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IN

V T R O

Vol . 10, No . 5 6, 1974

R E S P O N S E O F M A M M A L I A N C E LL S T O C O N T R O L L E D G R O W T H

R A T E S I N S T E A D Y S T A T E C O N T I N U O U S C U L T U R E 1

R . S I N C L A I R2

M c G i U U n i v e r s it y M o n t r e a l

S U M M A R Y

1 . M ouse LS c el ls g row i n c om pl e t e l y mi xe d s t e a d y-s t a t e c on t i nuous suspe ns ion

e he mos t a t ) c u l t u re i n de f i ne d me di um.

2 . Th e s t e a d y-s t a t e c onc e n t ra t i on o f c e ll s i s ma x i ma l a t a d i l u ti on ra t e o f 0 . 30 to

0.35 da y -1

3 . Gl uc ose c a n a c t a s the h mi t i ng su bs t ra t e fo r LS c e ll s und e r c he m os t a t c ondit ions_

4 . Th e g l ucose ox i da t i on ra t e pe r c el l doe s no t v a ry w i t h d i lu t i on ra t e .

5 . M a i n t e na nc e e ne rg y i s 19 p i c omol e s o f A TP pe r c el l pe r da y . G row t h e ne rgy i s

22 p i c omol e s o f A TP pe r c ell .

6 . S l owl y growi ng c e l l s c on t a i n more p ro t e i n a nd l e s s R NA pe r c e l l t ha n ra p i d l y

growing cel ls .

7 . Th e e ff ic iency of pro te in synthes i s decreases in slowly grow ing ce l l s , in whicb

a lowe r p rop or t i on o f ri bosome s is p re se n t i n t he f o rm of po l ysome s o r r i bosom a l

subuni t s .

8 . Ne wl y-ma de 18S R NA a ppe a rs e a r l y i n t he c y t op l a sm of r a p i d l y g rowi ng c e l l s ,

bu t i s gre a t ly de layed in s low ly grow ing ce ll s.

9 . Pu l se d a dd i t i ons o f a l i mi t i ng subs t ra t e t o s t e a dy-s t a t e popul a t i ons ma y l e a d t o

sync hron i z e d c e l l s t ha t ha ve a c on t ro l l e d i n t e rd i v i s i on t i me . He nc e c he mos t a t c u l t u re s

m a y b e use d t o i nve s t i ga t e t he i n t e rde pe nd e nc e o f e ve n t s i n t he c el l c ycl e.

I n h i s i n t r o d u c t o r y l e c t u r e t o t h e F i f t h I n t e r -

n a t io n a l S y m p o s i u m o n t h e C o n t i n u o u s C u l t u r e

of M i c ro-orga n i sms , P i t t (1 ) m a de t he fo ll owi ng

s t a t e m e n t : C h e m o s t a t c u l tu r e is t h e o n l y c o n -

c e i va b le wa y t o b r i ng und e r fu ll c on t ro l, g rowi ng

popu l a t ions o f e i t he r s ing l e t ype s o f o rga n i sms of

mi xe d c u l t u re s , c ons t i t u t e d f rom ba c t e r i a , a l ga e ,

p ro t oz oa ns , fung i , a n i ma l a nd p l a n t t i s sue c e l l s .

The u l t i ma t e ob j e c t i ve s o f suc h s t ud i e s a re t o

discover a l l the mechanisms of l iving ce l l s and

t he p rope r t i e s o f popul a t i ons , a nd t o i nve n t ne w

c ell b e h a v i o u r b y m e a n s o f m u t a n t o r g a n i sm s a n d

e xpl o i t a t i on o f me t a bo l i c r e gu l a t i on . Al mos t t he

whol e o f t he dyna mi c a s oppose d t o t he s t a t i c

mode l s o f c e l l s t ruc t u re a nd func t i on l i e be fore

u s a w a i ti n g d i s c o v e ry . T h e m o d e li n g a p p r o a c h ,

Presented a t the Twe nty-f i f th Annual M eet ing

of the Tissue Cul ture Assoc ia t ion in the Paul F .

Kruse , J r . Me mor i a l Sympos i um on S t e a dy-S t a t e

Cu lture o f Cel ls.

Send requests for reprints to Dr. R. Sinclair,

Dept . of Biology, McGi l l Univers i ty , Box 6070,

Stn. A, M ont rea l , H3C 3G1 Canada.

t ha t i s , t he t he ore t i c a l a s sumpt i ons a nd pre d i c -

t i ons a s to t he b e ha v i or o f popu l a t ions o f c e ll s i n

c he mos t a t c u l t u re i s we l l a dva nc e d , a nd t he de -

gre e o f soph i s t i c at i on o f t he c he m os t a t t e c hn i que

c ont i nue s t o i nc re a se . The l i t e ra t u re i s now re -

p l e t e wi t h de sc r i p t ions o f ma rv e l ous l y e ng i ne e re d

c he mos t a t s , c y t os t a t s , b i os t a t s , c y t oge ne ra t o r s ,

t u r b i d o s ta t s , a n d n e p h e l o s t a t s - - s y s t e m s t h a t a r e

des igned to grow ce ll s for indef ini te per iods a t

c ons t a n t a nd c on t ro l la b l e g rowt h ra t e s .

