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Spleen colony studies of leukemia L1210
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~' 1)1111 IIA,.I( I Y
VO[, I~ No. ti 19h8
SI'LI!]i~N' VI.
(.:OI, O N Y
S'I'UI)IF, S O F
I.~I~UKF, M I A
L ] '~,.,lO
Q u a z z t i t a l i o n o tize S u r v i v i n g I J o l ~ u l a t i o n o f I, v o z e n - l i z a w e ~ l L]21•O Cell~ U ~ i n g l. h c Nl~leen C o l o n y A s s a y ~'~ ' ".'4 K'f, (..IIAI(I " 'i' " " ISII)()I{E W()I)IN, /,"Ol.l,5, :\NI) C" . .I. I,:],;NSI , Eli
Lift, Nci,,cca I)/~.ixiu,,. Arll,,,. 1). Lilll(,., 111(.., Cambri,lgc, Ma.~.~acl~,scll.s (/21.}0 The t~'.~e of dyes (c~sitl, I.rypn~l 1)l~le ;tn(l 1~1~oI~ arrival and weiglmd '2° to 26 :-' before :~cri~lill~' or.n~c) ll:~,.~ gixt:n v:!ri:~l)h.' t'c,.~lllts fOr being l)laeed in eta experirnerlt. The mic.,~, wore ~llm!~tit~tlin~ Ih( r vietbilitv of froz!.,~l-lhnwed holzsc.d in sllspended c.a~es and had access 1,o !lonn,I :~l!d neol)l,'!slie cells, lCh!ek "t~ld Berenfood :ln(t chlorinated w, te.r ad libitum. The tmtttn' fmtn,I th.~t vi~!l•,ility lll(.,asl!r(,ll!oll|-8 wilh drinking wnit:r wax chlorinate(t (7 i(.) 10. ppna) c'osill or t r y l m n bltle ~lye exd~sion were ix!- io reduce tim l)oslirr',diation de'tths due to /),s'("l¢ doul o ?l aS. tlu(,no(,~l I~y (tv(. Ci)ll(:(!lll.rulioll, S01"Hlll e o l l c e l l i r a t i ~ , cell oo~.,onl, ratiorh an(l stt~i~!i~g time. Neo/)l.sm. Lymphocytic leukemic L1210 ceils .1~o ,,',,''''.,.... .1~:~.-.' slalo~I, il~:tl .rvt),t1!. 1olue perme~~- iJl ille log phase of growth wci'i'e'.})btained, from lfilily is l)rincilmlly ~ test, of tim into.griiv of D B A / 2 or C D F , (BALL~/Cg + D B A / 2 6 ) ~lm colt nw~l~r:~c and is :t truer tes~ ~t" via.- 1nice, wl~ich had been inomdated intraperitonetdly t>ilitv 'ti'ter l o w ltql~t)eraI~ll'C ])reservalio1~." 5 t(~ 7 .days previously. Cell eoungs were done l l:~tlmwav ct, ;~17 ¢,l~se!'ve(l ~lmt ;~cridine ornnge on n hemocytometer ~,i1(I. the (:ell suspension tmnl!~c'od lflmm,:lyn,qn~ic effects: o1~ tim cells un:v,s dihued with freezing medium 1o achieve a. loss lh~,y were cxa~nine~l r,~lfi~.ll','. fiJml cone(,ntrg|tion of 10" cells, per nil. ;l?lw 1, ~,'ivo vi:tbiliiv studies o~ frozen-ttmwed freezing 1)radium consisted of 80% 1Eagle's basnt ~!eol~lasrie c~lls I~:tve l_)ee~ q~lnlil,!tive, usually nwdium solution, la-,'<, calf' serum, and 0-c, /o t~ase(I o~ ;'lu!nor "takes," rt!te of tumor growth, glycerine. All (.ell suspensions were kept, nt ~)r I~c'reent;~go of lmst. surviv'tl. '~'v'*'''' These ice-water temperatures prior to use. sltl(tios indi¢'ate(I i.he presence of viable cells l,'reezi~~g procedure. Aliquots of 1 ml each }~t. did ~ t , (0mn~il:~le I.he i'raction of st~rviving were .,t..,tlcd~" , in ~.,.,..*~1',~,~ampules and frozen in a (,ells r'enmining. :\tl(Jltl])[. ~ tO Illt'.~tlSlll'e Llle s t l r Canal~.o cow,trolled t e m p e r a t u r e unit+ at a coolv i v i ~ fraction o[: froze~!-t}mwed cells i~ rive ing r,qte of 1°(23 per rain down to --'25°C and lmve 1.~ee~t li~nite~l ~'v' until lhe ~nenns of exlllCll /ll] :.1 l'tl{(-,* Of O G p e r r a i n d o w n t o --70°C, ploring such l)ossibili, ies were provided by at. wtiich tithe thev~ wore plae'r,~t in the liquid the Ieehniq~ws developed by Till an(1 MeCull.d~-~se of a Linde liquid nit.rogen ltefri~ern, t0r at. -- 195°C:. ]o,h"' *~ :,~(] by Bruce. et a]. :''q The experiments I)resented here ('on(:ern tim ehnnges in viability, Spleen, cob.my assay. The spleen e01ony (,xl)ressed in col()|!y-forming t~nils ( C F U ) , of ,aSs:n, has leech described in. detail, previousiv/'~' ~ leukemia 1,-12t0 cells during and after the Briefly, the. in,ravenous (IV) inoeulat.ion of freezing proce.d~re. L1210 cells into nonirradiated compatible CDF, mice gives rise tO maero,.cop~e ' ¢. " colonies in the ~[ATEIIlAI,S ANI) ~]ETII()DS .