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Optimum cooling and warming rates for the cryopreservation of cultured human keratinocytes and mouse L cells
516
ABSTRACTS,
25th ANNUAL
The cryopreservation of human granulocytes (PMNCs) has been investigated since the 1960s. Recovery of more demanding functions such as bacterial killing has not been obtained, although many different cooling procedures resulted in a numerical survival of this delicate type of cell. It was therefore considered to be necessary to first examine the conditions of the preparation with special regard to the addition and removal of two common cryoprotectants, dimethyl sulfoxide and trehalose, on the functional behavior of PMNCs in the absence of freezing and thawing. Besides counting the cells, viability was assayed in terms of phagocytosis and chemotaxis in combination with membrane integrity determinations by fluorescein diacetate and ethedium bromide. Heparin turned out to be a more suitable anticoagulant than ACD-B or CPDAl when a gravity leucapheresis procedure was used for the separation of the PMNCs. The concentration of the cryoprotectants was shown to be the most significant parameter investigated, whereas the time of exposure when varied between 5 and 60 min was of little importance for both additives. With increasing cryoprotectant concentration, the cell function decreased. Simple tests as cell number determination or cell membrane integrity determination did not document the loss of cell function. Phagocytosis or chemotaxis of the PMNCs was already reduced at low cryoprotectant concentrations. The temperature at which the cryoprotectant was added did not influence cell function when Me,SO was used. The addition of trehalose, on the other hand, turned out to be more beneficial at 22°C than at 4°C. 24. Mitogenic Response and Phenotype Expression of Frozen/Thawed Mononuclear Cells (MNC) Frozen in the Presence of l-O-Methylrat-glycerol. P. SCHUFF-WERNER,U. MILLER,
C. UNGER, Cl. A. NAGEL, AND H. EIBL (University Clinics, Department of Internal Medicine, Division of Hematology/Oncology, Robert-Koch-Str. 40, 3400-Gottingen, Federal Republic of Germany). Recently, I-0-methyl-rat-glycerol has been introduced as a new cryoprotective agent (CPA) for mononuclear cells (MNC). Any successful cryopreservation of peripheral mononuclear cells must preserve their viability and phenotypic and functional integrity. We therefore measured the mitogen-induced lymphocyte transformation of MNC before and after the freeze/ thaw process using I-0-methyl-rat-glycerol (I-0-MG) and dimethyl sulfoxide (Me,O) as cryoprotective agents. In parallel, a comparison of phenotypic markers of unfrozen and frozen-thawed cells was done by flow-cytometric analysis using the monoclonal T-cell markers CD 3, CD 4, and CD 8 as well as surface immunoglobulin (slg) as B-cell marker. Monocytes
MEETING
were identified by nonspecific esterase stain. The total cell recovery after freezing was in the range of 86% for I-0-MG and 90% for Me,SO, respectively. The lymphocyte transformation assay showed a good reproducibility of lymphocyte reactivity in cells frozen/ thawed in the presence of either CPA. The pan-T phenotype expression was not essentially altered by cryopreservation with both CPA, but there was a decrease in the percentage of SIG-positive B cells. The CD 4-positive cells decreased, whereas the CD 8positive increased, thus resulting in a decrease of the CD 4/CD 8 ratio. This effect was similar with both CPAs used. The portion of monocytes showed slight variations after cryopreservation in the different experiments, but in general both substances yielded comparable recovery rates of esterase-positive MNC. In conclusion, I-0-MG has comparable cryoprotective potency to Me,SO concerning the phenoptype and functional integrity of frozen/thawed MNC. 25. Optimum Cooling and Warming Rates for the Cryopreservation of Cultured Human Keratinocytes and Mouse L Cells. S. RANDOLPH
