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Osmotic tolerance of human peripheral blood lymphocytes and progenitor cells
ABSTRACTS,
676
19th ANNUAL
BARBAREEAND GARY N. SANDEN (Center for Disease Control, 1600 Clifton Rd., Atlanta, Georgia). Three types of bacteria, Pseudomonas aeruginosa, pneuStaphylococcus aureus, and Streptococcus moniae, were frozen and recovered under different conditions: harvesting from solid or liquid media, suspending the cells in different cryoprotectant solutions, and freezing at two different rates. No single set of conditions provided optimum recovery for all of these organisms. Survival was best (>95%) for P. aeruginosa when it was harvested from solid or liquid media, suspended in a mixture of serum-trehalose or serum-inositol, and frozen at 10”Clmin. Staphylococcus nureus survived better (>94%) harvested from either type of media, suspended in serum-trehalose, and frozen at l”C/min. Streptococcus pneumoniae responded better (>93%) when yarvested from a solid medium, suspended in serum inositol or 20% trehalose, and frozen at l”C/min. SESSION 6-CRYOPRESERVATION BLOOD 86. Cryopreservation
OF
COMPONENTS of PuriJed
Human Monocytes.
T. TAKAHASHI, M. F. HAMMETT, M. S. CHO, R. J. WILLIAMS, AND H. T. MERYMAN (ARC Blood Services Laboratories, Bethesda, Maryland 20814). Human monocytes were purified from buffy coat by centrifugal elutriation after the separation of mononuclear cells by Ficoll-Paque. Of the preparation 95 f 3% were monocytes. Cell suspensions were frozen by a two-step procedure in a solution consisting of 1 M Me,SO and 5% fetal calf serum in Hanks’ balanced salt solution. The cells were cooled at various rates in liquid nitrogen with storage at - 196°Cfor up to 4 months. A cooling rate of 1.25”C/min to a temperature below -50°C was found optimal. Following rapid thawing, sedimentation, and resuspension in cryoprotectant-free medium, no reduction in cell numbers or alterations of morphology were observed. Post-thaw assays included FDA hydrolysis, ethidium bromide exclusion, assay of surface receptors Fc and C3b by rosetting, bacterial killing, and chemotaxis. All were unaltered as compared to unfrozen controls. 10% (v/v) glycerol and ethylene glycol provided equally satisfactory cryoprotection. Studies of the effects of osmotic stress on monocytes show that they are more tolerant than granulocytes, unaffected by swelling or shrinking over the range 100 to 1500 mosm. Beyond these extremes, cells lost surface receptors and biological function. Monocytes could withstand volume reduction to 40% of isotonic volume and behaved as osmometers, exhibiting no volume plateau, unlike granulocytes which shrink less than predicted at os-
MEETING
molalities above about 600 mosm. This tolerance to osmotic stress, particularly to hyperosmotic stress, is the probable reason for the relative ease with which monocytes may be cryopreserved. (Supported in part by NIH Grants HL 27535 and RR05737.) 87. Osmotic Tolerance of Human Lymphocytes and Progenitor
Peripheral Blood Cells. P. LAW, P.
ALSOP, D. C. DOOLEY, AND H. T. MERYMAN (ARC Blood Services Laboratories, Bethesda, Maryland 20814). Human peripheral blood mononuclear cells were isolated with Ficoll-Paque from single units of whole blood. Monocytes were removed by attachment onto tissue culture plates and the remaining lymphocytes were at least 95% pure as judged by Wright stain. The cells were then exposed to various concentrations of Hanks’ balanced salt solution (HBSS) for 15 min at room temperature. Lymphocyte recovery was assessed with rH]thymidine uptake and the granulocytic-monocytic progenitor cells were assessed by colony formation in agar culture (CFU& The resulting isotope uptakes (fractions of maximum) were converted to fractions of lymphocytes remaining by comparison with a standard curve of cell density vs thymidine uptake. When the recoveries of lymphocytes and progenitor cells were plotted as a function of HBSS osmolality, the graph indicated that while progenitor cells in general survived better than mature lymphocytes following hyper- and hypotonic treatment, most of the points showed no statistically significant differences. The same is true for the enrichment ratio (the ratio of the recoveries of progenitor cells to those of lymphocytes). This suggests that osmotic stress may not be a useful procedure for the separation of peripheral blood stem cells from lymphocytes. (Supported in part by NIH Grants HL 27535 and RR05737.) 88. Relative Contributions of Cryobiological and Noncryobiological Factors to “Freezing” Injury in Human Granulocytes. PETER MAZUR,*
W. J. ARMITAGE,**’ AND JOHN FRIMt*’ (*Biology Division, Oak Ridge National Laboratory, and tUT-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830).