~pleen, and the number of colonies is linearly relnled to the .num:)er ' of Viable Cells in0eulated, A,imols. >ix'""to S.-t~'cc,,l-.-old- CD1;,~' -" (bALB/C-~ "> ' ' 9 In tl~es.': experiments, ampules containing l0 ° × D B A / 2 g ) n~ale, and M n a l e miee were ohtallied from vgtrio~.~s sutq-~liers ihrough t.he cells were removed during i]lo. freezing proCancer (.h~.~nother.tpy " ,tl Servme. .~. " C.,ca-. ee&~re at. --15, --30, --45, --60, nnd --195°C. ' ,', - N ~ atm.n, They were th,..t~'xed ral)idlv~ for 1 rain in a water ler. These a nimq]s were qtlg!rant.ined for 2 weeks "* bath at 3, C,. Received April 30, 1968. The contents of an ampule were dil~lted with * Supported by Contract, Pt1"-43-65-61 from ihe Chemotherapy Progrqm, National Cancer Insiituto, National Instit, utes of ]:t-eall h. * C,analco. Bethesda, Md "O
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* The confidence intervals represent one sbandard error of t,he n-man nnd are based or~ 20 mice per aSSay for each control group and 10 mice per :msay for each group of frozen-t.hw, ved LI_I0 9 cells. ]" ~ t C = grand qverage cff me~m colonies per spleen for 10 replicate experin~enis. .Earlc's balanced saline solut, im~ so t h a t an inocuhml of 300 ]2,1210 cells in 0.1. ml could be injected IV into each of 10 mice in nn expenm~ntal grotq) Three hundred nonfrozen Lt210 ceils were inoculated IV into groups of 20 mice. Six dnys later, the mice were killed, nnd their spleens were removed and placed in Bouin's fixative. Spleen colonies were counted 2-1 hrs l'tter. The results gtrd recorded, as colony-forming units, which can be defined as the n m n b e r of leukemic (:ells cap'd~le of forming splenic colehies and can be explc,_.sed ' " ",~" " as follows: •
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C l?U/a mp ule No. of cells/aml)ule X Mean no. of colm~ies/sph'.elt No. ot" cells inc)culated The C F U dr frozen-thawed cells was compared witll the O F U o f nonfrozen cells in each experiment,. ]{LSU~"L'rS As shown in Table 1, nn inlravenmls inocul'ltion of 300 non-frozen LJ210 cells into C D F , mice l~hMueed a pooled average count of 5.9 + 0.2 colonies per spleen (range 3.9 to 8.3) and agree d with our earlier re.port) ~ th'tt 1.5 to 3% of i.he injected le~Ikemie ceils formed macroscopic eolmfies in the spleen. Th.ere were no significant ,iifferenees in the n u m b e r of spleen cole, lies between control cells and those cells frozr.n to --15°C, .5.(3 ___ 0.2 colonies per spleen. The average number of spleen colonies showed
a progressive decline down to --(i0°C. Subsequenl, removal of L1210 cells from ilm liquid nitrogen refrigerator ,tt;ler storage for 24-ilrs at., --195°O demonst.rnted no filrl.ller decrease in the mean colony count'.per spleqn. The number of eoiony-t'onning ulliis per ampule was calculate(l, and the results are illust.rated in Figure 1. The average CI;'U for the 10 replicate experi{nm~ts "tre connected by a solid line. Tlmre werei no siglfifieant, differences in C F U or percentnge viability between controis and the cells frozen to --15°C. From --15 to - - 6 0 ° 0 the survival c~lrve decreased exponentially, demonstraling ec.'lhd:ir ir~jury. Tim average viable cell count was 1354, per '~mlmle. From - 6 0 to --195°C, there was no further reduelion inCF-U, sllggesiing the maximum cell destruction had been reached. .Drscussmx 5 [ a n y of the problems and limital:iozls encountered in delerrnining L1210 cell vial0ility by in vitro dy e u p t a k e methods have been overcmne 1)3" lneast;ring the ability o f t h e neoplastic cell to form spleen colonies after fi'eezing and thawing procedllres. Tiffs provides much more informqiion than mere cell membrane il~t
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1;[o. 1. ]{ecovcry of vmbl., L1210 cells during and after freezing procedures as estimated by colony-forming units, o , individual exl)eriments 20 mice per group n.t ,I°C and 10 mice per ~roup at other temperatures. ~ avera,.,m of the 10 experiments at. each point,.
lmtenl, inl, as estimated by the spleen colony assay, was 10% of its original value. It, would be ditticult, to estimate the number of viable L1210 cellsusing tile host, survival assay sinee tile variabiliLy of the a s h y is such that, one eallnot: distinguish 10-fold dit'ferenees?"~ ])uring tim lime in which we performed
these studies, Lewis et al. '~ reported that. murine hematopoietic stem cells could be cooled and thawed with a loss of only 5% of their trnnsplantar.ion potential as examined by spleen colony assay. This remn, rkabte repopulation etficiency was not obtained with 1,1210 cells, which su~,,ests~ . thal~ the neoplastic cell is more t
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i~mv~tu~c•, ~l('ln cell . l.~()u.ronele.~ " tins nlso holed Ill a | (,lip l)loro .l)rin~il,ive ~1~(., l)reserv(:d (.'ell ihe I)pller is 1110 ~l(,gr(,e 0f l)r('ser:':~li()ll. 111 ('olnlmrnl.ive viabilily slu(ties, !17:..4 of lh(, (.:ells were vi:tl)le fr()m a r)atient, with leukemic r('lie~loen(Iotheli()sis :~nd only 50:: fr()ln "[ lmlien~ w i i h n(:uie lyml~hntie leukemia. Ad(lit i(m,l (h,velol)~ents in the h n p r o v e d quaa(.ilalion i,~ '~'iro of vial)h.,, cells ean.l~e expe(.'.te(1 from ful uz'e i~x'('sl ign t.ions. ~I.'~MMAllY
T h e Sl:)leel~ eel(my ~ssav showed tirol, the 1)(,reen|:~ge of vinl_)le I.,1210 cells frozen I.o --15°(_i and i,hnwed w , s si~nih-tr 1:o l h a l for .n(ml'rozen leukemic cells as eslimate(I I)y colm~yformin~ unil.s (CF[7). At. l e m p e r ~ t u r e s l)etween --15 nn(t --(iO°C, lhe CF'LT (teerensed eXl)(mentialtv, dmnonstxatin,~ cellulnr inj~u'v during ~he freezing or l:hnvdng process. T h e surviving C]>U t'rnci.ion nt --(30°C wns 10% of lhe nonfrozen eoni.rnls., and furt.her reduction of ihe temper:~t~n'e io 195°C t'esul~ed in no f u r t h e r increase of cell destr~erion. 1~EI;'ER]r, N C E S 1. Bl,~ck, L., a m l ]~or~:,nt3aum, M. C. J:'aeto~,'s affee'tin~ tim dye exvlusio~; test. for coil vinbility. Exp. Cell. lies., 3.,5: 9.-1.'.3. 1964. 2.-]louron::le, B. A. Preservalion of lmmnn and norm':~l and ]eukmnie celts wit, h dimet, hyl snlfoxido at --80°0. Cryobiology, 3: 445-455, 19677 3. Bruce, W. ]~., and Meeker, ]3. E. ])iss(,minalion and grow(l~ of Iransptnnted iso]ogous mm'in(;..lyml',homa c,~?lls, a. N:tt,. Can<'c,r lns{.., 32: 1145-1159, 1964. 4, Bru('e, W. tt., and 1.h~tl Der Gang, ]-I.. A. A quantilative assay fen" the numl)c.:r of routine lyrnl~homa (,ells ('alto}d(' of lwoliforalion i~ ~-'i~'o. Nt:flure, 1..90: 79-80, 196,3.
