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Ultrastructural comparison of freeze-drying and freeze-substitution in preservation of the frozen state
CHYOBIOLOGY
14, 382-386
Ultrastructural
(1977)
Comparison of Freeze-Drying and Freeze-Substitution in Preservation of the Frozen State K. C. LIU
Department of Anatomy,
AND J. K. SHERMAN
Unitiersity of Arkansas for Medical Sciences, Little Rock, Arkansas
Correlation of cellular ultrastructure before freezing, while frozen, and after thawing provides a requisite basis for evaluation of functional ‘alterations which are induced during the formation and dissolution of ice (9). Th e respective roles of intracellular and extracellular ice figure prominently in theories of freezing injury (2, 3, 6, ll), often without correlative observations on the frozen state, especially in tissue cells. Freeze-drying and freeze-substitution are the techniques most commonly used by investigators, almost interchangeably, to preserve the areas in which ice forms in and around cells during freezing at various rates. Occasional statements have been made which suggest that these techniques may yield the same results in representation of the frozen state (4, 9). Results with muscular tissue indicate no difference in the size of intracellular ice spaces with both methods ( 12), as well as the unreliability of freeze-drying (5). There is little or no information on direct experimental comparisons of these methods with epithelial cells, however, especially on the ultrastructural level. Such information is required for a better interpretation of studies on the frozen state of import’ant tissues and organs. Freeze-etching, which theoretically may represent an even more valid picture of actual structure, but which pre--.Rcwivcd
June 28, 1976.
Copyright (: 1977 by Acadrmic I’ress. Inc. All rights of reproduction in any form reserved.
sents similar results in practice ( 12), has not “suplemented or replaced freeze-substitution and freeze-drying” in this cryobiological application ( 1, 9). The purpose of this brief communication is to present the results of a comparative evaluation of freeze-drying and freezesubstitution in the electron microscopic representation of ice in the frozen state in pancreatic cells. MATERIALS
AND
METHODS
The rationale and detailed description of methods used for freezing, freezedrying, freeze-substitution, and electron microscopy have been presented previously (8, 9). Only essentials are repeated here as a basis for comparison in a study which involved five experimental runs, each with three to five tissue samples. Freezing Small (2 X 3 mm) pieces of rat pancreatic tissue, 0.7 to 0.9 mm in thickness, were frozen on an aluminum boat. A rapid overall freezing rate of about 38”C/sec was achieved by direct immersion of the tissue samples in liquid nitrogen ( - 196°C ). They were kept at -196°C for 5 min. Freeze-Drying Frozen samples were transferred to a perforated aluminum basket immersed in
ISSN 0011-2240
BRIEF
COSlhi~~ICA’I’IONS
FIG. 1. Electron micrographs of rat pancreatic acinar cells before freezing (A) and in the frozen state, as preserved by freeze-drying (B and I)) and freeze-substitution (C and E) after freezing to -196°C at 38”Cj. sec. Clear spaces in B, C, 1), and E represent areas pwreticulum, viously occupied by ice. Nucleus, N; zymogen granules, ZC; rough endoplasmic RER. A, B, and C at X4500; U and E show HER at X4.5,000. Note that the representations are indistinguishable from each of the frozen state I)y freeze-drying and freeze-suhstitutiou other.
isopentanc ( -77°C) in a jar surrounded hy dry ice and carrid to the frc,ezc-clr)+lS apparatus. The basket was r~movccl from the isopentane, blotted on dry ice, and quickly lowered into the rcfrigrrator
~hatnl)u of the freeze-drying apparatus, which was maintuincd at -4.5”C during a May drying cycle under a xWxum of 10 L to 10~; torr. Driccl s~amplcs were immcrsecl in absolute alcohol for 1 hr of fixation prior
384
BRIEF
COMMUNICATIONS
FIG. 1. Continued
to further croscopy.
processing
for
electron
mi-
Freeze-Substitution Frozen samples were transferred to a substitution fluid of 1% osmic acid in equal parts of absolute alcohol and acctone, in polyethylene vials kept at -80 to -85°C in a mechanical freezer. The tissues were dehydrated and fixed during a 3week period with weekly agitations and
fluid changes. The fluid then was replaced with absolute acetone at -80 to -85°C and allowed to warm up to room temperature prior to preparation for ultraobservations. A mechanical structural freezer was used because of its availability in our laboratory. Actually, the use of an insulated box or a Dcwar container with dry ice in alcohol ( -78°C) is a much less expensive and equally effective alternative.
