The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
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The Journal of Visualized Experiments (JoVE) is a peer reviewed, PubMed-indexed video journal. Our mission is to increase the productivity of scientific research.
Department of Bioengineering, University of California, Berkeley
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Kurpinski, K., Li, S. Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain. J. Vis. Exp. (6), e242, doi:10.3791/242 (2007).
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 µm 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.
Day 0 - Sterilization before day of experiment
Day 1 - Assembly of stretch chambers
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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 cells1. 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.
The custom-built "stretch machine" depicted in this protocol has been used in various studies to investigate the effects of uniaxial cyclic strain on different cell types2,3. This machine is a versatile apparatus with several adjustable parameters that can be used for a variety of mechanical strain studies. The setup depicted herein represents a unique and robust method for delivering uniaxial cyclic strain to adherent cells in culture.
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| Name | Type | Company | Catalog Number | Comments |
| Fungizone | Reagent | Lonza Inc. | 17-836R | aliquot is 250 ug/mL, 100x, stored in -20C. |
| Kanamycin | Reagent | GIBCO, by Life Technologies | 15160-054 | 50 ug/mL final concentration. Store stock solution at 10 mg/ml in -20C. |
| Gentamicin | Reagent | Lonza Inc. | 17-518Z | 50 ug/mL final concentration. Store 1000x stocks at 50 mg/mL in -20C. |
| Pen/Strep | Reagent | GIBCO, by Life Technologies | 15140-122 | 1% final. (Also available from other companies) |
| Dulbecco’s Modified Eagle’s Medium | Reagent | GIBCO, by Life Technologies | 11966-025 | |
| 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). | ||||
1. Banes, A.J., Gilbert, J., Taylor, D., Monbureau, O. A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. J Cell Sci. 75, 35-42 (1985).
2. Park, J.S., Chu, J.S., Cheng, C., Chen, F., Chen, D., Li S. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells. Biotechnol. Bioeng. 88, 359-68 (2004).
3. Kurpinski, K., Chu, J., Hashi, C., Li S. Anisotropic mechanosensing by mesenchymal stem cells. Proc. Natl. Acad. Sci. USA 103, 16095-16100 (2006).
very nice - thought you might find this article of interest:
Winter LC, Annals of Biomedical Engineering, 30:1242, 2002
by the way - what were the conditions (gas, time, temp) of your plasma treatment -
cheers
Joel D. Bumgardner, PhD, Associate Professor (jbmgrdnr@memphis.edu)
1
ReplyPosted by: AnonymousJuly 14, 2008, 9:33 AM