Name: a method of targeted cell isolation via glass surface functionalization
Uploaded: 2016-09-20T08:00:00
Description: Watch this Scientific Journal Video about A Method of Targeted Cell Isolation via Glass Surface Functionalization at JoVE.com

We would like to thank the American Cancer Society, Illinois Division (282802) and the National Science Foundation CBET (1512598) for funding support. We also would like to thank Dr. Dianwen Zhang from the University of Illinois Beckman Institute for microscopy training. Finally, we would like to thank Jared Weddell, Stacie Chen, and Spencer Mamer for insightful discussions.

1. Cleaning the Glass Surface and Preparing Reagents
<ol>
	<li>Place a glass surface in an oxygen plasma machine for 5 min at 50% power to clean it.</li>
	<li>Prepare 2.5 ml 2% reconstituted (3-aminopropyl) triethoxysilane (APTES) solution, by adding 50 &#181;l of APTES and 2.45 ml of ethanol in a conical tube.</li>
</ol>
2. APTES and DSB Functionalization
<ol>
	<li>Add APTES solution to the surfaces. Pipette 150 &#181;l per well for 8 well plates. Pipette 100 &#181;l per well for 24 well plat...

<ol>
	<li>Erdbruegger, U., Haubitz, M., Woywodt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+endothelial+cells:+a+novel+marker+of+endothelial+damage.">Circulating endothelial cells: a novel marker of endothelial damage.</a> Clin. Chim. Acta. 373 (1-2), 17-26 (2006).</li><li>De Spiegelaere, W., Cornillie, P., Van Poucke, M., Peelman, L., Burvenich, C., Van den Broeck, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mRNA+expression+analysis+in+kidney+glomeruli+using+microdissection+techniques.">Quantitative mRNA expression analysis in kidney glomeruli using microdissection techniques.</a> Histol. Histopathol. 26 (2), 267-275 (2011).</li><li>Chen, S., Guo, X., Imarenezor, O., Imoukhuede, P. 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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=Detachment+of+captured+cancer+cells+under+flow+acceleration+in+a+bio-functionalized+microchannel.">Detachment of captured cancer cells under flow acceleration in a bio-functionalized microchannel.</a> Lab Chip. 9 (12), 1721-1731 (2009).</li><li>Privorotskaya, N., 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=Rapid+thermal+lysis+of+cells+using+silicon-diamond+microcantilever+heaters.">Rapid thermal lysis of cells using silicon-diamond microcantilever heaters.</a> Lab Chip. 10 (9), 1135-1141 (2010).</li><li>Park, K., Akin, D., Bashir, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+capture+and+lysis+of+vaccinia+virus+particles+using+silicon+nano-scale+probe+array.">Electrical capture and lysis of vaccinia virus particles using silicon nano-scale probe array.</a> Biomed. Microdevices. 9 (6), 877-883 (2007).</li><li>Galletti, G., Sung, M., Vahdat, L. <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+breast+cancer+and+gastric+cancer+circulating+tumor+cells+by+use+of+an+anti+HER2-based+microfluidic+device.">Isolation of breast cancer and gastric cancer circulating tumor cells by use of an anti HER2-based microfluidic device.</a> Lab Chip. 14 (1), 147-156 (2014).</li><li>Schudel, B. R., Choi, C. J., Cunningham, B. T., Kenis, P. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chip+for+combinatorial+mixing+and+screening+of+assays.">Microfluidic chip for combinatorial mixing and screening of assays.</a> Lab Chip. 9 (12), 1676-1680 (2009).</li><li>Lien, K. Y., Chuang, Y. 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=Rapid+isolation+and+detection+of+cancer+cells+by+utilizing+integrated+microfluidic+systems.">Rapid isolation and detection of cancer cells by utilizing integrated microfluidic systems.</a> Lab Chip. 10 (21), 2875-2886 (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.">Isolation of circulating tumor cells using a.</a> PNAS. 107 (35), 18392-18397 (2010).</li><li>Yu, M., Ting, D., Stott, S., Wittner, B., Ozsolak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing+of+pancreatic+circulating+tumour+cells+implicates+WNT+signalling+in+metastasis.">RNA sequencing of pancreatic circulating tumour cells implicates WNT signalling in metastasis.</a> Nature. 487 (7408), 510-513 (2012).