Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Podsumowanie

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

Streszczenie

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

Wprowadzenie

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital since CTCs are rare and heterogeneous. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful techniques for CTC isolation because they offer the necessary elements with high sensitivity¹. Rare number of tumor cells mixed with normal blood closely mimics real patient blood since 2-3 mL of real patient blood only contains 1-10 CTCs. To solve a critical experimental problem, instead of using a large number of tumor cells introduced in PBS or mixed with normal blood, th....

Protokół

Patient blood samples were supplied by Longhua Hospital Affiliated to Shanghai Medical University.The protocol follows the guidelines of Peking University Third Hospital's human research ethics committee. Informed consent was obtained from the patients for using the samples for research purposes.

1. Pre-experiment to check the capture efficiency with cultured tumor cells

1. Culture the tumor cells MCF-7, MDA-MB-231, and HeLa for determining the capture efficiency. Dilute the tumor cell suspension, count the number of tumor cells, and repeat until the desired number of cells in 1 mL of PBS is obtained.
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Wyniki

The whole setup includes a syringe pump, a syringe, and a microfluidic chip. The cell suspension in the syringe is connected to the syringe pump, and the cell suspension is introduced into the microfluidic chip to capture the cells. The capture efficiency for all the microfluidic chips utilized was around 90% or above. For the wave chip, we designed microstructures with varied gaps. The small gaps are used to capture the CTCs, and the big gaps are used to accelerate the flow rate. The cell suspension flows quickly in the.......

Dyskusje

The prognosis and early diagnosis of cancer have a significant effect on cancer treatment¹. CTC isolation with microfluidic chips offers a liquid biopsy with no invasion. However, CTCs are extremely rare and heterogeneous in the blood¹, which makes it challenging to isolate CTCs. CTCs have similar properties to the original tumor sources from which they originate. Thus, CTCs play a vital role in cancer metastasis¹.

The pr.......

Materiały

Name	Company	Catalog Number	Comments
Calcein AM	BIOTIUM	80011
calibrated microcapillary pipettes	Sigma- Aldrich	P0799
CD45-PE	BD Biosciences	560975
CK-FITC	BD Biosciences	347653	cytokeratin monoclonal antibody
DMEM	HyClone	SH30081.05
fetal bovine serum (FBS)	GIBCO,USA	26140
Hoechst 33342	Molecular Probes, Solarbio Corp., China	C0031
penicillin-streptomycin	Ying Reliable biotechnology, China
Red blood cells lysis (RBCL)	Solarbio, Beijing	R1010