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49. Viability of forty-two neoplasms after long term storage in liquid nitrogen at −195°C
ANNUAL M E E T I N G ABSTRA, OTS rain showed measurable amounts of SC'~-methio nine in the soluble protein fraction. This compound tonsil!ares one of the major organic St components in t,he protein fraction purified by centrifugalion ~+nd gel filtration. A ,t-week cold Irt,:~tmt:nt of hardy st.cdlings slmwed the l:,rutein fraction to contain :tbout 80% of Se%mcthioni:m found in a similar fraction from nontreated seedlings. A similar comt:,:trison of the extracts from the nonhardy cultivar sho~ed th:tt cold treatment resulted in about 135% of the levd observed on seedlings without cold exposure. A. 10-hr deacelimation period (25~C) of 4week reid-treated tissue resulted in e..~entially no <+hauge in Se~:'-methionine Jn the soluble protein fr'tetion of the hardy cultivar bul, a 40% decrease for (he nonhar(ly one. A!t,.'rnative explanations for the differential effect of low temperature on the Se%mr, thionine content of those cultivars are: 1) :alteration in the amino acid composition of the fl'a,:tion, 2) an :alteration in b:dgmce between Se re(luetion pathways aml the soluble protein Imol, trod 3) 'm inthtemee uf temper:tture on So uptake aml transport. 49. Viability of Forty-Two Neoplasms afler Long Term Storage in Liquid Nitrogen at --195°C. t ISlI)OI~E ~VoDINSKY. K. ~lE.~tYa',* ,,d'~l) C. a. Ke~SLEn* (Arthur D. Little, 1he., Cambridge, Massachuset,ts). Forly-two rodent neoplasms stored at -195~0 for periods up to 6V2 years have remairmd transi.';:,,tabte and lethal lo their inoculated hosts upon reconst, itut,ion. In general, the mean sm'vival times of mice or rats inoculatc.d with specific neoplasms were comparable after wifl~dratvat at each storage period. An ia rive bioassay which estimated the surviving cell population based on the kineti~.~ of exponentially growing leukemia L1210 cells resistant to cytosine arabinoside (L1210/ARA-C) demonst rat(,d that, there was a ~ % reduction of viable cells due to tl~e freezing procedure and no fl~rther losses due to storage at,--105°C. The correlation coefficient (r) between mean survival times and cell inocula of fred~ or froze,n-thawed L1210/ ARA-C tumor cells was calculated to be --0.998 and ~0.989. respectively, The doubling times of the fl'esh and frozen tumor cells were 11.0 and 1t~3 hrs (luring their exponential growth phase and the survival curve a l ~ showed that the growth rate of 'he viable fraction of frozen-ilmwed cells was simi1~,- to frosh unfrozen cells. The response of 14 drag-induced resistan~ neoplasms to chemotherapeutic qgenls h,~s remt~ined Supported by Contract P H 43-6.5-61 with the Cancer Chemotherapy National Service Center, X:ttioaal Canc(,r Institute, United States Public Hvalfh Service.
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unaltered after long term storage, snggesting that malignant, cells stored at tl~ese low temperatures ~how no major changes, in lheir ~'t'l'lt,'i:tr eom* ponents on which the growth-inhibiting drugs are presumed to act. 50. T h e Influence of Cryoproteetant "tVa~hout on EdeJna and Surviva| of F r o z e . Rat Skin A ~ t t o g r a f t s . ~ ]:~AI,Pr~ J~[A3tlI,TON, ][.)0NAT0 ].~Ross.t.,* axt~ Ros~=IA'~"S~3313mm.i~* (tin(risen Department uf Surgical Research, School of Medicine, University of Pennsylvania, arid the tlospito! of the University of Pennsylvania, Phitaddphia, Y'enasylvania 19104). Dimcfl~yl sulfoxide (DMSO) has toxic effects on ceils directly proportional to concentration and temperature, yet it remains a u~eful c~'oprotective agent for skin grafts. Since circulation in skin grafts is not reestablished for ,t8 to 72 hrs after grafting, residual DMSO in the thawed imphmted graft may exert a deleterious effecL for several hours until removed }.~" diffusion acro~ the graft bed. The purpose of this experi.'