A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Summary

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Abstract

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

Introduction

The mechanical microenvironment plays an important role in many cell functions such as proliferation, migration, and differentiation, which have a profound impact in the development and homeostasis of tissues and also in diseases^1-6. Over the years, a multitude of experimental tools have been used to mechanically stimulate cells or tissues and measure mechanical properties of biological tissues with the goal of increasing our understanding of basic mechanobiology and studying the onset and progression of diseases^6-17. However, one must often rely on several different experimental devices in order to achieve the goals of a particular study. This a....

Protocol

1. Mechanical Deformation of Single Cells

1. Fabrication of a PDMS Substrate with Embedded Fluorescent Beads
   Prior to fabrication of the substrate, fluorescent microspheres in water solution are resuspended in isopropanol to enhance bead mixing in the PDMS due to its hydrophobic nature.
   1. Pipette 500 μl of fluorescent microspheres into a 1.5 ml microcentrifuge tube and centrifuge at 16,200 x g for 10 min.
   2. Discard the supernatant and add 500 μl of isopropanol followed with 5 min of vortexing. Put the vial aside overnight in the dark in order to allow any large particles aggregates to sediment.
   3. The next morning, carefully re....

Results

Cell Stretching

The BAXS platform was used to investigate the mechanical response of the nucleus in single mouse myoblast cells (C2C12) exposed to a substrate deformation of 25%. Myoblast cells are found in muscle tissue and are constantly exposed to mechanical stretch and compression in vivo. The shape and mechanical properties of the cell nucleus have shown to play a major role in the regulation of gene expression and transcriptional activity^20,21 and als.......

Discussion

The BAXS platform presented here facilitates numerous experiments in the study of mechanobiology, from investigations of single cells to whole tissues. In addition, the platform is highly flexible and configurable, allowing for numerous mechanical stimulation experiments and multi-axial tensile testing. The platform also enables the maintenance of cells and tissues in physiological conditions and allows for simultaneous microscopy during stretching experiments. The two experiments described in the previous sections demon.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PDMS	Ellsworth Adhesives	184 SIL ELAST KIT 0.5KG	The ratio base to cross-linker used in this protocol is 20:1. Mix in a laminar hood to keep dust from contamining your
FluoSpheres fluorescent microspheres	Invitrogen	F8810	Keep away from light.
Linear voice coil	Moticont	LVCM-051-051-01	The motro comes in two pieces (magnet and coil). It has to be mounted on a ball bearing sytem to be functional.
Ball bearing slide	Edmund Optics	NT37-360	Miniature and Small Linear Motion Ball Bearing Slides
Linear positioning stage	Edmund Optics	38-960	Center Drive 1.25" Square Linear Translation Stages
Optical encoder	GSI microE systems	Mercury II 1600S - 0.5um resolution	reflective incremental encoder.
Motion controller	Galil	DMC-2143(DIN)-DC48 with AMP-20440	4 axis controller with a 4 axis amplifier
Load cell	Honeywell	31 low	miniature load cell with a range of 0-150 g
Insect minutiens pins (0.20 mm)	Pin Service Austerlitz Insect pins		Stainless steel pins that are bended in an opened triangle shape
SU-8 2050	Micro Chem	SU-8 2050	Permanent epoxy negative photoresist. Keep away from heat and light
Air-plasma treatment system	Glowresearch		Autoglow Oxygen Plasma System
Rat-tail collagen	Invitrogen	A10483-01	Collagen I, Rat Tail 5 mg/ml
Hoechst 33342	Invitrogen	R37605	DNA-specific fluorescent dye. Keep in the fridge.
Kapton (Polyimide Film) Insulated Flexible Heaters	omega.ca	KHLV-0504/(10)-P	28 V flexible heaters; can be supplied with a 24 V
1/16 DIN Autotune Temperature and Process Controllers	omega.ca	CN63200-R1-LV	Temperature controller; supply 24 V.
DMEM culture medium	Hyclone	SH3024301	Dulbecco’s Modified 30 Eagle Medium. Keep at 4 °C
Penicillin-Streptomycin	Hyclone	SV30010	Keep stock frozen. Keep working solution at 4 °C.
Fetal bovine serum (FBS)	Hyclone	SH3039603C	Keep frozen.
Trypsin 0.05%	Hyclone	SH30236.02	Keep frozen. Digestion of cell attachement proteins for subcultivation
Hepes	Wisent Inc	330-050-EL	HEPES-buffered salt solution
NaCl	Fisher Scientific	BP358-1	HEPES-buffered salt solution / Krebs physiological solution
KCl	Fisher Scientific	BP366-500	HEPES-buffered salt solution / Krebs physiological solution
MgSO4	Fisher Scientific	M65-500	HEPES-buffered salt solution / Krebs physiological solution
CaCl2	Fisher Scientific	C614-500	HEPES-buffered salt solution / Krebs physiological solution
Dextrose	Fisher Scientific	BP220-1	HEPES-buffered salt solution / Krebs physiological solution
NaHCO3	Fisher Scientific	BP328-1	Krebs physiological solution
KH2PO4	Fisher Scientific	BP362-500	Krebs physiological solution
Carbogen 95% O2/5% CO2	Lindle	DIN:02154749	Krebs physiological solution oxygenation
Nocodazole	Sigma	M1404	Microtubules depolymerization agent
Cytochalasin-D	Sigma	C8273	Actin filaments depolymerization agent
Anti-α-SMA-FITC	Sigma	F3777	Used to stain and quantify smooth muscle cells content
Picrosirius red stain	Fluka	43665	Used to stain and quantify collagen content