MAY, SHARONK. RUDIS, AND CARMELA. RuBIN (LifeCell Corporation, 3606-A Research Forest Drive, The Woodlands, Texas). The optimum combinations of cooling and warming rates were determined for cultured normal human epidermal keratinocytes (NHEK) derived from human foreskins and mouse L cells (NCTC 929). The cryopreservation agent consisted of 10% v/v DMSO in MCDB 153 medium with 0.15 M calcium, 10 t&ml EGF, 5 &ml insulin, 0.5 l&ml hydrocortizone, 0.4% v/v bovine pituitary extract, penicillin-streptomycin and amphotericin B (for NHEK), and 10% v/v DMSO in Eagle’s minimal essential medium/Earle’s balanced salt solution plus IO%0horse serum and nonessential amino acids (for NCTC 929). Controlled cooling rates were -0.3”C/min, - l”C/min, - lO”C/min, and - 20”C/min. Controlled warming rates were + l”C/min and +ZO”C/min in a programmed device, and + 173”C/min in a 37°C waterbath with agitation. Viability tests were based on trypan blue dye exclusion and staining with a combination of 0.67 FM acridine orange (stains live cells green) and 75 PM propidium iodide (stains dead cells red). The optimum coolingwarming rate combination for NCTC 929 cells was - l”C/min cooling and + 173”C/min warming, with 94-96% maximum viability obtained; lower viability levels were obtained with slower warming rates. The optimum cooling-warming rate combination for NHEK cells was - l”C/min cooling and + 173”C/min warming, with 73-91% maximum viability obtained (depending on the experiment). The level of viability of NHEK cells was found to be similar to levels in human partial-thickness skin such as that used for al-
ABSTRACTS, 25th ANNUAL lotransplantation, so that NHEK cells may be used as a model system for optimizing cryopreservation parameters for human skin. 26. Changes in the Resistance of Protoplasts and Regenerated Cells to Freezing and Osmotic Dehydration during Protoplast Culture of Marchantia polymorpha. Y. SUGAWARA, AND M.
TAKEUCHI (Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan). Resistance to freezing and osmotic dehydration of protoplasts and regenerated cells was studied in protoplast culture of Marchantia polymorpha in relation to cell wall regeneration and cell division. Protoplasts isolated from callus tissues were cultured on modified Murashige and Skoog’s medium containing 0.23 M mannitol [Z. Pflanzenphysiol. 109, 275(1983)]. Regeneration of the cell wall was initiated immediately in the protoplasts after the beginning of protoplast culture, and more than 90% of the protoplasts formed new, thin cell walls within 24 hr of culture in either light or dark conditions. Division of regenerated cells was observed some time after 48 hr among cells cultured in the light but not in the dark. The protoplasts and regenerated cells were suspended in culture media and frozen to various temperatures at a cooling rate of 0.5-0.8”Cimin. The frozen samples were thawed in water at 40°C. Survival rates at - 40°C of callus cells and freshly isolated protoplasts were less than 10%. The survival rate at -40°C of regenerated cells increased with time of culture, reaching a maximum level (6& 70%) by 18 to 20 hr of culture in the light or dark, and decreased thereafter to the initial level. About 40% of the regenerated cells cultured for 20 hr could survive freezing to - 196°C and grow again normally after thawing. The freezing resistance thus gained was rapidly lost by treatment with cell wall digesting enzyme. A similar trend was observed in the resistance of regenerated cells to osmotic dehydration in balanced salt solution (BSS): The survival rate of protoplasts in 1.O M BSS was about 30%. Whereas, with regenerated cells, no decrease in survival rate was observed even at 2.5 M BSS. Little or no difference between protoplasts and regenerated cells was observed in the degree of volumetric reduction at each concentration of BSS. However, the shapes of regenerated cells observed changing during osmotic contraction in BSS were very different from those of protoplasts. Electron microscopic observations by the freeze-fracture method showed that formation of multilamellar structures under the P face of the plasma membrane is induced in protoplasts by osmotic dehydration in 2.0 M BSS but not in regenerated cells. These data suggest that during the early stage of protoplast culture, the regenerated cell wall and/or some structure of the protoplast, especially in the plasma membrane, play an
MEETING
517
important role in the development of resistance to freezing and osmotic dehydration. 27. A Comparative Study of the Changes in the Morphology of Hyphae during Freezing and Viability upon Thawing for 19 Species of Fungi. G. J.