5, ll:~(Imwa3", W. I';.,N(,wl)y, I,..\., nl)~l (iilllens. ,). li. 'l'I)o .)r)'i~lim.c))'.~igo vi:~l)ilil.v J(',~l,al)l>li~'~l l() l~on~, l~inr)'()w e~'lls. I. C.()n'~,l,lion wi(l~ I.r3"l>a)| 1)hm ,111(1 eosin (13"t~ ex('lusic)~l aml lis,..'l~, r ulI~Ir, Ir:msform,ti~m. lqood, 2;~: 517-525, 19fi,l. 6. ll:msrl~lr|~ml r~,ll t3"pes afire" lwolon,m:d slori~e at. •--~,, v. (...:~ncor 1~,,~., 19" 6,;14--(~,)3, 1959. 7. l(li|w, 1., Ark(w, R. F.: An(h,rson, G., ,rid ,%'lmlmrlz, S. lh'~,sorvniion aml cl~:~rn(,l(:,rizalio~ of c~ll,ur~,~Itell .n¢l ;mim:fl l~n~ors. Canec.r C,hemolhm', l'leI~.,20: 57-72, Ii)62. g. lwwis, ,1. 1~., t'assov,.tv, M.. and q'ro)mugl~, 1:. l:]., .Jr. T h e |ra.nsrd,~l;tiilm ~,tth:i~,n(.y of m . r r o w (.ooh,d ~o --IO0"C :~t 2'"C l)er n~h> ~i~,. C-'~ry()l)ioh')g3!,3 : ,17-:52, 1966. 9, ~lo('l~ll(wl~, E. A., :rod Till, ,l. ]C. The sensitivi!y ~)J" (.ells from ]~m'mal Irene n~:trrt",w 1~
Re~.. I¢;: g22-$3'2, 1,962. 10. P('~g', ]3, 1';. Cyloh~gy of ]~mz~n t,(me m:u'r(m" Slll),ior.l('~t 1(~ lh~'~.~tim;s~,~I storage, at. --,,I ,1. A!fld. l'hx'si¢fl., 1#: 301-309. 196-1. 11. ,S~g:iul':~. N. l-'ro~en slor;~(, (>f 23 v~~riou.~ solid m~,t aseiles l.un~ors. ('.aIl,,;',r l~r.,~., 21 : ,1.qG--501, 1961. 12. Till..1. E., a~.i ~IrC, ullcwl~. E. A. A ~tireeL mo:~sur~,rnm~l, of ~I., rn(li'~lion s~,nsilivilv ~f norm,.~l mous~' l~on,, n~arrow crll.~. ]~:t,tinl. R-s., 1.~: 213-~222, 1961. 13. \V(>(tinsk3", l.. l",~l(~y, (?., .,~ml. K(,nsh,r, C, J. ],:s~imat.os (:d" t.h(. survivin.~ pot,ulntion of h:uk('tni:~ L'I210 ('ells 1>5" i,-"~,itro and i,-~,i~',, :~ssay.q (altair.). (?ryol>iol(t~y. 2: 312. 1966. 14. Wo~tinsky, I., Me, ahoy, K. I:...rid l((,n~lc, r, C. ,t. Vinl>ili~y of tiftv-fivc, m,,'>pl:~sn~s afoot s'toro,go i~ ]iq~fi(t nitro.ram. (.'.rynt~iol,)g.v, l : 217226, 1965. 1..5. Wodinsky, I., Swini,trski, J., ,m(1 ](ensh,r; (2'. ,I. St)lo(,n c o l o n y sia,lies ~f I(,uk(-,~i(- 1,1210. {. (;ro,,vtl~ kine, ii(.s of tylnl~lmc'y|ie ]..1210 ,.~,lts i'n '~,i~:,, as (h"u'|-mined t,v sl,]o(,r~ C(>]oIIy a'~sa3". L.'atu'er Clu'm(,ll~(,r. ]i('l~.. 51: .tt5--421. 1967. 16. Woclin~ky, l., gwiniarski, J., and Kenstel', C. ,l, Sl)lc'('n ('olcmy st,~di~!s of le~kemia l,1210, l]. ])iff(,.rent, ial sensit, ivil.i(,s of normal an~l leuI,:emi(: l)onc: ~m~rrow colony-forming cells Io single and divi(lod (t(~se therapy with (,yrosin(, m'al'~in()si(.le (NSC. 6a878). C::,ncm" Chemotlmr, iRep., al : ,123-429. 1967.
