result, hence the term ‘vitrication’. He also realised that thawing would have to be equally
rapid, for icecrystals’ might form‘during the réWàtrming process.
"
Using isopentane cooled to —-— 150 °C by liquid nitrogen as his refrigerant medium and f
thawing in warm saline, he successfully froze and thawed Vinegar eels, ox red cells and the
heart of an embryo chick. However, he found that
results were considerably improved
”
if lie performed an initial dehydration of his tissues. This he did in an attempt to improve
the freeZing rate, but it is of some'interest in view of further discoveries that he got the
best results if he used ethylene glycol for this dehydration.
Another milestone in the preservation of living nucleated cells by freezing was the redis—
covery by Polge, Smith and Parkes (1949) of the protective action of glycerol. Initially they
.
found that fowl sperm was preserved if ‘frozen‘ in the presence of glycerol, but later it was
found that this preservative e"ect also applied to red bloOd cells. The preservative action
of glycerol has been found to be fairly widespread, and the method has been applied to
the preservation of many living cells and has been particularly valuable in the laboratory
for the preservation of tissue culture cells.
A marked dierence between the vitrication and the glycerol technique is in the optimal
rate of freezing. For vitrication, freezing must be as rapid as possible; With the glycerol
î Ï "Ï E
technique a slow freeze of about 1 °C per minute seems to be optimal.
Great confusion has arisen from there being two optimal rates of cooling and from the
rival theories as to whether the freezing injury is due to ice-crystal damage or the osmotic
effects of salt concentrations or the denaturing e'ects of high salt concentrations. The
probable answer is
these mechanisms may befatal to the cell, but that different
cells vary as to their sènSitivityto the Various deStructive forces. Ice crystals possibly
disrupt cell nùclei. They may also alter the _permeability of a cell membrane if they grow
through the membrane pores from extracellular ice. Osmotic e”ects may well rupture a
'
delicate cell membrane while they have
eecton a tough cell membrana Fortunately ,
_
all the rival theories are agreed that thètwingshould be as rapid as possible.
,
'
the absence of an
rapid freeze and _thaw Would seem to give the
‘
best chanCe of
would minimisethe crystal_size and also the time to which the
cell
to high salt concentrations. Thé fact that iiithe presence of
slower freezeis optimal would suggest that either the glycerol can protect against destruc—
tive
is
having
,
ôii ice _..crystàl formaôäa- Ï
f
.,
'
…
'
[
Ü ] ; @ l
'
=
‘
'
The discovery of the mode ofact1onofprotectweadd1t1V6$lsOf the greatest importance
…
f°f
.
.…
lsworhngmfreezedrymgbyproîectmgagamstfœezmg1n1ury Orj
Ç<Ï>.f
.
,' j
_—
THE
AND DRYING OF NON—NUCLEATED CELLS
1 “The
cblf‘prov1des fan ”è>äœiieht“*iâi6cîiélîi‘bf‘thé studyof freezin‘g
‘
‘
,
'
‘
‘ AS already-stated, Luyethad some'suCcess following
WllthedlSC®V6fÿofg1ycemlpr0tecüwltwasSO®nfoundthatglycerolalsoproteètcd
…
“fi“ ‘thehuanredœfwfreezmgmiurybuttheopt1malrateoffreezrngwasfoundtobe
'
rapid, for icecrystals’ might form‘during the réWàtrming process.
"
Using isopentane cooled to —-— 150 °C by liquid nitrogen as his refrigerant medium and f
thawing in warm saline, he successfully froze and thawed Vinegar eels, ox red cells and the
heart of an embryo chick. However, he found that
results were considerably improved
”
if lie performed an initial dehydration of his tissues. This he did in an attempt to improve
the freeZing rate, but it is of some'interest in view of further discoveries that he got the
best results if he used ethylene glycol for this dehydration.
Another milestone in the preservation of living nucleated cells by freezing was the redis—
covery by Polge, Smith and Parkes (1949) of the protective action of glycerol. Initially they
.
found that fowl sperm was preserved if ‘frozen‘ in the presence of glycerol, but later it was
found that this preservative e"ect also applied to red bloOd cells. The preservative action
of glycerol has been found to be fairly widespread, and the method has been applied to
the preservation of many living cells and has been particularly valuable in the laboratory
for the preservation of tissue culture cells.
A marked dierence between the vitrication and the glycerol technique is in the optimal
rate of freezing. For vitrication, freezing must be as rapid as possible; With the glycerol
î Ï "Ï E
technique a slow freeze of about 1 °C per minute seems to be optimal.
Great confusion has arisen from there being two optimal rates of cooling and from the
rival theories as to whether the freezing injury is due to ice-crystal damage or the osmotic
effects of salt concentrations or the denaturing e'ects of high salt concentrations. The
probable answer is
these mechanisms may befatal to the cell, but that different
cells vary as to their sènSitivityto the Various deStructive forces. Ice crystals possibly
disrupt cell nùclei. They may also alter the _permeability of a cell membrane if they grow
through the membrane pores from extracellular ice. Osmotic e”ects may well rupture a
'
delicate cell membrane while they have
eecton a tough cell membrana Fortunately ,
_
all the rival theories are agreed that thètwingshould be as rapid as possible.
,
'
the absence of an
rapid freeze and _thaw Would seem to give the
‘
best chanCe of
would minimisethe crystal_size and also the time to which the
cell
to high salt concentrations. Thé fact that iiithe presence of
slower freezeis optimal would suggest that either the glycerol can protect against destruc—
tive
is
having
,
ôii ice _..crystàl formaôäa- Ï
f
.,
'
…
'
[
Ü ] ; @ l
'
=
‘
'
The discovery of the mode ofact1onofprotectweadd1t1V6$lsOf the greatest importance
…
f°f
.
.…
lsworhngmfreezedrymgbyproîectmgagamstfœezmg1n1ury Orj
Ç<Ï>.f
.
,' j
_—
THE
AND DRYING OF NON—NUCLEATED CELLS
1 “The
cblf‘prov1des fan ”è>äœiieht“*iâi6cîiélîi‘bf‘thé studyof freezin‘g
‘
‘
,
'
‘
‘ AS already-stated, Luyethad some'suCcess following
WllthedlSC®V6fÿofg1ycemlpr0tecüwltwasSO®nfoundthatglycerolalsoproteètcd
…
“fi“ ‘thehuanredœfwfreezmgmiurybuttheopt1malrateoffreezrngwasfoundtobe
'