</li><li>Sheng, W., Ogunwobi, O., Chen, T., 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=>Capture,+release+and+culture+of+circulating+tumor+cells+from+pancreatic+cancer+patients+using+an+enhanced+mixing+chip.">>Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip.</a> Lab Chip. 14 (1), 89-98 (2014).</li><li>Zheng, X., Cheung, L. S. L., Schroeder, J. A., Jiang, L., Zohar, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-performance+microsystem+for+isolating+circulating+tumor+cells.">A high-performance microsystem for isolating circulating tumor cells.</a> Lab Chip. 11 (19), 3269-3276 (2011).</li><li>Imoukhuede, P. I., Popel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+cell-to-cell+variation+of+vascular+endothelial+growth+factor+receptors.">Quantification and cell-to-cell variation of vascular endothelial growth factor receptors.</a> Exp. Cell Res. 317 (7), 955-965 (2011).</li><li>Imoukhuede, P. I., Popel, A. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell-by-cell+profiling+reveals+temporal+dynamics+of+VEGFR1+and+VEGFR2+membrane-localization+following+murine+hindlimb+ischemia.">Endothelial cell-by-cell profiling reveals temporal dynamics of VEGFR1 and VEGFR2 membrane-localization following murine hindlimb ischemia.</a> Am J Physiol Hear. Circ Physiol. 4 (8), H1085-H1093 (2013).</li><li>van Beijnum, J. R., Rousch, M., Castermans, K., van der Linden, E., Griffioen, A. W. <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+endothelial+cells+from+fresh+tissues.">Isolation of endothelial cells from fresh tissues.</a> Nat. Protoc. 3 (6), 1085-1091 (2008).</li><li>Imoukhuede, P. I., Popel, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+fluorescent+profiling+of+VEGFRs+reveals+tumor+cell+and+endothelial+cell+heterogeneity+in+breast+cancer+xenografts.">Quantitative fluorescent profiling of VEGFRs reveals tumor cell and endothelial cell heterogeneity in breast cancer xenografts.</a> Cancer Med. 3 (2), 225-244 (2014).</li><li>BD Biosciences. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CD Marker Handbook: Human and Mouse. , (2010).</li><li>Tanzeglock, T., Soos, M., Stephanopoulos, G., Morbidelli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+mammalian+cell+death+by+simple+shear+and+extensional+flows.">Induction of mammalian cell death by simple shear and extensional flows.</a> Biotechnol. Bioeng. 104 (2), 360-370 (2009).</li><li>Perritt, D., Wong, P., Macpherson, J. L., Henrichsen, K., Symonds, G., Pond, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Processing Blood. , (2014).</li><li>Fukuda, S., Schmid-Sch&#246;nbein, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrifugation+attenuates+the+fluid+shear+response+of+circulating+leukocytes.">Centrifugation attenuates the fluid shear response of circulating leukocytes.</a> J. Leukoc. Biol. 72 (July), 133-139 (2002).</li><li>dela Paz, N. G., Walshe, T. E., Leach, L. L., Saint-Geniez, M., D'Amore, P. 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J., 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=Tumor+Cells+Circulate+in+the+Peripheral+Blood+of+All+Major+Carcinomas+but+not+in+Healthy+Subjects+or+Patients+With+Nonmalignant+Diseases+Tumor+Cells+Circulate+in+the+Peripheral+Blood+of+All+Major+Carcinomas+but+not+in+Healthy+Subjects+or+Patients+With+Nonmalignant+diseases.&amp;#34.">Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant diseases.&amp;#34.</a> Clinical Cancer Research. 10, 6897-6904 (2005).</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+tumour+cells+in+cancer+patients+by+microchip+technology.">Isolation of rare circulating tumour cells in cancer patients by microchip technology.</a> Nature. 450 (7173), 1235-1239 (2007).</li><li>Chen, S., Weddel, J., Gupta, P., Conard, G., Parkin, J., Imoukhuede, P. 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Improvements in cell isolation techniques furthers scientific studies in structure-function relationships in neuroscience18, stem cell programming in regenerative biology, and angiogenic signaling in vascular biology19. Indeed, primary cell culture20 (e.g., HUVECs) in vascular biology is primarily done through the use of cell isolation techniques. Cell isolation was also recently used for quantitative flow (qFlow) cytometry analysis of plasma membrane receptors3,14,15,19,21<...

