Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

Summary

We present a new polyacrylamide hydrogel, called hydroxy-PAAm, that allows a direct binding of ECM proteins with minimal cost or expertise. The combination of hydroxy-PAAm hydrogels with microcontact printing facilitates independent control of many cues of the natural cell microenvironment for studying cellular mechanostransduction.

Abstract

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their extracellular matrix (ECM) environment. Eukaryotic cells constantly sense their local microenvironment through surface mechanosensors to transduce physical changes of ECM into biochemical signals, and integrate these signals to achieve specific changes in gene expression. Interestingly, physicochemical and mechanical parameters of the ECM can couple with each other to regulate cell fate. Therefore, a key to understanding mechanotransduction is to decouple the relative contribution of ECM cues on cellular functions.

Here we present a detailed experimental protocol to rapidly and easily generate biologically relevant hydrogels for the independent tuning of mechanotransduction cues in vitro. We chemically modified polyacrylamide hydrogels (PAAm) to surmount their intrinsically non-adhesive properties by incorporating hydroxyl-functionalized acrylamide monomers during the polymerization. We obtained a novel PAAm hydrogel, called hydroxy-PAAm, which permits immobilization of any desired nature of ECM proteins. The combination of hydroxy-PAAm hydrogels with microcontact printing allows to independently control the morphology of single-cells, the matrix stiffness, the nature and the density of ECM proteins. We provide a simple and rapid method that can be set up in every biology lab to study in vitro cell mechanotransduction processes. We validate this novel two-dimensional platform by conducting experiments on endothelial cells that demonstrate a mechanical coupling between ECM stiffness and the nucleus.

Introduction

Many aspects of the local cellular microenvironment (e.g., rigidity, pore size, nature of proteins, or cell-ligand density) provide a coordinate set of regulatory cues that control cellular processes such as motility, cell proliferation, differentiation, and gene expression. Modifications of the physicochemical properties of the extracellular environment can be perceived by cells and cause different physiological consequences, including deformation of cellular polarization, migration, and differentiation. It remains unclear, however, how cells translate ECM modifications into cellular biochemical signals. It is therefore of major importance to engineer controlled in vitro microenvironments that can reproduce the interactions between cells and their microenvironment for studying mechanotransduction pathways. To address this problem, we have recently introduced a novel method¹, called hydroxy-PAAm hydrogels, to easily generate two-dimensional soft matrices that permit to independently control important mechanotransduction cues: matrix stiffness, cell geometry and confinement, nature of the protein and cell-ligand density.

ECM directs cellular processes via gradients in morphogens (chemotaxis), adhesive proteins (haptotaxis), and stiffness (durotaxis). Over the last few decades, advanced in vitro platforms have been developed to isolate these extracellular cues in order to tease out how cells are able to translate biochemical and biophysical features into physiological processes^2-5. Electron-beam⁶, photolithography⁷, photochemical immobilization⁸, or plasma-assisted techniques⁹ have been developed to direct the growth of living cells on micropatterned substrates. Although these techniques have yielded important results, most of them do not allow discrimination between the individual influence of different cues on cell behavior and they require technical facilities that few laboratories can afford. Among these techniques, microcontact printing (µCP), has emerged as a robust and accessible method to create cell-adhesive micro-islands¹⁰. More recently, extensive efforts^11-14 have been made to develop µCP on hydrogels with tunable rigidities in order to reproduce the wide range of rigidities observed in living tissues. Among these works, polyacrylamide (PAAm) has become popular¹⁵ and is already one of the most commonly used polymer-based matrices for cell biomechanics assays.

PAAm surfaces are commonly functionalized with the heterobifunctional cross-linker N-sulfosuccinimidyl-6-[4'-azido-2'-nitrophenylamido] (sulfo-SANPAH) and ECM proteins are linked to the surface by UV activation of the sulfo-SANPAH nitrophenyl azide groups¹⁶. Another technique consists in coupling hydrazine to proteins that have been severely oxidized with periodate¹⁷. Hynd and coworkers introduced a technique for patterning biomimetic hydrogel surfaces with protein and peptides that requires photopolymerization in presence of an acroyl-streptavidin monomer¹⁸. More recently, Tseng et al. have reported a new micropatterning method¹⁹ based on deep UV exposure of PAAm through an optical quartz mask that requires to incubate activated PA gels with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) water solutions prior to add the protein. Despite the ability of these techniques to create homogeneous and reproducible proteins micropatterns, most of them suffer major limitations: long synthesis processes (e.g., dialysis, lyophilization, etc), expensive chemical compounds (e.g., hyaluronic acid, sulfo-SANPAH) or deep UV irradiation. In addition, these techniques do not allow independent modulation of substrate stiffness, micropattern geometry, ECM protein nature, and cell-ligand density.