~' 1)1111 IIA,.I( I Y
VO[, I~ No. ti 19h8
SI'LI!]i~N' VI.
(.:OI, O N Y
S'I'UI)IF, S O F
I.~I~UKF, M I A
L ] '~,.,lO
Q u a z z t i t a l i o n o tize S u r v i v i n g I J o l ~ u l a t i o n o f I, v o z e n - l i z a w e ~ l L]21•O Cell~ U ~ i n g l. h c Nl~leen C o l o n y A s s a y ~'~ ' ".'4 K'f, (..IIAI(I " 'i' " " ISII)()I{E W()I)IN, /,"Ol.l,5, :\NI) C" . .I. I,:],;NSI , Eli
Lift, Nci,,cca I)/~.ixiu,,. Arll,,,. 1). Lilll(,., 111(.., Cambri,lgc, Ma.~.~acl~,scll.s (/21.}0 The t~'.~e of dyes (c~sitl, I.rypn~l 1)l~le ;tn(l 1~1~oI~ arrival and weiglmd '2° to 26 :-' before :~cri~lill~' or.n~c) ll:~,.~ gixt:n v:!ri:~l)h.' t'c,.~lllts fOr being l)laeed in eta experirnerlt. The mic.,~, wore ~llm!~tit~tlin~ Ih( r vietbilitv of froz!.,~l-lhnwed holzsc.d in sllspended c.a~es and had access 1,o !lonn,I :~l!d neol)l,'!slie cells, lCh!ek "t~ld Berenfood :ln(t chlorinated w, te.r ad libitum. The tmtttn' fmtn,I th.~t vi~!l•,ility lll(.,asl!r(,ll!oll|-8 wilh drinking wnit:r wax chlorinate(t (7 i(.) 10. ppna) c'osill or t r y l m n bltle ~lye exd~sion were ix!- io reduce tim l)oslirr',diation de'tths due to /),s'("l¢ doul o ?l aS. tlu(,no(,~l I~y (tv(. Ci)ll(:(!lll.rulioll, S01"Hlll e o l l c e l l i r a t i ~ , cell oo~.,onl, ratiorh an(l stt~i~!i~g time. Neo/)l.sm. Lymphocytic leukemic L1210 ceils .1~o ,,',,''''.,.... .1~:~.-.' slalo~I, il~:tl .rvt),t1!. 1olue perme~~- iJl ille log phase of growth wci'i'e'.})btained, from lfilily is l)rincilmlly ~ test, of tim into.griiv of D B A / 2 or C D F , (BALL~/Cg + D B A / 2 6 ) ~lm colt nw~l~r:~c and is :t truer tes~ ~t" via.- 1nice, wl~ich had been inomdated intraperitonetdly t>ilitv 'ti'ter l o w ltql~t)eraI~ll'C ])reservalio1~." 5 t(~ 7 .days previously. Cell eoungs were done l l:~tlmwav ct, ;~17 ¢,l~se!'ve(l ~lmt ;~cridine ornnge on n hemocytometer ~,i1(I. the (:ell suspension tmnl!~c'od lflmm,:lyn,qn~ic effects: o1~ tim cells un:v,s dihued with freezing medium 1o achieve a. loss lh~,y were cxa~nine~l r,~lfi~.ll','. fiJml cone(,ntrg|tion of 10" cells, per nil. ;l?lw 1, ~,'ivo vi:tbiliiv studies o~ frozen-ttmwed freezing 1)radium consisted of 80% 1Eagle's basnt ~!eol~lasrie c~lls I~:tve l_)ee~ q~lnlil,!tive, usually nwdium solution, la-,'<, calf' serum, and 0-c, /o t~ase(I o~ ;'lu!nor "takes," rt!te of tumor growth, glycerine. All (.ell suspensions were kept, nt ~)r I~c'reent;~go of lmst. surviv'tl. '~'v'*'''' These ice-water temperatures prior to use. sltl(tios indi¢'ate(I i.he presence of viable cells l,'reezi~~g procedure. Aliquots of 1 ml each }~t. did ~ t , (0mn~il:~le I.he i'raction of st~rviving were .,t..,tlcd~" , in ~.,.,..*~1',~,~ampules and frozen in a (,ells r'enmining. :\tl(Jltl])[. ~ tO Illt'.~tlSlll'e Llle s t l r Canal~.o cow,trolled t e m p e r a t u r e unit+ at a coolv i v i ~ fraction o[: froze~!-t}mwed cells i~ rive ing r,qte of 1°(23 per rain down to --'25°C and lmve 1.~ee~t li~nite~l ~'v' until lhe ~nenns of exlllCll /ll] :.1 l'tl{(-,* Of O G p e r r a i n d o w n t o --70°C, ploring such l)ossibili, ies were provided by at. wtiich tithe thev~ wore plae'r,~t in the liquid the Ieehniq~ws developed by Till an(1 MeCull.d~-~se of a Linde liquid nit.rogen ltefri~ern, t0r at. -- 195°C:. ]o,h"' *~ :,~(] by Bruce. et a]. :''q The experiments I)resented here ('on(:ern tim ehnnges in viability, Spleen, cob.my assay. The spleen e01ony (,xl)ressed in col()|!y-forming t~nils ( C F U ) , of ,aSs:n, has leech described in. detail, previousiv/'~' ~ leukemia 1,-12t0 cells during and after the Briefly, the. in,ravenous (IV) inoeulat.ion of freezing proce.d~re. L1210 cells into nonirradiated compatible CDF, mice gives rise tO maero,.cop~e ' ¢. " colonies in the ~[ATEIIlAI,S ANI) ~]ETII()DS .