Current bench-top cell separation approaches (e.g., fluorescence activated cell sorting1, laser capture micro-dissection2, immuno-magnetic bead separation1) can take several hours of preparation and sorting. These large time scales can affect physiological response and expression levels, resulting in analyses that are not representative of the physiological response3. Systems are needed that can rapidly and efficiently isolate specific cell types without disrupting cell-surface receptor-levels in order to improve cell isolation and enrichment for biomedical applications. Therefore, the rationale for our approach is to develop a gentle approach for cell isolation.
The &#34;lab on a chip&#34; concept offers the promise of orders of magnitude quicker (hours-to-minutes) cell isolation, and most frequently involves capturing cells onto a surface and releasing cells or intracellular contents through physical4,5 or chemical methods6. Although these approaches offer a few advantages such as identifying protein7,8 expression, identifying RNA expression9-11, or even providing cells for in vitro culture12,13, many of these techniques cannot be translated to diagnostics such as cell receptor profiling due to their non-physiological environments. Enzymatic lifting agents such as collagenases can also affect these receptor quantities14,15, meaning cell receptor quantification techniques that use these lifting agents will not generate accurate physiological data. Cellular lysis prevents differentiation between the native surface receptors, and those which were previously internalized16. This protocol describes a fast and gentle approach for cell isolation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>(3-Aminopropyl) triethoxysilane (APTES)</td>
			<td>Acros Organics</td>
			<td>919-30-2</td>
			<td>Used to make 2% APTES solution</td>
		</tr>
		<tr>
			<td>Plasma Cleaner Pico</td>
			<td>Diener</td>
			<td>Model 1</td>
			<td>Cleans surfaces and allows for bonding of PDMS to glass</td>
		</tr>
		<tr>
			<td>d-Desthiobiotin (DSB)</td>
			<td>Sigma</td>
			<td>D20655</td>
			<td>Used as the releasing mechanism in the cellular capture surface.</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td>British Drug Houses (BDH)</td>
			<td>BDH1115-1LP</td>
			<td>Dissolves the DSB into solution</td>
		</tr>
		<tr>
			<td>1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)</td>
			<td>Thermo-Scientific</td>
			<td>5g: 22980 
			25g: 22981</td>
			<td>Activates Carboxylic Acids and allows binding of proteins to glass surface.</td>
		</tr>
		<tr>
			<td>uncoated 8-well culture slide</td>
			<td>BD Falcon</td>
			<td>Case of 24: 354118 
			Case of 96: 354108</td>
			<td>Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments</td>
		</tr>
		<tr>
			<td>Glass bottom 24-well plates</td>
			<td>MatTek</td>
			<td>P24G-0-13-F</td>
			<td>Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments</td>
		</tr>
		<tr>
			<td>Mercaptoethanol</td>
			<td>Science Lab</td>
			<td>60-24-2</td>
			<td>Used to quench reaction between EDC and DSB</td>
		</tr>
		<tr>
			<td>4-Morpholinoethanesulfonic acid hydrate 
			(MES Hydrate 99%)</td>
			<td>Fisher Scientific</td>
			<td>AC172590250</td>
			<td>Used to make 0.1 M MES Buffer for use in EDC reaction</td>
		</tr>
		<tr>
			<td>Precision Oven</td>
			<td>Thermo Scientific</td>
			<td>11-475-153</td>
			<td>Used in curing of PDMS and APTES layer.</td>
		</tr>
		<tr>
			<td>Titramax 1000 Shaker</td>
			<td>Heidolph</td>
			<td>13-889-420</td>
			<td>Used to ensure even distribution of APTES on surfaces.</td>
		</tr>
		<tr>
			<td>1X Streptavidin 5mg 
			[e7105-5mg]</td>
			<td>Proteo Chem</td>
			<td>9013-20-1</td>
			<td>Biotin-binding protein 
			May cause irritation</td>
		</tr>
		<tr>
			<td>5 cm Glass Dish</td>
			<td>Fisher Scientific</td>
			<td>08748A</td>
			<td>Used in HUVEC studies as well as future profiling studies.</td>
		</tr>
		<tr>
			<td>14 cm Petri Dish with Cover</td>
