A Versatile Method of Patterning Proteins and Cells

Summary

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Abstract

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

Introduction

In tissue engineering and biosensing, the ability to control the spatial organization of proteins and cells on a µm scale, has become increasingly important over the last four decades^1,2,3. Precise spatial organization of proteins and cells has allowed researchers to examine the interaction between cells and substrates containing similar or different types of cells, to guide cell growth, and to immobilize biomolecules for the fabrication of biosensors^4,5,6,7,8,9.

Current methods of patterning proteins include photopatterning and microcontact printing. Photopatterning utilizes light sensitive material which is crosslinked upon exposure to ultra violet (UV) light. UV light directed at a photomask (consisting of transparent areas with darker regions to prevent UV light transmission) causes crosslinking in specific regions which can then be used for subsequent attachment of biomaterials or cells^10,11. While this scheme is very accurate and allows for precise control of the topography of the culture surface, it is limited to UV-sensitive biomolecules that can be patterned by UV radiation¹². Microcontact printing is another popular method of patterning specific proteins^13,14. In this method, a poly-dimethyl siloxane (PDMS) stamp is treated with a variety of surface modification reagents before being soaked in a solution of the chosen biomolecular substrate. It is then gently pressed onto a glass coverslip or other surface thus "stamping" the biomolecule onto the culture surface. However, stamping is limited to the type of material that can be transferred as well as the wettability of biomolecules to the surface of the PDMS stamp¹⁵.

Direct patterning of cells can be more difficult and relies on complex methods such as switchable substrates, stencil based methods, or patterning with specific cell adhesion molecules^16,17. These methods are limited in their ability to pattern cells due to the lack of compatible cell adhesion substrates, incompatibility of the process to work with sensitive biological cells and constraints, inconsistency in reproducing the patterning, and complexity of the procedure. For example, with switchable substrates, custom substrates need to be designed for every cell type, to switch their adherence to specific cell types without degradation upon exposure to the UV light and heat used in process^17,18,19,20. Stencil based patterning methods are versatile in their ability to pattern cells; however, it is difficult to manufacture PDMS stencils at the appropriate thicknesses for use^16,21. Direct injection of cells into PDMS microfluidic channels have some advantages such as: 1) ease in fabrication of microfluidic channels and 2) suitability for many different cells and substrates. However, the prevalent issue of air bubble capture during the injection process due to the hydrophobicity of PDMS without the use of plasma cleaning, or other methods to decrease air bubbles, makes it difficult to consistently create patterned cells on glass or plastic surfaces²¹.

This work expands upon capillary micromolding^{22,23,24,25,26} and reports a method to inject protein and cell suspensions into microchannels. The method used here demonstrates the patterning of substrates and both direct and indirect patterning of specific cell types. This technique overcomes the high hydrophobicity of PDMS and eliminates the presence of bubbles during injection of either substrates or cells by taking advantage of the gas permeability of PDMS²⁷. This paper demonstrates the use of the technique with several different substrates and cell types. The article also highlights the fabrication of molds for soft lithography using conventional photolithography as well as a simple and low-cost adhesive tape method useful in resource limited settings^28,29.

Protocol

NOTE: Please consult all relevant material safety data sheets (MSDS) before use. Some of the chemicals used in this protocol are toxic and carcinogenic. Please use all appropriate safety practices (fume hood, glovebox) and personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes) when using toxic or acid/base materials.