MORRIS,D. SMITH, AND G. E. COULSON. The changes in morphology of 19 species of fungi during freezing were examined in relation to cooling rate and the presence and absence of glycerol using a cryomicroscope. The viability of fungal hyphae was determined after equivalent rates of cooling to - 196°C in the presence or absence of glycerol. All hyphomycetes, the ascomycete, Sordaria, the zygomycete, Mucor, and the basidiomycete, Schizophyllum, survived freezing and thawing in the absence of glycerol. Cryomicroscopy demonstrated that for these fungi the formation of intracellular ice at rapid rates of cooling was not lethal. Isolates from the Mastigomycotina and some Basidiomycetes required glycerol for survival. The morphological response of Phytophthora, Aschersonia alleyrodis and Volvariella volvacea differed from other isolates, with shrinkage occurring at all rates of cooling. 28. Survival and Stability of Microorganisms during Freeze-Drying K. A. MALIK (Deutsche Sam-
mlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, West Germany) Freeze-drying is a convenient method for the preservation and long-term storage of microorganisms; however, it is not suited to a variety of delicate microorganisms and even after 60 years of intensive development in this field, freeze-drying can still harm several microorganisms. During freeze-drying microorganisms have to undergo many stresses and DNA damage may result which could either cause total death of living cells or a paralysis by which the desired or useful qualities of the living cells could be lost or damaged. Several useful strains which are obtained as a result of gene manipulation, selective screening, mutations, and bear plasmids are mostly affected by freeze-drying and may lose their useful qualities after lyophilization. During freeze-drying gram-positive bacteria usually survive better than gram-negative bacteria. The age of a culture and the cell concentration to be freeze-dried could have a profound effect on the survival of microorganisms. A number of other characteristics may also influence the resistance of several species to freeze-drying as during several years of experimentation it has been observed that strains with spores and cysts are relatively resistant to freeze-drying whereas in particular large-celled gramnegative bacteria, PHB (poly-B-hydroxy butyric acid) filled bulky cells, spirillum-like large cells (due to their abnormal growth in the cell wall), and temporarily de-
ABSTRACTS,
25th ANNUAL
The cryopreservation of human granulocytes (PMNCs) has been investigated since the 1960s. Recovery of more demanding functions such as bacterial killing has not been obtained, although many different cooling procedures resulted in a numerical survival of this delicate type of cell. It was therefore considered to be necessary to first examine the conditions of the preparation with special regard to the addition and removal of two common cryoprotectants, dimethyl sulfoxide and trehalose, on the functional behavior of PMNCs in the absence of freezing and thawing. Besides counting the cells, viability was assayed in terms of phagocytosis and chemotaxis in combination with membrane integrity determinations by fluorescein diacetate and ethedium bromide. Heparin turned out to be a more suitable anticoagulant than ACD-B or CPDAl when a gravity leucapheresis procedure was used for the separation of the PMNCs. The concentration of the cryoprotectants was shown to be the most significant parameter investigated, whereas the time of exposure when varied between 5 and 60 min was of little importance for both additives. With increasing cryoprotectant concentration, the cell function decreased. Simple tests as cell number determination or cell membrane integrity determination did not document the loss of cell function. Phagocytosis or chemotaxis of the PMNCs was already reduced at low cryoprotectant concentrations. The temperature at which the cryoprotectant was added did not influence cell function when Me,SO was used. The addition of trehalose, on the other hand, turned out to be more beneficial at 22°C than at 4°C. 24. Mitogenic Response and Phenotype Expression of Frozen/Thawed Mononuclear Cells (MNC) Frozen in the Presence of l-O-Methylrat-glycerol. P. SCHUFF-WERNER,U. MILLER,