			<td>Sigma-Aldrich</td>
			<td>Z717231</td>
			<td>Used to hold samples being functionalized and transport them.</td>
		</tr>
		<tr>
			<td>MCF7-GFP cells</td>
			<td>Cell Biolabs</td>
			<td>AKR211</td>
			<td>Stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>RAW264.7 
			mouse macrophages</td>
			<td>ATCC</td>
			<td>TIB-71</td>
			<td>Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen</td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Life Technologies</td>
			<td>12605036</td>
			<td>Stored in 100mL at room temperature</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium</td>
			<td>Cell Media Facility at School of Chemical Sciences at UIUC</td>
			<td>50003PC</td>
			<td>Supplier: Corning</td>
		</tr>
		<tr>
			<td>Nonessential amino acids</td>
			<td>Cell Media Facility at School of Chemical Sciences at UIUC</td>
			<td>25-025-CI</td>
			<td>Already added into DMEM by facility. 
			Supplier: Corning</td>
		</tr>
		<tr>
			<td>10% fetal bovine serum</td>
			<td>Fisher Scientific</td>
			<td>03-600-511</td>
			<td>Stored in 500mL at &#60; -10&#8304;C</td>
		</tr>
		<tr>
			<td>1% Penicillin&#8211;Streptomycin</td>
			<td>Life Sciences Storeroom at UIUC</td>
			<td>17602e</td>
			<td>Supplier: VWR 
			Stored in 100 ml at 4&#8304;C</td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>12-565-58</td>
			<td>Small 23cm 50 pack</td>
		</tr>
		<tr>
			<td>Cell Dissociation Solution</td>
			<td>Corning</td>
			<td>MT-25-056CI</td>
			<td>Used to lift cells non-enzymatically for the use in cell experiments</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td>Used to count cells for quantification of cell solutions and capture and release effectivity.</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Amresco</td>
			<td>58-85-5</td>
			<td>Used to release cells from surface.</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Created from Recipe</td>
			<td>N/A</td>
			<td>Used to keep cells alive in suspension as well as wash surfaces of non-specific binding. (Adapted from Cold Spring Harbor Protocols): In 500 mL, use 4 g NaCl, .2 g KCl, .0402 g Na2PO4*7H2O, .03 g KH2PO4 and .5 g Glucose. Add DI water to get to 500 mL, filter, and then refrigerate.</td>
		</tr>
		<tr>
			<td>HLA-ABC Antibody</td>
			<td>BioLegend</td>
			<td>311402</td>
			<td>Antibody used to capture MCF7gfp cells</td>
		</tr>
		<tr>
			<td>hIgG Antibody</td>
			<td>BioLegend</td>
			<td>HP6017</td>
			<td>Antibody used to capture MCF7gfp cells</td>
		</tr>
		<tr>
			<td>MCF7 GFP cells</td>
			<td>Cell Biolabs</td>
			<td>AKR-211</td>
			<td>Luminal Breast Cancer line that has been transfected with green fluorescent protein.</td>
		</tr>
		<tr>
			<td>Assorted Conicals</td>
			<td>Thermo-Scientific</td>
			<td>15mL: 12-565-268</td>
			<td>50/15 mL plastic conicals for storing solutions and aliquots.</td>
		</tr>
		<tr>
			<td>Mini-Tube Rotators (End over End Mixer)</td>
			<td>Fisher Scientific</td>
			<td>05-450-127</td>
			<td>Used to incubate antibody and mix other cellular solutions in order to mix</td>
		</tr>
		<tr>
			<td>Axiovert 200M (Fluorescence Microscope)</td>
			<td>Zeiss</td>
			<td>N/A</td>
			<td>Zeiss Axiovert 200 M inverted florescence microscope.</td>
		</tr>
		<tr>
			<td>Zeba Desalting columns</td>
			<td>Thermo-Scientific</td>
			<td>PI-87770</td>
			<td>Used to purify newly biotinylated antibodies after the use of the Biotinylation Kit. Instructions provided at: http://www.funakoshi.co.jp/data/datasheet/PCC/89894.pdf</td>
		</tr>
		<tr>
			<td>EZ Link Sulfo NHS Low Weight Biotinylation Kit</td>
			<td>Thermo- Scientific</td>
			<td></td>
			<td>Used to biotinylate antibodies to allow them to integrate with the capture surface</td>
		</tr>
		<tr>
			<td>Plate Reader</td>
			<td>BioTek</td>
			<td>Synergy HTX Multimode Reader</td>
			<td>Used to quantitatively measure fluorescent intensity in the titration experiments.</td>
		</tr>
	</tbody>
</table>

