Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Summary

Here, a method that enables quick, efficient, and inexpensive preparation of polyacrylamide gels in a multiwell plate format is described. The method does not require any specialized equipment and could be easily adopted by any research laboratory. It would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Abstract

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 – 100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn’t require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses.

Introduction

Most tissues in the body are soft viscoelastic materials with a Young’s modulus ranging from 0.1 kPa for brain to 100 kPa for soft cartilage, yet, most in vitro cell research is conducted on tissue culture polystyrene (TCP) which has a modulus of ~1 GPa.¹ This stiffness mismatch greatly affects the way cells respond to their environment. A growing body of research is thus dedicated to elucidating the effect of substrate stiffness on the fate of various cell types,^2,3 including stem cells.⁴ As a result, multiple hydrogels have been developed to aid in the understanding of stiffness-dependent cell biology including polyacrylamide (PA),^5-7 polyethylene glycol (PEG),^8,9 polydimethylsiloxane (PDMS),¹⁰ and alginate.¹¹ While the evidence that substrate stiffness has a substantial impact on cell fate is growing, most studies are conducted on a small scale with a small number of samples. Systematic, multidimensional studies on the effect of substrate stiffness for an array of cell types or environmental conditions are rare.¹²

Several promising high-throughput hydrogel technologies have been developed, including PEG-based microarrays,¹³ microfluidic devices for the production of agarose hydrogel microbeads,¹⁴ or micro and nano-rods where stiffness is modulated by the diameter and height of the microrods.¹⁵ However, the technologies to prepare such substrates are sophisticated and available to limited number of laboratories. Much research involving stiffness modulated cell responses utilizes polyacrylamide (PA) gels which are not only inexpensive and simple to implement, but also exhibit a physiologically relevant range of Young’s modulus, namely 0.3 – 300 kPa.^16-22 However, existing methods to fabricate PA gels for cell culture are labor intensive and consequently prepared in small batches. Some of the difficulties associated with the preparation of PA gels as cell substrates stem from the requirement that the gels have to be prepared: 1) in the absence of oxygen to allow complete polymerization, 2) with a flat and smooth surface to permit uniform cell attachment and spreading, and 3) permanently affixed to the bottom of the cell culture dish to prevent floating.

Several groups have attempted to produce PA gels for cell culture in large batches. Semler et al. prepared thick sheets of PA gels which were then “cut” with a hole punch and placed into 96-well plates.²³ However, this method is limited to stiffer gels, i.e., > 1 kPa in Young’s modulus, because softer gels are “sticky”, difficult to cut, and easily damaged. Mih et al. developed a more sophisticated technique which allows the gels to be directly polymerized in a glass-bottom multiwell plate.⁶ This was achieved by pouring the gel solutions into functionalized glass-bottom plates and forming gels by “sandwiching” them with a custom coverglass array.⁶ Even though very promising, slight edge effects were still observed with this technique. Additionally, the technique requires a custom-designed array not immediately accessible to many laboratories as well as costly glass-bottom multiwell plates.

This paper describes a simple and inexpensive way to assemble PA gels in a multiwell plate that could be easily adopted by any laboratory. Here, a flexible plastic support is utilized, which has two sides — a hydrophobic one, which is repellent to PA gels, and a hydrophilic one, which covalently binds the PA gel upon deposition. Once PA gel sheets are deposited and permanently affixed to the flexible plastic support, it enables handling gels of any thickness or stiffness and cutting them into any desired shape. This approach not only produces custom plastic ‘coverslips’ in sizes not otherwise commercially available, but also obviates the necessity to pre-treat glass surfaces, either glass coverslips or the wells of costly glass-bottom multiwell plates, with a PA binding solution, which is a tedious and a time-consuming step. Lastly, uniform PA gels sheets can be prepared in large batches and stored de-hydrated for several months.

In summary, the assay presented here is an improvement over existing methods in several aspects. First, the process of multiwell plate assembly is efficient, and the overall cost of the required materials is low. Second, the hydrogels are produced in large batches in a single homogeneous gel film. Finally, only materials that are commercially available are required. The utility of the assay is illustrated by exploring the effect of substrate stiffness on cell morphology and spreading area.