Taking these limitations into account, we have developed a novel and simple acrylamide-based approach that allows immobilization of a variety of proteins and biomolecules on soft hydrogels and permits independent tuning of mechanotransduction cues in order to decipher their role on cellular functions. Instead of treating PAAm hydrogels with harsh chemical compounds, we introduce a commercial acrylamide monomer with hydroxyl groups during PAAm polymerization. This simple operation overcomes the intrinsic anti-adhesive property of PAAm hydrogels without any other technical requirements.

The presence of hydroxyl groups leads to a high affinity of hydroxy-PAAm hydrogels for proteins and biomolecules that form hydrogen-bonding interactions. In combination with µCP, hydroxy-PAAm hydrogels enable a rapid generation of two-dimensional culture platform with an independent control on matrix rigidity, type of ECM proteins, cell-ligand density and confined adhesiveness, which are envisioned to be a powerful platform for studying mechanotransduction.

The purpose of this protocol is to provide the necessary information for easily making hydroxy-PAAm hydrogels without any expertise in material sciences. The ultimate goal is to provide a means for researchers to ask physiologically relevant questions at the cellular and tissue levels that may lead to a better understanding of mechanotransduction pathways involved in pathophysiological mechanisms.

Protocol

1. Activating the Surface of Glass Coverslips

1. Place glass circular coverslips (25 mm diameter) in a Petri dish and smear 0.1 M NaOH solution on it for 5 min (chemical fume hood recommended).
2. Remove the NaOH solution and fully immerse coverslips with sterile ddH₂O for 20 min while gently rocking on a rocking plate in a sterile culture hood.
3. Drain sterile ddH₂O and repeat the step 1.2.
4. Remove the coverslips with sterile tweezers and place them in a new Petri dish with the activated face up.
5. Dry coverslips under a steady flow of high-purity nitrogen gas.
6. In a sterile culture hood, smear a thin layer of 3-(trimethoxysilyl)propyl acrylate (92%) on the activated side of the coverslip for 1 hr.
7. Extensively wash glass coverslips with 3 washes of sterile ddH₂O and immerse them in sterile ddH₂O in a new Petri dish.
8. Tap the Petri dish with Parafilm and place it on a rocker plate under gentle agitation for 10 min.
9. Remove the coverslips from ddH₂O with sterile tweezers with fine tips and place them in a new Petri dish with the activated face up.
10. Store at RT in a dry place with aluminum foil to avoid dust from sticking to the coverslips.

2. Preparation of Hydroxy-PAAm Hydrogels

1. Prepare a weight of 65 mg of N-hydroxyethyl acrylamide (HEA) in a 1.5 ml Eppendorf tube. It is important to prepare a fresh HEA solution.
2. Add 1 ml of 50 mM HEPES buffer to HEA and mix using a vortexer until the complete HEA dissolution.
3. Add 400 µl of 40% w/w in HEPES acrylamide solution and the required volume of 2% w/w in HEPES bis-acrylamide solution (see Table 1) to reach the desired hydrogel stiffness. Adjust with 50 mM HEPES to a final volume of 5 ml.
4. Mix the solution using a vortexer and degas it in a vacuum chamber for 20 min in order to reduce oxygen concentration within the solution, which prevents hydroxy-PAAm polymerization.
5. Under a sterile hood, filter the degased solution with a 0.2 µm pore size filter in order to sterilize it.
6. Activate circular glass coverslips (22 mm diameter) in a UV/ozone cleaner during 7 min.
7. Prepare 100 µl of 10% ammonium persulfate (APS) solution, that is 10 mg APS in 100 µl ddH₂O. It is important to prepare a fresh APS solution.
8. Add 2.5 µl of tetramethylenediamine (TEMED) and 25 µl of APS solution to the sterilized hydroxy-PAAm solution (step 2.5) to initiate the polymerization. Mix the solution by 3 successive pipettings without introduction of bubbles, under sterile conditions.
9. Under a sterile hood, place a 25 µl drop of the hydroxy-PAAm solution on a 25 mm coverslip (available from step 1.9) and immediately place a 22 mm glass coverslip (prepared on step 2.5) on top of the droplet to squeeze the hydroxy-PAAm solution. Center the 22 mm glass coverslip with sterile tweezers and smooth out any bubbles.
10. Allow hydroxy-PAAm hydrogels to polymerize at RT for 15 min. Invert manually the remaining hydroxy-PAAm solution in the Eppendorf tube to follow the completion of the polymerization process.
11. Fully immerse coverslips with sterile ddH₂O and carefully separate the 22 mm glass coverslips by introducing the edge of a razor blade between the 22 mm glass coverslips and the hydroxy-PAAm hydrogel layer.
12. Wash hydroxy-PAAm hydrogels with sterile PBS (3 exchanges of PBS) and let the gels fully immersed in sterile PBS to maintain hydration.
13. Store hydroxy-PAAm hydrogels in sterile PBS at 4 °C for up to 3 days.

