A Method of Targeted Cell Isolation via Glass Surface Functionalization

Summary

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.

Abstract

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.

Introduction

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 "lab on a chip" 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 physical^4,5 or chemical methods⁶. Although these approaches offer a few advantages such as identifying protein^7,8 expression, identifying RNA expression^9-11, or even providing cells for in vitro culture^12,13, 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 quantities^14,15, 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.

Protocol

1. Cleaning the Glass Surface and Preparing Reagents

1. Place a glass surface in an oxygen plasma machine for 5 min at 50% power to clean it.
2. Prepare 2.5 ml 2% reconstituted (3-aminopropyl) triethoxysilane (APTES) solution, by adding 50 µl of APTES and 2.45 ml of ethanol in a conical tube.

2. APTES and DSB Functionalization

1. Add APTES solution to the surfaces. Pipette 150 µl per well for 8 well plates. Pipette 100 µl per well for 24 well plates. Pipette 1.1 ml for 60 x 15 mm glass dishes. Cover the surfaces to prevent evaporation and uneven distribution of APTES solution. Place the surfaces on a platform shaker for 50 min at room temperature, which creates an even distribution of the APTES layer.
   NOTE: APTES is an aminosilane that forms the first layer of the surface. If using a different surface, heuristically determine the volume of solution needed to cover the surface.
2. Select the temperature for the oven, while the surfaces are on the shaker: Heat to 55 °C for 2 hr for the 8 well plates and 24 well plates. Heat glass only dishes to 90 °C for 1 hr.
3. Rinse the surfaces with ethanol.
   1. Administer the amount of ethanol required by referencing based on the surface used. Add 150 µl ethanol to each well for 8 well plates. Add 125 µl ethanol to each well for 24 well plates. Add 1.1 ml ethanol for glass dishes.
   2. Rinse the surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface. Rinse twice more using ethanol.
      NOTE: If any of the glass bottom well plates have any plastic in them, do not heat them above 65 °C, as the plastic will begin to melt and warp.
4. Dry with 100% nitrogen gas dispensed from a tank. Place the surfaces in the oven.
5. Prepare 2.5 ml of d-desthiobiotin (DSB) solution by combining 1.5 mg/ml DSB in 37.5 µl of dimethyl sulfoxide (DMSO), and 5 mg/ml 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in 2462.5 µl of the 0.1 M 4-morpholinoethanesulfonic acid hydrate (MES) (pH 6) buffer. Then combine both solutions.
6. Add 1 µl of 2-β mercaptoethanol, after 15 min, into the solution to quench the reaction between DSB and EDC. Remove hot APTES functionalized glass surfaces from the oven. Wait 5-10 min for the glass surfaces to cool.
7. Add MES buffer the surface to rinse, using amounts based on the surface. Add 150 µl MES buffer to each well for 8 well plates. Add 125 µl MES buffer to each well for 24 well plates. Add 1.1 ml MES buffer for glass dishes.
8. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface. Rinse twice more with MES buffer.
9. Apply the DSB solution to the surfaces to allow them to incubate. Add 150 µl DSB solution per well for 8 well plates. Add 100 µl DSB solution per well for 24 well plates. Add 1.1 ml for glass dishes DSB solution.
10. Place the DSB covered glass surfaces on a damp paper towel inside a Petri dish. Cover and incubate in a 4 °C fridge for 18-24 hr.

3. Streptavidin Functionalization

1. Rinse each glass surface three times with 1 ml of 1.0x Phosphate Buffered Saline (PBS). Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface. Dilute the streptavidin (SAv) stock solution to 0.4 mg/ml (recommended).
   NOTE: PBS is isotonic so it can be used as a medium for dilution and rinsing. PBS is used as a solvent for the SAv, and therefore, will not affect the ability of the SAv to bind to the surface.
2. Apply 0.4 mg/ml of SAv solution evenly to the surfaces so that a thin layer of forms at the bottom of the glass, select the amount of solution based on surface. For 8 well plates, add 150 µl 0.4 mg/ml SAv solution per well. For 24 well plates, add 100 µl 0.4 mg/ml SAv solution per well. For glass dishes, add 1.1 ml 0.4 mg/ml SAv solution.
3. Cover and move the plates to a 14 cm Petri dish to retain moisture. Incubate the Petri dish in refrigerator for 18-24 hr.
   NOTE: It is essential to utilize consistent volumes of APTES, DSB, and SAv on each surface.
4. Rinse each glass surface three times with 150 µl of PBS to remove SAv. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface.
5. Wet a paper towel with de-ionized water and place the paper towel flat in 14 cm Petri dish surrounding the plates to retain moisture in the wells. Cover the Petri dish containing the wells. Incubate the Petri dish in a 4 °C Biosafety level 1 (BSL-1) refrigerator until needed.