W h a t a r e w e l e a r n i n g a b o u t t h e d y n a m i c

mode l o f c e l l s t ruc t u re a nd func t i on t ha t shou l d

a c c o mp a ny the se t he ore t i c a l a nd t e c hn i c a l a d-

v a n c e s ? I t t u r n s o u t t h a t r a t h e r l i t t l e u s e h a s

be e n ma de of t he se e xc i t i ng t oo l s t o i nve s t i ga t e

physiologica l a l t e ra t ions in eukaryot ic ce l l s grow,

i ng a t d i f f e re n t r a t e s , de sp i t e t he f a c t t ha t t he y

do prov i de a wi de a ve nue t o t he s t u dy of re gu l a -

t i o n o f g e n e a c t i v i t y t h r o u g h t h e m a n i p u l a t i o n

of c on t ro l l a b l e e nv i ronme nt a l f a c t o r s . C ha nge s

i n ma mm a l i a n c el ls wi t h g row t h ra t e ha ve i nde e d

b e e n e x a m i n e d , b u t t h e e x p e r i m e n t a l s y s t e m s

295

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2 9 6 S I N C L A I R

u~ e d a re h i gh l y b i o l og i c a l a nd re l a t i ve l y unc on-

t ro l l e d , a nd i nc l ude , fo r e xa mpl e , r e ge ne ra t i ng

l i ve r , c e l l s r e l e a se d f rom c on t a c t i nh i b i t i on o r

s t a r v a t i o n , p h y t o h e m a g g l u t i n i n - s t i m u l a t e d l y m -

p h o c y t e s , v i r u s - t r a n s f o r m e d c e l l s , h e p a t o m a s o f

w t r y m g g r o w t h r a t e , a n d s i m i l a r s y s t e m s . G e n -

e ra l l y spe a k i ng , phys i o l og i c a l p ro f i l e s , fo r e xa m-

p l e , o f t he p ro t e i n - syn t he s i z i ng ma c h i ne ry , o r o f

t h e e n e r g y - p r o d u c h l g s y s t e m s , a r c e x am i n e d i n

t he qu i e sc e n t s t a t e , a nd c ompa re d w i t h c e l l s t ha t

h a v e b e e n s t im u l a t e d t o g r o w a n d d i v i d e a t s o m e

a r b i t r a r y a n d p o o r l y d e f in e d r a te . N o t o n l y is

t he more ra p i d ra t e i l l de f i ne d , i t i s o f t e n a t r a n -

s i t o ry s t a t e , a nd one w h i c h t he c e ll w i ll no t m a i n -

t a i n i n d e f i n i t d y . T h i s a g a i n e m p h a s i z e s t h e n e e d

fo r po l m l a t i ons o f ce ll s g row i ng i n c on t ro l l e d

s t e a d y - s t a t e , a n d f o r a n e x a m i n a t i o n o f t h e

c ha n g i ng phy s i o l og y o f suc h c el ls .

The g row t h o f c e l l s i n a e he mos t a t i s c on -

t r o l le d b y n u t r i e n t l i m i t a ti o n . A n y g r o w t h r a t e

te ss t h a n t h e m a x i m u m is a r e su l t o f s t a r v a t i o n

c ond i t i ons , o f c e l l s g row i ng more s l ow l y be c a use

s o m e l im i t i n g s u b s t r a t e is u n a v a i l a b l e w h e n

ne e de d . S t a rva t i on how e ve r , i s a r e l a t i ve t e rm.

A l l t he f a c t o r s r e qu i re d t o a l l ow ma mma l i a n c e l l s

t o e x p re s s th e i r m a x i m a l g r o w t h p o t e n t i a l a r e n o t

y e t k n o w n , a n d w e c a n n o t b e s u r e t h a t e v e n i n

t h e r i c h e s t m e d i u m t h e r e i s n o t s o m e f a c t o r , y e t

u n k n o w n , w h i c h , w h e n a d d e d ( o r r e m o v e d ) , w i l l

u rge t he c e l l s t o f a s t e r g row t h ra t e s . Th i s i s a

p r o b l em t h r o u g h w h i ch t h e d e s ig n o f m a m m a l i a n

c e l l g row t h me d i a ha s a l r e a dy e vo l ve d ; f i r s t , t he

m e d i u m i s d e s i g n e d t o c o n t a i n t h e m i n i m a l c o m -

p o n e n t s t o s u p p o r t g r o w t h , t h e n s u p p l e m e n t s a r e

a d d e d t o i n cr e a se t h e r a t e o f g r o w t h , a a d f in a ll y

t h e q u a l i t y o f g r o w t h b e c o m e s m o r e i m p o r t a n t

t h a n t h e q u a n t i t y , a n d m e d i a a r e c o n s t r u c t e d

fo r spe c i f ic p u rpose s .

The e x i s t e nc e , t he n , o f a l i m i t i ng subs t ra t e ,

doe s no t p re se n t a p ro b l e m ; t he re w i ll a lw : t ys be

a l i mi t , w he t he r i t be t he e x t r i n s i c nu t r i e n t , o r

t he i n t r i n s i c l i m i t a t i on i n t he c a pa c i t y o f t he c e l l

t o r e p roduc e i t s e l f f a s t e r t ha n a g i ve n ra t e . The

na t u re o f t he l i mi t i ng subs t ra t e , how e ve r , i s o f

g re a t s i gn i f i c a nc e , a nd ma y , a s t he mi c rob i o l o -

g i st s : d r e a d y k n o w , h a v e a p r o f o u n d e f fe c t o n t h e

phy s i o l og y o f t he c el l.

T h e g r o w t h o f m a m m a l i a n c ells i n s t e a d y - s t a t e

c on t i nuous c u l t u re w i l l be c ons i de re d unde r f i ve

t op i c s , e a c h o f w h i c h de ve l ops f rom t he p re c e d i ng

o n e : ( a ) D o m a m m a l i a n c ells a c h i e v e s t e a d y -

s t a t e i n t he e he m os t a t ? (b ) D o t he s t e a dy-s t ' ~ t e

c o n d i t i o n s a c h i e v e d c o n f o r m t o t h e t h e o r e t i c a l

p r e d i c t i o n s d e v e l o p e d f o r m i c r o o r g a n i s m s b y

M o n o d , N o v i c k , a n d H e r b e r t a n d t h e i r a s s o ci -

a t e s? ( c ) D oe s t he me t a bo l i c a c t i v i t y o f t he c e l l

c h a n g e a t t i m v a r i o u s g r o w t h r a t e s w h i c h c a n b e

a c h i e v ( ~ i n t he c he m os t a t ? (d ) D o t he c el ls r e -

m a i n s t r u c t u r a l l y t h e s a m e a t d i f f e r e n t g r o w t h

ra t e s '? ( e ) B y obse rv i ng a l t e ra t i ons i n t he c e ll s a t

d i f fe re n t g row t h ra t e s , i s i t pos s i b l e t o fo rm c on-

c l us i ons a bou t t he r e gu l a t i on o f gone a c t i v i t y

f r o m s u c h e x p e r i m e n t s ?

