Name: clinical microfluidic chip platform for the isolation of versatile circulating tumor cells
Uploaded: 2023-10-13T13:40:00
Description: Watch this Scientific Journal Video about Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells at JoVE.com

This research work was supported by the Anhui Natural Science Foundation of China (1908085MF197, 1908085QB66), the National Natural Science Foundation of China (21904003), the Scientific Research Project of Tianjin Education Commission (2018KJ154), the Provincial Natural Science Research Program of Higher Education Institutions of Anhui Province (KJ2020A0239), and the Shanghai Key Laboratory of Multidimensional Information Processing, East China Key Laboratory of Multidimensional Information Processing, East China Normal University (MIP20221).

Patient blood samples were supplied by Longhua Hospital Affiliated to Shanghai Medical University.The protocol follows the guidelines of Peking University Third Hospital&#39;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
<ol>
	<li>Culture the tumor cells MCF-7, MDA-MB-231, and HeLa for determining the capture efficiency....

<ol>
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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...

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, the use of rare number of tumor cells provides us with a low number of blood cells, which is closer to reality when performing an experiment.
Cancer is the leading cause of death in the world2. CTCs are tumor cells shed from the original tumor that circulate in the blood and lymphatic circulation systems3. When CTCs move to a new survivable environment, they grow as a second tumor. This is called metastasis and is responsible for 90% of deaths in cancer patients4. CTCs are vital for prognosis, early diagnosis, and for understanding the mechanisms of cancer. However, CTCs are extremely rare and heterogeneous in patient blood5,6.
Microfluidic chips offer a liquid biopsy that does not invade the tumor. They have the advantage of being portable, low cost, and having a cell-matched scale. The isolation of CTCs with microfluidic chips is classified mainly into two types: affinity-based, which relies on antigen-antibody binding7,8,9 and is the original and most widely used method of CTC isolation; and physical-based chips, which utilize size and deformability differences between tumor cells and blood cells10,11,12,13,14,15, are label-free, and are easy to operate. The advantage of microfluidic chips over alternative techniques is that the physical-based approach of big-ellipse microfilters firmly captures CTCs with high capture efficiency. The reason for this is that ellipse microposts are organized into slim tunnels of line-line gaps. The line-line gaps are different from the traditional point-point gaps formed by microposts such as rhombus microposts. Wave chip-based capturing of CTCs combines both physical property-based and affinity-based isolation. Wave chip-based capture involves 30 wave-shaped arrays with the antibody of anti-EpCAM coated on circular microposts. The CTCs are captured by the small gaps, and the big gaps are used to accelerate the flow rate. The missed CTCs have to pass the small gaps in the next array and are captured by the affinity-based isolation integrated inside the chip16.
The goal of the protocol is to demonstrate the counting of rare numbers of tumor cells and the clinical analysis of CTCs with microfluidic chips. The protocol describes the CTC isolation steps, how to obtain a low number of tumor cells, the clinical physical separation of small-ellipse filters, big-ellipse filters, and trapezoid filters, affinity modification, and enrichment17.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcein AM</td>
			<td>BIOTIUM</td>
			<td>80011</td>
			<td></td>
		</tr>
		<tr>
			<td>calibrated microcapillary pipettes</td>
			<td>Sigma- Aldrich</td>
			<td>P0799</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45-PE</td>
			<td>BD Biosciences</td>
			<td>560975</td>
			<td></td>
		</tr>
		<tr>
			<td>CK-FITC</td>
			<td>BD Biosciences</td>
			<td>347653</td>
			<td>cytokeratin monoclonal antibody</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30081.05</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>GIBCO,USA</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Molecular Probes, Solarbio Corp., China</td>
			<td>C0031</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Ying Reliable biotechnology, China</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cells lysis (RBCL)</td>
			<td>Solarbio, Beijing</td>
			<td>R1010</td>
			<td></td>
		</tr>
	</tbody>
</table>

clinical microfluidic chip platform for the isolation of versatile circulating tumor cells

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.

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...