3. Polydimethylsiloxane (PDMS) Microstamp Fabrication

NOTE: The fabrication of a silicon master is required prior to start the PDMS microstamp fabrication. This microfabrication of a silicon master can be done by lithographic techniques, which requires specialized equipment and training. Collaborations with a nanofabrication facility are encouraged to fabricate the silicon master. Alternatively, contact a company that fabricates custom-made microstructured silicon masters on demand. It is important to note that the fabrication of the silicon master needs only to be done once. Indeed, microstructured silicon masters can be used indefinitely to produce elastomeric stamps.

1. Mix PDMS and the curing agent in a 10:1 ratio in a plastic beaker and mix thoroughly using a pipette for 10 min.
2. Degas the PDMS mixture under vacuum to remove air bubbles that were formed during the step 3.1.
3. Place the microstructured silicon master in a Petri dish and cast a 10-mm thick layer of degassed PDMS mixture on it without forming bubbles.
4. Let cure the PDMS for 2 hr at 60 °C in an oven.
5. In a dust-free environment, peel-off the PDMS layer and excise 1 cm² microstamps with a scalpel.
6. Using forceps, place PDMS microstamps pattern-up in a Petri dish.

4. Micropatterning Hydroxy-PAAm Hydrogels

1. Place PDMS microstamps in an ethanol/water (50/50) solution and sonicate for 15 min.
2. Dry the stamps with a stream of nitrogen flow and place them pattern-up in a UV/Ozone cleaner (λ < 200 nm) for 7 min.
3. Under a sterile hood, place a 150-µl drop of a desired protein solution (e.g., 100 µg/ml laminin in PBS or 25 µg/ml fibronectin in PBS) onto the microstructured surface of a 1-cm² PDMS stamp.
4. Optional: Modify the concentration of the protein solution to modulate the cell-ligand density.
5. Spread the protein solution across the stamp surface by moving it with a sterile tip of a pipette toward each corner of the stamp.
6. Leave the protein solution to adsorb on the PDMS stamp for 60 min under a sterile hood. Turn off lamps to avoid protein damage.
7. Under a sterile hood, transfer hydroxy-PAAm coated coverslips (available from step 2.13) into a Petri dish.
8. Remove excess PBS from the surface of hydroxy-PAAm substrates with a low nitrogen stream under sterile conditions. Stop the procedure as soon as no evidence of standing water on the gel surface is observed. The gel should not be dried thoroughly at this stage.
9. Dry carefully the structured surface of the PDMS stamp with a steady flow of high-purity nitrogen gas.
10. Grasp the protein-coated stamp with dressing tissue forceps and place the structured surface in contact with the dried hydrogel surface. Apply brief pressure points with the tip of tweezers on the top of the PDMS stamp to ensure a good contact between stamp microfeatures and the hydrogel surface.
11. Leave the PDMS stamp on the hydrogel surface for 1 hr at RT.
12. Gently remove PDMS stamps from hydroxy-PAAm hydrogels with dressing tissue forceps and follow the step 4.1 to clean the stamp.
13. Wash extensively the stamped hydroxy-PAAm hydrogels by 3 exchanges of PBS (pH = 7.4) in sterile conditions for 10 min per exchange.
14. Optional: Additional micropatterns of other ECM proteins can be added to the hydroxy-PAAm surface by following steps 4.5 to 4.11.
15. Passivate non-printed zones with a sterile solution of BSA at 5 mg/ml in PBS during one night at 4 °C under a gentle agitation on a rocking plate.
16. Wash extensively by 3 exchanges of PBS (pH = 7.4) in sterile conditions for 10 min per exchange. At this stage, stamped hydroxy-PAAm hydrogels can be stored at 4 °C up to one week.

5. Cell Deposition on Micropatterned hydroxy-PAAm Hydrogels

1. Incubate coverslips in cell media for 30-45 min prior to plating cells.
2. Wash adherent cells cultured in a 75 cm² culture flask with sterile PBS at 37 °C and detach it with 3 ml of trypsin-EDTA or accutase for 10 min.
3. Transfer the desired amount of pre-warmed complete growth medium appropriate for your cell line into the flask containing the detached cells and centrifuge the cell suspension for 3 min at 650 x g.
4. Remove the supernatant with a micropipette and resuspend the cells in complete culture medium at 15-20,000 cells/ml.
5. Add 4 ml of the cell solution to a micropatterned coverslip (obtained from Step 5.1) and place the cell-covered coverslip in a culture incubator at 37 °C and 5% humidity for 1-2 hr.
6. Aspirate gently unattached cells and replace culture medium. Return the attached cells to the incubator and let them spread fully (3-6 hr, depending on the cell type).

