Shear stress $tau$ in this small sizes is usually measured in dyne/cm2 or N/m2 = Pa. The equations betweeen them: $1dyn/cm^2 = 10^{-5}N/cm^2 = 0.1N/m^2 = 0.1Pa$.
What kind of damages zygotes can suffer by pipetting?
Using scanning electron microscopy, we found open holes on the surface
of lysed eggs, indicating failure of the plasma membrane to reseal
after microinjection. No holes were seen in unlysed eggs, but many of
them had membrane alterations suggestive of healed punctures.
Even a small 1.2 dyn/cm2 shear stress induces apoptosis by pipetting zygotes. So zygotes have their critical shear stress level by 1.2 dyn/cm2 and pipetting involves greater forces than 1.2 dyn/cm2.
Shear stress at 1.2 dynes/cm2 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.
The critical shear stress level is somewhere between 0.01 and 1000 dyn/cm2 by animal cells depending on the cell type and species. (I think the average is somewhere about 50 dyn/cm2, but it is very hard to differentiate between articles mentioning critical shear levels and most lethal shear levels, so the range and the average might be lower.) The death constant (1/h) increases exponentially by increasing the shear stress.
An apparatus for the detailed investigation of the influence of shear
stress on adherent BHK cells was developed. Shear forces between 0.0
and 2.5 N m−2 were studied. The influence on cell viability, cell
morphology, cell lysis, and cell size was determined. Increasing shear
forces as well as increasing exposure duration caused increasing
changes in cell morphology and cell death. A “critical shear stress
level” was determined.
Shear stress related damage to a mouse hybridoma was examined by
Abu-Reesh and Kargi under laminar and turbulent conditions in a
coaxial cylinder Searle viscometer. Cells were exposed to 5 to 100
N/m2 shear stress levels for 0.5 to 3.0 h. At a given shear stress and
exposure time, turbulent shear was much more damaging than laminar
shear as also reported in the past for protozoa and plant cells. Under
turbulent conditions, damage occurred when shear stress exceeded 5
N/m2. Respiratory activity of the cells was damaged earlier than the
cell membrane, thus implying transmission of the stress signal to the
interior of the cell. Cell damage followed first-order kinetics both
in laminar and turbulent environments. For turbulent shear stress
levels of 5 to 30 N/m2, the death rate constant (kd) increased
exponentially with increasing stress level; the kd values varied over
0.1 to 1.0 1/h.
Subconfluent endothelial cultures continuously exposed to 1–5
dynes/cm2 shear proliferate at a rate comparable to that of static
cultures and reach the same saturation density (≃ 1.0–1.5 × 105
cells/cm2 ). When exposed to a laminar shear stress of 5–10 dynes/cm2
, confluent monolayers undergo a time-dependent change in cell shape
from polygonal to ellipsoidal and become uniformly oriented with flow.
Regeneration of linear “wounds” in confluent monolayer appears to be
influenced by the direction of the applied force. Preliminary studies
indicate that certain endothelial cell functions, including fluid
endocytosis, cytoskeletal assembly and nonthrombogenic surface
properties, also are sensitive to shear stress. These observations
suggest that fluid mechanical forces can directly influence
endothelial cell structure and function.
Shear stress above 0.25 dyne/cm(2) resulted in dramatic loss of
podocytes but not of proximal tubular epithelial cells (LLC-PK(1)
cells) after 20 h.
A series of careful studies has been made on blood damage in a
rotational viscometer. Specific attention has been focused on the
effects of solid surface interaction, centrifugal force, air interface
interaction, mixing of sheared and unsheared layers, cell-cell
interaction, and viscous heating. The results show that there is a
threshold shear stress, 1500 dynes/cm2, above which extensive cell
damage is directly due to shear stress, and the various secondary
effects listed above are negligible.
The shear stress threshold of some dinoflagellates (microalgae) is
even lower than that of erythrocytes (0.029 N/m2). For example, a
continuous laminar shear stress level of only 0.0044 N/m2 (equivalent
to a shear rate of 2.2 1/s) has proved lethal to the dinoflagellate
Gonyaulax polyedra.
Other cell types are not necessary as sensitive as animal cells and they don't necessary react with apoptosis (about 10 dyn/cm2) to shear stress, so you have to use necrotic (about 5000 dyn/cm2) forces to destroy them :
cell type size shear sensitivity
microbial cells 1-10μm low
microbial pellets/clumps up to 1cm moderate
plant cells 100μm moderate/high
plant cell aggregates up to 1-2cm high
animal cells 20μm high
animal cells on microcarriers 80-200μm very high
fungi cells 2-10μm moderate/high
Results show that Chinese Hamster Ovaries and Human Embryonic Kidney
cells will enter the apoptotic pathway when subjected to low levels of
hydrodynamic stress (around 2.0 Pa) in oscillating, extensional flow.
