermoFisher Scientific, Waltham, MA) with PBMC at a 1:1 ratio at room temperature followed by magnetic capture of bead-bound cells on DynaMag™-5 Magnet (ThermoFisher Scientific, Waltham, MA). Selected cells were resuspended in CAR-T culturing medium supplemented with 300 IU/mL IL-2 (SL Pharm, Beijing, China) and incubated at 37 ℃ with 5% CO2 for 44–52 h to complete T cell activation.
X vivo 15
Manufactured by Lonza
Sourced in United States, Switzerland, United Kingdom, Belgium, Germany, China, Denmark
X-VIVO 15 is a serum-free, protein-free, and chemically defined medium designed for the in vitro culture of a variety of cell types, including hematopoietic cells, epithelial cells, and fibroblasts. The medium is optimized to support cell growth and proliferation while maintaining cell function and viability.
Automatically generated - may contain errors
Lab products found in correlation
Glutamax, by Thermo Fisher Scientific (41 mentions)
Penicillin, by Thermo Fisher Scientific (19 mentions)
Streptomycin, by Thermo Fisher Scientific (18 mentions)
M csf, by Thermo Fisher Scientific (14 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (14 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (14 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (14 mentions)
Human ab serum, by Merck Group (13 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (13 mentions)
Rpmi 1640, by Thermo Fisher Scientific (12 mentions)
Human serum, by Merck Group (11 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (11 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (10 mentions)
Human ab serum, by Valley Biomedical (10 mentions)
N acetyl l cysteine, by Merck Group (9 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (8 mentions)
Retronectin, by Takara Bio (8 mentions)
Macsibeads from the t cell activation expansion kit, by Miltenyi Biotec (8 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (7 mentions)
360 protocols using x vivo 15
1
Optimizing CD3+ T Cell Isolation for CAR-T Therapy
2
Kasumi-3 Cell and CD34+ HPC Infection Protocol
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Kasumi-3 cells were infected as described before [10 (link),11 (link),25 (link)]. Briefly, cells were maintained in X-VIVO15 (Lonza) for 2 d and then infected at a multiplicity of 1.0 TCID50/cell by centrifugal enhancement (1000× g, 35 min, room temperature) at 5 × 105 cells/mL in X-VIVO15. Infected cells were allowed to recover overnight, and the next day, they were treated with trypsin to remove any virus that had not entered the cell. Cells were next cushioned onto Ficoll-Paque (GE Healthcare Life Sciences) to remove residual virus and debris, washed three times with PBS, and replated in X-VIVO15 at 5 × 105 cells/mL.
Isolation of CD34+ HPCs is described in detail elsewhere [24 (link)], and these cells were infected at a multiplicity of 2.0 TCID50/cell, as previously described [10 (link),11 (link),25 (link),28 (link)], in infection media consisting of IMDM supplemented with 10% BIT9500 serum substitute (Stem Cell Technologies), 2 mM L-glutamine, 20 ng/mL low-density lipoproteins, and 50 μM 2-mercaptoethanol. The following day, cultures were washed three times in PBS and replated in 0.4 μm-pore transwells (Corning) over irradiated murine stromal cells in hLTCM, as detailed above.
Isolation of CD34+ HPCs is described in detail elsewhere [24 (link)], and these cells were infected at a multiplicity of 2.0 TCID50/cell, as previously described [10 (link),11 (link),25 (link),28 (link)], in infection media consisting of IMDM supplemented with 10% BIT9500 serum substitute (Stem Cell Technologies), 2 mM L-glutamine, 20 ng/mL low-density lipoproteins, and 50 μM 2-mercaptoethanol. The following day, cultures were washed three times in PBS and replated in 0.4 μm-pore transwells (Corning) over irradiated murine stromal cells in hLTCM, as detailed above.
3
Comprehensive Cell Culture Media Formulations
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Leishmania culture medium: Equal parts SM and SDM-79 medium [30 (link),31 (link)] (pH 7.4) supplemented with 10% heat-inactivated FCS and 2 μg/mL hemin. mTeSR Plus medium: mTeSR Plus Basal Medium (pH 7.4) (STEMCELL Technologies) supplemented with mTeSR Plus 5x Supplement and 1% penicillin/streptomycin (Gibco). Complete RPMI medium: RPMI 1640 Glutamax (pH 7.4) (Gibco) supplemented with 10% heat-inactivated FCS (Gibco), 1% sodium pyruvate (Gibco), 1% penicillin/streptomycin, 25 mM HEPES (Gibco), 0.055 mM β-mercaptoethanol (Gibco). MDM medium: DMEM (pH 7.4) (Gibco) supplemented with 10% FBS, 1% Glutamax (Gibco), 0.055 mM β-mercaptoethanol, 1x MEM Non-Essential Amino Acids (Gibco) and 1% penicillin/streptomycin. X-Vivo 15 medium: X-Vivo 15 (pH 7.4) (Lonza) supplemented with 1% Glutamax, 0.055 mM β-mercaptoethanol and 1% penicillin/streptomycin.