Results

Figure 1A presents the co-polymerization of acrylamide (AAm) and bisacrylamide (bis-AAm) with N-hydroxyethylacrylamide (HEA) monomers containing a primary hydroxyl formed by random radical polymerization a hydrophilic network of polyacrylamide with embedded hydroxyl groups (hydroxy-PAAm). In this protocol, a weight 65 mg of HEA must be diluted in a volume of 1 ml of HEPES. Knowing that the density of HEA is roughly equal to one, we assume that we obtain a working volume of 1,065 µl (HEA+HE...

Discussion

Many in vitro observations in modern cell biology have been performed on rigid glass coverslips, often coated with a thin layer of ECM proteins or synthetic peptides containing the RGD sequence. However, such basic culture substrates do not recapitulate the whole physicochemical complexity of the ECM and thus do not provide an accurate model for studying cellular mechanotransduction processes. To tackle this problem, we propose a simple alternative to functionalize two-dimensional hydrogels with any desired amou...

Materials

Name	Company	Catalog Number	Comments
UV/Ozone Photoreactor	Ultra-Violet Products	Model PR-100
Rocking plate	IKAcWerke	Model KS 130 Basic
Vortexer	Scientific Industries	Model Vortex Genie2
Vacuum degassing chamber	Applied Vacuum Engineering	DP- 8-KIT
Parafilm	Sigma-Aldrich	P7793-1EA
Stainless steel forceps with fine tip	Sigma-Aldrich	Z225304-1EA
Dressing tissue forceps	Sigma-Aldrich	F4392-1EA
Petri dishes in polystyrene	Sigma-Aldrich	P5731-500EA
Aluminium foil, thickness 0.5 mm	Sigma-Aldrich	266574-3.4G
Isopore membrane filter (0.2 µm pore size)	Millipore	GTTP Filter code
Round glass coverslip (22 mm diameter)	Neuvitro	GG-22
Round glass coverslip (25 mm diameter)	Neuvitro	GG-25
Variable volume micropipette	Sigma-Aldrich	Z114820
Protein microcentrifuge tubes	Sigma-Aldrich	Z666505-100EA
Scalpel handles	Sigma-Aldrich	S2896-1EA
Scalpel blades	Sigma-Aldrich	S2771-100EA
Cell culture flasks (75 cm2)	Sigma-Aldrich	CLS430641
Ultrasonic bath tray, solid (stainless steel)	Sigma-Aldrich	Z613983-1EA
Polydimethylsiloxane	Dow Corning	Sylgard 184 silicone elastomer kit
Acrylamide (powder)	Sigma-Aldrich	A3553
N,N’-Methylenebis(acrylamide)	Sigma-Aldrich	146072
N-Hydroxyethylacrylamide	Sigma-Aldrich	697931
N,N,N’,N’-Tetramethylethylenediamine	Sigma-Aldrich	T9281
Amonium PerSulfate (APS)	Sigma-Aldrich	A3678
3-(Trimetoxysilyl)propyle acrylate	Sigma-Aldrich	1805
Human Plasma Fibronectin	Millipore	FC010
Laminin from EHS	Sigma-Aldrich	L2020
Sodium hydroxyde	Sigma-Aldrich	221465-25G
Double-distilled water (ddH2O)
Endothelial cell growth medium	Cells Applications	211K-500
Human Umbilical Vein Endothelial Cells (HUVEC)	Invitrogen	C-003-5C
Accutase	PAA laboratories	L11-007
HEPES buffer solution 1 M in H2O	Sigma-Aldrich	83264-500ML-F
Antibiotics-antimycotics	PAA laboratories	P11-002
Phosphate Buffer Saline solution	PAA laboratories	H15-002
Alexa Fluor 488 Phaloidin	Molecular Probes	A12379
Anti-vinculin antibody produced in mouse	Sigma-Aldrich	V9131
Goat anti-mouse antibody-tetramethylrhodamine	Molecular Probes	T-2762
Anti-Fibronectin (rabbit)	Sigma-Aldrich	F3648
Streptavidin	Sigma-Aldrich	41469
Anti-Laminin antibody (rabbit)	Sigma-Aldrich	L9393
Anti-rabbit IgG-FITC	Sigma-Aldrich	F7512
Trypsin-EDTA solution	Sigma-Aldrich	T3924-100ML
Absolute ethanol	Sigma-Aldrich	459844-2.5L