T h e e x p e r i m e n t s t h a t a r e r e v i e w e d h e r e c o n -

c o r n c o m p l e t e l y m i x e d s t e a d y - s t a t e c o n t i n u o u s

s u s p e n s i o n c u l t u r e s , a n d a l t h o u g h t h e y d o n o t

q u a l i f y u n d e r t h e s t r i c t d e f n i t i o n o f t h e c h e m o -

s t a t , t h is t e r m i s f r e q u e n t l y u s e d a s a n a b b r e -

v i a t i on , a nd i t s u se w i l l be c on t i nue d i n t h i s a r t i -

cle.

STEADY STATE CONDITIONS IN THE CHEMOSTAT

S om e o f t he e a r l ie s t a t t e m pt s t o g row c e ll s un -

d e r c h e m o s t a t c o n d i ti o n s w e r e r e p o r t e d b y C o h e n

a n d E a g l e ( 2 ) , w h o d e m o n s t r a t e d t h a t H e L a $8

c e l l s c ou l d be g row n c on t i nuous l y i n a s i mp l e

s y s t e m i n n o r m a l m e d i u m , a n d u n d e r f a v o r a b l e

c o n d i t i o n s c o u l d b e k e p t i n s t e a d y - s t a t e f o r a s

l ong a s 10 da ys . The y a l so de sc r i be d c ons i de ra b l e

osc i ll a ti ons in t he c el l pop u l a t i o n i n e x t e nde d c u l -

t u r e , w h i c h t h e y a s c r i b e d t o u n d e f i n e d c h a n g e s

i n t h e n u t r i e n t m e d i u m . W o r k f r o m o u r l a b o r a -

t o r y (3 ) , de sc r i be d a so l e ra c u l t u r e i n w h i c h

m e d i u m w a s a d d e d a t a c o n s t a n t r a t e t o a s t i r r e d

c u l t u r e a n d , w h e n t h e v o l u m e h a d r e a c h e d 3 t o

4 t i me s t he s t a r t i ng vo l ume , t he e xc e s s o f c e l l

s u s p e n s i o n w a s r e m o v e d a n d t h e c y c l e r e p e a t e d .

O ne suc h so l e ra c u l t u re o f s t r a i n L c e l l s i n de -

f i n e d m e d i u m w a s m a i n t a i n e d i n t h i s w a y f o r

m o r e t h a n 3 0 0 d ' t y s . T h i s w a s l a t e r d e v e l o p e d

i n t o a m o r e c o n v e n t i o n ' d c o n s t a n t v o l u m e a r -

r a n g e m e n t ( 4 ) , a n d i t w a s s h o w n t h a t , f r o m a

h i gh s t a r t i ng c onc e n t ra t i on o f c e l l s , i t w a s pos s i -

b l e t o r e a c h s t e a dy-s t a t e c e l l popu l a t i ons i n 5 t o

6 d a y s . P i r t a n d C a l l o w ( 5 ) , w o r k i n g w i t h m o r e

e l a b o r a t e l y m e c h , m i z e d e q u i p m e n t , s h o w e d t h a t

ERK a nd s t r a i n L c e l l s c ou l d a l so a c h i e ve s t e a dy-

s t a t e c ond i t i ons . Th i s i s be s t i l l u s t r a t e d i n a l a t e r

p a p e r ( 6 ) , w h i c h s h o w s o n e c u lt u r e i n w h i c h t h e

d i l u t i on ra t e w a s sh i f t e d re pe a t e d l y , a f t e r s t e a dy-

s t a t e ha d be e n c onf i rme d . In e ve ry c a se , 4 t o 8

da ys w e re re qu i re d t o r e a c h c ons t a n t c ond i t i ons .

P e r h a p s t h e m o s t p a i n s t a k i n g a n a l y s e s w e r e

c a r r ie d o u t b y M o s e r a n d V e c c h io ( 7 ) , w h o m a i n -

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C E L L B E H A V I O R I N S T E A D Y - ST A T E C U L T U R E 2 97

r a i n e d c u l t u r e s o f P 8 1 5 Y m o u s e a s e i t e s m a s t

c e l l s , i n a mod i f i e d c y t oge ne ra t o r fo r 50 t o 60

d a y s . S t e a d y - s t a t e c o n d i ti o n s w e r e a c h i e v e d o n l y

a f t e r a n a v e r a g e o f 2 0 d a y s , a n d t h e a p p r o a c h

t o s t e a d y - s t a t e w a s c h a r a c t e r i z e d b y r e g u l a r o s -

c i l l a t i ons i n c e l l c onc e n t ra t i ons . More r e c e n t l y ,

t h e n e p h e l o s t a t h a s b e e n m o d i f i e d t o p e r m i t t h e

g r o w t h o f m a m m a l i a n c e ll s ( 8 ) , a n d t h e d e v e l -

o p e r s o f t h is e q u i p m e n t h a v e d e m o n s t r a t e d t h a t ,

w h e n r e q u i r e d t o d o s o , t h e n e p h e l o s t a t i s c a -

pa b l e o f ma i n t a i n i ng H e L a c e ll s i n suspe ns i on a t

c o n s t a n t c o n c e n t r a t i o n s f o r u p t o 5 d a y s , t h e c o n -

c e n t r a t i o n b e i n g c o n t r o l l e d b y a p p r o p r i a t e d i l u -

t i o n w i t h f r e s h m e d i u m . N o i n f o r m a t i o n w a s

g i ve n , howe ve r , t o i nd i c a t e t he d i l u t i on r a t e s r e -

q u i r e d t o m a i n t a i n t h e s e c o n s t a n t c o n c e n t r a t i o n s .