4
Modulation of T Cell Proliferation
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PBTs were incubated with 1 µM Fludarabine (Tocris, Bristol), 1 µM Stattic (Cayman chemical, Ann Abor, Michigan), 10 µM STATV inhibitor (Cayman chemical, Ann Abor, Michigan) or 0.1% DMSO (Sigma Aldrich, St. Louis, Missouri) for 1 h at 37 °C, 5 % CO2 in serum-free medium (XVIVO-15, Lonza, Basel). These cells were washed once in serum-free medium (XVIVO-15, Lonza, Basel), before they were added to IFNγ-pretreated and SEB-loaded primary KCs and were cocultured at 37 °C, 5 % CO2. T cell proliferation was analyzed by destaining of CFDA using flow cytometry after 72 h.
5
Optimized PBMC Activation and Expansion
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PBMCs/T cells were cultured at 37°C with 5% CO2. Upon thawing, PBMCs were washed with PBS (300xg, 5min) and resuspended at a density of 2 × 106 cells/ml in X-VIVO™ 15 (Lonza) medium supplemented with 200U/ml rhIL-2 (Immunotools) and seeded into 24-well plates (1 ml/well). Adherent cells were allowed to attach for 4 h. After 4 h, non-adherent cells were collected, counted, adjusted to a cell density of 1 × 106 cells/ml with X-VIVO™ 15 (Lonza) medium supplemented with 200U/ml rhIL-2 (Immunotools) and re-seeded into 24-well plates (1ml/well). To each well, 5 µl of ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (StemCell Technologies) was added and the T cells activated for 72–96 h prior to gene editing.
6
Expanded Treg Cell Production Protocol
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Except where noted, protocol 3 followed the previously published protocol.38 Briefly, FACS-isolated cells were plated and activated with Dynabeads at a 4:1 bead-to-cell ratio. Cells were cultured either in X-VIVO 15 or in X-VIVO 15 customized by Lonza by substituting 100% of the glucose in the base medium with D-glucose (6,6-2H2, 99%) (Cambridge Isotope Laboratories, catalog no. DLM-349-MPT) supplemented with 10% human heat-inactivated pooled APB serum. On day 2, the culture volume was doubled, and IL-2 was added (300 IU/mL, Proleukin). Cells were resuspended, and fresh medium and IL-2 were added (600 IU/mL, Proleukin) on days 5, 7, 12, and 14, assuming consumption. On day 9, cells were restimulated with additional Dynabeads at a 1:1 ratio.
Additionally, the final release criteria for this protocol require that the final cell product be evaluated for purity (≤5% CD8+ cells, <100 beads/3 × 106 cells, and endotoxin ≤3.5 endotoxin units [EU]/mL), phenotype (≥95% CD4+ cells and ≥60% FOXP3+), sterility (negative for mycoplasma, anaerobic and aerobic bacteria, gram stain, fungal culture, potassium hydroxide [KOH] exam), and viability (≥85%).23
Additionally, the final release criteria for this protocol require that the final cell product be evaluated for purity (≤5% CD8+ cells, <100 beads/3 × 106 cells, and endotoxin ≤3.5 endotoxin units [EU]/mL), phenotype (≥95% CD4+ cells and ≥60% FOXP3+), sterility (negative for mycoplasma, anaerobic and aerobic bacteria, gram stain, fungal culture, potassium hydroxide [KOH] exam), and viability (≥85%).23
7
Flow Cytometric Analysis of TH17 Cells
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2.6 ml of venous blood were collected after informed consent during a routinely performed venipuncture or during routine placement of a peripheral venous catheter.
