Repetitive Tumor Challenge Assay: An In Vitro Culture Assay to Evaluate Chimeric Antigen Receptor (CAR) T Cells for Repetitive Tumor Killing Potential

Abstract

Source: Wang, D. et al. In Vitro Tumor Cell Rechallenge for Predictive Evaluation of Chimeric Antigen Receptor T cell Antitumor Function. J. Vis. Exp. (2019)

In this video, we demonstrate the repetitive tumor challenge assay to evaluate the CAR T cells’ repetitive tumor killing potential during high tumor cell loads. Further, the CAR T cells can be analyzed using flow cytometry to determine the different subpopulations of CAR T cells in the sample.

Protocol

1. Media Preparation Prepare neural stem cell media for culturing primary GBM cell lines: DMEM:F12, 1:50 B27, 5 µg/mL heparin, and 2 mmol/L L-glutamine; supplemented with 20 ng/mL epidermal growth factor (EGF) and 20 ng/mL basic fibroblast growth factor (FGF) twice a week (see Table of Materials). Prepare T cell media: X-VIVO 15 containing 10% fetal calf serum (FCS); supplemented with 70 IU/mL rhIL-2 and 0.5 ng/mL rhIL-15 every 48 h (see Table…

Representative Results

Figure 1: Schema and analysis strategy of repetitive challenge assay. (A) Schema and timeline of repetitive tumor challenge assay. For each well, CAR T cells were first co-cultured with PBT030-2 GBM cells (4,000 CAR+ cells, 16,000 tumor cells) and re-challenged with 32,000 tumor cells every other day (D2, D4 and D6). Analysis of tumor cell and CAR T cell number, as well as C…

Materials

0.05% Trypsin/0.53mM EDTA in HBSS without Calcium and Magnesium	Corning	MT25051CI
1 M Hepes	Irvine Scientific	9319
200 mM L-Glutamine	Cambrex Bio Science	17-605E
4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) – FluoroPure grade	Invitrogen	D21490
Accutase	Innovative Cell Technologies	AT104
Aldesleukin Proleukin (rhIL-2)	Novartis Oncology	NDC 0078-0495-61
Anti-CD137, PE	BD Biosciences	555956	4B4-1
Anti-CD19, PE-Cy7	BD Biosciences	557835	SJ25C1
Anti-CD3, PERCP	BD Biosciences	340663	SK7
Anti-CD4, FITC	BD Biosciences	340133	SK3
Anti-CD45, PERCP	BD Biosciences	340665	2D1
Anti-CD45RO, PE	BD Biosciences	561137	UCHL1
Anti-CD62L, APC	BD Biosciences	559772	DREG-56
Anti-CD69, APC	BD Biosciences	340560	L78
Anti-CD8, APC-Cy7	BD Biosciences	348793	SK1
Anti-IL-13, PE	BD Biosciences	340508	JES10-5A2
Anti-LAG-3, PE	eBiosciences	12-2239-41	3DS223H
Anti-PD-1, APC-Cy7	BioLegend	329921	EH12.2H7
Anti-TIM-3, APC	eBiosciences	17-3109-42	F38-2E2
B-27 Serum-Free Supplement (50x)	Invitrogen	17504-044
Corning 96 Well Clear Flat Bottom Polystyrene TC-Treated Microplates, Individually Wrapped, with Lid, Sterile (Product #3596)	Corning Life Sciences	3596
Defined fetal bovine serum,HI IR	Hyclone Labs	SH30070.03IH
DMEM F-12 50:50 (1x) without Glutamine	MediaTech, Inc.	15-090-CV
DMEM High Gluc w/o L-Glu, Na Pyr 1 L	Invitrogen	11960051
DMEM-Ham's F12 50:50 Mixture with L-glutamine and 15 mM HEPES	Fisher Scientific	MT10092CV
HBSS	Irvine Scientific	9224
Heat-Inactivated FCS	Hyclone	SH30070.03
Heparin Sodium(1,000 U/mL)	American Pharmaceutical	401811B
PBS 1x W/CA & MG	Irvine Scientific	9236
PBS with 1 mM EDTA (No Ca2+ or Mg2+)	VWR Scientific Products	PB15009A
rhIL-15 Working Dilution (10 ng/µL)	CellGenix, US Operations	tF0297
Sodium Azide (NaN3)	Sigma	S8032
Recombinant human EGF	R&D Systems	236-EG-200
Recombinant human FGF	R&D Systems	233-FB