1. Fabrication of Master Molds for Soft Lithography using Photolithography

1. Draw the layout of the microchannel using a computer-aided design (CAD) drawing tool.
2. Print the layout on a blank mask plate using laser mask writer.
3. Rinse a 4 inch silicon wafer with acetone followed by isopropanol and dry with a nitrogen air gun to ensure that no solvent is left on the wafer.
4. Dehydrate the wafer by baking it on a hot plate at 200 °C for 5 min.
5. Select a negative photoresist designed for the desired height of the microchannels. For example, use negative photoresist SU-8 50 to obtain microchannels with a height of 50 µm.
6. Allow the wafer to cool to room temperature, and then align the wafer on the chuck of a spin coater.
7. Dispense 4 mL (1 mL/inch diameter of the wafer) of negative photoresist onto the center of the wafer.
8. Spin coat the wafer with a two-step spin cycle using a spinner. First, apply a spread cycle of 500 rpm with an acceleration of 100 rpm/s for 10 s. Then apply a spin cycle of 2,000 rpm with an acceleration of 300 rpm/s² for 30 s to obtain a 50 µm coating of photoresist on a wafer.
9. Soft bake the wafer in two steps on a hot plate according to the manufacturer's directions. For a 50 µm coating of photoresist, first pre-bake the wafer at 65 °C for 6 min and then immediately ramp up the hot plate temperature to post-bake the wafer at 95 °C for 20 min.
10. Allow the wafer to cool to room temperature and then load the wafer onto the lithography stage.
11. Align the mask on the wafer using mask aligner and expose the wafer to UV light (as per the manufacturer's instructions) at an intensity of 1.5 mW/cm² for 146 s to apply the total UV dose of 220 mJ/cm² required for a 50 µm thick layer of photoresist.
12. Apply post exposure bake to the wafer in two steps on a hot plate. For a 50-µm coating of photoresist, first pre-bake the wafer at 65 °C for 1 min and immediately ramp up the hot plate temperature to post-bake it at 95 °C for 5 min.
13. Develop the wafer in by submerging the wafer in SU-8 developer and gently shaking until the features become clear on the wafer. Develop the wafer for ~6 min for the 50 µm thick photoresist.
14. Rinse off excess developer with isopropanol and ethanol, then dry the wafer with a nitrogen air gun.
15. Hard bake the silicon wafer on a hot plate at 150 °C for 15 min.
16. Place the wafer in a desiccator with 25 µL of the silanizing agent tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane.
    NOTE: The silanizing agent is corrosive, thus should be used in an acid/base fume hood, with appropriate personal protective equipment (safety glasses, gloves, lab coat, full length pants, closed-toe shoes).
17. Apply the vacuum for 5 min and expose the wafer to the vapors of silane for 30 min without releasing the vacuum.
18. Transfer the wafer into a suitably sized Petri dish.

2. Fabrication of Master Molds for Soft Lithography Using Adhesive Tape

1. Draw the layout of the microchannel using a CAD drawing tool and print the drawing on white paper.
2. Clean a glass slide large enough to accommodate the design with isopropanol and then dry the slide with an air gun.
3. Attach adhesive tape onto the cleaned glass slide. Be careful not to trap any air bubbles.
4. Tape the sides of the glass slide onto the paper with the design, with the tape side up.
5. Use a scalpel to cut the tape on the glass slide using the paper design as a reference and then peel off tape from unwanted areas of the glass slide.
6. Rinse the glass slide with isopropanol and then dry with an air gun. Bake the glass slide in an oven at 65 °C for 30 min.
7. Gently roll over the tape with a rubber roller to remove air bubbles and allow the slide to cool to room temperature.

3. Soft Lithography Fabrication of the PDMS Devices

1. Mix PDMS elastomer and its curing agent in a ratio of 10:1 (w:w), stir the mixture vigorously, and then degas the mixture by placing it into a vacuum chamber until no air bubbles appear in the mixture.
2. Pour the mixture onto a silanized silicon mold or adhesive tape mold in a petri dish to obtain a ~2 mm thick layer of PDMS and degas until all air bubbles disappear.
3. Cure the PDMS in an oven at 65 °C for 2 h.
4. Use a scalpel to cut the PDMS layer at least 5 mm away from the features and then peel the cured PDMS off the mold using tweezer.
5. Punch a single inlet hole with a 1 mm biopsy punch anywhere on the microchannel, preferably at the end of a network of microchannels as seen in Figure 2a and 4b.
6. Clean the PDMS cast with adhesive tape to remove dust particles adsorbed onto the device. Sterilize the PDMS microchannel device (PDMS cast) by rinsing with a solution of 70% ethanol followed by sterile deionized (DI) water, and expose to UV for 30 min.
7. Keep the sterile PDMS device in a sterile petri dish until use.

