Isolation of donor T cells, in vitro activation, transduction, expansion, phenotyping, and functional analysis
CAR T cells have to be developed, optimized and validated in a pre-clinical setting by small-scale benchtop experiments. This application protocol describes a complete workflow for the engineering of CAR T cells for research. It includes isolation of donor T cells, followed by activation, gene-transfer of the CAR construct, CAR T cell expansion as well as the phenotyping and analysis of the final CAR T cell product.
Supplement TexMACS™ Medium with MACS® Cytokines, Human IL-7, premium grade (500 IU/mL) and Human IL-15, premium grade (84 IU/mL).
▲ Note: Make sure to freshly add Human IL-7, premium grade and Human IL-15, premium grade to the T cell medium for effective T cell expansion.
Prepare buffer by diluting the MACS® BSA Stock Solution 1:20 with autoMACS® Rinsing Solution or PBS/EDTA.
▲ Note: Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.
Prepare the 10 mM CFSE stock solution in 1 mL of DMSO. Use CFSE at a final concentration of 1 µL by diluting the stock solution 1:10,000 in PBS.
Freshly prepare the following master mixes for each sample.
▲ Note: Store master mix in the dark in the refrigerator (2–8 °C) until use. Do not store for extended periods.
Table 2: Master mix for immune cell composition antibody panel.
Table 3: Master mix for transduction efficiency antibody panel.
|CAR DR||PE||to be defined|
|Buffer||Fill up to 100 µL with buffer|
The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)). PBMCs should be isolated by density gradient centrifugation, e.g., using Ficoll-Paque™.
Resuspend cell pellet in an appropriate amount of PBS-EDTA and proceed to magnetic labeling.
▲ Note: PBMCs may be stored in the refrigerator overnight in PBS containing 0.5% BSA or autologous serum. Do not store cells longer than one day in the refrigerator. Wash at least once before proceeding magnetic labeling and resuspend cells in an appropriate buffer. For details see MACS® Cell Separation Reagents data sheets.
8. Place the reagent into the appropriate position on the reagent rack.
9. Go to the Separation menu and select the position for each sample. Select Cust_MB from the Labeling submenu (the correct labeling, separation, and wash protocols will be selected automatically).
10. Enter sample volume (min. 120 µL) into the Volume submenu. Press Enter.
11. Select Run.
12. The positive fraction containing CD4+ and CD8+ T cells is collected in row C.
13. Remove positive fraction and proceed with section "(Optional) Analysis of purity before and after T cell enrichment".
Table 4: Optimal surface density when working with purified T cells.
|Culture plate||Growth area||Max. working|
|Total T cell|
|T Cell TransAct|
to add per well
|96 well||0.31 cm2||0.2 mL||0.3×106||2 μL|
|48 well||1 cm2||1 mL||1×106||10 μL|
|24 well||2 cm2||2 mL||2×106||20 μL|
|12 well||4 cm2||4 mL||4×106||40 μL|
|6 well||10 cm2||5 mL||5×106||50 μL|
For CAR T cell phenotyping, e.g. to assess activation, differentiation, proliferation, and exhaustion of CAR T cells, please feel free to directly contact our experts at firstname.lastname@example.org. All CAR T cell phenotyping panels are compatible with MACSQuant® Express Modes.
(Optional) Target cell labeling for killing assay
MACSPlex Assays are designed for determining concentrations of soluble analytes in a single sample. The analysis is based on MACSPlex Capture Beads, which display defined fluorescence properties and can be identified using standard flow cytometry techniques (figure 4).
The MACSPlex Cytokine 12 Kit, human is used to detect GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and TNF-α.