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient (PAA, Linz, Austria) and were stimulated in a 12-well plate in X-VIVO 15 (X-VIVO 15 w/o Gentamycin and Phenol Red, Lonza, Velvier, Belgium) with Ionomycin (Sigma Aldrich Chemie, Steinheim, Germany) for 4 h (37°C, 5% CO2). Brefeldin A (eBioscience, San Diego, CA, USA) was added after 3 h. After staining these cells with anti-CD3 (FITC) and anti-CD4 (PE) antibodies (R&D Systems, Minneapolis, MN, USA), the PBMCs were fixated, permeabilized, and stained intracellularly with anti-IL-17A antibody (APC) (Flow Cytometry Fixation and Permeabilization Buffer Kit I, R&D Systems, Minneapolis, MN, USA).
Flow cytometry was performed on a 4-color FacsCalibur flow cytometer (BD Biosciences) using summit 4.3 software (Beckman Coulter, Krefeld, Germany). Gates were preset and the measurements were performed blinded for sample identity. TH17 cells were defined as CD3+CD4+IL-17Aic+ (22 (link)) and were evaluated as percentage of TH lymphocytes (CD3+CD4+) (Figure1 ).
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradient (PAA, Linz, Austria) and were stimulated in a 12-well plate in X-VIVO 15 (X-VIVO 15 w/o Gentamycin and Phenol Red, Lonza, Velvier, Belgium) with Ionomycin (Sigma Aldrich Chemie, Steinheim, Germany) for 4 h (37°C, 5% CO2). Brefeldin A (eBioscience, San Diego, CA, USA) was added after 3 h. After staining these cells with anti-CD3 (FITC) and anti-CD4 (PE) antibodies (R&D Systems, Minneapolis, MN, USA), the PBMCs were fixated, permeabilized, and stained intracellularly with anti-IL-17A antibody (APC) (Flow Cytometry Fixation and Permeabilization Buffer Kit I, R&D Systems, Minneapolis, MN, USA).
Flow cytometry was performed on a 4-color FacsCalibur flow cytometer (BD Biosciences) using summit 4.3 software (Beckman Coulter, Krefeld, Germany). Gates were preset and the measurements were performed blinded for sample identity. TH17 cells were defined as CD3+CD4+IL-17Aic+ (22 (link)) and were evaluated as percentage of TH lymphocytes (CD3+CD4+) (Figure
8
T-cell Receptor Activation Assay
9
CFSE-based Cytotoxicity Assay for T Cell-Mediated Killing
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Carboxyfluorescein diacetate succinimidyl ester (CFSE)-based cytotoxicity analyses were performed as described.53 KA2/TRP2 or FEMX-I cells were washed with PBS, resuspended at 1 × 107 cells ml−1, labeled with 10 μm CFSE (Sigma-Aldrich, Schnelldorf, Germany) for 10 min at 37 °C, washed and resuspended in X-VIVO-15 (Lonza) at 5 × 106 cells ml−1. The T cells expanded with SmartDC or SmartDC-TRP2 cultures were harvested, washed and resuspended in X-VIVO-15 (Lonza) at 5 × 104 cells ml−1. T cells and KA2/TRP2/CFSE or FEMX-I/CFSE targets were mixed in various E:T ratios and incubated at 37 °C and 5% CO2 for 8 h. After co-culture, T cells were labeled with fluorochrome-conjugated antibodies (CD8-PE-Cy7 and CD3-APC; BD Biosciences). Fluorochrome-labeled microbeads were added to facilitate the quantification of the CD3−/CD8−/CFSE+ not lysed melanoma cells by flow cytometry. The percentage-specific lysis was calculated by the following formula: % specific lysis=100−(CD8−CFSE+ live target cells in the test sample/CD8−CFSE+ live target cells in the control sample with no T cells) × 100. The acquisition was carried out in FACS LSR II (BD Biosciences) and the results were analyzed using Flowjo software v. 7.6.4 (Treestar Inc.).
10
Inducing Non-Myeloid Differentiation in EBs
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In order to induce non-myeloid differentiation at day 4, the formed EBs were transferred to T75 flasks in factory medium (FM) X-VIVO 15™ (Lonza) supplemented with GlutaMax 1% (Gibco), b-mercaptoethanol (Gibco), M-CSF 100 ng/mL (PeproTech) and IL-3 25 ng/mL (PeproTech). After day 14, medium was refreshed every 4 days. At day 22 onwards, iMϕ precursors were isolated and plated on 6-well plates in order to differentiate into iMφs. The differentiation medium (DM) used for the terminal differentiation is X-VIVO 15™ (Lonza) supplemented with GlutaMax 1% (Gibco), b-mercaptoethanol (Gibco), and M-CSF 100 ng/mL (PeproTech) for 7 days30 (link).
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