~pleen, and the number of colonies is linearly relnled to the .num:)er ' of Viable Cells in0eulated, A,imols. >ix'""to S.-t~'cc,,l-.-old- CD1;,~' -" (bALB/C-~ "> ' ' 9 In tl~es.': experiments, ampules containing l0 ° × D B A / 2 g ) n~ale, and M n a l e miee were ohtallied from vgtrio~.~s sutq-~liers ihrough t.he cells were removed during i]lo. freezing proCancer (.h~.~nother.tpy " ,tl Servme. .~. " C.,ca-. ee&~re at. --15, --30, --45, --60, nnd --195°C. ' ,', - N ~ atm.n, They were th,..t~'xed ral)idlv~ for 1 rain in a water ler. These a nimq]s were qtlg!rant.ined for 2 weeks "* bath at 3, C,. Received April 30, 1968. The contents of an ampule were dil~lted with * Supported by Contract, Pt1"-43-65-61 from ihe Chemotherapy Progrqm, National Cancer Insiituto, National Instit, utes of ]:t-eall h. * C,analco. Bethesda, Md "O
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* The confidence intervals represent one sbandard error of t,he n-man nnd are based or~ 20 mice per aSSay for each control group and 10 mice per :msay for each group of frozen-t.hw, ved LI_I0 9 cells. ]" ~ t C = grand qverage cff me~m colonies per spleen for 10 replicate experin~enis. .Earlc's balanced saline solut, im~ so t h a t an inocuhml of 300 ]2,1210 cells in 0.1. ml could be injected IV into each of 10 mice in nn expenm~ntal grotq) Three hundred nonfrozen Lt210 ceils were inoculated IV into groups of 20 mice. Six dnys later, the mice were killed, nnd their spleens were removed and placed in Bouin's fixative. Spleen colonies were counted 2-1 hrs l'tter. The results gtrd recorded, as colony-forming units, which can be defined as the n m n b e r of leukemic (:ells cap'd~le of forming splenic colehies and can be explc,_.sed ' " ",~" " as follows: •
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C l?U/a mp ule No. of cells/aml)ule X Mean no. of colm~ies/sph'.elt No. ot" cells inc)culated The C F U dr frozen-thawed cells was compared witll the O F U o f nonfrozen cells in each experiment,. ]{LSU~"L'rS As shown in Table 1, nn inlravenmls inocul'ltion of 300 non-frozen LJ210 cells into C D F , mice l~hMueed a pooled average count of 5.9 + 0.2 colonies per spleen (range 3.9 to 8.3) and agree d with our earlier re.port) ~ th'tt 1.5 to 3% of i.he injected le~Ikemie ceils formed macroscopic eolmfies in the spleen. Th.ere were no significant ,iifferenees in the n u m b e r of spleen cole, lies between control cells and those cells frozr.n to --15°C, .5.(3 ___ 0.2 colonies per spleen. The average number of spleen colonies showed
a progressive decline down to --(i0°C. Subsequenl, removal of L1210 cells from ilm liquid nitrogen refrigerator ,tt;ler storage for 24-ilrs at., --195°O demonst.rnted no filrl.ller decrease in the mean colony count'.per spleqn. The number of eoiony-t'onning ulliis per ampule was calculate(l, and the results are illust.rated in Figure 1. The average CI;'U for the 10 replicate experi{nm~ts "tre connected by a solid line. Tlmre werei no siglfifieant, differences in C F U or percentnge viability between controis and the cells frozen to --15°C. From --15 to - - 6 0 ° 0 the survival