BRIEF CO\ISII’XICATIOSS
3s.i
Frc:. 1. Continued
Electron
Microscopy
and freeze-substituted, Freeze-dried, control tissues were infiltrated with propylenc oxide, cmbeddcd in Epon-Aralditc ( 1: 1)) thin-sectioned, mowted on copper grids > and double-stained with ura~~yl acetate and lead citrate. Observations and photographs were made with a Sicwcwc IA Elmiskop. RESULTS AND DISCUSSIOiY
The patterns of size, shape, location, and variable distribution of ice, as well as its relation to organelles, were identically represented by both methods (Figs. 1B and C). ICC artifacts in these rapidl) frozen tissues ranged in size from a fraction of 1 to 3 pm, with some variation from cell to cell, from nucleus to cytoplasm, and regionally within each cell, especially in the cytoplasm. Ice formations were smallest in the arca of the rough cndoplasmic reticulum where they were net-like and largest around the zymogen granules. The potentially destructive eff cct of intracellular ice in and around the rough endo( 10) was similarly plasmic reticulum demonstrated by freeze-drying and frwzcsubstitution (Figs. 1D and E).
Identical results in representation of the frozen state with freeze-drying and freezesubstitution are not predictable. Dchydration by freeze-drying depends upon sublimination of ice by molecular transfer from the solid to gaseous state without passing through the fluid state, while frwzc-substitution involws the solubility of water in the solid state in an unfrozcw orgmic solvent which replaces it at subfreezing in tempcraturcs. In freeze-drying the temperature of frozen tissues is raised 151”C, from -196 in liquid nitrogen to -4.5”C, during its sublimation or drying period of 3 days. The increase is only 111°C when frozen tissues are transferred to the substitution fluid at -85°C whew dehydration by frcczc-substitution is a I lowed to continue for 2 wwks. No diffcrcwccs due to recrystallization (7) uiidcr such dissimilar time-temperature conditions wcw noted by clcctron microscopy. This may reflect the limitations of the tcchniqucs of observation, however, rather than the abscncc of diffcwnws in the ultrastructure of the frozcw state. The absence of detectable differences in wprwcntation of the frozen state by these two mcthocls suggests thcb validity of their ilitcr~hanjieuble usu for cryobiological in-
3SG
BRIEF
COhlhlUh’ICATIONS
vestigations. There are factors otht,r thall visible results, howcvclr, which \vill determine the choice by investigators of one technique over the other. WC found no differences in the preparation for electron microscopy or in the results and their reproducibility. Greater care usually is required, however, in handling frozen tissues during transfers to and from the glass drying chamber and in other manipulations in freeze-drying. Less than $100 will establish a freeze-substitution system, unless the additional convcnicncc, but considcr,able expense, of a mechanical freezer is desired. A freeze-drying apparatus, even one like the simplified “homemade” unit we use, now costs about $1000. Freeze-drying, however, is a much more rapid method for dehydration. Time required in both mcthods can be reduced by preliminary evuluation of each type of specimen. The size of the freeze-drying chambcr( s) and the geometry of the apparatus relative to cfficient drying in a vacuum limit the number of samples in each run. In freeze-substitution the container space for immersion of vials in the refrigerant is easily enlarged with other containers to accommodate, for example, 100 instead of only the 5 to 10 samples in the freeze-drying unit. Both freeze-drying and freeze-substitution require the addition of excess dry ice to maintain the temperature (-78°C) of the trap and the substitution fluid, respectively, unless a mechanical freezer is used in freeze-substitution. More frequent additions of dry ice to the organic solvent mixture, however, are required to maintain a temperature of -40 to -50°C in the specimen chamber of the freeze-drying apparatus during sublimation. We have found freeze-substitution to be more convenient and less complicated, especially to a newly oriented laboratory worker. The relatively long dehydration time is of little consequence as other laboratory activities are performed in the interim. It has been reported that frccze-
substitutiolr is prcfcrrcd bccausc~ frcc~zcdrying also is not rcproducibl~~ enough to be trustrd as a routine proccdurc, at least under the frcxrzc-drying conditions in one laboratory (5). \Ve have noted such variations in freeze-drying only in specimens pretreated with cryoprotective agents (glycerol and dimethysulfoxide) in which, at least for glycerol, questionable sublimation may compromise the results (9). REFERENCES
1. Bank, H. Freeze injury as visualized
in tissue cultured cells hy freeze-etching. Exp. Cell
Res. 85, 367-376 ( 1974 ). 2. Luyet, B. J., and Gehenio, P. M. “Life
3.
