tissue by the nitrogen decompression method. Although most
of the work reported by these authors was done with rat tissue,
they also treated spleen, white cells, lymph nodes, tumors,
thymus and other tissues to establish the general applicability
of the method. Their results clearly demonstrated that cells
can be disrupted by this method with minimum physical and
chemical damage to the components.
H & C obtained complete disruption at pressures of 1300 psi
and above, while pressures below 700 psi left whole cells and
clumps of cells in the homogenate. At pressures between 800 and
1000 psi, cell-free homogenates were produced with nuclei intact.
A hand press was used to pre-mince tissues prior to treatment
in the vessel. The condition of the nuclei was found to be
dependent upon the composition of suspending buffer solution.
Good results were obtained using isotonic solutions while nuclei
swelling and rupture were observed in cells suspended in very
dilute solutions. This was attributed to osmotic swelling which
H & C found could be controlled by adding inorganic salts such
as sodium chloride or organic solutes such as sucrose or
glycerol. The nuclei were extremely fragile when the suspending
medium contained no calcium, but the presence of as little as
0.0002M calcium chloride was found to stabilize the nuclei.
Magnesium acetate is also useful for this purpose.
To determine the extent of damage to labile cells, H & C
studied Deoxyribonucleoprotein, DNP, because of its suscepti-
bility to chemical and physical stress, obtaining recoveries of over
90% DNP from the nuclear fraction with excellent preservation of
the material. They also compared the enzyme activities of mito-
chondrial suspensions prepared by the nitrogen decompression
method with suspensions produced in a Potter-Elvehjem homog-
enizer. No differences in enzyme activities were detected.
Dowben, Gaffey and Lynch
2,3
used the nitrogen decom-
pression technique to prepare polyribosomes from L Cells, fibro-
blasts, human fetal cells from amniotic fluid, rat livers and muscle
from chick embryos. Using 600 psi pressure they obtained better
than 99.9% rupture and recovered more than 95% of the nuclei
intact. Polysome yield was two to three times greater than when
the cells were homogenized in a Dounce tissue grinder.
In addition, they had better defined and more reproducible
profiles. Significantly greater activities, as measured by amino
acid incorporation, were also reported.
Short, Maines and Davis
4
compared the nitrogen decom-
pression method with the Potter-Elvehjem types of Teflon pestle
and glass tube homogenizers for preparing microsomal fractions
for drug metabolism studies. The decompression method consis-
tently produced over twice as much microsomal protein per gram
of tissue as the pestle and tube fractionation. Enzyme activity per
milligram of microsomal protein was found to be essentially the
same for both methods, but it must be remembered that nitrogen
decompression yielded over twice as much microsomal protein
per gram of starting material.
Under microscopic examination the homogenates produced
by the decompression method were found to be cell-free, while
numerous cell clumps were observed in the pestle and tube
Equilibrium
Sufficient time must be allowed for the nitrogen to dissolve
and come to equilibrium within the cells. Periods as short as five
minutes may be sufficient for small samples, while longer times
up to thirty minutes may be required for large samples. Stirring
with a magnetic bar placed in the bottom of the vessel will accel-
erate this process, particularly when working with large samples;
it will also hold the cells in a uniform suspension. Since the
vessel is made of a non-magnetic stainless steel, the stirring bar
can be driven by simply placing the vessel on a magnetic stirring
plate. If cooling is required to protect the sample, the vessel can
be pre-cooled before it is charged, or ice can be packed around
the inner sample holder, or the vessel can be packed in ice or
held in a cold room during the equilibration period.
Disruption and Collection
The actual disruption process does not occur as cells are pres-
surized with nitrogen. Instead, it occurs at the discharge valve at
the instant of decompression as the sample passes from the high
pressure environment inside the vessel to atmospheric pressure
on the outside. A rapid flow rate is not required in order to attain
maximum disruption since disruption occurs on an individual cell
basis and is independent of the rate at which cells are released
from the vessel. At all flow rates, this process is assisted by the
vigorous agitation which develops as the homogenate flows
through the valve.
Any suitable container can be used to collect the sample as it
is released from the delivery tube. A side arm suction flask works
well for this purpose. Simply insert the delivery tube into the flask
and close the neck with a cotton wad or other stopper but leave
the side arm open to release the excess nitrogen. To prevent
splattering in the receiver and freezing in the discharge tube, it
is well to close the discharge valve after the bulk of the sample
has been recovered, then release the remaining nitrogen through
the inlet valve. The inlet valve can also be used to release the
pressure should it become necessary to abort a test after the
vessel has been pressurized. All pressure within the vessel must
be released before attempting to open the vessel.
Some investigators working with small samples of very fragile
cells have found that such materials can be treated satisfactorily
by decompression within the vessel without running the sample
through the discharge valve. In these special cases, the pres-
surized sample is allowed to remain in the vessel while the excess
nitrogen is released through the inlet valve. After the pressure
has been reduced to atmospheric, the vessel can be opened
to recover the sample. Although this procedure is effective
for certain types of cells, in most cases it is best to release the
sample through the discharge valve to utilize the instantaneous
decompression which occurs as cells pass through the valve.
Applications and Techniques
Mammalian Cells
Hunter and Commerford
1
published a paper in 1961 which
has become a basic “cookbook” for the disruption of mammalian
P a r r I n s t r u m e n t C o m p a n y
6
w w w . p a r r i n s t . c o m
Cell Disruption Vessels
