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53. The viability of cell cultures following long-term storage in liquid nitrogen
A N N U A L M E E T I N G ABSTI),ACTS 217-226, 1965) resulted in a 41% recovery of viable ('ells with the trypan blu(. method anti 71% viability count with the acridine orange fluorescent technique. In rive studies showed that the mean survival times of BDFt mice were similar when aliquots of tiw control leukemia cells and the L1210 cells kept at 0 ° for intervals up to 24 hrs were implanted intraperitoneally. Mice which receiw:d aliquots of (.ells frozen 1o -195°C demonstrated that 93% of |he ('oils were nonviable with a concomitant increase in their mean survival times although the increase was not statistically significant. Spleen colony assays showed that cell viability was not impaired between 0 ° and - 1 5 ° C and the number of colonies decreased exponentially from --15 to - 6 0 ° C . Tiae surviving fraction was 15% and was stable from -60°C. The surviving fraction was 15% and was stable from --60 to -1950C. These fimlings indicated that the in vivo spleen colony assay in nonirradiated mice was a much more precise technique for measuring the number of viable cells remaining after freezing than was the mean survival time assay.
53. The Viability of Cell Cuhures Following Long.Term Storage in Liquid Nitrogen. 2~ ARTHUR E. GREgNE, BaLU ATIIREYA, I-IERNDON B. Lmm, A.~DLEWIS L. COmZLL (South Jersey Medical Research Foundation, Camden, New Jersey, and Harrison Department of Surgical Research, University of Pennsyh'ania, Philadelphia, Pennsylvania). The use of liquid nitrogen for storage of biological materials has increased in recent years because it has been observed that deterioration of cell cultures occurs through slow enzyme activity whcn stored in CO._, cabinets where the temperature varies between --50 ° and -79°C. Recently Stulberg and Bernmn have reported possible deteriora(ion of viability, changes in L D H activity, and increase in generation time of cell cultures (hwing storage in liquid nitrogen, and we have, therefore, tested the viability of 23 frozen lots of cells comprising 16 different cell lines which have been in liquid nitrogen storage in our laboratory from 570 to 1,530 days. The criteria used for the evaluation of (:ell viability were 1) dye exclusion of trypan blue, 2) plating efficiency, and 3) cell growth in roller tubes and milk dilution bottles. Excellent growth in roller tubes and milk dilution bottles
s'Supported by USPlCIS CA-04953-07 and the John A. Hartford Foundation.
313
was observed in all 23 lots as shown by daily observation and scoring on a 1 to 4-plus basis and by (,'ell counts a f a r 1-week incubation in the re(;every vessel. Plating efficiency decreased in 60% and increased in 40% of the 23 lots, and these resuits were the least reproducible of the 3 assay methods. By the trypan blue test, viability was the same or increased 4 to 11% in 16 lots and was decreased 17 and 22% in 2 lots. In this assay method, 11% increase or decrease of the origimd viability is assumed to be experimental error of the method, and on this basis only 2 lots showed a greater decrea,se in viability. Additional testing after greater intervals of time will show whether loss of viability is occurring, but at tim m o m e n t we believe that none of the tests used to date show a ~gnificant loss in viability beyond the "inherent margin of error of the assay methods during storage of cells for up to 4 years in liquid nitrogen.
54. Freezing of Human Kidney Cell Suspensions. PAUL J. PRICE, BRm'¢DA P. ANDERSON,* AND M e a T y R. BZLL (Microbiological Associates Inc., Bethesda, Maryland). H u m a n embryonic, fetal, or neonatal (within 48 hrs after birth) kidneys were processed by enzymatic digestion aml frozen as primary suspensions. Sixty suspensions were frozen by wrapping the 16-ram screwcap test tubes with cotton, sealing them in a Styrofoam container, and placing the container directly into liquid nitrogen vapor. During the same time period, 100 suspensions were frozen at a controlled rate using a Linde BF-3-2 freezing apparatus (-I°C per rain from 0 ° to -30°C and --10°C per rain to -150°C) and stored in liquid nitrogen vapor. Test cultures were reconstituted within 2 months after freezing and again at various iute~vals ranging from 1 m o n t h to 1 ½ years. The relative merits of the two methods were evaluated with respect to number of days to culture confluency. A comparison was also nmde between the number of prefreeze and postfreeze days to confluency. The results ~howed that there was only a small difference between the number of days to confluency .(8.7 days for .the uncontrolled freezing as compared with 9.1 days for the controlled) when tested within 2 months. However, upon more prolonged storage the number of days to confluency in the uncontrolled frozen suspensions increased substantially as compared with the co~trolled freezing which varied from a reduction in the number of days (1 case) to an increa~ of 4 days. There was an average increase of 2.3 days to confluency between the posffreeze and prefreeze cultures.
