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. 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.
	<ol>
		<li>Culture the cells in a cell culture flask with a starting cell number of ~ 1 x 105 cells in 1 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Incubate in a humidified atmosphere at 37 &#176;C with a 5% CO2 atmosphere.</li>
		<li>When the cell lines grow as adherent monolayers to 95% confluence, detach them from the culture dishes with 0.25% trypsin solution for 2 min as described in Chen et al.16.</li>
		<li>Stain the tumor cells with calcein AM. Put 3 &#181;L of calcein AM in the culture dish, and keep the dish in the incubator for 30 min. Then,digest all the cells with trypsin.</li>
		<li>Count the cultured tumor cells in PBS with a cell counting chamber, and dilute until 100 tumor cells per 1 mL of PBS are obtained. 
		NOTE: In order to precisely enumerate cell number, take 50 &#181;L of the obtained cell suspension to see whether the number of tumor cells in it is five or not with a microscope. Decide the volume of cell suspension that needs to be taken to obtain 100 tumor cells according to the actual number of tumor cells in 50 &#181;L of cell suspension.</li>
	</ol>
	</li>
	<li>Introduce the cell suspension containing 100 tumor cells per 1 mL of PBS into the microfluidic chip using a syringe with a syringe pump at varied flow rates of 0.5 mL/h, 1 mL/h, 2 mL/h, 3 mL/h, 4 mL/h, and 5 mL/h. Obtain the capture efficiency for the various flow rates, and determine the optimal flow rate.
	<ol>
		<li>Count the number of tumor cells captured on the chip and flowing out from the outlet. Calculate the capture efficiency as below: 
		&#8203;Capture efficiency = Number of cells captured/(Number of cells captured + number of cells flowing out) &#215; 100%</li>
		<li>Repeat to obtain the capture efficiency for different numbers of tumor cells from 10 to 100 (10, 20, 30, 40, 50, 60, 70, 80, 90, 100).</li>
	</ol>
	</li>
	<li>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 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 tumor cells diluted in 1 mL of PBS. Detect and enumerate the number of tumor cells for each sample after their capture on the chip. 
	NOTE: The hollow needle is around 3 cm in length with a 1 mm external diameter.</li>
	<li>Perform clinical pre-experiment testing for tumor cells spiked into normal blood samples.
	<ol>
		<li>Stain the tumor cells with calcein AM, and then enumerate 100 tumor cells in 5 &#181;L of PBS. Spike these cells into 1 mL of whole normal blood samples. Introduce these cells into the chip, and enumerate the number of tumor cells captured on the chip with green immunofluorescence. Perform in vivo enumeration on the chip as described above, and calculate the capture efficiency after capture.</li>
		<li>Repeat step 1.4.1 for nine additional concentrations of tumor cell numbers from 10 to 90 (10, 20, 30, 40, 50, 60, 70, 80, 90).</li>
		<li>Enumerate 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 tumor cells as described in step 1.3 without staining, spike into 1 mL of whole normal blood, introduce these samples into the microfluidic chip, and capture the tumor cells on the chip.</li>
		<li>Stain on the chip with Hoechst, CK-FITC, and CD45-PE. Put 3-5 &#181;L of fluorescent dye into 20-30 &#181;L of PBS, and then introduce this solution onto the microfluidic chip with a syringe. Enumerate the number of tumor cells captured on the chip with both blue and green immunofluorescence to determine the capture efficiency.</li>
	</ol>
	</li>
	<li>Perform modification of the microfluidic chip for the affinity-based capture of anti-EpCAM. 
	NOTE: Since the wave chip combines both affinity-based and physical property-based isolation, modify the chip with agents.
	<ol>
		<li>Modify the surface of the chip with 100 &#181;L of 4% (v/v) 3-mercaptopropyl trimethoxysilane in ethanol at room temperature for 45 min. Introduce this solution into the chip carefully and slowly in case it destroys the internal structure of the chip, especially the bonding of the top surface and substrate glass. 
		NOTE: The chip surface is modified using mercaptosilane. The interactions that occur on the chip are determined by chemical bonds. Chemical bonds are established to realize the bonding of the antibody modified with the antigen. Various reagents are modified inside the chip to establish chemical bonds that connect with each other. The last reagent to be modified is an anti-epithelial adhesion molecule (anti-EpCAM). The tumor cell surface antigen of EpCAM combines with anti-EpCAM inside the chip to realize the capture of the CTCs.</li>
		<li>Wash with ethanol 3x. Add 100 &#181;L of the coupling agent N-y-maleimidobutyryloxy succinimide ester (GMBS, 1 &#181;M) onto the chip, and allow it to interact for 30 min.</li>
		<li>Wash with PBS 3x. Use a syringe to introduce 1 mL of PBS onto the chip to wash.</li>
		<li>Treat the chip with 30-40 &#181;L of 10 &#181;g/mL neutravidin at room temperature for 30 min, leading to immobilization of the cells onto the GMBS, and then flush with PBS to remove excess avidin.</li>