Protocol

1. Preparation of Hydrogel-associated Solutions and Aliquots

1. Preparation of polyacrylamide gel precursor solution.
   1. Prepare polyacrylamide gel precursor solution by mixing acrylamide (A) (40% w/v, M_r 71.08 g/mol), the crosslinker bisacrylamide (B) (2% w/v, M_r 154.17 g/mol), and de-ionized water in the volume percentages specified in Table 1.
      NOTE: These solutions can be prepared in large batches and stored at 4 °C for up to several months.
      1. CAUTION: Acrylamide is toxic upon inhalation or ingestion, particularly, when in powder form: thus, preferably use 40% w/v solution to reduce toxicity risks. Handle only while wearing protective clothing, such as gloves, goggles, and a lab coat. Store in a light-resistant, tightly closed container in the fridge (<23 °C, well-ventilated area).
      2. CAUTION: Bis-acrylamide is toxic upon inhalation or ingestion, particularly, when in powder form: thus, preferably use 2% w/v solution to reduce toxicity risks. Handle only while wearing protective clothing, such as gloves, goggles, and a lab coat. Store in a light-resistant, tightly closed container in the fridge (<4 °C, well-ventilated area).
2. Preparation and usage of N-Sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate (Sulfo-SANPAH, M_r 492.40 g/mol) aliquots. CAUTION: Sulfo-SANPAH causes serious eye irritation; handle with gloves and proper eye or face protection. Store at -20 °C upon receipt and prior to aliquoting.
   1. To store sulfo-SANPAH: dissolve Sulfo-SANPAH in dimethylsulfoxane (DMSO) at 50 mg/ml. Aliquot the stock solution into 50 micro centrifuge tubes of 20 μl per tube. Flash freeze on dry ice or in liquid nitrogen (an optional but preferred step to preserve maximum crosslinker efficiency) and store at -80 °C.
      NOTE: The aliquots can be stored for several months.
   2. To use sulfo-SANPAH: thaw the aliquot briefly and dilute in 480 μl of de-ionized water. Use immediately. Sulfo-SANPAH hydrolyzes quickly in water: therefore take caution to perform all of the above steps quickly.
3. Preparation of ammonium persulfate (M_r 228.18 g/mol) aliquots.
   NOTE: Ammonium persulfate causes eye, skin and respiratory irritation. Wear appropriate personal protective equipment when handling. Handle ammonium persulfate powder in a chemical fume hood. Store powder in a dry, well-ventilated place. Once aliquoted, the dilute solution could be used on the work bench.
   1. Dissolve ammonium persulfate in deionized water to achieve a final ammonium persulfate concentration of 10% w/v. Aliquot and store at -20 °C. Thaw immediately prior to use.
4. Preparation of Collagen Type I solution.
   1. Prepare 0.2 mg/ml collagen solution by diluting the stock solution in 1x phosphate buffered saline (PBS) of pH 7.4. Store on ice briefly or use immediately upon dilution.