53. The Viability of Cell Cuhures Following Long.Term Storage in Liquid Nitrogen. 2~ ARTHUR E. GREgNE, BaLU ATIIREYA, I-IERNDON B. Lmm, A.~DLEWIS L. COmZLL (South Jersey Medical Research Foundation, Camden, New Jersey, and Harrison Department of Surgical Research, University of Pennsyh'ania, Philadelphia, Pennsylvania). The use of liquid nitrogen for storage of biological materials has increased in recent years because it has been observed that deterioration of cell cultures occurs through slow enzyme activity whcn stored in CO._, cabinets where the temperature varies between --50 ° and -79°C. Recently Stulberg and Bernmn have reported possible deteriora(ion of viability, changes in L D H activity, and increase in generation time of cell cultures (hwing storage in liquid nitrogen, and we have, therefore, tested the viability of 23 frozen lots of cells comprising 16 different cell lines which have been in liquid nitrogen storage in our laboratory from 570 to 1,530 days. The criteria used for the evaluation of (:ell viability were 1) dye exclusion of trypan blue, 2) plating efficiency, and 3) cell growth in roller tubes and milk dilution bottles. Excellent growth in roller tubes and milk dilution bottles
s'Supported by USPlCIS CA-04953-07 and the John A. Hartford Foundation.
313
was observed in all 23 lots as shown by daily observation and scoring on a 1 to 4-plus basis and by (,'ell counts a f a r 1-week incubation in the re(;every vessel. Plating efficiency decreased in 60% and increased in 40% of the 23 lots, and these resuits were the least reproducible of the 3 assay methods. By the trypan blue test, viability was the same or increased 4 to 11% in 16 lots and was decreased 17 and 22% in 2 lots. In this assay method, 11% increase or decrease of the origimd viability is assumed to be experimental error of the method, and on this basis only 2 lots showed a greater decrea,se in viability. Additional testing after greater intervals of time will show whether loss of viability is occurring, but at tim m o m e n t we believe that none of the tests used to date show a ~gnificant loss in viability beyond the "inherent margin of error of the assay methods during storage of cells for up to 4 years in liquid nitrogen.
54. Freezing of Human Kidney Cell Suspensions. PAUL J. PRICE, BRm'¢DA P. ANDERSON,* AND M e a T y R. BZLL (Microbiological Associates Inc., Bethesda, Maryland). H u m a n embryonic, fetal, or neonatal (within 48 hrs after birth) kidneys were processed by enzymatic digestion aml frozen as primary suspensions. Sixty suspensions were frozen by wrapping the 16-ram screwcap test tubes with cotton, sealing them in a Styrofoam container, and placing the container directly into liquid nitrogen vapor. During the same time period, 100 suspensions were frozen at a controlled rate using a Linde BF-3-2 freezing apparatus (-I°C per rain from 0 ° to -30°C and --10°C per rain to -150°C) and stored in liquid nitrogen vapor. Test cultures were reconstituted within 2 months after freezing and again at various iute~vals ranging from 1 m o n t h to 1 ½ years. The relative merits of the two methods were evaluated with respect to number of days to culture confluency. A comparison was also nmde between the number of prefreeze and postfreeze days to confluency. The results ~howed that there was only a small difference between the number of days to confluency .(8.7 days for .the uncontrolled freezing as compared with 9.1 days for the controlled) when tested within 2 months. However, upon more prolonged storage the number of days to confluency in the uncontrolled frozen suspensions increased substantially as compared with the co~trolled freezing which varied from a reduction in the number of days (1 case) to an increa~ of 4 days. There was an average increase of 2.3 days to confluency between the posffreeze and prefreeze cultures.