4.
5. 6. 7.
8.
9.
10.
11.
12.
and Death at Low Temperatures.” Biodynamics, Normandy, hlissouri 1940. Mazur, P., Lribo, S. P., and Chu, E. H. Y. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Erp. Cell Res. 71, 345-355 (1972). Menz, L. J., and Luyet, B. J. An electron microscope study of the distribution of ice in sin& muscle fibers frozen rapidly. Biotlynan~ica 8, 261-294 ( 1961). Menz, L. Personal commlmication ( 1975). Meryman, H. T. (Ed.) “Cryobiology.” Academic Press, New York, 1966. Pyrdc, J. A., and Jones, G. 0. Properties of vitreous water. Nature (London) 170, 685688 ( 1952). Shxman, J. K. Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fert. Steril. 13, 49-64 (1964). Sherman, J. K., and Kim, K. S. Correlation of cellular ultrastructure before freezing, while frozen, and after thawing in assessing freeze-thaw-induced injury. Cryobiology 4, 61-74 ( 1967). Sherman, J. K., and Liu, K. C. Relation of ice formation to ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticu!um. Cryobiology 13, 599-608 ( 1976). Smith, A. U. Effects of low temperatures on living cells and tissues. In “Biological Applications of Freezing and Drying (R. J. C. Harris, Ed. ), Chap. 1. Academic Press, New York, 1954. Whittaker, D. K. Ice crystals formed in tissue during cryosurgery. II. Electron microscopy. Cryobiology 11, 202-217 ( 1974).
14, 382-386
Ultrastructural
(1977)
Comparison of Freeze-Drying and Freeze-Substitution in Preservation of the Frozen State K. C. LIU
Department of Anatomy,
AND J. K. SHERMAN
Unitiersity of Arkansas for Medical Sciences, Little Rock, Arkansas
Correlation of cellular ultrastructure before freezing, while frozen, and after thawing provides a requisite basis for evaluation of functional ‘alterations which are induced during the formation and dissolution of ice (9). Th e respective roles of intracellular and extracellular ice figure prominently in theories of freezing injury (2, 3, 6, ll), often without correlative observations on the frozen state, especially in tissue cells. Freeze-drying and freeze-substitution are the techniques most commonly used by investigators, almost interchangeably, to preserve the areas in which ice forms in and around cells during freezing at various rates. Occasional statements have been made which suggest that these techniques may yield the same results in representation of the frozen state (4, 9). Results with muscular tissue indicate no difference in the size of intracellular ice spaces with both methods ( 12), as well as the unreliability of freeze-drying (5). There is little or no information on direct experimental comparisons of these methods with epithelial cells, however, especially on the ultrastructural level. Such information is required for a better interpretation of studies on the frozen state of import’ant tissues and organs. Freeze-etching, which theoretically may represent an even more valid picture of actual structure, but which pre--.Rcwivcd
June 28, 1976.
Copyright (: 1977 by Acadrmic I’ress. Inc. All rights of reproduction in any form reserved.