F r o m t h e f o r e g o i n g e v i d e n c e , i t m a y b e c o n -

e l u d e d t h a t m a m m a l i a n c e l l s c a p a b l e o f g r o w i n g

i n s u s p e n s i o n c a n p r o b a b l y r e a c h s t e a d y - s t a t e

c ond i t i ons wi t h i n a l i m i t e d r a nge o f d i l u t i on

r a t e s . T h i s r a n g e v a r i e s f r o m j u s t b e l o w t h e

ma x i ma l g row t h r a t e o f t he c el ls , u sua l l y n o t l e ss

t h a n a d o u b l i n g t i m e ( t~ ) o f 24 h r , d o w n t o

g r o w t h r a t e s o f a b o u t o n e - f i f th o f t h a t , i.e . d o u -

b l i ng onc e e ve ry 120 t o 150 h r . I t i s ge ne ra l l y

a g r e ed t h a t v e r y s lo w g r o w t h r a te s h a v e n o t y e t

b e e n a c h i e v e d c o n s i s t e n t l y ; m o r e o v e r , s u c h

g r o w t h r a t e s m a y w e ll b e a c c o m p a n i e d b y a s i g -

n i f i c a n t p r o p o r t i o n o f c e l l d e a t h s . A p p r o a c h t o

s t e a dy -s t a t e i s o f t e n c ha ra c t e r i z e d by a n o sc i l l a -

t o r y p a t t e r n , t h e a n a l y s i s o f w h i c h p r o m i s e s t o

b e f a s c in a t i n g , w h e n t h e t e c h n i ca l m e t h o d s a r e

su f f i c i e n t l y soph i s t i c a t e d t o ob t a i n o sc i l l a t i ons

wi t h r e gu l a r i t y .

CELL CONCENTR TION V RI TIONS WITH

GROWTH R TE

A r e t h e s t e a d y - s t a t e c o n c e n t r a t i o n s r e a c h e d i n

c o n t i n u o u s c u l t u r e i n a c c o r d w i t h t h e p r e d i c ti o n s

b a s e d o n M i c h a e l i s - M e n t e n k i n e t i c s , a n d b e s t

d e v e l o p e d b y H e r b e r t , E l l s w o r t h a n d T e l l in g

(9 ) ? Th i s i s a qu e s t i on a ske d b y f e w i nve s t i -

g a t o r s g r o w i n g m a m m a l i a n c ells . E a r l y w o r k

f r o m P i r t ' s l a b o r a t o r y ( 5 ) s h o w e d s t e a d y - s t a t e

c ell c o n c e n t r a t i o n s w i t h n o o b v i o u s p a t t e r n ; b u t

l a t e r , a m o r e s a t i s f a c t o r y r e l a t i o n s h i p w a s o b -

t a i n e d t h a t d i s p l a y e d r e a so n a b l e c o n f o r m i t y , b u t

w i t h c o n s i d e r a b l y r e d u c e d c o n c e n t r a t i o n s a n d

l o w d i l u ti o n r a t e s ( 6 ) . D e s p i t e t h e m a i n t e n a n c e

b y m o u s e a s c it e s m a s t c ells o f p r o l o n g e d s t e a d y -

s t a t e c e l l c onc e n t ra t i ons , t he se c onc e n t ra t i ons

f l uc t ua t e d wi t h d i l u t i on r a t e i n a n i nc ons i s t e n t

m a n n e r , v a r y i n g f r o m 1 4 • 1 0~ ce ll s pe r ml a t

~t. = 32 hr , 18 • 10~ ce ll s pe r m l a t t~ = 53 hr ,

9 • 10~ pe r m l a t t . = 57 h r , a nd 1 .8 • 10~

c el ls pe r m l a t t , = 122 h r (7 ) . In ou r own

l a b o r at o r y ., i t w a s s h o w n t h a t c o n f o r m i t y w a s

m a i n t a i n e d f o r a l i m i t e d n u m b e r o f d i l u t i o n

ra t e s , fo r s t r a i n L c e ll s g rowi ng i n de f i ne d m e -

d i u m ( 4 ) . M o r e r e c e n t l y , w e h a v e u s e d L S c el ls

a n d h a v e o b t a i n e d f u r t h e r e v i d e n c e t h a t t h e s e

c el ls do , be h a ve p re d i c t a b l y i n c on t i nuou s c u l t u re ,

a n d a p p e a r t o d i s p l a y tw o m o d i f i ca t io n s o f t h e

c l a s si c a l m i c ro o rga n i sms p a t t e r n . F i g . 1 i l lu s -

t r a t e s c e l l n u m b e r , D N A ( a s d e o x y a d e n o s i n e ,

d A ) a n d p r o t e i n p l o t t e d a g a i n s t d i l u t i o n r a t e , o r

ra t i o , a l ong t he ho r i z on t a l a x i s , t r a ns l a t e d i n t o

doub l i ng t i me a l ong t he uppe r a x i s . The c e l l s a re

LS c e l l s , de r i ve d by P a u l f rom mouse L c e l l s , a nd

s e le c te d b e c a u s e o f t h e i r p o o r a t t a c h m e n t t o g la s s

s u r f a c e s . T h e m e d i u m i s t h e B i r c h a n d P i t t

f o r m u l a t i o n ( 1 0 ) , c o n t a i n i n g t h e n o r m a l s p e c -

t r u m o f d e f i n e d c o m p o n e n t s , a l t h o u g h c o m p a r a -

t i v e l y r i ch i n a m i n o a c i d s. I t s s o le m a c r o m o l e c u -

l a r c o n s t i t u e n t i s l o w m o l e c u l a r w e i g h t m e t h y l -

ce l lu lose . These resul t s , which i l lus t ra te the be -

h a v i o r o f s e m i c o n t i n u o u s c u l t u r e s, h a v e n o t r e -

q u i r e d t h e u s e o f a n y s o p h i s t i c a t e d , a u t o m a t e d

e q u i p m e n t b e y o n d a r e l i a b l e a l a r m c l o c k . C u l -

t u r e s w e r e s e t u p i n m a g n e t i c a l ly s t i r r e d F l o r e n c e

f l asks , ga s se d c on t i n uou s l y wi t h 5 C O2 i n a i r ,

a nd po r t i on s o f t he c e ll su spe ns i on w e re r e p l a c e d

w ~ t h f r e s h m e d i u m a t 8 - h r i n t e r v a l s , t o a c h i e v e

t he d i l u t i on r a t i o s no t e d a l ong t he a bsc i s s a .