4. Substrate Patterning

1. Place the sterile PDMS cast using tweezers onto a sterile glass coverslip or petri dish and apply gentle pressure using the tip of the tweezers.
   NOTE: The PDMS cast makes a conformal but reversible seal with the glass coverslip or petri dish forming microchannels without use of any adhesive.
2. Place a droplet (20 - 40 µL) of the substrate solution on the inlet completely covering the inlet hole. Pattern Poly-D-Lysine (PDL) by placing a 20 µL droplet of PDL in sodium tetraborate buffer on the inlet of the microchannels.
3. Put the petri dish in a vacuum of ~254 mmHgA (equivalent to house vacuum in biological laboratories) for 10 min, and observe air coming out of the channel in the form of bubbles.
4. Release the vacuum and observe the substrate solution flowing into the microchannel.
5. Incubate the dish at the appropriate conditions for substrate adherence. See Tables 1 and 2 for incubation conditions (these vary depending on the substrate). For PDL patterning, incubate for 1 h at 37 °C.
6. Carefully peel off the PDMS stamp using sterile tweezers and wash the pattern on glass coverslip thrice with DI water.
7. Add a 1% bovine serum albumin (BSA) solution to the petri dish to cover the dish or the glass coverslip and incubate overnight at 37 °C.
   NOTE: This will reduce non-specific adherence in uncoated regions.
8. Aspirate off the 1% BSA solution, and the patterned substrate is now ready to use.

5. Indirect Patterning of Cells

1. Prepare the cell suspension in serum-free culture medium. The cell type and cell density are listed in Table 1.
2. Add the cell suspension to the patterned Petri dish and completely submerge the patterned region in cell suspension.
3. Incubate the petri dish at 37 °C in an incubator for 15 min to allow cells to attach to the patterned substrate.
4. Aspirate the excess cell suspension from the Petri dish and wash the patterned cells three times with phosphate buffered saline (PBS) while gently shaking for 10 s to remove unattached cells.
5. Add the appropriate culture medium to the patterned cell cultures and incubate the patterned cells at 37 °C in a CO₂ incubator.

6. Direct Patterning of Cells

NOTE: This technique is an alternative to the indirect cell patterning described in step 5. However, unlike in step 5, in this technique cells are patterned on tissue culture surfaces with or without substrate coating.

1. Sterilize the microfluidic device by rinsing with a solution of 70% of ethanol followed by DI water.
2. Soak the device overnight in a solution of 1% BSA to prevent the cell adhesion to the PDMS.
3. Dry the device at room temperature and attach it to the bottom of the tissue culture-treated petri dish.
4. Prepare the cell suspension in serum-free culture media. The cell type and cell density are listed in Table 2.
5. Place a droplet (4 - 8 µL) of the cell suspension, enough to fill the microchannels, on the inlet completely covering the inlet hole.
6. Put the petri dish in a vacuum for 10 min and observe air coming out of the channel in the form of bubbles. Release the vacuum and observe the cell suspension flowing into the microchannel.
7. Incubate the Petri dish in an incubator to promote cell adhesion as per the incubation conditions (depend on type of cell patterned) mentioned in Table 2. Add PBS to the petri dish submerging the PDMS device.
8. Peel the PDMS carefully off the petri dish with tweezers and wash the pattern with PBS. Add cell culture media to the Petri dish and place the cell culture in the incubator.

Results

This method allows the patterning of proteins and indirect patterning of cells using dead-end microfluidic channels with dimensions as small as 10 µm and equipment available in almost all biological laboratories once the master mold is made. This technique can be utilized with PDMS microfluidic channels created using traditional soft photolithography, or with PDMS microfluidic channels created with adhesive tape fabrication (Figure 1)^28,<...