sents similar results in practice ( 12), has not “suplemented or replaced freeze-substitution and freeze-drying” in this cryobiological application ( 1, 9). The purpose of this brief communication is to present the results of a comparative evaluation of freeze-drying and freezesubstitution in the electron microscopic representation of ice in the frozen state in pancreatic cells. MATERIALS
AND
METHODS
The rationale and detailed description of methods used for freezing, freezedrying, freeze-substitution, and electron microscopy have been presented previously (8, 9). Only essentials are repeated here as a basis for comparison in a study which involved five experimental runs, each with three to five tissue samples. Freezing Small (2 X 3 mm) pieces of rat pancreatic tissue, 0.7 to 0.9 mm in thickness, were frozen on an aluminum boat. A rapid overall freezing rate of about 38”C/sec was achieved by direct immersion of the tissue samples in liquid nitrogen ( - 196°C ). They were kept at -196°C for 5 min. Freeze-Drying Frozen samples were transferred to a perforated aluminum basket immersed in
ISSN 0011-2240
BRIEF
COSlhi~~ICA’I’IONS
FIG. 1. Electron micrographs of rat pancreatic acinar cells before freezing (A) and in the frozen state, as preserved by freeze-drying (B and I)) and freeze-substitution (C and E) after freezing to -196°C at 38”Cj. sec. Clear spaces in B, C, 1), and E represent areas pwreticulum, viously occupied by ice. Nucleus, N; zymogen granules, ZC; rough endoplasmic RER. A, B, and C at X4500; U and E show HER at X4.5,000. Note that the representations are indistinguishable from each of the frozen state I)y freeze-drying and freeze-suhstitutiou other.
isopentanc ( -77°C) in a jar surrounded hy dry ice and carrid to the frc,ezc-clr)+lS apparatus. The basket was r~movccl from the isopentane, blotted on dry ice, and quickly lowered into the rcfrigrrator
~hatnl)u of the freeze-drying apparatus, which was maintuincd at -4.5”C during a May drying cycle under a xWxum of 10 L to 10~; torr. Driccl s~amplcs were immcrsecl in absolute alcohol for 1 hr of fixation prior
384
BRIEF
COMMUNICATIONS
FIG. 1. Continued
to further croscopy.
processing
for
electron
mi-
Freeze-Substitution Frozen samples were transferred to a substitution fluid of 1% osmic acid in equal parts of absolute alcohol and acctone, in polyethylene vials kept at -80 to -85°C in a mechanical freezer. The tissues were dehydrated and fixed during a 3week period with weekly agitations and
fluid changes. The fluid then was replaced with absolute acetone at -80 to -85°C and allowed to warm up to room temperature prior to preparation for ultraobservations. A mechanical structural freezer was used because of its availability in our laboratory. Actually, the use of an insulated box or a Dcwar container with dry ice in alcohol ( -78°C) is a much less expensive and equally effective alternative.
BRIEF CO\ISII’XICATIOSS
3s.i
Frc:. 1. Continued
Electron
Microscopy
and freeze-substituted, Freeze-dried, control tissues were infiltrated with propylenc oxide, cmbeddcd in Epon-Aralditc ( 1: 1)) thin-sectioned, mowted on copper grids > and double-stained with ura~~yl acetate and lead citrate. Observations and photographs were made with a Sicwcwc IA Elmiskop. RESULTS AND DISCUSSIOiY
The patterns of size, shape, location, and variable distribution of ice, as well as its relation to organelles, were identically represented by both methods (Figs. 1B and C). ICC artifacts in these rapidl) frozen tissues ranged in size from a fraction of 1 to 3 pm, with some variation from cell to cell, from nucleus to cytoplasm, and regionally within each cell, especially in the cytoplasm. Ice formations were smallest in the arca of the rough cndoplasmic reticulum where they were net-like and largest around the zymogen granules. The potentially destructive eff cct of intracellular ice in and around the rough endo( 10) was similarly plasmic reticulum demonstrated by freeze-drying and frwzcsubstitution (Figs. 1D and E).
