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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly-sensitive+capture+of+circulating+tumor+cells+using+micro-ellipse+filters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis:+A+question+of+life+or+death.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-selective+collection+of+circulating+tumor+cells+using+Vortex+technology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+circulating+tumor+cells+from+patients+with+localized+and+metastatic+prostate+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+circulating+tumor+cells+using+a+microvortex-generating+herringbone-chip.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+rare+circulating+tumor+cells+in+cancer+patients+by+microchip+technology.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+radial+flow+microfluidic+device+for+ultra-high-throughput+affinity-based+isolation+of+circulating+tumor+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdevice+for+the+isolation+and+enumeration+of+cancer+cells+from+blood.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+circulating+cell+sorting+by+deformability+using+a+microfluidic+gradual+filter.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+microfluidics+combined+with+active+and+passive+approaches+for+continuous+cell+separation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+circulating+tumour+cells+by+physical+means+in+a+microfluidic+device:+A+review.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoswitchable+membranes+based+on+peptide-modified+nanoporous+anodic+alumina:+Toward+smart+membranes+for+on-demand+molecular+transport.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+microfluidic+platform+for+physical+and+immunological+detection+and+capture+of+circulating+tumor+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+antibody-coated,+physical-based+microfluidic+chip+with+wave-shaped+arrays+for+isolating+circulating+tumor+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+circulating+tumour+cell+enrichment+technologies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inertia-deformability+hybrid+CTC+chip:+Design,+clinical+test+and+numerical+study.">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 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 ...

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

Research

JoVE Journal

Bioengineering

1.0K 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

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

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 Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

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 ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.