F e e d i n g a t i n t e rv a l s w a s c o n t i n u e d f o r 1 0 t o 1 2

d a y s u n t i l w e w e r e s at is f ie d t h a t s t e a d y - s t a t e h a d

b e e n r e a c h e d . I t i s e v i d e n t t h a t t h e s t e a d y - s t a t e

c o n c e n t r a t i o n o f c e l ls g o e s t h r o u g h a d e f i n it e

m a x i m u m a t a b o u t 0 . 3 3 d a y -1, w i t h a p r o n o u n c e d

de c l i ne i n t he va l ue s o n e a c h s i de o f t h i s m a x i -

m u m . T h e n e a r c o n s t a n t o r h m : i z 0 n t a l r e l a t i o n -

sh i p , p re d i c t e d a nd e xpe r i me n t a l l y ve r i f i e d i n

m i c r o o r g a n i s m s , h a s d i s a p p e a r e d . A t h i g h d i l u -

t i on r a t e s t he de c l i ne i s no t a s s t e e p a s t he p re -

d i c t ed p a t t e r n , w h i c h r e q u i r es a p r e c i p i t o u s d r o p

t o t he c r i t i c a l d i l u t i on r a t e , a b ov e wh i c h on Iy

w a s h o u t o c c u r s . T h i s p h e n o m e n o n h a s b e e n

n o t e d t o a le s s er d e g r e e w i t h m i c r o o r g a n i s m s a n d

h a s b e e n a t t r i b u t e d t o w a l l g r o w t h ( 9 , 1 1 ) , a n d

Co t h e a c c u m u l a t i o n o f t o x ic b y - p r o d u c t s ( 1 1 ) .

W e h a v e n o t e n c o u n t e r e d s i g n i f i c a n t i n t e r f a c e

g rowt h wi t h t h i s pa r t i c u l a r c e l l sy s t e m, a nd

whi l e e xpe r i me n t s ha ve no t be e n c a r r i e d ou t , t o

e x a m i n e t h e p o s s i b i l i t y o f t o x i c b y - p r o d u c t s , i t

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:FIG. 1 . S tead y-s ta te concen t ra t ions of ce ll numbers , D N A , and prote in a t va r iou s d i lu-

t i on r a t i o s. LS m ouse c e ll s we re g rown se mi c on t i nuous l y i n de f ine d me d i um , a n d a pp rop r i a t e

f ra c t i ons we re r e p l a c e d a t 8 -h r i n t e rva l s fo r 10 t o 12 da ys . P o i n t s r e p re se n t me a ns o f t he

t h re e s a mpl e s t a ke n du r i ng 1 da y .

wou l d c e r t a i n l y be d i f f i c u l t t o v i sua l i z e , i n ma m-

ma l i a n c e ll s, a g re a t e r a c c um ul a t i on o f tox i ns a t

h i g h t h a n a t l o w d i l u t io n r a t e s . A m o r e l ik e l y

e x p l a n a t i o n i s a m o r e f u n d a m e n t a l o n e , b a s e d

o n t h e m o d e l o f c ell g r o w t h p r o p o s e d b y W i l l ia m s

( 1 2 ) . H e r e g a r d e d c e l l g r o w t h a s a t w o - s t a g e

p r o c e s s : u p t a k e o f n u t r ie n t s i n t o t h e c ell , a n d

s y n t h e s i s a n d p r o c e s s in g o f m a c r o m o l e c u l e s f r o m

t h e n u t r i e n t p o o l . O n th i s b a s is , th e s t e a d y - s t a t e

c onc e n t ra t i on o f c e l l s wi t h r e spe c t t o d i l u t i on

r a t e t u r n e d o u t t o b e a l i n e a r r e l a t i o n s h i p , t h e

c e l l c onc e n t ra t i on de c l i n i ng s t e a d i l y wi t h i nc re a s -

i n g d i l u ti o n r a t e . I f n u t r i e n t u p t a k e f o l l o w e d

M i c h a e l i s - M e n t e n k i n e t i c s , a n d h a d s u p e r i m -

p o s e d o n i t a c o n s t a n t t i m e p e r i o d f o r m a c r o -

m o l e c u l a r s y n t h e s i s , t h e n t h i s m a y w e l l l e a d t o

a r e l a ti o n s h i p c lo s e r t o t h a t f o u n d e x p e r i m e n -

ta l ly .

