Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood

Published: April 30, 2023

Abstract

Source: Chen S. et al. Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization. J. Vis. Exp. (2018)

In this video, we demonstrate the isolation of EpCAM-expressing epithelial cancer cells from peripheral blood samples. The technique involves the use of a medical-grade wire functionalized with EpCAM antibodies to capture the target cells. The captured cells are then subsequently extracted into a buffer solution.

Protocol

NOTE: This protocol describes the isolation of HT-29 cells (human colon cancer cell line) from PBS or from artificial mixtures of HT-29 cells and peripheral blood. The same experiment was performed with two additional cell lines (LNCaP and VCaP) and can theoretically be performed with all cells expressing EpCAM. 1. Preparation of Target Cells Cell culture and labeling of cells NOTE: In this …

Representative Results

Figure 1: Visualizing labeled cells. (A,E) Cells before charging: Cells are labeled with CFSE and counterstained with a DNA intercalating dye showing a range of CFSE (green) signal intensity. (B,F) Labeled cells captured on the wire. (C,G) Wire after cell-detachment …

Açıklamalar

The authors have nothing to disclose.

Materials

Gilupi Release Buffer Gilupi GIL083 Used to detach cells from the wire
McCoy's 5A Medium (1x) suppl. with L-Glutamine Thermo Fisher Scientific 16600-082 Culture medium used for HT-29 cells, adapt culture medium to cell line used for the experiments
HEPES Buffer Solution (1 M) PAA S11-001 Supplement for McCoy's 5A Medium
Foetal Bovine Serum Gold PAA A15-151 Supplement for McCoy's 5a Modified Medium
Penicillin-Streptomycin (100x) Biowest L0018-100 Supplement for McCoy's 5a Modified Medium
Phosphate Buffered Saline (1x PBS) Thermo Fisher Scientific 10010-015
Accutase Biowest L0950-100 Cell detachment solution (cell culture)
Water bath GFL Type 1003 Used for prewarming solutions
Incubator Thermo Fisher Scientific Used to incubate cells (cell culture)
50 mL reaction tubes VWR 525-0403
Roller mixer Stuart Scientific SRT6D Used to attach cells to the wire while keeping the cells from sedimenting
CellTrace CFSE Cell Proliferation Kit Thermo Fisher Scientific C34554 Used to label living cells (cytoplasmic label)
Hoechst 33342 H3570 Thermo Fisher Scientific Used to stain DNA (nucleic counterstain)
Centrifuge (equipped for holding 15 mL and 50 mL reaction tubes) Thermo Fisher Scientific Heraeus Megafuge 40R Used for pelleting cells
Observer Z1 (Fluorescence/laser microdissection microscope) Carl Zeiss Microimaging Inverted (immunofluorescence) microscope capable of laser microdissection; Fluorescence microscopes must be able to detect Hoechst 33342 (DAPI filter) and CFSE (FITC filter)
Bovine Serum Albumin (BSA) Sigma-Aldrich A4503-50G Used for coating glass/plastic surfaces
MS1 Minishaker Sigma-Aldrich Z404039 Used for vortexing
Vacutainer EDTA (or Na-heparin) tubes BD 366450 (or 366480) Used as reaction tube for ex vivo capture of target cells
Detektor CANCER03 DC03 Gilupi GIL003 Functionalized wire capable of isolating and detaching EpCAM-positive cells (also referred to as catch&release, C&R)
Syringe needles (G20) VWR 613-0554 Used to puncture rubber caps in order to allow the non-functional part of the C&R to lead through it
Neubauer chamber Roth T729.1 Used to count cells
Pasteur pipettes 150 mm Volac D810 Used for the low-volume application of cells to the C&R
Grease pen Dako S2002 Used on glass slides to hold cell suspension in place
Sterile syringe filter (0.2 µm) Corning 431219 For filtering freshly prepared Gilupi Release Buffer
Delfia PlateShaker Perkin Elmer 1296-003 Used for agitation during cell detachment
1.5 mL tubes Eppendorf 30,125,150
Ampli1 WGA Kit Silicon Biosystems To be ordered via Silicon Biosystems
Axiovert M200 equipped with Mikromanipulator MMJ and CellTram vario Zeiss/Eppendorf Use micromanipulation at hand
Microcapillaries (25 µm in diameter), CustomTip Type I Eppendorf 930001201 Used for micromanipulating cells
0.2 mL PCR tubes Biozym 710920
Cytocentrifuge and equippment Hettich Universal 32 Use cytocentrifuge at hand
Thermo cycler Bio-Rad DNA Engine Dyad Every thermo cycler with heated lid can be used
Microcentrifuge Roth CX73.1 Use desk-top centrifuge at hand
MembraneSlide 1.0 PET Zeiss 415101-4401-050 Membrane-coated glass slides used for laser microdissection

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Bu Makaleden Alıntı Yapın
Functionalized Wire-based Target Cell Isolation: An Ex Vivo Technique to Isolate Cancer Cells from Spiked Peripheral Blood. J. Vis. Exp. (Pending Publication), e20395, doi: (2023).

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