C. UNGER, Cl. A. NAGEL, AND H. EIBL (University Clinics, Department of Internal Medicine, Division of Hematology/Oncology, Robert-Koch-Str. 40, 3400-Gottingen, Federal Republic of Germany). Recently, I-0-methyl-rat-glycerol has been introduced as a new cryoprotective agent (CPA) for mononuclear cells (MNC). Any successful cryopreservation of peripheral mononuclear cells must preserve their viability and phenotypic and functional integrity. We therefore measured the mitogen-induced lymphocyte transformation of MNC before and after the freeze/ thaw process using I-0-methyl-rat-glycerol (I-0-MG) and dimethyl sulfoxide (Me,O) as cryoprotective agents. In parallel, a comparison of phenotypic markers of unfrozen and frozen-thawed cells was done by flow-cytometric analysis using the monoclonal T-cell markers CD 3, CD 4, and CD 8 as well as surface immunoglobulin (slg) as B-cell marker. Monocytes
MEETING
were identified by nonspecific esterase stain. The total cell recovery after freezing was in the range of 86% for I-0-MG and 90% for Me,SO, respectively. The lymphocyte transformation assay showed a good reproducibility of lymphocyte reactivity in cells frozen/ thawed in the presence of either CPA. The pan-T phenotype expression was not essentially altered by cryopreservation with both CPA, but there was a decrease in the percentage of SIG-positive B cells. The CD 4-positive cells decreased, whereas the CD 8positive increased, thus resulting in a decrease of the CD 4/CD 8 ratio. This effect was similar with both CPAs used. The portion of monocytes showed slight variations after cryopreservation in the different experiments, but in general both substances yielded comparable recovery rates of esterase-positive MNC. In conclusion, I-0-MG has comparable cryoprotective potency to Me,SO concerning the phenoptype and functional integrity of frozen/thawed MNC. 25. Optimum Cooling and Warming Rates for the Cryopreservation of Cultured Human Keratinocytes and Mouse L Cells. S. RANDOLPH
MAY, SHARONK. RUDIS, AND CARMELA. RuBIN (LifeCell Corporation, 3606-A Research Forest Drive, The Woodlands, Texas). The optimum combinations of cooling and warming rates were determined for cultured normal human epidermal keratinocytes (NHEK) derived from human foreskins and mouse L cells (NCTC 929). The cryopreservation agent consisted of 10% v/v DMSO in MCDB 153 medium with 0.15 M calcium, 10 t&ml EGF, 5 &ml insulin, 0.5 l&ml hydrocortizone, 0.4% v/v bovine pituitary extract, penicillin-streptomycin and amphotericin B (for NHEK), and 10% v/v DMSO in Eagle’s minimal essential medium/Earle’s balanced salt solution plus IO%0horse serum and nonessential amino acids (for NCTC 929). Controlled cooling rates were -0.3”C/min, - l”C/min, - lO”C/min, and - 20”C/min. Controlled warming rates were + l”C/min and +ZO”C/min in a programmed device, and + 173”C/min in a 37°C waterbath with agitation. Viability tests were based on trypan blue dye exclusion and staining with a combination of 0.67 FM acridine orange (stains live cells green) and 75 PM propidium iodide (stains dead cells red). The optimum coolingwarming rate combination for NCTC 929 cells was - l”C/min cooling and + 173”C/min warming, with 94-96% maximum viability obtained; lower viability levels were obtained with slower warming rates. The optimum cooling-warming rate combination for NHEK cells was - l”C/min cooling and + 173”C/min warming, with 73-91% maximum viability obtained (depending on the experiment). The level of viability of NHEK cells was found to be similar to levels in human partial-thickness skin such as that used for al-
ABSTRACTS, 25th ANNUAL lotransplantation, so that NHEK cells may be used as a model system for optimizing cryopreservation parameters for human skin. 26. Changes in the Resistance of Protoplasts and Regenerated Cells to Freezing and Osmotic Dehydration during Protoplast Culture of Marchantia polymorpha. Y. SUGAWARA, AND M.