T h e d e c l i n e f o u n d a t l o w d i l u t i o n r a t e s h a s

b e e n a t t r i b u t e d t o t h e r e d u c e d a v a i la b i l it y o f

e ne rgy . S t a t e d s i mp l y , a s t he c e ll s g row m ore

s l o w l y , m o r e o f t h e a v a i l a b l e e n e r g y m u s t b e

c ha nne l e d i n t o ma i n t e na n c e o f t he c el l, a nd l e s s

i s a va i l a b l e fo r g rowt h p roc e s se s . S i nc e t he r a t e

o f g row t h i s f ixe d, t h i s l e a ds t o r e du c e d s t e a dy -

s t a t e c e l l c onc e n t ra t i ons . The o re t i c a l a na l ys i s o f

t h i s pos s i b i l i t y ha s be e n e xp l o re d by P i r t (13 ) ,

a n d b y W a s e a n d H o u g h ( 1 4 ), a n d e x p e r im e n t a l

obse rv a t i ons und e r d i f f e re n t O~ c onc e n t ra t i on s

h a v e b e e n m a d e b y K i l b u r n e t al . ( 1 5 ) . B y m e a -

s u r i n g t h e r a t e s o f g l y c o l y s is a n d o x i d a t i o n i n

ba t c h c u l t u re s un de r d i f f e re n t O~ t e ns i ons , i t wa s

s h o w n t h a t , e v e n a t c o m p a r a t i v e l y h i g h g r o w t h

ra t e s ( e . g . t . ---- 1 .15 da y ) , some 65 o f t he c e l l' s

e n e r g y p r o d u c t i o n w a s u s e d f o r m a i n t a i n i n g t h e

c e l l , a s d i s t i nc t f rom t he e ne rgy r e qu i re d t o syn -

t he s i z e ma t e r i a l fo r a ne w da ugh t e r c e l l . Th i s

wa s c on t ra s t e d wi t h t he ba c t e r i a l c e l l wh i c h use d

l es s t h a n o n e - t e n t h o f it s e n e r g y p r o d u c t i o n f o r

m a i n t e n a n c e , d e s p i t e t h e f a c t t h a t i t r e q u i r e d a

m u c h g r e a te r ( b y 1 0 t im e s ) a m o u n t o f e n e r g y

p e r u n i t o f d r y w e i g h t f o r m a i n t e n a n c e . C e l l s

g r o w i n g m o r e s l o w ly w ill r e q u i r e a n e v e n g r e a t e r

p r o p o r t i o n o f t h ei r e n e r g y p r o d u c t i o n f o r m a i n -

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C E L L B E H A V I O R I N S T E A D Y - ST A T E C U L T U R E 2 9 9

t e n a n c e p u r p o s e s , a n d f u r t h e r e v i d e n c e o n t h i s

p r o b l e m i s p r e s e n t e d b e l o w .

I t m u s t b e a d d e d h e r e t h a t t h e d e c l in e i n

s t e a dy -s t a t e c e l l c onc e n t ra t i ons a t l ow d i l u t i on

ra t e s ma y a l so be a r e su l t o f c e l l de a t h . The o -

re t i c a l c onse que nc e s o f a c ons t a n t l o s s o f c e l l

v i a b i l i t y h a v e b e e n d i s c u s s e d b y S i n c l a i r a n d

T o p i w a l a ( 1 6 ) , w h o i n t ro d u c e d i n t o t h e s t e a d y -

s t a t e e q ua t i on , t he t e rm - -3 ,x , t he r a t e o f l o s s o f

c ells b y d e a t h , a n d s h o w e d t h a t t h e s t e a d y - s t a t e

c onc e n t ra t i on o f c e l l s wi l l de c l i ne t o a n i nc re a s -

i n g e x t e n t a t l o w g r o w t h r a t e s . M a m m a l i a n c ell

p o p u l a t i o n s a r e a l w a y s s u b je c t t o a p r o p o r t i o n

o f c e l l d e a t h s , b u t w e h a v e n o t f o u n d t h i s a

p rob l e m wi t h t h i s c e l l sy s t e m. Dye e xc l us i on

r o u t i n e l y s h o w s a v i a b i l i t y o f o v e r 9 0 % , a n d

e ve n i n t he s l owe s t c u l t u re s i t ha s r a re l y f a l l e n

be l ow 85% .

E N E R G Y ~ r A N D G R O W T H ] :~ A TE

Of t he p r i nc i pa l c a use s o f t he de c l ine , we re -

g a r d e n e r g y d e p r i v a t i o n a s p r e d o m i n a n t . T h i s i s

s h o w n i n F i g . 2 , i l l u s t r a t i n g t h e s t e a d y - s t a t e

c o n c e n t r a t i o n o f g l u c o s e a n d l a c t a t e i n t h e

g r o w t h m e d i u m . A t l o w d i l u t i o n r a t e s , n o g l u -

c o se is d e t ec t a b l e i n t h e o u t p u t m e d i u m u n t i l a

d o u b l i n g t i m e o f 2 d a y s ; a t s h o r t e r d o u b l i n g

t i m e s t h e m o r e r a p i d l y g r o w i n g c e l l s a r e n o t

us i ng a l l t he a va i l a b l e g l uc ose , a nd t he i nc re a s i ng

a m o u n t d e t e c t e d m i r r o r s t h e d e c l i n i n g s t e a d y -

s t a t e c onc e n t ra t i on o f c e l l s . Th i s i s e v i de nc e t ha t

m a m m a l i a n c ells c a n g r o w i n s t e a d y - s t a t e , w i t h

g l u c o se a s t h e l i m i t in g n u t r i e n t . L a c t a t e i s n o t

p r o d u c e d b y s l o w l y g r o w i n g c e l l s ; t h e m a x i m a l

c o n c e n t r a t i o n i s f o u n d i n t h e c u l t u r e i n w h i c h

t he s t e a dy -s t a t e c e l l c onc e n t ra t i on i s a l so h i gh -

e s t . T h e s e s i m p l e n u t r i e n t m e a s u r e m e n t s m a y b e

c o n v e r t e d t o m e t a b o l i c q u o t i e n t s ( F i g . 3 ) , b y

n o r m a l i z i n g a n d m u l t i p l y i n g b y t h e d i l u ti o n r a t e ,

a n d r e v e a l t h a t t h e g l u c o s e c o n s u m p t i o n p ~ r

mi l l i on c e l l s pe r da y i nc re a se s a l mos t l i ne x : l y

w i t h d i l u t i o n r a t e , a s d o e s l a c t a t e o u t p u t , ~ p -

p e a r i n g t o r e fl e ct t h e m o r e r a p i d r a t e o f g r o ~ t h

of the cel ls .

I t t u r n s o u t , h o w e v e r , t h a t w h e n t h e n m t a -

bo li c quo t i e n t o f O~ c on sum pt i o n i s me a su re d ,

t h i s doe s no t r e ve a l t he s a me l i ne a r i nc re a se

wi t h d i l u t i on r a t e . O xyge n is supp l i e d t o t he

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Fro. 2 . S tead y-s ta te concen t ra t ions of glucose an d lac ta te a t va r iou s d i lu t ion ra t ios .

