Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Summary

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Abstract

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

Introduction

Directed cell migration is a fundamental property of many cells and is a key aspect of many normal physiological processes, including embryonic development, defense against infection and wound healing. In addition, cell migration also plays a prominent role in many diseases such as vascular disease, tumour cell metastasis and chronic inflammation^1,2. While the classical states of cell migration - polarization, protrusion extension, formation of adhesion, force generation and rear retraction - are generally accepted^3,4 , the elucidation of spatiotemporal mechanisms by which signal integration is coordinated has been more challenging.

The extracellular matrix (ECM) serves as a substrate for cell adhesion, and through inherent chemical⁵ and physical⁶ diversity, provides directional cues for cell navigation^7,8. In addition to these adhesive cues⁹, soluble factors^10-12 such as chemokines and growth factors can induce directed cell migration through chemoattractant receptors and their downstream motogenic signalling. Currently, it is not known whether engagement and signalling through adhesion receptors (i.e. integrins) or chemoattractant receptors, such as receptor tyrosine kinase (RTK), are dominant. Nor is it known whether hierarchies of receptor systems are cell type specific.

Live cell microscopy gives a wealth of information that is not accessible in bulk assays and can be combined with microfluidic devices to generate gradients of immobilized ^13,14 and soluble cues^{15 16}. The method described here utilizes a series of simple and established steps for surface modification in conjunction with a commercially available microfluidic chamber to create an imaging assay for cell migration that can easily be implemented in a cell biology laboratory (Figure 1). The assembled microfluidic device has optical qualities that are comparable to glass¹⁷ and the gradients of diffusible molecules are stable for at least 16 hr. The system can be used for epifluorescence and advanced microscopy techniques. Unlike other chemotaxis setups¹⁸, this system is suitable for recording slowly migrating, adherent cells. Importantly, the system is modular and easily allows the introduction of alternative adhesive or soluble migration cues and examination of various cell types.

Protocol

1. Passivation of Glass Coverslips with PLL-PEG-biotin

This step is designed to passivate the surface so that cells adhere and migrate onto specific regions on the glass coverslip that are created with microcontract printing (Step 2-3a) or dip pen lithography (Step 3b). For the passivation, a co-polymer of poly-L-lysine (PLL) and polyethylene glycol (PEG) is used in which 20% of PEG molecules are grafted to biotin (PLL-PEG-biotin).

1. To clean glass coverslips (18 mm x 18 mm), submerge them in a rack in 100% ethanol and place the rack in a sonicating bath for 30 min. Dry the glass coverslips in air or under a stream of nitrogen.
2. Next, sonicate the glass coverslips for 30 min in 1 M NaOH. Afterwards rinse the glass surfaces by carefully dipping the rack in a beaker filled with 1.5 L Milli-Q H₂O. Repeat the rinsing process three times using fresh Milli-Q H₂O each time. Dry the glass coverslips in an oven at 60 °C.
3. Place half of the clean glass coverslips individually in a humid chamber, which is made of a 24-well plate partially filled with H₂O. Drop 15 μl of PLL-PEG-biotin (1 mg/ml) in PBS on each glass coverslip. Take the other half of the remaining clean glass coverslips and carefully sandwich the PEG solution. Leave the coverslips for 1 hr in the humid chamber. Slide the sandwiched coverslips gently away from each other without scraping the PEG-coated surface and rinse them with Milli-Q H₂O. The water should slide off easily on the PEG-treated side. If necessary, air-dry the glass coverslips on paper towel with the treated surface facing upward. Store the glass coverslips in a vacuum desiccator until further use.

2. Fabrication of Stamps for Microcontact Printing

We describe two processes to pattern streptavidin tracks onto the passivated glass coverslips. The first one, microcontact printing, typically requires the fabrication of a silicon master from which a PDMS stamp is cast. In our case, the pattern on the PDMS stamp are 100 μm-wide lines that are spaced 290 μm center-to-center with a feature height of 25 μm. Below, we describe briefly how to fabricate a suitable silicon master (Step 2.1-2.3) and PDMS stamp (Step 2.4). However, there are multiple protocols for microcontact printing described in the literature^19-23, which are suitable for printing protein patterns onto glass coverslips. Masters can also be custom made and purchased from commercial sources.

1. First, clean the silicon wafer (4" in diameter) by incubating it in piranha solution (3:1 v:v of sulphuric acid and 30% hydrogen peroxide) for 20 min. Rinse the wafer thoroughly 10 times with MilliQ H₂O. Dry the wafer in an oven at 150 °C O/N.
2. Rinse the clean and dried silicon wafer with 100% acetone, followed by 100% isopropyl alcohol. Next spin-coat 2 ml of GM1070 SU-8 photoresist (Gersteltec Sarl) onto the wafer at 3,100 rpm for 40 sec to create a 25 μm thick layer. The sample is then baked on a hot plate at 65 °C for 5 min followed by a further 5 min at 95 °C.
3. Expose the SU-8 photoresist to UV light for 60 sec under a photomask with the desired pattern (for example using a Quintel Q6000 mask aligner) and repeat the baking process describe in 2.2. Then the sample is developed by immersing the wafer in SU-8 developer (Gersteltec Sarl) for 4 min, rinsed in 100% isopropyl alcohol and dried under nitrogen flow. The area of the SU-8 photoresist that was not exposed to UV light is thus removed, creating the inverse pattern of the PDMS stamp.
4. The next step is to cast a PDMS stamp from the silicon master. First, thoroughly mix 1 part (w/w) of the curing agent with 10 parts of elastomer prepolymer (Sylgard 184). To remove any air bubbles, degas the mixture in a desiccator attached to a vacuum line for 1 hr. Next, pour the PDMS elastomer onto the silicon master inside a Petri dish to about 1 cm in height and cure the master and PDMS mixture in an oven at 70 °C for 1 hr. Finally, the PDMS stamp is removed from the silicon master and cut to size.

