I'm curious how much damage is potentially inflicted by shear stress by pipetting. I know that with syringes for stem cell injection cause a lot of damage. However, to what extend does this happen with P20 and P200 pipet tips? Understandably the shear modulus of bacterial cells are significantly different from cancer cells which will be different from stem cells.
This is an excellent question, I have been training people to culture cells for about 12 years and students have a hard time grasping this and appreciating the importance etc.
What cells are usually experiencing during pipetting is analogous to a crowd of people trying to fit through the doorway of a building. Shearing during pipetting is certainly a legitimate concern in cell culture. You will notice its negative effect on viability most explicitly when pipetting cells in freezing medium (containing DMSO) following a thaw. Until their DMSO concentration drops their membranes are weaker & more fluid. That's why you pipette the frozen cells drop-wise to the fresh medium, to be especially gentle at this point.
Prokaryotic cells such as the above mentioned TOP10 cells are treated with Calcium chloride and glycerol which has essentially the same effect on their walls and membranes. Hence pipetting should be delicately performed after thawing these cells as well.
With that being said, if you compare the sensitivity of prokaryotic and eukaryotic cells to shearing, eukaryotic cells are profoundly more sensitive.
For individuals using competent bacteria for sub-cloning and other routine uses, killing a small percentage of of your cells is not that important. However if you are using the transformation to generate cDNA libraries it's very important that you have a titer sufficiently representing the transcriptome the library is composed of.
In these instances using a more premium competent cell, adhering to correct temperatures and minimizing pipetting is essential. This is why many are taught to swirl the DNA the with the competent cells, rather than the harsher alternative: pipetting up-and-down.
The four most important factors that contribute to cellular shearing are, and in order form most to least contributory:
Because the make up if the cell itself is so influential on the amount of damage, and because of the enormous variety of cells; designing a representative experiment to assess damage would be very difficult and laborious.
Finally, the variety of tips and seriologicals is also enormous. However if one were to attempt to assess this it could be done:
Choose a representative variety of cell lines, choose a representative variety of pipettes. One would probably want to use a robotic pipette to be able to evaluate incrementally assigned speeds and pressures. Look at different culture concentrations, phases of the growth curve, time between pipetting and analysis, distance between the exiting fluid and the culture etc, etc.
I think a very successful analysis method would be using propidium iodide and FACS.
After your experiment which will cost a lot of time and $, I think you will find common sense rules: keep pipetting to a minimum and use wider tips whenever possible.
It's an easy experiment to do. Take your cells aliquot them into 10 microfuge tubes, and pipette each suspension increasing amount of times, stain with trypan blue and count.
The most important factors will be which pipette-type you use; I would expect a p1000 to cause more damage then a p200 then a p20 due to velocity of the fluid. Also the most important factor will be the skill of the scientist, if you pipette slowly it should decrease stress as opposed to pipetting quickly.
In my experience it depends on the pipette-type and the skill of the operator. The only way to answer this for you is to try it empirically.
Anecdotally I have not observed any cell death upon pipetting of E. coli DH5alpha or TOP10, however as competent cells, mixing by pipetting up and down is discouraged due to the compromised cell wall.
This question would be better served in the physics se or chemistry. I don't think we have a generic engineering where fluid dynamics could be described in a different method than physics but if we did it should also be answered there.
Indeed the osmotic or liquid pressure in and of itself would cause changes possibly damages to the cell. Then other eexothermic and endothermic reactions to the chemicals due to force of impact like a chem or friction burn.
I'm sure the other requirements of conservation of energy which I half-hazardly ignored.
I found this interesting paper which focuses on the shear stress in general.