G row th con di t ions were the same as in F ig . 1 , So, inpu t conc ent ra t ion of g lucose .

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Fro. 3. Metabolic quotients of tota l glucose used, lactate produced, glucose oxidized,

and oxy gen used by L S mo use ce ll s grow n in suspension a t va r iou s d i lu t ion ra tios9

c el ls b y c o n t i nuo us ga s s i ng wi t h 5% C O~ i n a i r ,

a n d b y u s i n g t h e F l o r e n c e f l a sk w i t h a r e l at i v e ly

h i gh su r fa c e t o vo l ume ra t i o , we f i nd i t i s pos -

s ib l e t o m a i n t a i n 8 0 t o 9 0 % o x y g e n s a t u r a t i o n

i n a l l t he c u l t u re s e xc e p t i n t he de nse s t c u l t u re s ,

w h e r e i t m a y f a ll to 6 0 t o 6 5% . O x y g e n p a r t ia l

p re s su re s o f 80 t o 160 mm Hg usua l l y a l l ow c e l l s

t o e x p r e s s t h e i r m a x i m a l g r o w t h ( 1 7 - 1 9 ) , a n d

t h e g a s s i n g m e t h o d i s c o n s i d e r e d t o m a i n t a i n a

n o n l i m i t in g s u p p l y o f 02 . O x y g e n u p t a k e r a t e s

w e r e m e a s u r e d b y a n e x t e r n a l m e t h o d . S a m p l e s

o f c ell su s p e n s io n w e r e t r a n s f e r r e d f r o m t h e c o n -

t i n u o u s c u l t u r e t o a s t a n d a r d C l a r k e l e c t r o d e

p o l a r o g r a p h a s s e m b l y , a n d a f t e r 3 r a i n e q u i li b r a -

t i on wi t h 5% C 02 i n a i r, t he r a t e o f O~ c on -

s u m p t i o n w a s m e a s u r e d . T h u s , f o r e a c h s t e a d y -

s t a t e p o p u l a t i o n o f c e l l s , t h e m a x i m u m c a p a c i t y

f o r O ~ u p t a k e w a s m e a s u re d . T h i s w a s f o u n d t o

b e r e m a r k a b l y s t a b l e ( F i g . 3 ) a n d , e x c e p t f o r

t he s l owe s t g row i ng c el ls , r e ma i ne d c on s t a n t a t

4 .0 t o 4 .2 mi c rom ol e s o f 02 up t a k e pe r l 0 s c ei ls

p e r d a y ( 1 8 ) . I f w e c a l c u l a t e t h e q u o t i e n t o f

g l u co s e t h a t is c o n s u m e d , b u t w h i c h d o e s n o t

a p p e . q r a s l a c t a t e i n t h e m e d i u m , t h a t i s , t h e

g l uc ose wh i c h i s p re sume d t o be ox i d i z e d , t h i s

re su l t s i n a g l uc ose ox i d i z e d quo t i e n t t h a t i s

a l so ne a r l y c ons t a n t , 0 . 67 t o 0 .74 mi e romol e s pe r

10~ c ells p e r d a y . A s s u m i n g a n u p t a k e o f s ix

mol e c u l es o f 0~ pe r mo l e c u l e o f g l uc ose ox id i z e d ,

t he se f i gu res a re su rp r i s i ng l y c o ns i s t e n t .

I t now be c ome s pos s i b l e t o c a l c u l a t e i n a

c o m p l e t e l y d i f f e r e n t w a y t h e r e l a t i v e a m o u n t s

o f e n e r g y u s e d i n m a i n t e n a n c e a n d i n g r o w t h .

The d i a g ra ms show t ha t , s i nc e ox i da t i on i s r e l a -

t i v e l y c o n s t a n t , e x t r a e n e r g y u s e d b y t h e m o r e

r a p i d l y g r o w i n g c ells i s o b t a i n e d f r o m t h e p r o -

d u c t i o n o f la c t a te , a l t h o u g h t h i s s o u r c e a m o u n t s

t o le ss t h a n 5 % o f t h e t o t a l r e q u i r e m e n t o f t h e

c e l l , e xc e p t i n t he c a se o f t he mos t r a p i d l y g row-

i ng c e i l s . Assumi ng 38 mo l e c u l e s o f ATP pe r

g l uc ose mo l e c u l e ox i d i z e d , a nd 2 mo l e c u l e s o f

A T P f ro m g l yc o l ys i s , i t is pos s i b l e t o c a l c u l a t e

t h e t o t a l e n e r g y p r o d u c t i o n o f th e c ell. T h e n ,

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D I L U T I O N R A T E , D A Y - !

EzG. 4 . A c t iv i t i e s of g lucose -6-pho spha te dehydrogen ase , cytoplasm ic mal ic deh ydroge nase ,

and lac ta te dehydrogenase in LS mouse ce l l s a t va r ious d i lu t ion ra t ios .