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.

Using this protocol we show cell capture (Figure 3A) and cell release (Figure 3C) of MCF7GFP cells as well as live cell controls (Figure 4). We quantified the cell capture as 60% and 80% were released (Figure 3C). When we extended this approach to a mixture of RAW 264.7 macrophages and MCF7GFP cells, 50% of RAW macrophages were captured (Fig. 3D) and 80% of RAW macrophages were release with 20 mM biotin (...

Watch this Scientific Journal Video about A Method of Targeted Cell Isolation via Glass Surface Functionalization at JoVE.com

A Method of Targeted Cell Isolation via Glass Surface Functionalization

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 ...

The overall goal of this glass surface functionalization process is to allow for the gentle capture and release of cells of interest. This method can help answer key questions in the field of tumor angiogenesis, enabling screening of cell biomarkers that indicate drug resistance. The main advantage of this technique is that it is completely customizable, for virtually any cell type of interest.As long as there is an antibody specific to the cell type, the surface can be adapted for its capture. Though this method can provide insight into cancer angiogenesis, it may also be applied to other diseases as purified samples are necessary for the development of more personalized treatment regimens. We first had the idea for this method when we were trying to develop a reversible release mechanism for capturing cells, using the interaction between desthiobiotin and the adivin family.After cleaning a glass surface in an oxygen plasma machine for five minutes according to the text protocol, prepare 2.5 milliliters of 2%reconstituted 3-Aminopropyl triethoxysilane, or APTES solution, by combining 50 microliters of APTES and 2.45 milliliters of ethanol in a conical tube. Pipette the APTES solution onto the glass surfaces, then cover the surfaces and place them on a platform shaker at room temperature for 50 minutes to evenly distribute the APTES layer. In the meantime, preheat the oven to 55 degrees Celsius for the eight-and 24-well plates, or 90 degrees Celsius for the glass dishes.After removing the plates from the shaker, use ethanol to rinse the glass surfaces according to the text protocol. With 100%nitrogen gas, dry the surfaces. Then place the eight-and 24-well plates in the oven for two hours, or the glass dishes in the oven for one hour.Next, prepare 2.5 milliliters of d-Desthiobiotin, or DSB solution, by mixing 1.5 milligrams per milliliter of DSB in 37.5 microliters of DMSO, and adding five milligrams per milliliter of EDC to 2, 462.5 microliters of 0.1 Molar MES buffer, pH 6. Then combine the two solutions. After 15 minutes, add one microliter of BME to quench the reaction between DSB and EDC.Remove the hot APTES functionalized glass surfaces from the oven, and allow them to cool for five to 10 minutes. Add MES buffer to the glass surfaces to rinse them by holding the pipette at a 70 degree angle so that the tip is not directly pointed at the surface. Then discharge and draw the buffer from a fixed point, such as the corner of the well.Then, use the MES buffer to rinse two more times. Apply DSB solution to the glass surfaces. Transfer them to a damp paper towel inside a Petri dish, cover the dish, and incubate in the fridge for 18 to 24 hours.After the incubation at four degrees Celsius, use PBS to rinse each glass surface three times, as demonstrated earlier in this video. Then, dilute Streptavidin, or SAV stock solution, to 0.4 milligrams per milliliter, and evenly apply it to the glass surfaces so that a thin layer forms. After incubating the glass for 18 to 24 hours, use 150 microliters of PBS to rinse each surface three times.Then wet a paper towel with deionized water, and place it flat in a 14 centimeter Petri dish surrounding the plates to retain moisture in the wells. Cover the Petri dish with the glass surfaces, and place it in a four degrees Celsius biosafety Level 1 fridge until needed. To carry out cell capture, aspirate the medium from T175 flasks of cells.Then use PBS to rinse the cells and aspirate the buffer. Add 10 milliliters of non-enzymatic lifting agent, such as Cell Dissociation Solution, to the flask. Then incubate at 37 degrees Celsius for six