Robust Isolation and Multiplexed Identification of Circulating Rare Cells via Continuous Centrifugal Microfluidics and Hydrogel Immunocytochemistry

Summary

This study presents a continuous centrifugal microfluidic workflow for efficient isolation and identification of circulating rare cells. The system enables precise liquid control and minimizes cell loss, allowing simultaneous analysis of multiple CRC subtypes, including CTCs and cCAFs, for improved diagnostic accuracy.

Abstract

Circulating rare cells (CRCs), including circulating tumor cells (CTCs) and circulating cancer-associated fibroblasts (cCAFs), are promising biomarkers for cancer diagnosis and prognosis. However, their extremely low abundance and phenotypic heterogeneity make efficient isolation and multiparametric analysis technically challenging. Here, we present a continuous centrifugal microfluidic workflow for the sequential isolation and identification of CRCs from whole blood. The cartridge comprises five interconnected chambers with controllable valves that enable precise handling of density gradient medium and blood layers during centrifugation. Based on a previously validated enrichment platform, the present workflow integrates hydrogel-based cell immobilization with multiplexed immunocytochemistry to support the downstream identification and characterization of multiple CRC subtypes, including CTCs and cCAFs. Following isolation, cells are immobilized within a hydrogel matrix to support cell retention during repeated immunostaining and imaging-based analysis. Using this workflow, both CTCs and cCAFs were identified from the same sample, demonstrating the feasibility of integrated analysis of multiple circulating rare-cell populations within a single liquid-biopsy workflow. This platform provides a practical and reproducible approach for downstream rare-cell characterization in liquid biopsy applications.

Introduction

Circulating rare cells (CRCs) are defined as cells that exist in the bloodstream at extremely low frequencies compared to normal blood components such as red blood cells, white blood cells, and platelets. CRCs originate from diverse sources, such as byproducts of biological processes, tissue remodeling, wound healing, or vascular injury. Representative examples include circulating endothelial cells, circulating fetal cells, megakaryocytes, erythroblasts, and various progenitor or stem cells^1,2. While the existence of CRCs alone does not necessarily reflect a pathological state, elevated levels frequently ....

Protocol

All procedures involving human blood samples from patients and healthy donors were conducted in accordance with the guidelines of the Institutional Review Board (IRB) of Severance Hospital. The study protocol was approved by the Institutional Review Board of Severance Hospital (IRB No. 4-2013-0059). The reagents and the equipment used are listed in the Table of Materials.

1. Device preparation

1. Press the power switch to turn on the instrument.
2. Engage the laser interlock switch, then turn on the laser power switch.
3. Launch the o....

Results

Figure 1A shows an image of a single cartridge used for rare cell isolation. The cartridge consists of five individual chambers connected by microchannels and controllable valves that regulate liquid flow. The structure of the cartridge is as follows: (1) a plasma chamber that collects plasma separated from whole blood; (2) an initial chamber in which whole blood is separated into plasma, red blood cell, and PBMC layers; (3) a mixing chamber where isolated PBMCs are combined with leukocyte i.......

Discussion

CRCs are important biomarkers with potential value for both cancer diagnosis and prognosis. To improve the clinical utility of CRC analysis, however, it is important to capture a broader range of phenotypically diverse CRC populations and to support parallel multiparametric characterization¹³. In addition, growing evidence in onco-immunology indicates that immune components and the tumor microenvironment play critical roles in shaping therapeutic response, highlighting the need to move beyond tumo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15 mL Conical Tube	SPL	50015
16% Formaldehyde (w/v), Methanol-free	Thermo Scientific	28908
Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L)	Jackson immunoresearch	711-545-152
Alexa Fluor 594 AffiniPure Donkey Anti-Goat IgG (H+L)	Jackson immunoresearch	705-585-003
Bovine Serum Albumin	GenDepot	A0100-010
CD45 antibody	Biolegend	304060
CellTracker Green CMFDA	Invitrogen	C7025
CTCD	CTCELLS	CTD-C103	Disc cartridge
CTCD rack	CTCELLS	CTDR-CM3	Disc rack
CTCeptor	CTCELLS	CTM-C2203	CTCeptor
CTR-S Reagent A	CTCELLS	CTR-S1001	density gradient media
CTR-S Reagent B	CTCELLS	CTR-S1002	leukocyte isolation beads
FAP antibody	Invitrogen	PA5-99313
Hoechst 33342	Thermo Scientific	62249
HypICC Photoinitiator	CTCELLS	CTR-G1002
HypICC Solution A	CTCELLS	CTR-G1001
HypICC Unit	CTCELLS	CTH-V01	HypICC chamber
Latex glove	Ansell	93743080
Magnetic stand			Fabricated in-house using a 3D printer
Manual Pipetting  (0.5-10 µL)	Eppendorf	3120000020
Manual Pipetting  (100-1000 µL)	Eppendorf	3120000062
Manual Pipetting  (10-100 µL)	Eppendorf	3120000046
Manual Pipetting  (20-200 µL)	Eppendorf	3120000054
Manual Pipetting  (2-20 µL)	Eppendorf	3120000038
pancytokeratin antibody	Invitrogen	53-9003-82
PBS (Phosphate Buffered Saline Solution)	Welegene	LB001-02
SMA antibody	Invitrogen	PA5-18292
Tip, 10 µL	TipOne	9104020333
Tip, 1250 µL	TipOne	9104020740
Tip, 200 µL	TipOne	9104020422
Triton X-100	Signma	X100-100ML
Whole Blood			Stored at 4-8 °C by collecting in EDTA
β actin antibody	abcam	ab8226