TAKEUCHI (Department of Regulation Biology, Faculty of Science, Saitama University, Urawa, Japan). Resistance to freezing and osmotic dehydration of protoplasts and regenerated cells was studied in protoplast culture of Marchantia polymorpha in relation to cell wall regeneration and cell division. Protoplasts isolated from callus tissues were cultured on modified Murashige and Skoog’s medium containing 0.23 M mannitol [Z. Pflanzenphysiol. 109, 275(1983)]. Regeneration of the cell wall was initiated immediately in the protoplasts after the beginning of protoplast culture, and more than 90% of the protoplasts formed new, thin cell walls within 24 hr of culture in either light or dark conditions. Division of regenerated cells was observed some time after 48 hr among cells cultured in the light but not in the dark. The protoplasts and regenerated cells were suspended in culture media and frozen to various temperatures at a cooling rate of 0.5-0.8”Cimin. The frozen samples were thawed in water at 40°C. Survival rates at - 40°C of callus cells and freshly isolated protoplasts were less than 10%. The survival rate at -40°C of regenerated cells increased with time of culture, reaching a maximum level (6& 70%) by 18 to 20 hr of culture in the light or dark, and decreased thereafter to the initial level. About 40% of the regenerated cells cultured for 20 hr could survive freezing to - 196°C and grow again normally after thawing. The freezing resistance thus gained was rapidly lost by treatment with cell wall digesting enzyme. A similar trend was observed in the resistance of regenerated cells to osmotic dehydration in balanced salt solution (BSS): The survival rate of protoplasts in 1.O M BSS was about 30%. Whereas, with regenerated cells, no decrease in survival rate was observed even at 2.5 M BSS. Little or no difference between protoplasts and regenerated cells was observed in the degree of volumetric reduction at each concentration of BSS. However, the shapes of regenerated cells observed changing during osmotic contraction in BSS were very different from those of protoplasts. Electron microscopic observations by the freeze-fracture method showed that formation of multilamellar structures under the P face of the plasma membrane is induced in protoplasts by osmotic dehydration in 2.0 M BSS but not in regenerated cells. These data suggest that during the early stage of protoplast culture, the regenerated cell wall and/or some structure of the protoplast, especially in the plasma membrane, play an
MEETING
517
important role in the development of resistance to freezing and osmotic dehydration. 27. A Comparative Study of the Changes in the Morphology of Hyphae during Freezing and Viability upon Thawing for 19 Species of Fungi. G. J.
MORRIS,D. SMITH, AND G. E. COULSON. The changes in morphology of 19 species of fungi during freezing were examined in relation to cooling rate and the presence and absence of glycerol using a cryomicroscope. The viability of fungal hyphae was determined after equivalent rates of cooling to - 196°C in the presence or absence of glycerol. All hyphomycetes, the ascomycete, Sordaria, the zygomycete, Mucor, and the basidiomycete, Schizophyllum, survived freezing and thawing in the absence of glycerol. Cryomicroscopy demonstrated that for these fungi the formation of intracellular ice at rapid rates of cooling was not lethal. Isolates from the Mastigomycotina and some Basidiomycetes required glycerol for survival. The morphological response of Phytophthora, Aschersonia alleyrodis and Volvariella volvacea differed from other isolates, with shrinkage occurring at all rates of cooling. 28. Survival and Stability of Microorganisms during Freeze-Drying K. A. MALIK (Deutsche Sam-
mlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, West Germany) Freeze-drying is a convenient method for the preservation and long-term storage of microorganisms; however, it is not suited to a variety of delicate microorganisms and even after 60 years of intensive development in this field, freeze-drying can still harm several microorganisms. During freeze-drying microorganisms have to undergo many stresses and DNA damage may result which could either cause total death of living cells or a paralysis by which the desired or useful qualities of the living cells could be lost or damaged. Several useful strains which are obtained as a result of gene manipulation, selective screening, mutations, and bear plasmids are mostly affected by freeze-drying and may lose their useful qualities after lyophilization. During freeze-drying gram-positive bacteria usually survive better than gram-negative bacteria. The age of a culture and the cell concentration to be freeze-dried could have a profound effect on the survival of microorganisms. A number of other characteristics may also influence the resistance of several species to freeze-drying as during several years of experimentation it has been observed that strains with spores and cysts are relatively resistant to freeze-drying whereas in particular large-celled gramnegative bacteria, PHB (poly-B-hydroxy butyric acid) filled bulky cells, spirillum-like large cells (due to their abnormal growth in the cell wall), and temporarily de-