Setup of the assay in a 96-well plate
Design your assay using two columns of the MACSPlex Filter Plate for the standards. Add each of the seven standard samples in duplicates next to each other. Standards should be run in order from the lowest concentration (blank control: 0 pg/mL) to the highest concentration (stock solution: 10,000 pg/mL or 50,000 pg/mL). Start with the unknown sample in the next column of the plate. For more details, refer to the respective data sheet, section ”Protocol for the assay using the MACSPlex Filter Plate”.
|B1/B2||MACSPlex Cytokine 12 Standard (3.2 pg/mL)||1:3125 (Dilution5; 1:55)|
|C1/C2||MACSPlex Cytokine 12 Standard (16 pg/mL)||1:625 (Dilution4; 1:54)|
|D1/D2||MACSPlex Cytokine 12 Standard (80 pg/mL)||1:125 (Dilution3; 1:53)|
|E1/E2||MACSPlex Cytokine 12 Standard (400 pg/mL)||1:25 (Dilution2; 1:52)|
|F1/F2||MACSPlex Cytokine 12 Standard (2 ng/mL)||1:5 (Dilution1; 1:51)|
|G1/G2||MACSPlex Cytokine 12 Standard (10 ng/mL)||Stock solution (Dilution0; 1:50)|
|A3–H12||Add unknown samples|
Flow cytometer setup
The kit includes MACSPlex Setup Beads for flow cytometer setup. MACSPlex Setup Beads are not required when using the MACSQuant Analyzer or MACSQuant Analyzer 10 but for all other flow instruments that can detect FITC, PE, and APC.
▲ Note: The kits are not suitable for use with the MACSQuant VYB.
Setup of the MACSQuant® Instrument
Calibrate the MACSQuant® Instrument using MACSQuant Calibration Beads (# 130-093-607). For details, refer to the data sheet of the MACSQuant Calibration Beads. After successfully completing the calibration, the MACSQuant Instrument is ready for measurement. No further steps are required as all necessary setup steps are performed automatically during calibration.
Setup of other flow cytometers
The analysis of MACSPlex Cytokine 12 Kit, human requires a flow cytometer with a blue (e.g. 488 nm) and a red (e.g. 635 nm) laser, which can detect FITC, PE, and APC. For setting up these cytometers use MACSPlex Setup Beads (included in the kit). For instructions on the setup procedures of other flow cytometers, please refer to the application note "General instructions for MACSPlex Cytokine Kits" available on the product page at www.miltenyibiotec.com/130-101-740 in the "Library" section.
Flow cytometric acquisition and data analysis using the MACSQuant® Express Mode
To perform the acquisition and data analysis of the MACSPlex Cytokine 12 Kit, human with the MACSQuant® Instrument it is recommended to use the Express Modes MACSPlex_Standard and MACSPlex_Sample to achieve automated measurement and data analysis. For details refer to the special protocol “Data acquisition and analysis of MACSPlex Cytokine Kits using the MACSQuant® Analyzer Express Modes” available at www.miltenyibiotec.com/130-099-169 in the "Library" section. The minimum version number of the Express Mode package needed to run the assay on the MACSQuant Instrument can be found there as well. To check the version number of your Express Mode package available on your MACSQuant Instrument please select Help> Info> expressModes within the MACSQuantify™ Software. The version number of the Express Mode package is increasing with each Express Mode updates. Make sure the MACSQuant Instrument contains an Express Mode package with at least the same or higher version number than the special protocol is marked with.
For results refer to figure 9 in section “Results”.
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In the EU, the CliniMACS System components are available as CE-marked medical devices for their respective intended use, unless otherwise stated. The CliniMACS Reagents and Biotin Conjugates are intended for in vitro use only and are not designated for therapeutic use or direct infusion into patients. The CliniMACS Reagents in combination with the CliniMACS System are intended to separate human cells. Miltenyi Biotec as the manufacturer of the CliniMACS System does not give any recommendations regarding the use of separated cells for therapeutic purposes and does not make any claims regarding a clinical benefit. For the manufacturing and use of target cells in humans the national legislation and regulations – e.g. for the EU the Directive 2004/23/EC (“human tissues and cells”), or the Directive 2002/98/EC (“human blood and blood components”) – must be followed. Thus, any clinical application of the target cells is exclusively within the responsibility of the user of a CliniMACS System.
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Table 1: Antibodies used for analysis of immune cell composition and transduction efficiency.
▲ Note: Antibodies can be replaced/included according to your respective needs. For additional antibodies refer to www.miltenyibiotec.com/antibodies.
▲ Note: For additional CAR T cell phenotyping panels, e.g. to assess activation, differentiation, proliferation, and exhaustion of T cells, consult our experts at email@example.com. All panels are compatible with MACSQuant® Express Modes.