2. Hydrogel Preparation (Refer to Figure 1)

1. Preparation of hydrophobic glass slides.
   1. Place a few drops of a hydrophobic solution on a glass plate and use tissue paper to spread across the surface. Let air dry and wipe with a tissue paper again to even out the hydrophobic coating.
      NOTE: Store hydrophobic solution in a flammable cabinet at RT. Wear personal protective equipment when handling. Work in a well-ventilated area.
2. Preparation of flexible plastic support.
   1. Cut the flexible plastic support to match the size of the hydrophobic-coated glass plate.
   2. Mark the hydrophobic side of the flexible plastic support by lightly scratching the surface with a sharp tool such as a scalpel.
      NOTE: Once the gel — which is fully transparent — dries, the scratch marks will help distinguish which flexible plastic support side contains the gel.
      NOTE: The flexible plastic support has a hydrophobic and a hydrophilic side. Once deposited, polyacrylamide permanently adheres to the hydrophilic side of the plastic support which facilitates easy subsequent hydrogel handling.
3. Gel Preparation (example volumes are given for 5 ml gel precursor solutions).
   NOTE: 5 ml would be sufficient to produce ~40 gels when the gel initial thickness is 0.5 mm. More gels can be produced if the gel thickness is reduced.
   1. Place 4972.5 μl of polyacrylamide precursor solution of desired final concentration (Table 1) into a 50 ml conical tube. Place in a degassing chamber for 30 min with the cap opened.
      NOTE: Oxygen acts as a free radical trap and when present, it inhibits polymerization.
   2. Add 25 µl of 10% w/v ammonium persulfate (refer to point 1.3) to achieve a final ammonium persulfate concentration of 0.05%.
   3. Add 2.5 µl of N,N,N’,N’-tetramethylethylenediamine (TEMED, M_r 116.24 g/mol) to the degassed gel solution to achieve a final TEMED concentration of 0.5%.
      NOTE: Store TEMED in a flammable cabinet at RT. Handle in a chemical fume hood when wearing appropriate personal protective equipment. Keep tightly closed under inert gas, as it is highly air and moisture sensitive.
   4. Mix the solution gently by pipetting up and down 3 – 5 times. Do not vortex to avoid oxygen diffusion into the gel precursor solution.
   5. Pipette the gel solution onto the hydrophilic side of the flexible plastic support and sandwich with hydrophobic-coated glass slide. Separate the two slides with silicone spacers of desired thickness (e.g., 0.5 mm). Leave a small amount (~100 μl) of polymer precursor solution in the 50 ml conical tube to use as an indicator of gelation.
      NOTE: Any spacer could be used. For example, a single strip of Parafilm gives a final swollen gel thickness of 100 – 120 μm.
   6. Place another glass plate atop the flexible plastic support/gel sandwich to ascertain an even hydrogel surface upon polymerization.
   7. Let the gel polymerize for 45 min, although less time can be used for gels of higher % wt if desired. To ascertain that the gel has polymerized, observe the remaining solution in the 50 ml conical tube; if the remaining solution has gelled, then it is likely that gelation on the glass plate has been initiated as well. However, note that premature opening of the mold will prevent complete polymerization.
   8. Once the gel is formed, peel off the flexible plastic support with the covalently attached polyacrylamide gel on top, and set gel-side-up to air dry.
      NOTE: Convection or heat drying can also be used to accelerate this step. Once dried onto the flexible plastic support, the gel can be stored indefinitely.
      1. Mark any bare spots of the flexible plastic support, where polyacrylamide gels did not form due to bubbles. Mark while the gel is still hydrated as this will blend in with the flexible plastic support once the gel is dry.

3. Multiwell Plate Assembly, Collagen Coating, and Sterilization

1. Multiwell plate assembly.
   1. Once dried, cut PA gels into desired shapes.
      1. For a 96-well plate, use a heavy-duty hole punch with a diameter of ~6 mm. Use a heavy-duty paper cutter to cut gels into square or rectangular shapes. Alternatively, use scissors.
   2. Prepare approximately 500 μl of PDMS per 96-well plate according to the manufacturer’s instructions. To glue the gels to the bottom of a multiwell plate, place a small droplet (~5 μl) of polydimethylsiloxane (PDMS) at the center of the each well. Using forceps, place one polyacrylamide gel in each well, flexible plastic support side down. To allow PDMS to cure, leave the assembled plate at 37 °C for a minimum of 4 hr.
2. Collagen coating of polyacrylamide gels.
   1. With a transfer pipette, place a small amount (7 – 8 μl) of sulfo-SANPAH (refer to point 1.2.2) in each well and swirl from side to side to coat the gel surface evenly. Work promptly since sulfo-SANPAH is not stable in water.
   2. Place the well plate under a high intensity UV lamp (intensity = 37 mW, λ = 302 – 365 nm) for 5 min. Rinse the gels with PBS to remove excess sulfo-SANPAH.
   3. Pipette 50 µl of 0.2 mg/ml Collagen Type I solution (refer to point 1.4) into each well. Leave the plate covered at RT for at least 2 hr or O/N at 4 °C.
      NOTE: To speed up the process of collagen coating, a transfer pipette can be used to add the collagen solution. Typically, 1 – 2 droplets of solution should be enough to completely cover the gel surface.
   4. Leave the well plate at RT for at least 2 hr to allow collagen bonding.
   5. Rinse with PBS to remove excess collagen solution and sterilize under UV (λ = 200 nm) in a tissue culture hood for 2 hr.
   6. Soak the gels in complete medium (refer to section 4.1 for medium composition) O/N to hydrate and equilibrate. Use for cell seeding immediately or store in the fridge for up to 2 days.

