144
FREEZE—DRYING
were writing this paper, it occurred to us that two of our observations might be related
,
profoundly to the known protective effects of glycols on cells. Thus, our observations on
microtubules and ribosomes in osmotically dehydrated eggs suggest that the regionsin the
immediate vicinityof microtubules may have special properties with respect to water in
that water in theseregions may not be subject to crystallizationin the destructive sense
during rapid freezing. Also, if _glycols do have the…effect of stimulating the formation of.
"
microtubules in cells other than marine eggs (denitely unproven, as yet) than at least one
—
effect of glycols and DMSO in protection againSt-freezing maybe to create many regions
(mierotubules) where comp0nents such as ribosomes (which are especially destroyed by ice
formationin cells) are protected from irreparable damage. Although such an e‘ect cannot
Î;;Î
beîthe only protective action in cells, it may be important in determining how glycols and
DMSO are applied to cells and what concentrations must be used. We hope to have some
information on this in the near future.…
‘
.
,
‘
‘
.
'
Recently, 'Malhotra, et al. (1965) and van Herreveld, et al., (1965), introduced a technique
of freezing and»_substitutig which inîvolves rapid attening (squashing) of fresh tissue on a
silverÿrod; held at liquid nitrogen temperature. Many of the detailed results on
;ÎÏ,
and ;cther;;çomponentsresemble those given here. We had worked with a
similar
and found that without squashing the tissue, freezing
was very pOor unless a drop of propane wetted the metal surface (Rebhun and Gagné,
1962). Although, freezing appeared successful under these latter circumstances, control over
water evaporation was not possible and the technique was abandonned. Since statistics on
successful freezings were not given, by Malhotra, et al., and van Herreveld, et al., and since
their techniques appear to this investigatorto be grossly subject to the possibility of evapo—
ration from the tissue (they
Only about 10 microns of surface tissue are ice—crystal—
free), it is not clear that Vitreous freezing of cells and intracellular space has been attained
in their case , although it cannot…»be denitely denied. Considerably more work will be
.
necessary before this
evaluated with thatreported here.
…
…
…
_
Three further -:remarks are :necessary.,First, why:
get appareny excellent
preSe1tvatidn of cellularcomponents-when so much water is removed? We obviouycannot
answer this question
suggest that it may be tied- up with the àquestion of bound
water in
cell.
sense each cellular component ’may be sitting in its
own environment in;‘,Which
water,is not free to crystallize if
by itself,
thiS—- W&ÎÇI ? is 110t Signicany= affected by therem0val of large amounts of the free: water.
ÎËUS5.WhŒf signicant amountser ,thelatter aïe removed, nozfurthet water is available for
Ç5tëtallizaüon
crystal free. Thisi may also account :for‘ the
'
rgäèÿsoh.thatstructures“ such
Land microtubules .don0tæseem to be smaller (in
thanthasameastrÿë‘eturcs:m chemically
,
», —; _J The
wl‘èlîÇSsiQ
,
th@::Ôonsiderableasimilètrity:;betweert the morphology pf
protective,=agents'gandtthose
with…
may
due, to ; the fact %that ebbthâ techniques “preserve‘îï
reahty1nthe @Çll
‘î,Ï£h©lÏÇf0t6é are
‘ChékaS…i on,;çthe
smarlar1ty maybe
aa,similaritelementfin
,
both processeswmchmaymfact
Thuschemlcaï xaüvesayact ab,y
«while
freezesubsütut1on(asdonehere)ayactthroughapalal Separation e…0jf£îthese,step‘s,îire.gä
‘?V‘ôîîûaÿbt‘««'àbïeî0dahY@hfeüe
cross,!
,
1mkneXîïfsohls separatmn0fthetwc>processes
:nfæ,
_
'
FREEZE—DRYING
were writing this paper, it occurred to us that two of our observations might be related
,
profoundly to the known protective effects of glycols on cells. Thus, our observations on
microtubules and ribosomes in osmotically dehydrated eggs suggest that the regionsin the
immediate vicinityof microtubules may have special properties with respect to water in
that water in theseregions may not be subject to crystallizationin the destructive sense
during rapid freezing. Also, if _glycols do have the…effect of stimulating the formation of.
"
microtubules in cells other than marine eggs (denitely unproven, as yet) than at least one
—
effect of glycols and DMSO in protection againSt-freezing maybe to create many regions
(mierotubules) where comp0nents such as ribosomes (which are especially destroyed by ice
formationin cells) are protected from irreparable damage. Although such an e‘ect cannot
Î;;Î
beîthe only protective action in cells, it may be important in determining how glycols and
DMSO are applied to cells and what concentrations must be used. We hope to have some
information on this in the near future.…
‘
.
,
‘
‘
.
'
Recently, 'Malhotra, et al. (1965) and van Herreveld, et al., (1965), introduced a technique
of freezing and»_substitutig which inîvolves rapid attening (squashing) of fresh tissue on a
silverÿrod; held at liquid nitrogen temperature. Many of the detailed results on
;ÎÏ,
and ;cther;;çomponentsresemble those given here. We had worked with a
similar
and found that without squashing the tissue, freezing
was very pOor unless a drop of propane wetted the metal surface (Rebhun and Gagné,
1962). Although, freezing appeared successful under these latter circumstances, control over
water evaporation was not possible and the technique was abandonned. Since statistics on
successful freezings were not given, by Malhotra, et al., and van Herreveld, et al., and since
their techniques appear to this investigatorto be grossly subject to the possibility of evapo—
ration from the tissue (they
Only about 10 microns of surface tissue are ice—crystal—
free), it is not clear that Vitreous freezing of cells and intracellular space has been attained
in their case , although it cannot…»be denitely denied. Considerably more work will be
.
necessary before this
evaluated with thatreported here.
…
…
…
_
Three further -:remarks are :necessary.,First, why:
get appareny excellent
preSe1tvatidn of cellularcomponents-when so much water is removed? We obviouycannot
answer this question
suggest that it may be tied- up with the àquestion of bound
water in
cell.
sense each cellular component ’may be sitting in its
own environment in;‘,Which
water,is not free to crystallize if
by itself,
thiS—- W&ÎÇI ? is 110t Signicany= affected by therem0val of large amounts of the free: water.
ÎËUS5.WhŒf signicant amountser ,thelatter aïe removed, nozfurthet water is available for
Ç5tëtallizaüon
crystal free. Thisi may also account :for‘ the
'
rgäèÿsoh.thatstructures“ such
Land microtubules .don0tæseem to be smaller (in
thanthasameastrÿë‘eturcs:m chemically
,
», —; _J The
wl‘èlîÇSsiQ
,
th@::Ôonsiderableasimilètrity:;betweert the morphology pf
protective,=agents'gandtthose
with…
may
due, to ; the fact %that ebbthâ techniques “preserve‘îï
reahty1nthe @Çll
‘î,Ï£h©lÏÇf0t6é are
‘ChékaS…i on,;çthe
smarlar1ty maybe
aa,similaritelementfin
,
both processeswmchmaymfact
Thuschemlcaï xaüvesayact ab,y
«while
freezesubsütut1on(asdonehere)ayactthroughapalal Separation e…0jf£îthese,step‘s,îire.gä
‘?V‘ôîîûaÿbt‘««'àbïeî0dahY@hfeüe
cross,!
,
1mkneXîïfsohls separatmn0fthetwc>processes
:nfæ,
_
'