Name: preparation of hydroxy-paam hydrogels for decoupling the effects of mechanotransduction cues
Uploaded: 2014-08-28T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

This work was supported by the Belgian National Foundation for Scientific Research (F.R.S.-FNRS) through &#8220;MIS Confocal Microscopy&#8221;, &#8220;Cr&#233;dit aux Chercheurs&#8221; grants and the &#8220;Nanomotility FRFC project&#8221; (no. 2.4622.11). T.G. doctoral fellowship is supported by the Foundation for Training in Industrial and Agricultural Research (FRIA). The authors gratefully acknowledge Sylvain Desprez for mechanical characterization and G&#233;raldine Circelli for confocal imaging.

1. Activating the Surface of Glass Coverslips
<ol>
	<li>Place glass circular coverslips (25 mm diameter) in a Petri dish and smear 0.1 M NaOH solution on it for 5 min&#160;(chemical fume hood recommended).</li>
	<li>Remove the NaOH solution and fully immerse coverslips with sterile ddH2O for 20 min&#160;while gently rocking on a rocking plate in a sterile culture hood.</li>
	<li>Drain sterile ddH2O and repeat the step 1.2.</li>
	<li>Remove the coverslips with sterile tweezers and place them in a n...

<ol>
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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...

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 processes2-5. 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 (&#181;CP), has emerged as a robust and accessible method to create cell-adhesive micro-islands10. More recently, extensive efforts11-14 have been made to develop &#181;CP 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&#39;-azido-2&#39;-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 &#181;CP, 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.

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			<td height="64" style="height:64px;width:216px;">UV/Ozone Photoreactor</td>
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			<td height="43" style="height:43px;width:216px;">Rocking plate</td>
			<td style="width:71px;">IKAcWerke</td>
			<td style="width:119px;">Model KS 130 Basic</td>
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			<td height="43" style="height:43px;width:216px;">Vortexer</td>
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			<td height="85" style="height:85px;width:216px;">Vacuum degassing chamber</td>
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			<td style="width:119px;">DP- 8-KIT</td>
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			<td height="43" style="height:43px;width:216px;">Parafilm</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P7793-1EA</td>
			<td style="width:167px;"></td>
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			<td height="43" style="height:43px;width:216px;">Stainless steel forceps with fine tip</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">Z225304-1EA</td>
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			<td height="43" style="height:43px;width:216px;">Petri dishes in polystyrene</td>
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			<td height="43" style="height:43px;width:216px;">Round glass coverslip (22 mm diameter)</td>
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			<td height="43" style="height:43px;width:216px;">Round glass coverslip (25 mm diameter)</td>
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			<td height="43" style="height:43px;width:216px;">Variable volume micropipette</td>
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			<td style="width:119px;">Z114820</td>
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			<td style="width:119px;">S2896-1EA</td>
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			<td height="43" style="height:43px;width:216px;">Name of the Reagent</td>
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			<td height="64" style="height:64px;width:216px;">Polydimethylsiloxane&#160;</td>
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			<td height="43" style="height:43px;width:216px;">N,N&#8217;-Methylenebis(acrylamide)</td>
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			<td style="width:119px;">146072</td>
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			<td style="width:119px;">697931</td>
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			<td height="43" style="height:43px;width:216px;">N,N,N&#8217;,N&#8217;-Tetramethylethylenediamine</td>
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			<td style="width:119px;">T9281</td>
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			<td height="43" style="height:43px;width:216px;">Amonium PerSulfate (APS)</td>
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			<td style="width:119px;">A3678</td>
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			<td height="26" style="height:26px;width:216px;">Double-distilled water (ddH2O)</td>
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		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Endothelial cell growth medium</td>
			<td style="width:71px;">Cells Applications</td>
			<td style="width:119px;">211K-500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Human Umbilical Vein Endothelial Cells (HUVEC)</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C-003-5C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Accutase</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">L11-007</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HEPES buffer solution 1M in H20</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">83264-500ML-F</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Antibiotics-antimycotics</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">P11-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Phosphate Buffer Saline solution</td>
			<td style="width:71px;">PAA laboratories</td>
			<td style="width:119px;">H15-002</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Alexa Fluor 488 Phaloidin</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">A12379</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-vinculin antibody produced in mouse</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">V9131</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Goat anti-mouse antibody-tetramethylrhodamine</td>
			<td style="width:71px;">Molecular Probes</td>
			<td style="width:119px;">T-2762</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Fibronectin</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">F3648</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Streptavidin&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">41469</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Laminin antibody</td>
			<td rowspan="2" style="width:71px;">Sigma-Aldrich</td>
			<td rowspan="2" style="width:119px;">L9393</td>
			<td rowspan="2" style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">(rabbit)</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Anti-rabbit IgG-FITC</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">F7512</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Trypsin-EDTA solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3924-100ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Absolute ethanol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">459844-2.5L</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

preparation of hydroxy-paam hydrogels for decoupling the effects of mechanotransduction cues

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.

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&#160;of HEPES. Knowing that the density of HEA is roughly equal to one, we assume that we obtain a working volume of 1,065 &#181;l&#160;(HEA+HE...

Watch this Scientific Journal Video about Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues at JoVE.com

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

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.

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

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