4. Cell Seeding on PA Stiffness Assay

NOTE: Although typical for common mammalian cell lines, the protocol outlined in this section is specifically used with the breast cancer MDA-MB-231 cell line (see Figures 4 and 5).

1. Collect cells from tissue culture flask by exposure to 5% trypsin/EDTA for 5 min at 37 °C. Use ~80 μl of trypsin/EDTA per each cm² of culture flask area; for example, use 2 ml of trypsin/EDTA for a T25 cell culture flask. Re-suspend collected cells in complete medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at a desired final cell concentration. Taking into account cell doubling time as well as the length of time the cells will be seeded on the assay, choose an appropriate sub-confluent cell concentration. Ensure that added medium is enough to completely submerge the hydrogels.
   NOTE: Since the hydrogels have been pre-equilibrated in media (refer to step 3.2.6) 100 μl volume should be sufficient. Typical medium volumes used for multiwell plates should be sufficient since the gels have been pre-equilibrated in media in the previous step.
   NOTE: The assay would be appropriate for any attachment-dependent cell type.
   1. Count cell number under an inverted microscope by using a hemacytometer. Load 10 µl of cell suspension into each hemacytometer port and average the cell count from at least 8 quadrants. To get the final cell concentration, multiply the cell count by 10⁴.
      NOTE: Best cell count results are obtained when cell number in each hemacytometer quadrant is 20 – 50.
2. Culture the cells onto the PA stiffness assay in a humidified incubator at 37 °C and 5% CO₂. Change media every 2 – 3 days. Collect cells when needed by exposure to 5% trypsin/EDTA at 37 °C for 5 min. Use ~80 μl of trypsin/EDTA per cm² of tissue culture flask. During all cell manipulation steps, take special care not to disrupt the hydrogel surface: aspirate or pipette medium by slightly tipping the multiwell plate sideways and touching the pipette tip onto the side wall of each well, as opposed to touching the hydrogel.
   NOTE: Except for taking special care not to damage the hydrogel surface during standard tissue culture handling, the cells seeded onto the stiffness assay can be manipulated the same way as if they were seeded onto a regular multiwell plate.

5. Imaging of Cells Seeded onto PA Stiffness Assay

1. Image cells directly on the PA stiffness assay.
   NOTE: Any microscope — inverted, fluorescent, or confocal, can be used for cell imaging.
   NOTE: The flexible plastic support used to construct the stiffness assay is fully transparent and does not autofluoresce or interfere with cell imaging. However, even though the flexible plastic support itself is transparent, imaging capabilities will be limited by the working distance of the objective. The flexible plastic support has a thickness of 0.23 mm and a typical working distance of a 10X objective is ~4 mm, sharply decreasing for higher magnifications.
   1. For live cell imaging, position PA stiffness assay in microscope plate holder and image. Keep imaging sessions under 2 hr, or use a microscope equipped with an environmental chamber for longer imaging times.
   2. When live cell imaging is not the goal, fix cells in 4% formaldehyde solution supplemented with 0.1% detergent. Soak cells in fixative at RT for a minimum of 2 hr. Rinse with PBS twice. Discard fixative waste in a designated waste container.
      NOTE: Cells can be imaged immediately or stored submerged in PBS at 4 °C for up to 2 weeks. CAUTION: Formaldehyde is toxic upon inhalation and contact. Handle with gloves in a chemical fume hood.
      NOTE: Cell fixation protocol has to be chosen based on the subsequent cell staining and handling protocols, because certain fixatives damage some cell proteins.
   3. For fluorescent imaging, stain fixed cells with desired cell stain directly in the multiwell plate. Image immediately.