minutes.After six minutes, add 10 milliliters of cold HBSS to dilute the lifting agent. Then pipette 20 microliters of the cells and use a hemocytometer to count them. Centrifuge the cell suspension at 500 x g, in four degrees Celsius, for five minutes.And use HBSS to resuspend the cells to one times 10 to the sixth cells per milliliter. Pipette up and down to resuspend the cells in solution, and reduce cellular clumping that may reduce antibody binding. Then, split the cellular suspension into separate control and experimental solutions.Add the biotinylated antibodies to the respective cells solutions and incubate on an end-over-end mixer at four degrees Celsius for 30 minutes. Use 150 microliters of HBSS to wash the functionalized glass in eight-well plates three times, as demonstrated earlier in the video. Add the cell solution to the wells, and incubate on ice on the shaker for 45 minutes.In the meantime, prepare a 20 millimolar solution of biotin in sterile HBSS. Then, after the incubation, gently use 150 microliters of HBSS to rinse the cell solution from the glass. After repeating the rinse two more times, add 150 microliters of the biotin solution to each respective release well, and incubate for 20 minutes to allow the reaction to proceed.Recollect nonspecifically bound cells by using HBSS to wash the cells. Then, proceed to fluorescence imaging and analysis according to the text protocol. Include images of live cells in a flask as a control.In this experiment, MCF7GFP cells were exposed to the functionalized surface. 60%of the cells were captured using the HLA-ABC antibody. Upon exposure to 20 millimolar biotin, 80%of the captured cells were released.As shown here when the MCF7GFP cells were mixed with RAW 264.7 cells, 50%of the RAW macrophages were captured, and the addition of 20 micromolar biotin subsequently released 80%of the captured RAW cells. This figure illustrates that the positive control RAW macrophages exhibit no fluorescence activity. However, the negative control MCF7GFP cells are positive for GFP fluorescence.Because excess antibody can decrease cell capture, the antibody concentration was optimized by titrating from zero to 10, 000 nanograms per milliliter of HLA antibody. The results showed that the ideal antibody concentration was between 100 and 1, 000 nanograms per milliliter. In addition, cell optimization experiments determined that the ideal concentration of cells that can be captured is between one times 10 to the fifth, and one times 10 to the sixth cells, since quantities below that number are lower than the background.Once mastered, this technique can be done in three days, with less than six hours of experimental time, not including the overnight incubations. While attempting this procedure, it's important to remember to collect the cell washes during the experiments, as they can give vital information about the effectiveness of the surface. Following this procedure, other methods like flow cytometry can be performed in order to answer additional questions, like what receptor expression levels the cells of interest may have.After its development, surface functionalization has paved the way for researchers in the field of biomaterials to explore biosafe integration of materials, such as, implants in patients for a variety of diseases and injuries. After watching this video, you should have a good understanding of how to surface functionalize glass surfaces to capture and release cells of interest, allowing for downstream analysis. Don't forget that working with live cells in sodium azide can be extremely hazardous, and precautions such as proper training, PPE, and disposal, should always be taken while performing this procedure.

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Research

JoVE Journal

Cancer Research

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer. 
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions. 
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2. 
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.

Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.

Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. ...

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

Surface-enhanced Resonance Raman Scattering Nanoprobe Ratiometry for Detecting Microscopic Ovarian Cancer via Folate Receptor Targeting