In contrast, necrotic death prevails when the cells are exposed to
hydrodynamic stresses around 1.0 Pa in simple shear flow or around
500 Pa in extensional flow.
The shear sensitivity is not determined only by cell type and species, there are many other factors involved:
- type of cell and species
- composition and thickness of cell wall when present
- size and morphology of cell
- the intensity and nature of shear stress, whether turbulent or laminar, or associated with interfaces (e.g. during bubble rise and rupture)
- growth history, both short-term (e.g. starvation) and long term adaptation
- growth medium (trace elements, vitamins, carbon and nitrogen sources)
- growth rate
- growth stage
- type and concentration of shear protective agents if present
Cells can be very sensitive to shear stress caused by turbulent flow, while not so sensitive to shear stress caused by laminar flow.
On the basis of laminar flow viscometriy measurements, a critical
shear stress level of 80-200 N/m2 has been suggested for Morindata
citrifolia cells.
... while for Daucus carota a shear stress level of 50 N/m2 has been
associated with cell damage. In other study, carrot cells in a laminar
flow Couette viscosimeter lost the ability to grow and divide in the
shear stress range of 0.5-100 N/m2. The intracellular enzyme activity
was impaired at shear stress levels above 3000N/m2, but significant
lysis did not occur until a shear stress level of 10.000 N/m2 applied
over a prolonged perioud (>1h).
In contrast to the behavior in laminar flow, the cells were quite
sensitive to turbulent impeller agitation. Impeller tip speeds of ~1.1
m/s lysed a significant proportion of the cells within 40min.
The bubble damage is severe (1000 cells by a single 3.5mm size bubble) because of the cell adherence to the interface of the bubble and the strong forces involved (>1000 dyn/cm2 by stirred bioreactors). The adhesion and so the damage can be reduced with surfactants.
It is proposed that when cells are either attached to, or very near, a
rupturing bubble, the hydrodynamic forces associated with the rupture
are sufficient to kill the cells.
All experiments were conducted with Spodoptera frugiperda (SF-9)
insect cells, in TNM-FH and SFML medium, with and without Pluronic
F-68. Experiments indicate that approximately 1050 cells are killed
per single, 3.5-mm bubble rupture in TNM-FH medium and approximately
the same number of dead cells are present in the upward jet. It was
also observed that the concentration of cells in this upward jet is
higher than the cell suspension in TNM-FH medium without Pluronic F-68
by a factor of two. It is believed that this higher concentration is
the result of cells adhering to the bubble interface. These cells are
swept up into the upward jet during the bubble rupture process.
Finally, it is suggested that a thin layer around the bubble
containing these absorbed cells is the “hypothetical killing volume”
presented by other researchers.
For a hybridoma line, reported that exposure to laminar shear stress
(208 N/m2) in unaerated flow in a cone and plate viscometer led to
substantial loss in cell count and viability within 20 min. At a
constant 180 s exposure, increasing shear stress over 100-350 N/m2
linearly enhanced cell disruption, with >90% of the cells being
destroyed at 350 N/m2 stress level. Shear stres levels associated with
bubble rupture at the surface of a bioreactor may range over 100-300
N/m2. These values are remarkably consistent with shear rates that
damaged hybridomas in unaerated laminar flow experiments.
Smaller hole pipettes cause more damage.
We also examined aspects of the gene transfer procedure that might
influence survival such as the size of injection pipettes and their
taper relative to zygote diameter, possible toxicity of the injection
medium, the timing of injection, and immediate vs. delayed pipette
withdrawal. The only factors that significantly affected cell
viability were pipette size and taper, and timing of injection in
relation to first cleavage. This suggests that zygote viability
correlates inversely with the size of the hole produced by the
injection pipette and that damage to the membrane is less successfully
repaired as the fertilized egg readies itself for division.
It is hard to find anything about the level of shear stress by pipetting. It can be certainly more than 1 dyn/cm2. It has a short duration (at most a few seconds). I think the following factors can influence the shear stress levels by pipetting:
- pipette type
- flow speed (faster flow can be more likely turbulent)
- bubble formation
Probably more factors are involved but I am not a pipetting expert. ;-) I agree with the others, it surely depends on the personal skills e.g. an amateur can create huge bubbles by pipetting, which can kill a lot of cells by formation and disruption...
I agree with Artem that this is an experiment to do especially if the result is important for you. What you need to create a model about pipette damage, are the shear stress levels by pipetting and the critical shear stress levels of the cells. I think it is hard to design and experiment in which you can measure the shear stress levels in your pipettes and there is no flow model for pipetting as far as I know, so it can be a good topic for a thesis or a diploma work.