Watch this Scientific Journal Video about Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells at JoVE.com

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

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.

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...

This protocol described a method for red tumor cell enumeration, and the clinical CTC isolation. To begin, culture the tumor cells MCF-7, MDA-MB-231, and HeLa in a cell-culture flask at one times 10 to the fifth in one-millimeter of DMEM, supplemented with 10%fetal bovine serum, and 1%penicillin-streptomycin. Incubate the cells at 37 degrees Celsius with 5%carbon dioxide.When the cell lines grow as adherent monolayers to 95%confluence, detach them from the culture dish with 0.25%trypsin solution for two minutes. Stain the tumor cells by adding three-microliters of Calcein AM, and place the dish in the incubator for 30 minutes. At the end of incubation, digest all the cells with trypsin.Count the culture tumor cells in PBS with a cell-counting chamber, and dilute until 100 tumor cells per one-milliliter of PBS are obtained. Next, introduce the diluted cell suspension into the microfluidic chip using a syringe with a syringe pump at varied flow rates. Obtain the capture efficiency for the various flow rates, and determine the optimal flow rate.Count the number of tumor cells captured on the chip, and flowing out from the outlet. Calculate the capture efficiency using the given formula. Repeat the procedure to obtain the capture efficiency for different numbers of tumor cells, from 10 to 100.Test and validate the microfluidic chip for rare numbers of tumor cells. Inject these 10 sample suspensions into the microfluidic chips using a hollow needle made with a micropipette puller to aspirate diluted tumor cells. Detect and enumerate the number of tumor cells for each sample after their capture on the chip.Next, stain the tumor cells with Calcein AM, and enumerate 100 tumor cells in five-microliters of PBS. Spike these cells into one-milliliter of whole, normal blood sample. Introduce these cells into the chip, and enumerate the number of tumor cells captured on the chip with green immunofluorescence.Perform in vivo enumeration as described, and calculate the capture efficiency after capture. Enumerate one to 10 tumor cells without staining. Spike these cells into one-milliliter of whole, normal blood.Introduce these samples into the microfluidic chip, and capture the tumor cells. Add three to five-microliters of fluorescent dye into 20 to 30-microliters of PBS, and introduce this solution onto the chip with a syringe. Enumerate the number of tumor cells captured on the chip with blue and green immunofluorescence to determine the capture efficiency.Next, modify the surface of the chip with 100-microliters of 4%3-mercaptopropyl trimethoxysilane and ethanol at room temperature for 45 minutes for the affinity-based capture of anti-epithelial cell adhesion molecule. At the end of incubation, wash the chip three times with ethanol. Then add 100-microliters of the coupling agent GMBS, and allow it to interact for 30 minutes.At the end of incubation, use a syringe to wash the chip three times with one-milliliter of PBS. Treat the chip with 30 to 40-microliters of 10-micrograms per milliliter neutravidin at room temperature for 30 minutes, leading to immobilization of the cells onto the GMBS, and then flush with PBS to remove excess avidin. Modify the chip with three-microliters of anti-biotinylated epithelial cell adhesion molecule antibody, and incubate overnight.All the tumor cells were captured around the array of the wave chip without any cells missing, indicating the high-capture efficiency of the microfluidic chip. Tumor cells stained with Hoechst and Calcein AM are shown here. CTCs from a gastric cancer patient captured using small ellipse micro-filters are shown here.Also, CTCs from a colorectal cancer patient captured using trapezoid micro-filters, and emitting both blue and green fluorescence, are shown. High-capture efficiency and viability were observed for tumor cells captured using big ellipse micro-filters. Clinical immunofluorescence analysis of colorectal CTCs captured on the microfluidic chip recognized CTCs as DAPI-positive, CK-positive, CD45-negative, and the WBCs as DAPI-positive, CK-negative, CD45-positive.Colorectal tumor cells cultured on the big ellipse chip after capture, and colorectal tumor cells captured on the big ellipse micro-filters are shown here. This method of modifying with aptamer provides a new approach to enrich CTC isolation.

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