Results

Polyacrylamide (PA) hydrogels are widely used to test stiffness-dependent cell responses.^17,24 By mixing various concentrations of acrylamide (A) and bis-acrylamide (B) one can make PA gels that span the stiffness range of most soft tissues in the body — 0.3 – 300 kPa Young’s modulus.¹ However, preparation of polyacrylamide gels is tedious and time consuming, often limiting their usefulness in “high-throughput” applications such as for example drug screening.¹² Here, a simple and rapid me...

Discussion

Polyacrylamide gels, originally developed for electrophoresis,²⁸ are now routinely used as cell culture substrates to study the effects of substrate stiffness on cell morphology, motility, and communication^3,24,29 among other cell characteristics. Polyacrylamide allows manipulation of substrate stiffness to encompass the stiffness of all soft tissues in the body (0.3 – 300 kPa)¹ with a simple change in polymer precursor concentration (Figure 2, Table 1, also see ref...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
40% Acrylamide	Bio-Rad	161-0140
2% Bis-acrylamide	Bio-Rad	161-0142
Ammonium Persulfate	Bio-Rad	161-07000
TEMED	Sigma Aldrich	T9281
Sulfo-SANPAH	Thermo Scientific	22589
Collagen Type 1, from Rat tail, 3.68 mg/ml	BD Biosciences	354236
Dimethyl sulfoxide (DMSO)	Fisher Scientific	BP231-100
Hydrophobic solution — Repel Silane	GE Healthcare Bio-Sciences	17-1332-01
PBS (1x), pH 7.4	HyClone	SH30256.01
Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]	Elsworth Adhesives	3097358-1004
Tyrpsin/EDTA (10x)	Sigma Aldrich	44174
RPMI-1640 Medium (1x)	HyClone	SH30027-02
Fetal Bovine Serum	HyClone	SH30073-03
Penicillin Streptomycin	MP Biomedicals	1670046
Detergent: Triton-X	Sigma Aldrich	T8787
Formaldehyde 37% Solution	Sigma Aldrich	F1635
Bovine Serum Albumin (BSA)	Sigma Aldrich	A2153
BSA-based cell adhesion blocking kit — ECM Cell Adhesion Array Kit	Chemicon International	ECM540
Disposable lab equipment
flexible plastic support — GelBond PAG Film for Polyacrylamide Gels	GE Healthcare Bio-Sciences	309819
Glass Plates	Slumpys	GBS4100SFSL
50 ml conical tubes	Fisher Scientific	3181345107
15 ml conicals tubes	FALCON	352097
Micro centrifuge tubes	Fisher Scientific	2 ml: 02681258
96-well plate (flat bottom)	Fisher Scientific	12565501
Disposable Pipettes (1 ml, 2 ml, 5 ml, 10 ml, 25 ml, 50 ml)	Fisher Scientific	1 ml: 13-678-11B, 2 ml: 05214038, 5 ml (FALCON): 357529, 10 ml: 13-678-11E, 25 ml: 13-678-11, 50 ml: 13-678-11F
Glass Transfer Pipettes	Fisher Scientific	5 3/4": 1367820A, 9":136786B
Pipette Tips (1-200 μl, 101-1000 μl)	Fisher Scientific	2707509
Plastic Standard Disposable Transfer Pipettes	Fisher Scientific	13-711-9D
Parafilm	PARAFILM	PM992
Powder Free Examination Gloves	Quest	92897
Silicone spacers — Silicone sheet, 0.5 mm thick/13 cm x 18 cm	Grace Bio-Labs	JTR-S-0.5
Large/non-disposable lab equipment
Light and Flourescent Microscope (Axiovert 200M)	Zeiss	3820005619
Microscope Software	Zeiss	AxioVision Rel. 4.8.2
UV oven	UVITRON	UV1080
Vacuum chamber/degasser	BelArt	999320237
Vacuum pump for degasser	KNF Lab	5097482
Tissue Culture Hood	NUAIRE	NU-425-600
Chemical Fume Hood	KEWAUNEE	99151
Inverted Microscope (Axiovert 25)	Zeiss	663526
Incubator	NUAIRE	NU-8500
Pipette Aid	Drummond Scientific Co.	P-76864
Hemacytometer	Bright-Line	383684