c~lrve decreased exponentially, demonstraling ec.'lhd:ir ir~jury. Tim average viable cell count was 1354, per '~mlmle. From - 6 0 to --195°C, there was no further reduelion inCF-U, sllggesiing the maximum cell destruction had been reached. .Drscussmx 5 [ a n y of the problems and limital:iozls encountered in delerrnining L1210 cell vial0ility by in vitro dy e u p t a k e methods have been overcmne 1)3" lneast;ring the ability o f t h e neoplastic cell to form spleen colonies after fi'eezing and thawing procedllres. Tiffs provides much more informqiion than mere cell membrane il~t
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1;[o. 1. ]{ecovcry of vmbl., L1210 cells during and after freezing procedures as estimated by colony-forming units, o , individual exl)eriments 20 mice per group n.t ,I°C and 10 mice per ~roup at other temperatures. ~ avera,.,m of the 10 experiments at. each point,.
lmtenl, inl, as estimated by the spleen colony assay, was 10% of its original value. It, would be ditticult, to estimate the number of viable L1210 cellsusing tile host, survival assay sinee tile variabiliLy of the a s h y is such that, one eallnot: distinguish 10-fold dit'ferenees?"~ ])uring tim lime in which we performed
these studies, Lewis et al. '~ reported that. murine hematopoietic stem cells could be cooled and thawed with a loss of only 5% of their trnnsplantar.ion potential as examined by spleen colony assay. This remn, rkabte repopulation etficiency was not obtained with 1,1210 cells, which su~,,ests~ . thal~ the neoplastic cell is more t
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i~mv~tu~c•, ~l('ln cell . l.~()u.ronele.~ " tins nlso holed Ill a | (,lip l)loro .l)rin~il,ive ~1~(., l)reserv(:d (.'ell ihe I)pller is 1110 ~l(,gr(,e 0f l)r('ser:':~li()ll. 111 ('olnlmrnl.ive viabilily slu(ties, !17:..4 of lh(, (.:ells were vi:tl)le fr()m a r)atient, with leukemic r('lie~loen(Iotheli()sis :~nd only 50:: fr()ln "[ lmlien~ w i i h n(:uie lyml~hntie leukemia. Ad(lit i(m,l (h,velol)~ents in the h n p r o v e d quaa(.ilalion i,~ '~'iro of vial)h.,, cells ean.l~e expe(.'.te(1 from ful uz'e i~x'('sl ign t.ions. ~I.'~MMAllY
T h e Sl:)leel~ eel(my ~ssav showed tirol, the 1)(,reen|:~ge of vinl_)le I.,1210 cells frozen I.o --15°(_i and i,hnwed w , s si~nih-tr 1:o l h a l for .n(ml'rozen leukemic cells as eslimate(I I)y colm~yformin~ unil.s (CF[7). At. l e m p e r ~ t u r e s l)etween --15 nn(t --(iO°C, lhe CF'LT (teerensed eXl)(mentialtv, dmnonstxatin,~ cellulnr inj~u'v during ~he freezing or l:hnvdng process. T h e surviving C]>U t'rnci.ion nt --(30°C wns 10% of lhe nonfrozen eoni.rnls., and furt.her reduction of ihe temper:~t~n'e io 195°C t'esul~ed in no f u r t h e r increase of cell destr~erion. 1~EI;'ER]r, N C E S 1. Bl,~ck, L., a m l ]~or~:,nt3aum, M. C. J:'aeto~,'s affee'tin~ tim dye exvlusio~; test. for coil vinbility. Exp. Cell. lies., 3.,5: 9.-1.'.3. 1964. 2.-]louron::le, B. A. Preservalion of lmmnn and norm':~l and ]eukmnie celts wit, h dimet, hyl snlfoxido at --80°0. Cryobiology, 3: 445-455, 19677 3. Bruce, W. ]~., and Meeker, ]3. E. ])iss(,minalion and grow(l~ of Iransptnnted iso]ogous mm'in(;..lyml',homa c,~?lls, a. N:tt,. Can<'c,r lns{.., 32: 1145-1159, 1964. 4, Bru('e, W. tt., and 1.h~tl Der Gang, ]-I.. A. A quantilative assay fen" the numl)c.:r of routine lyrnl~homa (,ells ('alto}d(' of lwoliforalion i~ ~-'i~'o. Nt:flure, 1..90: 79-80, 196,3.