		<li>Modify the chip with 3 &#181;L of anti-biotinylated EpCAM antibody at a concentration of 10 &#181;g/mL in 100 &#181;L of PBS with 1% (w/v) BSA. Keep this overnight.</li>
	</ol>
	</li>
</ol>
2. Clinical experiment on the chip to enumerate the circulating tumor cells (CTCs)
<ol>
	<li>Pre-process clinical cancer patient blood samples using red blood cells lysis (RBCL) solution, or introduce 2-3 mL of the blood sample directly onto the microfluidic chip using a syringe.
	<ol>
		<li>Perform pre-processing, which takes around 30 min. Collect whole blood samples in anticoagulation tubes. Add 6-9 mL of RBS lysis solution into 2-3 mL of blood. Centrifuge at 111 x g for 5-8 min at room temperature, and discard the upper layer of red clear liquid.</li>
	</ol>
	</li>
	<li>Capture, stain, recognize, and enumerate the cells on the chip as described below.
	<ol>
		<li>Capture the cells on the chip as described in step 1.Stain the chip for the CTCs captured using Hoechst, CK-FITC, and CD45-PE. CK-FITC is a specific stain for tumor cells, and CD45-PE is for white blood cells.</li>
		<li>Add 3 &#181;L of CK-FITC to 20 &#181;L of PBS. Introduce this into the syringe, and pump the diluted CK-FITC onto the chip. Allow to stain for 30 min. Introduce 300 &#181;L of PBS onto the chip to wash the chip.</li>
		<li>Add 3 &#181;L of CD45-PE to 20 &#181;L of PBS. Introduce this into the syringe, and pump the diluted CD45-PE onto the chip. Allow to stand for 30 min. Introduce 300 &#181;L of PBS onto the chip to wash the chip.</li>
		<li>Identify the CTCs with an inverted fluorescence microscope at 20x or 40x magnification. CTCs emit both blue and green fluorescence, and white blood cells (WBCs) emit both blue and red fluorescence. Identify the CTCs with both blue and green fluorescence and the WBCs with both blue and red fluorescence.</li>
		<li>From the immunofluorescence images, enumerate the number of CTCs captured on the chip. Enumerate the CTCs as Hoechst+/CK-FITC+/CD45&#8722; and the WBCs as Hoechst+/CK-FITC&#8722;/CD45+.</li>
	</ol>
	</li>
	<li>Enrich the captured CTCs by flushing with PBS through the syringe in the reverse direction onto the microfluidic chip to collect the CTCs captured on the chip from the inlet. Use a syringe with 1 mL of PBS, introduce it from the outlet to enrich the CTCs captured on the chip within 2-3 min, and collect them from the inlet. Use 1 mL of PBS for each washing step, and repeat 3x.</li>
	<li>Stain the tumor cells or CTCs captured on the microfluidic chip as described below.
	<ol>
		<li>Stain using calcein AM. Add 5 &#181;L of calcein AM to 20 &#181;L of PBS, stain the cells for 30 min, then centrifuge at 111 x g for 2 min at room temperature to obtain tumor cells, and suspend in 1 mL of PBS.</li>
		<li>To identify the cellular nuclei, add 20 &#181;L of DAPI solution (10 &#181;L of DAPI reagent in 20 &#181;L of PBS) to the chip at the optimal flow rate of 1 mL/h for big-ellipse microfilters and 1.5 mL/h for trapezoid ones. Pass through 20 &#181;L of anti-cytokeratin stock solution (3 &#181;L of anti-cytokeratin antibody in 20 &#181;L of PBS) to react with the chip for 30 min.</li>
		<li>Stain the WBCs captured on the chip. After the CTCs have been captured on the chip, add 25 &#181;L of anti-CD45 antibody solution (5 &#181;L of anti-CD45 solution in 20 &#181;L of PBS) to the chip, and allow to stain for 30 min. Wash with PBS, and identify the epithelial cells with both blue and green fluorescence.</li>
	</ol>
	</li>
	<li>Perform immunofluorescence identification with fluorescence microscopy as described below.
	<ol>
		<li>Use a fluorescence microscope and excite the sample with the blue laser. Find the cells emitting blue fluorescence, which are nucleate cells. Use one of the following magnifications: 10x, 20x, or 40x. Find a clear field for the tumor cell. For the blue laser source, use an excitation plate wavelength of 420-485 nm and an emission plate wavelength of 515 nm.</li>
		<li>Without moving the samples, use another laser source. Rotate the fluorescence microscope and excite the sample with the green laser. Find the cells emitting green fluorescence, which is indicative of tumor cells. Take images of the same field with the same magnification with this green laser source. The cells that emit both blue and green fluorescence are recognized as tumor cells. For the green laser source, use an excitation plate wavelength of 460-550 nm and an emission plate wavelength of 590 nm</li>
		<li>Without moving the samples, use another laser source. Rotate the fluorescence microscope and excite the sample with the red laser. Find the cells emitting red fluorescence. Take images of the same field with the same magnification with this red laser source. The cells that emit both blue and red fluorescence are recognized as white blood cells.</li>
		<li>Save the image for obtained by using each of the laser sources above. Take several images of the same field with different colored lights.</li>
		<li>Use ultraviolet light with an excitation plate wavelength of 330-400 nm and an emission plate wavelength of 425 nm. Use purple light with an excitation plate wavelength of 395-415 nm and an emission plate wavelength of 455 nm.</li>
	</ol>
	</li>
	<li>Calculate the capture efficiency as in step 1.2.1.</li>
</ol>

