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 contro....

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 hoo....

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+HEPES). As presen.......

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

List of materials used in this article

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