4. Cell Capture and Release

1. Start with T175 flask(s) of cells intended for the experiment. Aspirate the media from flask(s). Wash out the remaining media with 5 ml of room temperature PBS. Aspirate PBS from the flask(s).
2. Add 10 ml of non-enzymatic lifting agent, such as Cell Dissociation Solution, to the T-175 flask of cells. Put the flask in the incubator for 6 min to allow for lifting of the cells from the flask.
3. After 6 min, add 10 ml of cold Hank's Balanced Salt Solution (HBSS; See List of Materials) to inactivate the lifting agent. Take out 20 µl of cells to count number of cells in solution using a hemacytometer.
   NOTE: Depending on the functionalized surfaces used in the capture experiment, the number of cells needed varies. For functionalized 8 well plates, use 300,000 cells per well. For functionalized glass dishes, use 1.1 million cells. For functionalized 24 well plates, 125,000 cells are recommended. More information on cell counting is found in the Supplement.
4. Repeat steps 4.1- 4.3 for another flask of cells, if fewer cells are present than needed for the experiment. Combine cell suspensions and recount cells to get total number of cells in solution.
5. Spin down cell suspension in a centrifuge at 500 x g for 5 min at 4 °C to get a pellet of concentrated cells. Find the appropriate amount of HBSS to add to the cell suspension to get a concentration of 1 x 10⁶ cells per ml, then aspirate the supernatant from the spun down cells, and add the calculated volume. This volume can be calculated using the equations in the Supplement.
6. Pipette up and down (triturate) to resuspend the cells in solution and reduce cellular clumping in solution that may reduce antibody binding. Split the cellular solution into separate control and experimental solutions. View Supplemental File for components and calculations.
7. Add the biotinylated antibodies to the respective cell solutions as described in the Supplemental File. Incubate for 30 min at 4 °C on an end-over-end mixer.
   NOTE: For MCF7GFP cells, 0.5 mg/ml hIgG or 0.5 mg/ml HLA-ABC antibodies are recommended. For RAW macrophages, 1 mg/ml mCD11b are recommended at half the volume to account for the dilution differences. For Human Umbilical Vein Endothelial Cells (HUVECs), 0.5 mg/ml hCD31 is recommended.
8. Wash the functionalized glass surface with HBSS. For this experiment, an 8 well plate is advised.
   1. Add 150 µl HBSS to each well for 8 well plates. Rinse twice more using HBSS. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface. Keep cold at 4 °C.
9. Add cell solutions to the wells and wait 45 min for the cells to incubate on ice on the shaker. Make the solution of biotin in sterile HBSS by using calculated volumes as described in the Supplement.
10. Remove the cell solution from the glass wells by using HBSS.
    1. Gently pipette 150 µl HBSS into each well. Then pipette out the HBSS. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface.
    2. Repeat two more times. After washing, add HBSS to the wells to keep the cells wet.
11. Add 150 µl of 20 mM biotin solution to each respective release well, and then wait 20 min to allow for reaction.
12. Recollect non-specifically bound cells by washing with HBSS as stated above, and then if using fluorescently labeled cells, proceed to fluorescence image the cells. Also image live cells in a flask (as a control, to compare with the cells in the well). If using green fluorescent protein (GFP) transfected cells, the excitation will be 470 nm, and the emission will be 515 nm.

5. Antibody Optimization: Antibody Titration

1. Begin by lifting the cells from the T75 or T175 flask as explained in step 4.1- 4.3.
   NOTE: These volumes are calibrated for 24 well plates, but can be changed to suit any glass functionalized surfaces through heuristic testing. A representative antibody titration is shown in Figure 1.
2. Count cells using the hemacytometer, and then spin down the cell solution in a conical tube at 500 x g for 5 min at 4 °C. Reconstitute cells to 1 million cells per ml, using the calculations described in the Supplement.
3. Split the 1 million cells per ml solution into six different solutions of 500 µl in different centrifuge tubes for the antibody (Ab) solutions.
   1. Dilute the antibody stock solution to 10 µg/ml, 1 µg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml. Create the controls by using 100 µl of Stain Buffer (PBS + 1% sodium azide + 1% BSA), and 500 µl of cells for the control with no Ab in it. For the blank control (No Ab and no cells), use a solution of 300 µl PBS.
      NOTE: CAUTION: Sodium azide is extremely toxic, explosive, and extreme care must be taken when using it. Please consult the material safety data sheet (MSDS) and use the appropriate safety equipment.
4. Combine and incubate the Ab solutions with the cell solutions in the end-over-end mixer at 4 °C for 30 min. Rinse the functionalized 24 well plates with HBSS three times. Rinse the surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface.
5. Aliquot 125 µl of each sample solution into the appropriate APTES, DSB, and SAv functionalized well. Incubate at 4 °C or on ice for 45 min on the glass surface. Rinse the surface with HBSS three times to remove non-specifically attached cells. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface. Do NOT rinse the bottom row, as those are controls.
6. Add 150 µl of HBSS to each washed well, put the 24 well plates on ice, and then use a plate reader to measure fluorescence of GFP cells (Excitation 485 nm / Emission 528 nm) .

