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

Day 0 - Sterilization before day of experiment

1. Put materials into plastic tub and sterilize with 70% alcohol for 2 hours:
   • Chambers
   • Lids
   • Frames (all 3 pieces)
   • Screws
   • Rubber gaskets
   • Forceps
   • Scissors
   • Hex wrenches
2. Clean membranes with Aquet soap and distilled water.
3. Sonicate membranes in 70% alcohol for 10 minutes.
4. Place the clean membranes into plastic square dishes. If using patterned membranes, ensure that the patterned side is face up (spray alcohol onto one side of the membrane and watch for the liquid to run down the grooves).
5. Leave mem....

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.

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Materials

List of materials used in this article

Name	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).