The inability of human granulocytes to retain high functional viability after freezing appears not to be due to improper cooling and warming rates. Few cells will even survive contact with 2 M glycerol and subsequent dilution in the absence of freezing. This detrimental effect of glycerol is seen only if cells are incubated at 37°C for about 60 min prior to the survival assay [chemotaxis or fluorescein diacetate (FDA)]. In contrast, nearly 100%of cells in phosphate-buffered saline
676
19th ANNUAL
BARBAREEAND GARY N. SANDEN (Center for Disease Control, 1600 Clifton Rd., Atlanta, Georgia). Three types of bacteria, Pseudomonas aeruginosa, pneuStaphylococcus aureus, and Streptococcus moniae, were frozen and recovered under different conditions: harvesting from solid or liquid media, suspending the cells in different cryoprotectant solutions, and freezing at two different rates. No single set of conditions provided optimum recovery for all of these organisms. Survival was best (>95%) for P. aeruginosa when it was harvested from solid or liquid media, suspended in a mixture of serum-trehalose or serum-inositol, and frozen at 10”Clmin. Staphylococcus nureus survived better (>94%) harvested from either type of media, suspended in serum-trehalose, and frozen at l”C/min. Streptococcus pneumoniae responded better (>93%) when yarvested from a solid medium, suspended in serum inositol or 20% trehalose, and frozen at l”C/min. SESSION 6-CRYOPRESERVATION BLOOD 86. Cryopreservation
OF
COMPONENTS of PuriJed
Human Monocytes.
T. TAKAHASHI, M. F. HAMMETT, M. S. CHO, R. J. WILLIAMS, AND H. T. MERYMAN (ARC Blood Services Laboratories, Bethesda, Maryland 20814). Human monocytes were purified from buffy coat by centrifugal elutriation after the separation of mononuclear cells by Ficoll-Paque. Of the preparation 95 f 3% were monocytes. Cell suspensions were frozen by a two-step procedure in a solution consisting of 1 M Me,SO and 5% fetal calf serum in Hanks’ balanced salt solution. The cells were cooled at various rates in liquid nitrogen with storage at - 196°Cfor up to 4 months. A cooling rate of 1.25”C/min to a temperature below -50°C was found optimal. Following rapid thawing, sedimentation, and resuspension in cryoprotectant-free medium, no reduction in cell numbers or alterations of morphology were observed. Post-thaw assays included FDA hydrolysis, ethidium bromide exclusion, assay of surface receptors Fc and C3b by rosetting, bacterial killing, and chemotaxis. All were unaltered as compared to unfrozen controls. 10% (v/v) glycerol and ethylene glycol provided equally satisfactory cryoprotection. Studies of the effects of osmotic stress on monocytes show that they are more tolerant than granulocytes, unaffected by swelling or shrinking over the range 100 to 1500 mosm. Beyond these extremes, cells lost surface receptors and biological function. Monocytes could withstand volume reduction to 40% of isotonic volume and behaved as osmometers, exhibiting no volume plateau, unlike granulocytes which shrink less than predicted at os-
MEETING
molalities above about 600 mosm. This tolerance to osmotic stress, particularly to hyperosmotic stress, is the probable reason for the relative ease with which monocytes may be cryopreserved. (Supported in part by NIH Grants HL 27535 and RR05737.) 87. Osmotic Tolerance of Human Lymphocytes and Progenitor
Peripheral Blood Cells. P. LAW, P.
ALSOP, D. C. DOOLEY, AND H. T. MERYMAN (ARC Blood Services Laboratories, Bethesda, Maryland 20814). Human peripheral blood mononuclear cells were isolated with Ficoll-Paque from single units of whole blood. Monocytes were removed by attachment onto tissue culture plates and the remaining lymphocytes were at least 95% pure as judged by Wright stain. The cells were then exposed to various concentrations of Hanks’ balanced salt solution (HBSS) for 15 min at room temperature. Lymphocyte recovery was assessed with rH]thymidine uptake and the granulocytic-monocytic progenitor cells were assessed by colony formation in agar culture (CFU& The resulting isotope uptakes (fractions of maximum) were converted to fractions of lymphocytes remaining by comparison with a standard curve of cell density vs thymidine uptake. When the recoveries of lymphocytes and progenitor cells were plotted as a function of HBSS osmolality, the graph indicated that while progenitor cells in general survived better than mature lymphocytes following hyper- and hypotonic treatment, most of the points showed no statistically significant differences. The same is true for the enrichment ratio (the ratio of the recoveries of progenitor cells to those of lymphocytes). This suggests that osmotic stress may not be a useful procedure for the separation of peripheral blood stem cells from lymphocytes. (Supported in part by NIH Grants HL 27535 and RR05737.) 88. Relative Contributions of Cryobiological and Noncryobiological Factors to “Freezing” Injury in Human Granulocytes. PETER MAZUR,*
W. J. ARMITAGE,**’ AND JOHN FRIMt*’ (*Biology Division, Oak Ridge National Laboratory, and tUT-Oak Ridge Graduate School of Biomedical Sciences, Oak Ridge, Tennessee 37830).
The inability of human granulocytes to retain high functional viability after freezing appears not to be due to improper cooling and warming rates. Few cells will even survive contact with 2 M glycerol and subsequent dilution in the absence of freezing. This detrimental effect of glycerol is seen only if cells are incubated at 37°C for about 60 min prior to the survival assay [chemotaxis or fluorescein diacetate (FDA)]. In contrast, nearly 100%of cells in phosphate-buffered saline