Identical results in representation of the frozen state with freeze-drying and freezesubstitution are not predictable. Dchydration by freeze-drying depends upon sublimination of ice by molecular transfer from the solid to gaseous state without passing through the fluid state, while frwzc-substitution involws the solubility of water in the solid state in an unfrozcw orgmic solvent which replaces it at subfreezing in tempcraturcs. In freeze-drying the temperature of frozen tissues is raised 151”C, from -196 in liquid nitrogen to -4.5”C, during its sublimation or drying period of 3 days. The increase is only 111°C when frozen tissues are transferred to the substitution fluid at -85°C whew dehydration by frcczc-substitution is a I lowed to continue for 2 wwks. No diffcrcwccs due to recrystallization (7) uiidcr such dissimilar time-temperature conditions wcw noted by clcctron microscopy. This may reflect the limitations of the tcchniqucs of observation, however, rather than the abscncc of diffcwnws in the ultrastructure of the frozcw state. The absence of detectable differences in wprwcntation of the frozen state by these two mcthocls suggests thcb validity of their ilitcr~hanjieuble usu for cryobiological in-
3SG
BRIEF
COhlhlUh’ICATIONS
vestigations. There are factors otht,r thall visible results, howcvclr, which \vill determine the choice by investigators of one technique over the other. WC found no differences in the preparation for electron microscopy or in the results and their reproducibility. Greater care usually is required, however, in handling frozen tissues during transfers to and from the glass drying chamber and in other manipulations in freeze-drying. Less than $100 will establish a freeze-substitution system, unless the additional convcnicncc, but considcr,able expense, of a mechanical freezer is desired. A freeze-drying apparatus, even one like the simplified “homemade” unit we use, now costs about $1000. Freeze-drying, however, is a much more rapid method for dehydration. Time required in both mcthods can be reduced by preliminary evuluation of each type of specimen. The size of the freeze-drying chambcr( s) and the geometry of the apparatus relative to cfficient drying in a vacuum limit the number of samples in each run. In freeze-substitution the container space for immersion of vials in the refrigerant is easily enlarged with other containers to accommodate, for example, 100 instead of only the 5 to 10 samples in the freeze-drying unit. Both freeze-drying and freeze-substitution require the addition of excess dry ice to maintain the temperature (-78°C) of the trap and the substitution fluid, respectively, unless a mechanical freezer is used in freeze-substitution. More frequent additions of dry ice to the organic solvent mixture, however, are required to maintain a temperature of -40 to -50°C in the specimen chamber of the freeze-drying apparatus during sublimation. We have found freeze-substitution to be more convenient and less complicated, especially to a newly oriented laboratory worker. The relatively long dehydration time is of little consequence as other laboratory activities are performed in the interim. It has been reported that frccze-
substitutiolr is prcfcrrcd bccausc~ frcc~zcdrying also is not rcproducibl~~ enough to be trustrd as a routine proccdurc, at least under the frcxrzc-drying conditions in one laboratory (5). \Ve have noted such variations in freeze-drying only in specimens pretreated with cryoprotective agents (glycerol and dimethysulfoxide) in which, at least for glycerol, questionable sublimation may compromise the results (9). REFERENCES
1. Bank, H. Freeze injury as visualized
in tissue cultured cells hy freeze-etching. Exp. Cell
Res. 85, 367-376 ( 1974 ). 2. Luyet, B. J., and Gehenio, P. M. “Life
3.
4.
5. 6. 7.
8.
9.
10.
11.
12.
and Death at Low Temperatures.” Biodynamics, Normandy, hlissouri 1940. Mazur, P., Lribo, S. P., and Chu, E. H. Y. A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells. Erp. Cell Res. 71, 345-355 (1972). Menz, L. J., and Luyet, B. J. An electron microscope study of the distribution of ice in sin& muscle fibers frozen rapidly. Biotlynan~ica 8, 261-294 ( 1961). Menz, L. Personal commlmication ( 1975). Meryman, H. T. (Ed.) “Cryobiology.” Academic Press, New York, 1966. Pyrdc, J. A., and Jones, G. 0. Properties of vitreous water. Nature (London) 170, 685688 ( 1952). Shxman, J. K. Improved methods of preservation of human spermatozoa by freezing and freeze-drying. Fert. Steril. 13, 49-64 (1964). Sherman, J. K., and Kim, K. S. Correlation of cellular ultrastructure before freezing, while frozen, and after thawing in assessing freeze-thaw-induced injury. Cryobiology 4, 61-74 ( 1967). Sherman, J. K., and Liu, K. C. Relation of ice formation to ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticu!um. Cryobiology 13, 599-608 ( 1976). Smith, A. U. Effects of low temperatures on living cells and tissues. In “Biological Applications of Freezing and Drying (R. J. C. Harris, Ed. ), Chap. 1. Academic Press, New York, 1954. Whittaker, D. K. Ice crystals formed in tissue during cryosurgery. II. Electron microscopy. Cryobiology 11, 202-217 ( 1974).