3. Patterning Streptavidin or Adhesive Cues onto Passivated Glass Coverslips

After passivating glass coverslips, protein patterns are printed with microcontact printing (Step 3a) or dip-pen lithography (Step 3b). In our case, streptavidin is printed as lines, which form the basis for RGD gradients (Step 4). The same procedure can also be used for printing matrix proteins to create adhesive patterns.

An alternative method to microcontact printing for the patterning of biomolecules onto surfaces is dip pen lithography. In dip pen lithography, a cantilever is used to transfer a small volume of solution onto the surface. The size of the cantilevers, viscosity of the solution, contact time of the cantilever on the surface, hydrophobicity of the surface and humidity in the printing chamber determine the size of the printed features. The advantage of this method is that different patterns can be printed for each experiment, as there is no need to fabricate masters and stamps. The disadvantage is that it takes longer to print a given pattern and that dip pen lithography requires a specialist instrument that may not be available in many laboratories. The dip pen lithography instrument that we used was the NanoeNabler from Bioforce Nanoscience. Here, we printed small dots that were <10 μm in diameter, for which we used SPT10 cantilevers.

3a. Microcontact Printing (μCP) Technique

1. To clean and make the PDMS stamp more hydrophilic, treat the patterned side of the PDMS stamps in an UV and ozone (for example, UV Ozone Procleaner from Bioforce Nanoscience) for 1.5 hr. Alternatively, use a plasma cleaner for a shorter time for the same purpose. This process creates an oxidized PDMS surface²⁴, which improves the printing process.
2. Immediately after the ozone or plasma treatment, place and spread 10 μl streptavidin (1 mg/ml in PBS) onto the PDMS stamps and leave for 1 hr in a humid chamber (such as a partially filled 6-well plate, see Step 1.3). If tracks should be visible under the fluorescent microscope, use streptavidin that is conjugated to a fluorophore such as streptavidin-AlexaFluor350.
3. Remove excess streptavidin from the stamp with tissue paper and air dry the stamp for approximately 1 min. Lightly press the stamp onto the PLL-PEG-biotin-coated glass coverslip.
4. If fluorescent streptavidin was used in Step 3.a2, examine the pattern using under an epifluorescence microscope. Store printed surfaces in a desiccator.

3b. Dip Pen Lithography

1. First, mix 1 part of 1 mg/ml streptavidin or streptavidin-AlexaFluor350 with 10 parts of glycerol (v/v) and load 5 μl of the mixture onto the cantilever. Then begin the printing process as described in the operation manual of the dip pen lithography instrument. To reduce the spot size to approximately 5 μm, adjust printing speed, contact time of the cantilever on the surface or vertical distance of the cantilever to the surface. In the example shown in Figure 2c, dots were printed in a line with row and column separation set to 10-15 μm. This distance is sufficient to prevent dots from merging into each other but allows the viewing of multiple dots in a single field of view with most microscope settings.
2. Store printed surfaces in a desiccator.

4. Creating Adhesive Gradients onto Streptavidin-pattern Surfaces

We create adhesive gradients of biotin-RGD onto glass coverslips that are coated with streptavidin (Figure 3) or contain streptavidin patterns on passivated glass coverslips (Figure 5). To create a surface gradient, we used a commercially available microfluidic device (called sticky-Slide Chemotaxis ^3D from Ibidi) that creates a stable solution gradient by passive diffusion. To compete for streptavidin binding sites, we employed two opposing gradients of biotin-RGD and biotin that was not conjugated to RGD (see Figure 1).

1. Adhere the streptavidin-coated or -patterned glass coverslips onto the sticky side of the sticky-Slide Chemotaxis ^3D device. To ensure that there is no leakage for several hours, the edges of the device are sealed with a thin layer of warmed Vaseline and with a second layer of a mixture of 1 part of Vaseline to 1 part of paraffin wax (w/w).
2. Fill the channel of the device with 6 μl PBS in the channel. Then fill the two reservoirs with 70 μl of 0.03 μg/μl biotin-4-fluorescein in PBS and 70 μl of 0.03 μg/μl biotin-RGD in PBS, respectively. Incubate the samples at RT in the dark for 1 hr.
3. Remove the biotin solutions and carefully rinse the surface while still attached to the microfluidic device twice with PBS. Mark the direction of the adhesive gradient and if needed, store the device filled with PBS at 4 °C for but ensure that the device does not dry out. If required, analyze the adhesive gradient with a fluorescent microscope.