Discussion

While conventional photolithography is a well-established technique for the creation of molds for soft lithography, the equipment, materials, and skills necessary to use conventional photolithography are not readily available to most laboratories. For laboratories without access to these resources, we have presented adhesive tape fabrication as a method of creating molds with relatively simple features for microfluidic devices. This method allows any laboratory to create and utilize microfluidic devices for research purp...

Materials

Name	Company	Catalog Number	Comments
CorelDRAW X4 CAD Drawing Tools	Corel Corporation, Canada	X4 Version 14.0.0.701	CAD tool used to draw the layout of the microfluidic device
Laser Printer HP	Hewlett Packard, CA	1739629	Used to print the layout of microfluidic device for adhesive tape technique
Bel-Art Dessicator	Fisher Scientific, MA	08-594-16B	Used to degass the PDMS mixture
Adhesive Scotch Tape	3M Product, MN	Tape 600	Used to fabricate adhesive tape Master
PDMS Sylgard 184	Dow Corning, MI	1064291	Casting polymer
Petri Dish	Fisher Scientific, MA	08-772-23	Used to keep the mold to cast with PDMS
Stainless steel Scalpel (#3) with blade (# 11)	Feather Safety Razor Co. Ltd. Japan	2976#11	Used to cut the PDMS
Tweezers	Ted Pella, CA	5627-07	Used to handle the PDMS cast during peeling
Glass slides	Fisher Scientific, MA	12-546-2	Used as surface to pattern the Substrate
Glass slides	Fisher Scientific, MA	12-544-4	Used as surface to pattern the Substrate
Rubber Roller	Dick Blick Art Materials, IL	40104-1004	Used to attach adhesive tape on glass without trapping air bubbles
Laser Mask Writer	Heidelberg Instruments, Germany	DWL66fs	Used to fabricate quartz mask used in photolithography fabrication process
EVG Mask Aligner (Photolithography UV exposure tool)	EV Group, Germany	EVG 620T(B)	Used to expose the photoresist to UV light
Spin Coater Headway	Headway Research Inc, TX	PWM32-PS-CB15PL	Used to spin coat the photoresist on silicon wafer
Photoresists SU-8 50	MicroChem, MA	Y131269	Negative photoresist used for mold fabrication
SU-8 Devloper	MicroChem, MA	Y020100	Photoresist developer
Tridecafluoro-1,1,2,2-Tetrahydrooctyl-1-Trichlorosilane	UCT Specialties, PA	T2492-KG	Coat mold to avoid PDMS adhesion
Isopropanol	Sigma-Aldrich, MO	190764	Cleaning Solvent
Ethanol	Sigma-Aldrich, MO	24102	Sterilization Solvent
Poly-D-Lysine hydrobromide (PDL)	Sigma-Aldrich, MO	P0899-10MG	PDL solution is made at 0.1 mg/mL in Sodium Tetraborate Buffer
Laminin	Sigma-Aldrich, MO	L2020	Laminin aliquoted into 10 µL aliquots and diluted to 20 µg/µL in PBS prior to use
BSA	Fisher Scientific, MA	BP1605100	Cell culture
C2C12 Myoblast cell lline	ATCC, VA	CRL-1722	Used to demonstrate C2C12 patterning
PC12 Cell Line	ATCC, VA	CRL-1721	Used to demonstrate PC12 patterning
Collagen type 1, rat tail	BD Biosciences	40236	Cell culture
DMEM	GIBCO, MA	11965-084	Cell culture
Horse Serum, heat inactivated	Fisher Scientific, MA	26050-070	Cell culture
Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC)	Sigma-Aldrich, MO	P1951	To label cells
Calcein-AM live dead cell Assay kit	Invitrogen, MA	L-3224	Cell viability Assay
Biopsy Hole Punch	Ted Pella, CA	15110-10	Punched hole in PDMS