Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain
Summary
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.
Abstract
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.
Protocol
Discussion
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.
…Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Fungizone | Reagent | Lonza | 17-836R | aliquot is 250 ug/mL, 100x, stored in -20C. |
| Kanamycin | Reagent | Gibco | 15160-054 | 50 ug/mL final concentration. Store stock solution at 10 mg/ml in -20C. |
| Gentamicin | Reagent | Lonza | 17-518Z | 50 ug/mL final concentration. Store 1000x stocks at 50 mg/mL in -20C. |
| Pen/Strep | Reagent | Gibco | 15140-122 | 1% final. (Also available from other companies) |
| Dulbecco’s Modified Eagle’s Medium | Reagent | Gibco | 11966-025 |
References
- 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).
- 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, 359-368 (2004).
- Kurpinski, K., Chu, J., Hashi, C., Li, S. Anisotropic mechanosensing by mesenchymal stem cells. Proc. Natl. Acad. Sci. U S A. 103, 16095-16100 (2006).
