Name: mechanical stimulation of stem cells using cyclic uniaxial strain
Uploaded: 2007-07-29T00:00:00
Description: Watch this Scientific Journal Video about Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain at JoVE.com

<p class="jove_title">Day 0 - Sterilization before day of experiment</p>
<ol>
<li>Put materials into plastic tub and sterilize with 70% alcohol for 2 hours: 
<ul>
<li>Chambers</li>
<li>Lids</li>
<li>Frames (all 3 pieces)</li>
<li>Screws</li>
<li>Rubber gaskets</li>
<li>Forceps</li>
<li>Scissors</li>
<li>Hex wrenches</li>
</ul>
</li>
<li>Clean membranes with Aquet soap and distilled water.</li>
<li>Sonicate membranes in 70% alcohol for 10 minutes.</li>
<li>Place the clean membranes into plastic square dishes. If using patterned membranes, ensure...

<ol>
	<li>Banes, A. J., Gilbert, J., Taylor, D., Monbureau, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+vacuum-operated+stress-providing+instrument+that+applies+static+or+variable+duration+cyclic+tension+or+compression+to+cells+in+vitro.">A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro.</a> <em style='font-style: italic'>J Cell Sci</em>. <b>75</b>, 35-42 (1985).</li><li>Park, J. S., Chu, J. S., Cheng, C., Chen, F., Chen, D., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+equiaxial+and+uniaxial+strain+on+mesenchymal+stem+cells.">Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells.</a> <em style='font-style: italic'>Biotechnol. Bioeng</em>. <b>88</b>, 359-368 (2004).</li><li>Kurpinski, K., Chu, J., Hashi, C., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anisotropic+mechanosensing+by+mesenchymal+stem+cells.">Anisotropic mechanosensing by mesenchymal stem cells.</a> <em style='font-style: italic'>Proc. Natl. Acad. Sci. U S A</em>. <b>103</b>, 16095-16100 (2006).</li></ol>

<p class="jove_content">Banes et al. first reported the use of a system for mechanical stimulation of cells in vitro by using a flexible elastomeric substrate to deliver mechanical force to cells<sup>1</sup>. Since this time, many variations on this design have been conceived and utilized. Several mechanical stretch systems are commercially available under the name "Flexercell" (Flexcell International Corp.), while some labs use custom-built devices. In this video protocol we have described the setup and use of one such device in our lab.</p>
...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fungizone</td>
<td>Reagent</td>
<td>Lonza</td>
<td>17-836R </td>
<td>aliquot is 250 ug/mL, 100x, stored in -20C.</td>
</tr>
<tr>
<td>Kanamycin</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15160-054</td>
<td>50 ug/mL final concentration. Store stock solution at 10 mg/ml in -20C.</td>
</tr>
<tr>
<td>Gentamicin</td>
<td>Reagent</td>
<td>Lonza</td>
<td>17-518Z </td>
<td>50 ug/mL final concentration. Store 1000x stocks at 50 mg/mL in -20C. </td>
</tr>
<tr>
<td>Pen/Strep</td>
<td>Reagent</td>
<td>Gibco</td>
<td>15140-122</td>
<td>1% final. (Also available from other companies)</td>
</tr>
<tr>
<td>Dulbecco's Modified Eagle's Medium</td>
<td>Reagent</td>
<td>Gibco</td>
<td>11966-025</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
			Notes on media:
These uniaxial stretch experiments are prone to contamination due to the complexity of bioreactor setup.  To combat this, we have tried various combinations of antibiotics and antifungal agents. The 1% fungizone usually necessary, along with a combination of antibiotics.  Either use a Pen/Strep combination, OR use a combination of Kanamycin and Gentamicin (but DO NOT try using a combination/cocktail  of three or more antibiotics together as it may lead to resistant mutant bacteria, and may also be detrimental to the cells).		</div>
		

mechanical stimulation of stem cells using cyclic uniaxial strain

<p class="jove_content">The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.</p>

Watch this Scientific Journal Video about Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain at JoVE.com

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

<p class="jove_content">It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.</p>

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

Research

JoVE Journal

Biology

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Patterning of Embryonic Stem Cells Using the Bio Flip Chip

Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such ...

Live Cell Response to Mechanical Stimulation Studied by Integrated Optical and Atomic Force Microscopy

To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to ...

Analysis of Pluripotent Stem Cells by using Cryosections of Embryoid Bodies

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst-stage early mammalian embryos 1. A crucial stage in the ...

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

Micro-Mechanical Characterization of Lung Tissue Using Atomic Force Microscopy

Matrix stiffness strongly influences growth, differentiation and function of adherent cells1-3.  On the macro scale the stiffness of tissues and organs ...

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

Omprogrammering humane somatiske celler i induceret pluripotente stamceller (IPSC&#39;er) under anvendelse af retroviral vektor med GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Mekanisk stimulering af chondrocyt-agarose Hydrogeler

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Mekanisk Stimulation-induceret Calcium bølgeudbredelse i cellemonolag: eksempel med Bovin hornhindeendothelceller

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...