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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.

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It might be possible to compare P200 with P20 on the cheap (anybody up for a crowd-sourced experiment on HeLa or E. coli?), but I suspect you actual question asks for absolute numbers, and is therefore almost impossible to answer. It is hard to envisage any non-pipette method to deliver 100% viable cells, and even harder to think of alternatives (measure free DNA after pipeting? how can that be converted to cell counts?). User137 suggests something decent, but still short of absolute. In the absence if evidence, all the answers you will receive are bound to be speculative "expert opinion". –  Nick Sandor Sep 1 at 1:36

4 Answers 4

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 freeing 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:

  1. Diameter of the bore in the pipette tip, the smaller the more shearing.
  2. Speed at which the suspension is passing through the opening of the tip. The faster the more damaging.
  3. Size and rigidity of the cell. Larger cells are more prone to damage. Cells lacking a murein wall are less prone to damage.
  4. Concentration of cells. Cultures that are more concentrated are more prone to damage.

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.

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This is a good explanation but does not actually give a numerical representation of percent death to no harm caused by the palleting. –  caseyr547 Aug 31 at 2:56
    
Good point Here I will rephrase as it seems to be getting quite a number votes –  rhill45 Sep 2 at 1:04

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.

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Well, then that will heavily be based on operator error. Anecdotally, I've heard that it is the other way; a P1000 would causes less damage than a P20. –  bobthejoe Jun 13 '12 at 6:42
    
Artem is right: the only way to say is empirically. The P1000 will influence the cell damage likely because of the velocity. In contrast, the P200 and P20 could give some damage due to the narrow tip hole. –  Gianpaolo R Jun 13 '12 at 18:36
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Give it a try and post the results. I do believe that operator skill is the single most important factor. –  Artem Jun 18 '12 at 21:50
    
We used to use a peristaltic pump to plate HepG2 cells into 384 and 1536 plates. When we moved to primary hepatocytes the pump killed too many and had to move to hand plating with an 8 channel pipette. So cell type matters a lot. –  user137 Jul 30 at 23:17
    
Naw this isn't going to measure the correct....just naw –  caseyr547 Aug 31 at 2:54

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.

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Good pointIt's pretty hard to kill bacteria mechanically period, the competent cells are more sensitive though and concern comes when building cDNA library's mostly as you need a very representative number of colonies –  rhill45 Aug 30 at 16:57
    
This is a fine observation. –  caseyr547 Aug 31 at 2:56

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.

Pipetting causes shear stress and elevation of phosphorylated stress-activated protein kinase/jun kinase in preimplantation embryos.

Xie Y1, Wang F, Puscheck EE, Rappolee DA.

Shear stress at 1.2 dynes/cm(2) induces stress-activated protein kinase/jun kinase phosphorylation that precedes and causes apoptosis in embryos (Xie et al., 2006b, Biol Reprod). Pipetting embryos is necessary for many protocols, from in vitro fertilization to collecting embryos prior to analyzing gene expression by microarrays. We sought to determine if pipetting upregulates phosphorylated MAPK8/9 (formerly known as stress-activated protein kinase/jun kinase/SAPK/JNK1, 2). We found that phosphorylated MAPK8/9, a marker of MAPK8/9 activation, is upregulated in a dose-dependent manner by pipetting. Whereas embryos with the zona pellucida removed were more sensitive to stress-induced lethality mediated by 1.2 dynes/cm(2) shear force, phosphorylated MAPK8/9 was induced at lower numbers of pipet triturations in hatched embryos at E4.5. E4.5 embryos were more sensitive to induction of MAPK8/9 than unhatched embryos at E2.5 or E3.5. E3.5 embryos also showed a pipetting dose-dependent induction of FOS protein (formerly known as c-fos), a marker of shear stress in many cell types. Phosphorylated MAPK8/9 measured in ex vivo embryos from E1.5 to E4.5 were expressed at low levels. Embryos that had been pipetted sufficiently to induce phosphorylated MAPK8/9 and FOS had the same number of cells as untreated embryos 24 hr later. This suggests that rapid phosphorylation of MAPK8/9 due to transient shear stress does not mediate long-term negative biological outcomes. But, it is possible that techniques requiring multiple handling events would induce MAPK8/9 and cause biological outcomes or that other biological outcomes are affected by low amounts of transient shear stress. This study suggests that embryo handling prior to experimental measurement of signal transduction phosphoproteins, proteins and mRNA should be performed with care. Indeed, it is likely that shear stress may cause rapid transient changes in hundreds of proteins and mRNA.

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@rhill45 yeah the question was about cell shear stress damage during pipetting..the other answered seemed to buy into the only damage coming from the pal tip..but i wanted to point out that the shear stress from the pipetted liquid is equally likely to damage/destroy the cell. –  caseyr547 Sep 1 at 23:21
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@rhill45 also if your going to learn php you should invest time in asp because many industries see saw between the two. –  caseyr547 Sep 1 at 23:43
    
Thanks @caseyr547's It's a mind reading point you make, I manage our departments website at the university here and everything is in asp. It sucks though cause the site uses master pages I dont have access to, and thus can't test anything I write. Asp is next –  rhill45 Sep 2 at 0:59

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