5, ll:~(Imwa3", W. I';.,N(,wl)y, I,..\., nl)~l (iilllens. ,). li. 'l'I)o .)r)'i~lim.c))'.~igo vi:~l)ilil.v J(',~l,al)l>li~'~l l() l~on~, l~inr)'()w e~'lls. I. C.()n'~,l,lion wi(l~ I.r3"l>a)| 1)hm ,111(1 eosin (13"t~ ex('lusic)~l aml lis,..'l~, r ulI~Ir, Ir:msform,ti~m. lqood, 2;~: 517-525, 19fi,l. 6. ll:msrl~l
Re~.. I¢;: g22-$3'2, 1,962. 10. P('~g', ]3, 1';. Cyloh~gy of ]~mz~n t,(me m:u'r(m" Slll),ior.l('~t 1(~ lh~'~.~tim;s~,~I storage, at. --,,I ,1. A!fld. l'hx'si¢fl., 1#: 301-309. 196-1. 11. ,S~g:iul':~. N. l-'ro~en slor;~(, (>f 23 v~~riou.~ solid m~,t aseiles l.un~ors. ('.aIl,,;',r l~r.,~., 21 : ,1.qG--501, 1961. 12. Till..1. E., a~.i ~IrC, ullcwl~. E. A. A ~tireeL mo:~sur~,rnm~l, of ~I., rn(li'~lion s~,nsilivilv ~f norm,.~l mous~' l~on,, n~arrow crll.~. ]~:t,tinl. R-s., 1.~: 213-~222, 1961. 13. \V(>(tinsk3", l.. l",~l(~y, (?., .,~ml. K(,nsh,r, C, J. ],:s~imat.os (:d" t.h(. survivin.~ pot,ulntion of h:uk('tni:~ L'I210 ('ells 1>5" i,-"~,itro and i,-~,i~',, :~ssay.q (altair.). (?ryol>iol(t~y. 2: 312. 1966. 14. Wo~tinsky, I., Me, ahoy, K. I:...rid l((,n~lc, r, C. ,t. Vinl>ili~y of tiftv-fivc, m,,'>pl:~sn~s afoot s'toro,go i~ ]iq~fi(t nitro.ram. (.'.rynt~iol,)g.v, l : 217226, 1965. 1..5. Wodinsky, I., Swini,trski, J., ,m(1 ](ensh,r; (2'. ,I. St)lo(,n c o l o n y sia,lies ~f I(,uk(-,~i(- 1,1210. {. (;ro,,vtl~ kine, ii(.s of tylnl~lmc'y|ie ]..1210 ,.~,lts i'n '~,i~:,, as (h"u'|-mined t,v sl,]o(,r~ C(>]oIIy a'~sa3". L.'atu'er Clu'm(,ll~(,r. ]i('l~.. 51: .tt5--421. 1967. 16. Woclin~ky, l., gwiniarski, J., and Kenstel', C. ,l, Sl)lc'('n ('olcmy st,~di~!s of le~kemia l,1210, l]. ])iff(,.rent, ial sensit, ivil.i(,s of normal an~l leuI,:emi(: l)onc: ~m~rrow colony-forming cells Io single and divi(lod (t(~se therapy with (,yrosin(, m'al'~in()si(.le (NSC. 6a878). C::,ncm" Chemotlmr, iRep., al : ,123-429. 1967.