<ol><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly-sensitive+capture+of+circulating+tumor+cells+using+micro-ellipse+filters%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Highly-sensitive capture of circulating tumor cells using micro-ellipse filters.</a> Scientific Reports. 7 (1), 610(2017).</li>
<li>World Health Organization Cancer report. , Available from: 
 <a target="_blank" href="https://www.who.int/news-room/fact-sheets/detail/cancer">https://www.who.int/news-room/fact-sheets/detail/cancer</a> (2022).</li>
<li>Pantel, K., Brakenhoff, R. H., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection%2C+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Detection, clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature Reviews Cancer. 8 (5), 329-340 (2008).</li>
<li>Mehlen, P., Puisieux, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metastasis%3A+A+question+of+life+or+death%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Metastasis: A question of life or death.</a> Nature Reviews Cancer. 6 (6), 449-458 (2006).</li>
<li>Sollier, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size-selective+collection+of+circulating+tumor+cells+using+Vortex+technology%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">Size-selective collection of circulating tumor cells using Vortex technology.</a> Lab on a Chip. 14 (1), 63-77 (2014).</li>
<li>Stott, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+circulating+tumor+cells+from+patients+with+localized+and+metastatic+prostate+cancer%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Isolation and characterization of circulating tumor cells from patients with localized and metastatic prostate cancer.</a> Science Translational Medicine. 2 (25), 23(2010).</li>
<li>Stott, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+circulating+tumor+cells+using+a+microvortex-generating+herringbone-chip%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Isolation of circulating tumor cells using a microvortex-generating herringbone-chip.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (43), 18392-18397 (2010).</li>
<li>Nagrath, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+rare+circulating+tumor+cells+in+cancer+patients+by+microchip+technology%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Isolation of rare circulating tumor cells in cancer patients by microchip technology.</a> Nature. 450 (7173), 1235-1239 (2007).</li>
<li>Murlidhar, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+radial+flow+microfluidic+device+for+ultra-high-throughput+affinity-based+isolation+of+circulating+tumor+cells%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">A radial flow microfluidic device for ultra-high-throughput affinity-based isolation of circulating tumor cells.</a> Small. 10 (23), 4895-4904 (2014).</li>
<li>Tan, S. J., Yobas, L., Lee, G. Y. H., Ong, C. N., Lim, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microdevice+for+the+isolation+and+enumeration+of+cancer+cells+from+blood%5BTitle%5D)+AND+%22Biomedical+Microdevices%22%5BJournal%5D)">Microdevice for the isolation and enumeration of cancer cells from blood.</a> Biomedical Microdevices. 11 (4), 883-892 (2009).</li>
<li>Preira, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Passive+circulating+cell+sorting+by+deformability+using+a+microfluidic+gradual+filter%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">Passive circulating cell sorting by deformability using a microfluidic gradual filter.</a> Lab on a Chip. 13 (1), 161-170 (2013).</li>
<li>Yan, S., Zhang, J., Yuan, D., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hybrid+microfluidics+combined+with+active+and+passive+approaches+for+continuous+cell+separation%5BTitle%5D)+AND+%22Electrophoresis%22%5BJournal%5D)">Hybrid microfluidics combined with active and passive approaches for continuous cell separation.</a> Electrophoresis. 38 (2), 238-249 (2017).</li>
<li>Patil, P., Madhuprasad Kumeria, T., Losic, D., Kurkuri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+circulating+tumour+cells+by+physical+means+in+a+microfluidic+device%3A+A+review%5BTitle%5D)+AND+%22RSC+Advances%22%5BJournal%5D)">Isolation of circulating tumour cells by physical means in a microfluidic device: A review.</a> RSC Advances. 5 (109), 89745-89762 (2015).</li>
<li>Kumeria, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoswitchable+membranes+based+on+peptide-modified+nanoporous+anodic+alumina%3A+Toward+smart+membranes+for+on-demand+molecular+transport%5BTitle%5D)+AND+%22Advanced+Materials%22%5BJournal%5D)">Photoswitchable membranes based on peptide-modified nanoporous anodic alumina: Toward smart membranes for on-demand molecular transport.</a> Advanced Materials. 27 (19), 3019-3024 (2015).</li>
<li>Mahesh, P. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+microfluidic+platform+for+physical+and+immunological+detection+and+capture+of+circulating+tumor+cells%5BTitle%5D)+AND+%22Biosensors%22%5BJournal%5D)">Recent advances in microfluidic platform for physical and immunological detection and capture of circulating tumor cells.</a> Biosensors. 12 (4), 220(2022).</li>
<li>Chen, H., Cao, B., Chen, H., Lin, Y. -S., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combination+of+antibody-coated%2C+physical-based+microfluidic+chip+with+wave-shaped+arrays+for+isolating+circulating+tumor+cells%5BTitle%5D)+AND+%22Biomedical+Microdevices%22%5BJournal%5D)">Combination of antibody-coated, physical-based microfluidic chip with wave-shaped arrays for isolating circulating tumor cells.</a> Biomedical Microdevices. 19 (3), 66(2017).</li>
<li>Rushton, A. J., Nteliopoulos, G., Shaw, J. A., Coombes, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+circulating+tumour+cell+enrichment+technologies%5BTitle%5D)+AND+%22Cancers%22%5BJournal%5D)">A review of circulating tumour cell enrichment technologies.</a> Cancers. 13 (5), 970(2021).</li>
<li>Chen, H., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+inertia-deformability+hybrid+CTC+chip%3A+Design%2C+clinical+test+and+numerical+study%5BTitle%5D)+AND+%22Journal+of+Medical+Devices%22%5BJournal%5D)">An inertia-deformability hybrid CTC chip: Design, clinical test and numerical study.</a> Journal of Medical Devices. 12 (4), 041004(2018).</li>
</ol>

