A Widely Accepted Method
Cell disruption by rapid decompression from a pressure vessel
has been used for many years by investigators who wanted
to overcome the limitations imposed by other cell disruption
procedures. Although the technique is not new, interest in the
decompression method and many new applications for it have
grown rapidly following the introduction of convenient pressure
equipment such as the Parr Cell Disruption Vessel.
Many Applications
The nitrogen decompression method is particularly well suited
for treating mammalian and other membrane-bound cells. It has
also been used successfully for treating plant cells, for releasing
virus from fertilized eggs and for treating fragile bacteria. It is not
recommended for untreated bacterial cells, but this restriction can
be eliminated by using various pretreatment procedures (p. 7) to
weaken the cell wall. Yeast, fungus, spores and other materials
with tough walls do not respond well to this method.
How It Works
The principle of the method is quite simple. Large quantities
of nitrogen are first dissolved in the cell under high pressure
within a suitable pressure vessel. Then, when the gas pressure
is suddenly released, the nitrogen comes out of the solution as
expanding bubbles which stretch the membranes of each cell
until they rupture and release the contents of the cell.
Why It Is So Effective
It’s a gentle method.
Although sometimes referred to as
“explosive decompression,” nitrogen decompression is actually
a gentle method for homogenizing or fractionating cells since
the chemical and physical stresses which it imposes upon the
sub-cellular components are held to an absolute minimum. It is
much more protective of delicate enzymes and organelles than
ultrasonic and mechanical homogenizing methods. In fact, it
compares favorably to the controlled disruptive action obtained
in a Teflon and glass mortar and pestle homogenizer, but it does
the job faster and more uniformly, with the added ability to treat
large samples quickly and conveniently.
There is no heat damage.
While other disruptive methods
depend upon friction or a mechanical shearing action which
generates heat, the nitrogen decompression procedure is accom-
panied by an adiabatic expansion which cools the sample instead
of heating it. In addition, the entire cycle can be conducted at low
temperature by pre-chilling or by operating the vessel in an ice
bath. The vessel can also be filled with ice to keep the sample
cool during the processing period.
There is no oxidation.
The blanket of inert nitrogen gas
which saturates the cell suspension and the homogenate offers
excellent protection against oxidation of any labile cell compo-
nents. Although other gases: carbon dioxide, nitrous oxide,
carbon monoxide and compressed air have been used in this
technique, nitrogen is preferred because of its non-reactive
nature and because it does not alter the pH of the suspending
medium. In addition, nitrogen is preferred because it is generally
available at low cost and at pressures suitable for this procedure.
Any suspending medium can be used.
The suspending
medium can be chosen for its compatibility with the end use of
the homogenate and without regard for its adaptability to the
disruptive process. This offers great flexibility in the preparation
of cell suspensions and produces a clean homogenate which
does not require intermediate treatment to remove contaminates
which might be introduced when using other disruption methods.
Each cell is exposed only once.
Once released, subcellular
substances are not exposed to continued attrition which might
denature the sample or produce unwanted damage. There is no
need to watch for a peak between enzyme activity and percent
disruption.
The product is uniform.
Since nitrogen bubbles are
generated within each cell, the same disruptive force is applied
uniformly throughout the sample, thus ensuring unusual uni-
formity in the product. Cell-free homogenates can be produced.
It’s Easy To Apply
Use any sample size.
Cell disruption by this method is inde-
pendent of sample size or concentration. Any size sample from a
few cc’s to five hundred can be treated equally well in a Parr Cell
Disruption Vessel with excellent recovery of the starting material.
In addition, a wide variety of materials can be treated with the
opportunity for scale-up work where labile cell components or
organelles are involved.
Easy to control.
The degree of cell fractionization is easily
controlled by adjusting the nitrogen pressure. High pressures
which dissolve large quantities of nitrogen within the cell usually
produce total homogenization. Or, moderate pressures can
be employed to reduce the disruptive forces and thus release
nuclei, active mitochondria and other organelles intact. Operating
conditions can also be adjusted to homogenize suspensions of
subcellular components such as nuclei and mitochondria that are
normally difficult to disrupt because of their small size.
Special skills are not required.
The few simple steps
required to operate the Parr vessel are easily learned. After oper-
ating conditions have been established, uniform and repeatable
results can be obtained from run to run, usually within less than
twenty minutes – even with large samples.
A rapid and effective way to:
• Homogenize cells and tissues
• Release intact organelles
• Prepare cell membranes
• Release labile biochemicals
• Produce uniform and repeatable homogenates
without subjecting the sample to extreme
chemical or physical stress.
P a r r I n s t r u m e n t C o m p a n y
2
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Cell Disruption by Nitrogen Decompression
