I am working with conditionally immortalized human podocytes, and would like to subject them to some form of mechanical stress/pressure to recreate an environment that is 'comparable' to the glomerulus.
I'm trying to ascertain whether the idea is feasible, i.e. equipment, costing etc. The methods I've looked at seem quite complicated and refer to expensive software and specialised equipment. I'm hoping that someone can give me some advice; what is essential, what's not.
Any advice would be greatly appreciated.
I have been involved in experiments whereby mechanical strain (stretching) has been applied to bone cells. You are right in saying that the equipment can be expensive and over complicated.
I suspect podocytes would be subject to static biaxial stretch (tension) via pressure on the basement membrane in the glomerulus - in which case deforming cells by inducing a dome shape (use an upturned watch glass) on the bottom of a culture plate would be a place to start. Has the level of mechanical deformation (strain) in vivo ever been measured or calculated?
To ascertain whether podocytes respond to mechanical stimulation, and thereby giving you a stronger argument to pursue that line of investigation, it should not cost much to make your own system - a row of upturned watch glasses, a cassette to hold the culture plates and a method of reproducibly forcing the plates down onto the glasses (a weight), or pushing the glasses up to the plates. You could get a quasi dose:response by altering the amount of weight applied.
Good luck.
Sorry for this,
I have not got any experience with cells subjecting to mechanical stress. I have an experience about the topography and the viscoelastic (Young's modulus and hysteresis) and electrical passive (membrane capacitance, C(m)) properties of the C2C12 cell surface.
The article is 'Role for stress fiber contraction in surface tension development and stretch-activated channel regulation in C2C12 myoblasts' by Sbrana F, Sassoli C, Meacci E, Nosi D, Squecco R, Paternostro F, Tiribilli B, Zecchi-Orlandini S, Francini F, Formigli L. Am J Physiol Cell Physiol. 2008 Jul;295(1):C160-72. Epub 2008 May 14 and is visible on Pubmed.
Good work!!
Alessia
Hi Emma,
Simon Rawlinson is right, the capital outlay for the equipment etc is expensive, as are the consumables. It would be better to do a pilot experiment as he suggests, the other thing you might think about however is not just the amount of deformation (i.e. strain), but also the number of times deformation is applied!
Good luck
A P.I. I use to work for has a machine that does this. Here is his site [http://www.georgiahealth.edu/centers/vbc/black.html]
, search on the page (control-f) for "Shear". Hope this helps.
I think methods have been published in the fiels of phospoho-calcic see ASBMR or Journal of bone and mineral research
best regards, AD
I have experience with plant cells and protoplasts and the mechanical stress was different levels of gravity force. If it help you in your project, let me know. Intactness and viability were our main measures.
Well, I applied pressure on my cell line (corneal epithelial cells) in a different way. My technique was to utilize a u-tube manometer incorporated into the pressure system which also included the input of gas mix and a pressure box to contain the gas. In a sense, the u-tube manometer allowed for a back pressure to be placed onto the cells which were contained in the said pressure box with the gas mix flowing through the apparatus. The pressure is set with the difference in water (or any other liquid, if desired) column heights of the u-tube manometer. It was fun with physics meeting biology =)
In a fixed bed cell culture, cells attached on the bottom, tolerate a different hydrostatic pressure than upper cells if the volume or height to be adjusted for it.
Thank you so much everyone...all your comments are invaluable. Very much appreciated.
I don't know what you are able to grow your cells in (petri dishes, flasks, etc), but if you have access to/know of an Atomic Force Microscope group, you can subject the cells to friction forces and mechanical loading at various loads and with great precision.
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