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic spread has already occurred. However, ovarian cancer has a unique pattern of metastatic spread, in that tumor implants are initially contained within the peritoneal cavity. This feature could enable, in principle, the complete resection of tumor implants with curative intent. Many of these metastatic lesions are microscopic, making them hard to identify and treat. Neutralizing such micrometastases is believed to be a major goal towards eliminating tumor recurrence and achieving long-term survival. Raman imaging with surface enhanced resonance Raman scattering nanoprobes can be used to delineate microscopic tumors with high sensitivity, due to their bright and bioorthogonal spectral signatures. Here, we describe the synthesis of two &#39;flavors&#39; of such nanoprobes: an antibody-functionalized one that targets the folate receptor &#8212; overexpressed in many ovarian cancers &#8212; and a non-targeted control nanoprobe, with&#160;distinct spectra. The nanoprobes are co-administered intraperitoneally to mouse models of metastatic human ovarian adenocarcinoma. All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center. The peritoneal cavity of the animals is surgically exposed, washed, and scanned with a Raman microphotospectrometer. Subsequently, the Raman signatures of the two nanoprobes are decoupled using a Classical Least Squares fitting algorithm, and their respective scores divided to provide a ratiometric signal of folate-targeted over untargeted probes. In this way, microscopic metastases are visualized with high specificity. The main benefit of this approach is that the local application into the peritoneal cavity &#8212; which can be done conveniently during the surgical procedure &#8212; can tag tumors without subjecting the patient to systemic nanoparticle exposure. False positive signals stemming from non-specific binding of the nanoprobes onto visceral surfaces can be eliminated by following a ratiometric approach where targeted and non-targeted nanoprobes with distinct Raman signatures are applied as a mixture. The procedure is currently still limited by the lack of a commercial wide-field Raman imaging camera system, which once available will allow for the application of this technique in the operating theater.

Ovarian cancer forms metastases throughout the peritoneal cavity. Here, we present a protocol to make and use folate-receptor targeted surface-enhanced resonance Raman scattering nanoprobes that reveal these lesions with high specificity via ratiometric imaging. The nanoprobes are administered intraperitoneally to living mice, and the derived images correlate well with histology.

Ovarian cancer represents the deadliest gynecologic malignancy. Most patients present at an advanced stage (FIGO stage III or IV), when local metastatic ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Isolation of Stem-like Cells from 3-Dimensional Spheroid Cultures

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident stem cells are relatively quiescent with niche restraints in adult tissues, they enter the cell cycle in anchor-free three-dimensional (3D) culture and undergo both symmetric and asymmetric cell division, giving rise to both stem and progenitor cells. The latter proliferate rapidly and are the major cell population at various stages of lineage commitment, forming heterogeneous spheroids. Using primary normal human prostate epithelial cells (HPrEC), a spheroid-based, label-retention assay was developed that permits the identification and functional isolation of the spheroid-initiating stem cells at a single cell resolution.
HPrEC or cell lines are two-dimensionally (2D) cultured with BrdU for 10 days to permit its incorporation into the DNA of all dividing cells, including self-renewing stem cells. Wash out commences upon transfer to the 3D culture for 5 days, during which stem cells self-renew through asymmetric division and initiate spheroid formation. While relatively quiescent daughter stem cells retain BrdU-labeled parental DNA, the daughter progenitors rapidly proliferate, losing the BrdU label. BrdU can be substituted with CFSE or Far Red pro-dyes, which permit live stem cell isolation by FACS. Stem cell characteristics are confirmed by in vitro spheroid formation, in vivo tissue regeneration assays, and by documenting their symmetric/asymmetric cell divisions. The isolated label-retaining stem cells can be rigorously interrogated by downstream molecular and biologic studies, including RNA-seq, ChIP-seq, single cell capture, metabolic activity, proteome profiling, immunocytochemistry, organoid formation, and in vivo tissue regeneration. Importantly, this marker-free functional stem cell isolation approach identifies stem-like cells from fresh cancer specimens and cancer cell lines from multiple organs, suggesting wide applicability. It can be used to identify cancer stem-like cell biomarkers, screen pharmaceuticals targeting cancer stem-like cells, and discover novel therapeutic targets in cancers.

Using human primary prostate epithelial cells, we report a novel biomarker-free method of functional characterization of stem-like cells by a spheroid-based label-retention assay. A step-by-step protocol is described for BrdU, CFSE, or Far Red 2D cell labeling; three-dimensional spheroid formation; label-retaining stem-like cell identification by immunocytochemistry; and isolation by FACS.

Despite advances in adult stem cell research, identification and isolation of stem cells from tissue specimens remains a major challenge. While resident ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the &#34;Murine Ommaya.&#34; As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

Here, we describe a murine xenograft model that functionally resembles an Ommaya reservoir in patients. We developed the Murine Ommaya to study novel therapeutics for the universally fatal leptomeningeal disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...