6. Cell Optimization: Cell Titration

1. Lift the cells as mentioned in steps 4.1 -4.3.
2. Count cells using a hemacytometer. Spin down cell solution in conical tube at 500 x g for 5 min at 4 °C. Reconstitute cells to 1 million cells per ml.
   NOTE: More information on the use of the hemacytometer can be found in the Supplement.
3. Pipette 1.6 ml from the cell solution for the 1.6 million cells stock and place in a conical tube. Pipette 800 µl of cells from the cell solution for the 800,000 cells stock and place in a conical tube. Pipette 80 µl from the cell stock for the 80,000 cells stock and place in a conical tube. Pipette 8 µl from the cell stock for 8,000 cells stock and place in a conical tube.
   NOTE: Concentrations are given per 8 well plate measurements, replicate as needed. An example of plate output is shown in Figure 2.
4. Take the four stock solutions and spin in a centrifuge at 500 x g for 5 min at 4 °C. Re-suspend all stock solutions in 400 µl of HBSS.
5. Add 1.6 µl of 100 ng/ml antibody to the 1.6 million cell stock, 0.8 µl to the 800,000 cell stock, and 0.5 µl to all other stocks. Incubate the antibody for 30 min in the end-over-end mixer for 30 min at 4 °C. Wash the functionalized glass surface with HBSS three times.
   NOTE: The functionalized 8 well plate is recommended for microscopy quantification, while the functionalized 24 well plate is recommended for plate reader quantification. Antibody concentration for 8,000 cell stock was not reduced to allow the limiting factor of the capture surface to not be the lack of antibodies in solution, but rather the capturing properties of the surface.
6. Apply 150 µl of the sample solutions to each well. Apply the 1.6 million cell stock to the first column of wells on the left. Apply the 800,000 cell stock to the second column from the left. Apply the 80,000 cell stock to the third column from the left. Finally apply the 8,000 cell stock to the last column. Incubate for 45 min at 4 °C on the shaker.
   NOTE: The 1.6 million cell stock serves as the highest concentration to be tested, and is double the amount of cells that is used typically in cell separation experiments (600,000 cells per well) The 800,000 cell stock serves as the control for the experiment as it is the typical cellular concentration that is used in cell separation experiments (3,000,000 cells per well). The 80,000 cell stock serves as a ten-fold dilution to test for lower concentrations for cellular capture (30,000 cells per well). The 8,000 cell stock serves as a hundred fold dilution to use as a test to check the lower limit of cellular separation (3,000 cells per well).
7. Rinse with HBSS three times. Rinse surface by discharging and drawing the liquid from a fixed point such as the corner of the well. Hold the pipette at approximately a 70° angle so the tip is not directly pointed into the surface. Collect all washes.
8. Image the cells on a fluorescence microscope, if using 8 well plates, take pictures of each surface, and apply 150 µl biotin to each surface for one hour at 4 °C on the shaker. After one hour, rinse biotin release wells with HBSS 3 times as before. Collect all washes from wells for counting with the hemocytometer and image the release wells.
9. Apply 150 µl of 20 mM excess biotin solution to appropriate biotin release wells for 1 hr, if using 24 well plates, and then rinse those wells 3 times with HBSS. Quantitate 24 well plates using a plate reader. Use a hemacytometer to count cells from all collected well washes.

7. Image Analysis

Note: The FIJI software package (http://fiji.sc/Fiji) is recommended for image analysis. Initially, the images were converted into grayscale image, and then the brightness/contrast was altered to bring out the cells.

1. Load up the image, and convert to grayscale by clicking the image tab then scrolling down to "Type" and then clicking "8 bit".
2. Increase the contrast of the image by clicking the image tab and then scrolling to "Adjust". Click on "Brightness and Contrast", and then use the scroll bars to make the cells stand out. If using fluorescence images, invert the images to make the cells more defined.
3. Load a plugin onto ImageJ called ITCN (http://rsb.info.nih.gov/ij/plugins/itcn.html) to analyze the images.
4. Open ITCN. When a dialog box appears that prompts the user with several parameters to estimate the size of the cell, set the minimum width of the cell of interest first. Click the "Detect Dark Peaks" option if the image is fluorescent and the cells are darkened.
5. Set the threshold value at 2 initially, and then hit the "Count" button. A newly counted version of the picture will appear with red dots delineating where cells have been counted.
6. Adjust the threshold and width parameter to get more accurate cellular data by defining the size of a "cell". Note: The number of cells counted will be in a dialog box on the right. Changing parameters will give different number of cells counted.
7. Continue to iterate through the threshold and width parameters until a good estimate is reached for the number of cells.

