Microfluidic, marker-free isolation of circulating tumor cells from blood samples

Abstract

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d.

Circulating tumor cell technologies

“…The antibodies used for selection are typically tethered to either the device surface or a magnetic substance (i.e., (Miltenyi et al, 1990;Pluim et al, 2012) MagSweeper EpCAM High purity, can process WB, 9 mL/h throughput Kim et al, 2014;Lohr et al, 2014;Powell et al, 2012;Talasaz et al, 2009 (Harb et al, 2013). mF, IM CTC-iChip EpCAM, Size Pos/neg enrichment, size-based separation debulks WB, inertial focusing aids in magnetic deflection, 8 mL/h (Karabacak et al, 2014;Ozkumur et al, 2013) IM, in vivo GILUPI CellCollectorä EpCAM Can process large volumes of blood (Saucedo-Zeni et al, 2012) Immunoaffinity e Negative Enrichment Antibodies targeting leukocyte-associated antigens are tethered to magnetic particles or the device surface deplete unwanted background cells.…”

Microfluidic device with integrated microfilter of conical-shaped holes for high efficiency and high purity capture of circulating tumor cells

“…Indeed, it is known that tumor cells of epithelial origin may undergo epithelial to mesenchymal transition 21,22 . In contrast, the non-affinity methods, including centrifuging deflection [23][24][25][26][27] , dielectrophoretic separation [28][29][30] and size-based filtration 12,[31][32][33][34][35][36][37][38][39][40][41][42][43][44] , are able to isolate both epithelial and mesenchymal phenotypes, which are more appropriate for analyses of tumor heterogeneity, tumor drug resistance, etc. However, these approaches are technologically biased and it is often difficult to reach a trade-off between capture efficiency, purity and cell viability which are all important criteria for both fundamental and clinical studies.…”

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

“…This field has been propelled by remarkable technological advances for efficiently isolating single cells 28, 29, and the reduction of the detection levels of protocols for profiling gene expression 30, 31, histone marks 25, 26, or chromatin accessibility 32, 33 in individual cells. These techniques allow researchers to overcome the limitations of population studies, in which averaging the measurements over a heterogeneous population of cells masks the variability at the epigenetics and transcriptional levels among cells within a culture or tissue 26, 34, 35.…”