.nent ~,'~ to determine the possible value of z~emoving reshlua] I)MSO with a post-thaw saline wash. Methods. Two 3 by 3 cm full thickness ekin grafts fl'om each of 77 Sprague-I)awley rat~u were taken. All grafts were soaked for 1 hr in a chilled ~olution of 0, 10, or 20% DMSO. The grafts from 33 animals were soaked but not frozen. The grafts from 4.t animals were soaked, frozen to - 6 5 ° C at 2 ° to 3~C per rain, and t.hawed at .i0~ to 450C per rain, One of each of the 77 pairs of grafts was given a 1-hr saline washout soak to remove residual DMSO. All grafts were imphmted ,'m autografts. Weights were recorded for all grafts before and after each ~)ak, and 14 graft,s were removed after 24 hrs of implantation for weight determination. The areas of the remaining 140 graft8 were measured 2 weeks later. Conctw~imv~. Surviv,'fl, 'as judged by areas 2 weeks following implantation, was not increased by DMSO washout in frozen aml unfrozen grafts, E(t,~ma of the graft& as judged by weight gai:a afler 24 hrs of implantalion, was not diminished by the saline washout soak. 51. The Relative Oatigenieity of Fresh and Frozen-Preserved Skin Homografls, ~ 811).~1¢'¢ APPle:t,* Axr~ I~AI,,PH Ha.'~tluroN (Harrison Departmeat of Surgical :Research, UnivcrsiW of P e r t . sylvania, and the tt0spital of the Unive~ikv of Pennsylvania, Plfiladelphia , Pennsylvania), Transplantation immunity, develops fl~ response to antigens released from skin homografts. The quantit)" and timing of rete~,se of antigens probably =Supported by a grant from ti~e John A, Hartford Foundatio~, inc.
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unaltered after long term storage, snggesting that malignant, cells stored at tl~ese low temperatures ~how no major changes, in lheir ~'t'l'lt,'i:tr eom* ponents on which the growth-inhibiting drugs are presumed to act. 50. T h e Influence of Cryoproteetant "tVa~hout on EdeJna and Surviva| of F r o z e . Rat Skin A ~ t t o g r a f t s . ~ ]:~AI,Pr~ J~[A3tlI,TON, ][.)0NAT0 ].~Ross.t.,* axt~ Ros~=IA'~"S~3313mm.i~* (tin(risen Department uf Surgical Research, School of Medicine, University of Pennsylvania, arid the tlospito! of the University of Pennsylvania, Phitaddphia, Y'enasylvania 19104). Dimcfl~yl sulfoxide (DMSO) has toxic effects on ceils directly proportional to concentration and temperature, yet it remains a u~eful c~'oprotective agent for skin grafts. Since circulation in skin grafts is not reestablished for ,t8 to 72 hrs after grafting, residual DMSO in the thawed imphmted graft may exert a deleterious effecL for several hours until removed }.~" diffusion acro~ the graft bed. The purpose of this experi.'.nent ~,'~ to determine the possible value of z~emoving reshlua] I)MSO with a post-thaw saline wash. Methods. Two 3 by 3 cm full thickness ekin grafts fl'om each of 77 Sprague-I)awley rat~u were taken. All grafts were soaked for 1 hr in a chilled ~olution of 0, 10, or 20% DMSO. The grafts from 33 animals were soaked but not frozen. The grafts from 4.t animals were soaked, frozen to - 6 5 ° C at 2 ° to 3~C per rain, and t.hawed at .i0~ to 450C per rain, One of each of the 77 pairs of grafts was given a 1-hr saline washout soak to remove residual DMSO. All grafts were imphmted ,'m autografts. Weights were recorded for all grafts before and after each ~)ak, and 14 graft,s were removed after 24 hrs of implantation for weight determination. The areas of the remaining 140 graft8 were measured 2 weeks later. Conctw~imv~. Surviv,'fl, 'as judged by areas 2 weeks following implantation, was not increased by DMSO washout in frozen aml unfrozen grafts, E(t,~ma of the graft& as judged by weight gai:a afler 24 hrs of implantalion, was not diminished by the saline washout soak. 51. The Relative Oatigenieity of Fresh and Frozen-Preserved Skin Homografls, ~ 811).~1¢'¢ APPle:t,* Axr~ I~AI,,PH Ha.'~tluroN (Harrison Departmeat of Surgical :Research, UnivcrsiW of P e r t . sylvania, and the tt0spital of the Unive~ikv of Pennsylvania, Plfiladelphia , Pennsylvania), Transplantation immunity, develops fl~ response to antigens released from skin homografts. The quantit)" and timing of rete~,se of antigens probably =Supported by a grant from ti~e John A, Hartford Foundatio~, inc.