Results

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 (...

Discussion

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 receptors^{3,14,15,19,21<...}

Materials

Name	Company	Catalog Number	Comments
(3-Aminopropyl) triethoxysilane (APTES)	Acros Organics	919-30-2	Used to make 2% APTES solution
Plasma Cleaner Pico	Diener	Model 1	Cleans surfaces and allows for bonding of PDMS to glass
d-Desthiobiotin (DSB)	Sigma	D20655	Used as the releasing mechanism in the cellular capture surface.
dimethyl sulfoxide (DMSO)	British Drug Houses (BDH)	BDH1115-1LP	Dissolves the DSB into solution
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)	Thermo-Scientific	5g: 22980 25g: 22981	Activates carboxylic acids and allows binding of proteins to glass surface.
uncoated 8-well culture slide	BD Falcon	Case of 24: 354118 Case of 96: 354108	Used in cellular experiments involving Zeiss fluorescence microscope such as initial capture and release quantification experiments
Glass bottom 24-well plates	MatTek	P24G-0-13-F	Used in cellular experiments involving the plate reader such as antibody and cellular titration experiments
Mercaptoethanol	Science Lab	60-24-2	Used to quench reaction between EDC and DSB
4-Morpholinoethanesulfonic acid hydrate (MES Hydrate 99%)	Fisher Scientific	AC172590250	Used to make 0.1 M MES Buffer for use in EDC reaction
Precision Oven	Thermo Scientific	11-475-153	Used in curing of PDMS and APTES layer.
Titramax 1000 Shaker	Heidolph	13-889-420	Used to ensure even distribution of APTES on surfaces.
1x Streptavidin 5 mg [e7105-5mg]	Proteo Chem	9013-20-1	Biotin-binding protein. May cause irritation.
5 cm Glass Dish	Fisher Scientific	08748A	Used in HUVEC studies as well as future profiling studies.
14 cm Petri Dish with Cover	Sigma-Aldrich	Z717231	Used to hold samples being functionalized and transport them.
MCF7-GFP cells	Cell Biolabs	AKR211	Stored in liquid nitrogen
RAW264.7 mouse macrophages	ATCC	TIB-71	Gifted to us from Smith lab at the University of Illinois. Stored in liquid nitrogen.
TrypLE	Life Technologies	12605036	Stored in 100 ml at room temperature
Dulbecco’s modified Eagle medium	Cell Media Facility at School of Chemical Sciences at UIUC	50003PC	Supplier: Corning
Nonessential amino acids	Cell Media Facility at School of Chemical Sciences at UIUC	25-025-CI	Already added into DMEM by facility. Supplier: Corning.
Cell scraper	Fisher Scientific	12-565-58	Small 23 cm 50 pack
Cell Dissociation Solution	Corning	MT-25-056CI	Used to lift cells non-enzymatically for the use in cell experiments
Hemacytometer	Hausser	02-671-54	Used to count cells for quantification of cell solutions and capture and release effectivity.
Biotin	Amresco	58-85-5	Used to release cells from surface.
HBSS	Created from Recipe	N/A	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, 0.2 g KCl, 0.0402 g Na2PO4•7H2O, 0.03 g KH2PO4 and 0.5 g glucose. Add DI water to get to 500 ml, filter, and then refrigerate.
HLA-ABC Antibody	BioLegend	311402	Antibody used to capture MCF7gfp cells
hIgG Antibody	BioLegend	HP6017	Antibody used to capture MCF7gfp cells
MCF7 GFP cells	Cell Biolabs	AKR-211	Luminal Breast Cancer line that has been transfected with green fluorescent protein.
Assorted Conicals	Thermo-Scientific	15mL: 12-565-268	50/15 ml plastic conicals for storing solutions and aliquots.
Mini-Tube Rotators (End over End Mixer)	Fisher Scientific	05-450-127	Used to incubate antibody and mix other cellular solutions in order to mix
Axiovert 200M (Fluorescence Microscope)	Zeiss	N/A	Zeiss Axiovert 200 M inverted florescence microscope.
Zeba Desalting columns	Thermo-Scientific	PI-87770	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
EZ Link Sulfo NHS Low Weight Biotinylation Kit	Thermo- Scientific		Used to biotinylate antibodies to allow them to integrate with the capture surface
Plate Reader	BioTek	Synergy HTX Multimode Reader	Used to quantitatively measure fluorescent intensity in the titration experiments.