5. Labelling of Cells with Fluorophores for Live Cell Imaging

Cells are labelled with fluorescent dyes that assist cell tracking under the fluorescent microscope. Common probes are fluorescent organelle markers, such Syto 64 Red, which labels the cell nucleus. To do so, cells are first washed three times with PBS, then incubated with 1 μM Syto 64 Red in culture media for 45 min and finally washed a further three times with PBS. Please note that cells should not be kept in PBS for long periods of time.

Alternative dyes for cell tracking are CellTracker series that stain the cytoplasm and are retained during cell migration and division. Transfection of cells with fluorescent fusion protein is particularly useful to record the subcellular distributions of a protein of interest during migration. However, fluorescent proteins typically photo-bleach faster than organic dyes so that long-term observations may not be possible.

6. Loading of Cells with a Soluble Gradient into the Microfluidics Device

1. Count fluorescently labelled cells and split into two tubes of equal cell numbers. Wash and resuspend one tube of cells with media containing the chemoattractant, such as low glucose Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). Wash and resuspend the other tube of cells in media without the chemoattractant, i.e. DMEM with 0% FBS. The final concentration of cells in each tube should be 5 x 10⁵ cells/ml.
2. Remove all the solutions from the microfluidic device (from Step 4.3) and place it on the microscope stage (see Step 7). Load 70 μl of cells (5 x 10⁵ cells/ml in 0% FBS DMEM) into one reservoir and 70 μl of HeLa cells (5 x 10⁵ cells/ml in 10% FBS DMEM) into another. Loosely place a tape over the reservoirs to avoid evaporation. If cells on the surface are pre-incubated prior to imaging, it is advisable that the cells are loaded into the device in growth media without the gradient. The device is then placed on the microscope stage and the media replaced with gradient media i.e. DMEM without FBS in one reservoir and DMEM with 10% FBS in the other.

7. Live Cell Imaging and Data Analysis

1. Switch on the microscope (Nikon Ti-E) and warm up the stage at least 1 hr before starting the experiment.
2. Ensure that the channel of the microfluidic device is in the field of view and cells are in focus. Select image acquisition parameter such as exposure times and fluorescent filters, image sequence of bright field and fluorescence that captures cells and tracks as well as time points and recording length (e.g. every 15 min for 16 hr).
3. Analyze data with appropriate softwares such as ImageJ Manual Tracking or Ibidi Chemotaxis and Migration Tool.

Results

To understand how cell integrate migratory signals²⁵, we have developed a method to image cells with fluorescence microscopy that migrate in an environment with competing adhesive and soluble gradients (Figure 1). Adhesive tracks that contained fluorescent streptavidin and biotinylated RGD were created with microcontact printed tracks and dip pen lithography (Figure 2). Successful microcontact printing is indicated by the line profile of the fluorescence intensity across the t...

Discussion

In this protocol, we used a commercially available microfluidic device (sticky-Slide Chemotaxis ^3D from Ibidi) to study the effects of chemically modified surfaces and chemoattractant gradients on cell migration. This microfluidic setup does not require flow because the gradient is established by diffusion along the length of the channel. This is important because flow could differentially affect slowly migrating cells such as fibroblasts that form stable focal adhesions and fast migrating cells like most leuk...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Coverglass staining outfits	Thomas Scientific	8542 E40	Coverslip rack
Oven	Binder		ED 53 series
Silicon wafer	Silicon Quest	708-007	Boron doped <100> wafer, 4" diameter, 500 μm, single side polished
GM1070 SU-8 photoresist	Gersteltec Sarl
SU-8 developer	Gersteltec Sarl
Sylgard 184 curing agent	Dow Corning
Sylgard 184 elastomer prepolymer	Dow Corning
PLL-PEG-biotin (20%)	SuSos AG	PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-Biotin (20%)	1 mg/ml in PBS
Fluorescein	Sigma	46955	1 mM in PBS
Streptavidin-AlexaFluor350	Invitrogen	S-11249	1 mg/ml in PBS
Biotin-4-fluorescein	Invitrogen	B-1370	0.03 μg/μl in PBS
Biotin-RGD	GenScript	SC1208	0.03 mg/ml in PBS
Syto 64 Red	Invitrogen	S-11346	1 μM in PBS
Sticky slide chemotaxis 3D	Ibidi	80328
200 μl Greiner yellow bevelled tip	Greiner Bio-One	739261
Vaseline	Sigma	16415
Paraffin wax, mp 55-57 °C	Sigma	327204
Nano eNabler 10 μm cantilever	BioForce	SPT-S-C-10s
Image J software	National Institute of Health		rsbweb.nih.gov/ij/download.html
Manual Tracking plugin	Fabrice Cordelières		rsb.info.nih.gov/ij/plugins/track/track.html
Chemotaxis and Migration Tool	Ibidi GmbH		www.ibidi.com/applications/ap_chemotaxis.html