The authors have nothing to disclose.

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><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 ...

clinical-microfluidic-chip-platform-for-isolation-versatile

Research

JoVE Journal

Bioengineering

1.1K Views.  Anhui University of Technology. 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.

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

A Method of Targeted Cell Isolation via Glass Surface Functionalization

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual nuances in expression from patient to patient. Current methodologies that try to separate cells to fill this niche result in disruption of physiological expression, making the separation technique useless as a diagnostic tool. In this protocol, we describe the functionalization and optimization of a surface for the cellular capture and release. This functionalized surface integrates biotinylated antibodies with a glass surface functionalized with an aminosilane (APTES), desthiobiotin and streptavidin. Cell release is facilitated through the introduction of biotin, allowing the recollection and purification of cells captured by the surface. This release is done through the targeting of the secondary moiety desthiobiotin, which results in a much more gentle release paradigm. This reduction in harsh reagents and shear forces reduces changes in cellular expression. The functionalized surface captures up to 80% of cells in a single cell mixture and has demonstrated 50% capture in a dual-cell mixture. Applications of this technology to xenografts and cancer separation studies are investigated. Quantification techniques for surface verification such as plate reader and ImageJ analyses are described as well.

This protocol describes customizable surface functionalization of the desthiobiotin, streptavidin, and APTES system in order to isolate specific cell types of interest. In addition, this manuscript covers the applications, optimization, and verification of this process.

One of the limiting factors to the adoption and advancement of personalized medicine is the inability to develop diagnostic tools to probe individual ...

Cancer Research

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Medicine

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...