w h e r e E i s t h e t o t a l e n e r g y p r o d u c e d ; Era t h e

a m o u n t u s e d i n m a i n t e n a n c e ; E~ t h e a m o u n t

u s e d i n g r o w t h ; x , t h e c el l c o n c e n t r a t i o n ; a n d D ,

t he d i l u t i on r a t e . Thu s , i f E i s p l o t t e d a ga i ns t

D , t h e i n t e r c e p t i s a m e a s u r e o f E , ~ a n d t h e

s l ope i s a me a su re o f E~ . R e gre s s i on a na l ys i s

s h o w s t h e m a i n t e n a n c e e n e r g y t o b e 1 9 m i c r o -

mol e s o f A T P pe r 106 c el ls pe r da y , a n d t h e

g r o w t h e n e r g y , i.e . th e e n e r g y t o m a k e 1 0~ cells,

is 22 m i c ro m o l e s o f A T P . T h e s a m e p a r a m e t e r s

m e a s u r e d f o r L S c ells g r o w n i n b a t c h c u l t u r e

u n d e r l i m i t i n g c o n c e n t r a t i o n s o f 0 2 w e r e s h o w n

t o be 17 a nd 23 mi c rom ol e s o f A T P pe r 106 c el ls ,

r e s p e c t iv e l y ( 1 5 ) . I t i s e v i d e n t t h a t , i n m o r e

ra p i d l y g rowi ng c e l l s , t he r e s i de nc e t i me i s

sho r t e r , a nd l e s s e ne rgy pe r c e l l i s r e qu i re d fo r

m a i n t e n a n c e , t h u s m o r e i s a v a i l a b l e f o r g r o w t h ,

a nd g l yc o l ys i s ne ve r ge ne ra t e s a s i gn i f i c a n t pa r t

o f t h e t o t a l e n e r g y i n t h i s s y s t e m .

A n u m b e r o f e n e r g y - a s s o c i a t e d e n z y m e s h a v e

be e n me a su re d wi t h i n t he c e l l s , a nd t he se a re

s h o w n i n F i g . 4 . C y t o p l a s m i c m a l i c d e h y d r o g e n -

a s e ( E C 1 . 1 . 1 . 3 7 ) ( M D H ) t e n d s t o d e cr ea s e

w i t h i n c r e a s e d g r o w t h r a t e . G l u c o s e - 6 - p h o s p h a t e

d e h y d ro g e n as e (E C 1 . 1 . 1 . 4 9 ) ( G - 6 - P D H ) r e -

m a i n s c o n s t a n t a t l o w d i l u t i o n r a t e s , b u t i n -

c re a se s a s t he c e l l g rowt h r a t e i nc re a se s , a nd

p r o b a b l y r e f l e c t s t h e i n c r e a s i n g d e m a n d f o r p e n -

rose fo r nuc l e i c a c i d syn t he s i s . La c t a t e de hydro -

g e n as e ( E C 1 . 1 . 1 . 2 7 ) ( L D H ) b e h a v e s i n w h a t

a p p e a r s t o b e a n a n o m a l o u s m a n n e r , d e c r e a s i n g

b y 4 - f o l d as t h e g r o w t h r a t e i n c re a se s . W h y

shou l d s l owl y g rowi ng c e l l s , wh i c h a re no t p ro -

d u c i n g l a c t a t e i n t o t h e m e d i u m , h a v e r e l a ti v e l y

h i g h c o n c e n t r a t i o n s o f l a c t a t e d e h y d r o g e n a s e ?

T h i s w o u l d s e e m t o b e a n a r t i f a c t o f t h e s e m i -

c on t i nuous f e e d i ng s c he du l e . Hour l y a na l ys i s o f

t h e m e d i u m a f t e r a n o r m a l p r o p o r t i o n a l c h a n g e ,

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3 0 2 S I N C L A I R

h a s s h o w n t h a t l a c t a te i s i m m e d i a te l y g e n e r a te d

i n t o t he me di um a f t e r f e e d i ng , a nd i n s l owl y

growi ng c e l l s t h i s l a c t a t e i s l a t e r r e move d f rom

t h e m e d i u m b y t h e c e l ls . T h u s , b e c a u se t h e y a r e

chro nica l ly glucose deficient , n ewly ad ded glu-

c ose i s i mm e di a t e l y c onve r t e d t o qu i c k e ne rg y

by glycolys i s , and the lac ta te i s then s lowly

t a k e n u p a g a i n a n d o x id iz ed . T h e r e f o re , t h e L D H

a c t i v i t y m u s t b e m a i n t a i n e d t o c a t a l y z e t h i s

me t a bo l i c r e qu i re me nt , a nd i t mi gh t be p re d i c t e d

t ha t i n a fu l l y c on t i nuous c u l tu re , LD H a c t i v i t y

woul d be m uc h l owe r i n s l owl y growi ng c el ls .

Th i s be ha v i or i s r e mi n i sc e n t o f e a r l i e r work

by Gl i nos e t a l . (20) , who showe d t ha t s t r a i n L

c e l l s ma i n t a i ne d i n s t a t i ona ry pha se wi t hou t r e -

n e w a l o f m e d i u m , h a d a n L D H a c t i v i ty o f a b o u t

1 .0 un i t p e r mg pro t e i n , a va l ue s imi l a r t o t ha t o f

e xpone nt i a l l y g rowi ng c e l l s , a nd t he a c t i v i t y i n -

c re a se d t o 8 t o 10 t i me s t ha t va l ue whe n t he

me di um wa s re p l a c e d e a c h da y . The l a t t e r c e l l s

showe d a s i mi l a r r e l e a se o f l a c t a t e i n t o t he me -

d i um a f t e r f e e d i ng , fo l l owe d by a l a t e r a nd more

g r a d u a l u p t a k e o f t h a t l a c t a t e . S i m i l a r e n z y m e

a c t i v i ti e s i n oxy ge n- l i mi t e d ba t c h c u l t u re s o f LS

cel l s have been invest iga ted by Se l f e t a l . (21) ,

w h o s h o w e d t h a t G - 6 - P D H i n c r e a s e d s l i g h t l y

wi t h i nc re a s i ng 08 t e ns ion a nd grow t h ra t e ;

M D H d e cr ea s ed , an d L D H r e m a in e d n e a rl y c o n -

s t a n t . I n a s t u d y o f M o r r i s h e p a t o m a t i s s u e s ,

C r i s s (22) h a s shown t ha t r a p i d l y g rowi ng ,

p o o r l y d i ff e r en t ia t e d h e p a t o m a s h a v e M D t t a c -

t i v i ti e s t ha t a re l owe r t ha n i n the m ore s lowl y

growi ng h i gh l y d i f fe re n t i a t e d he pa t oma s , whi c h

i n t u rn a re l owe r t ha n i n norm a l li ve r . S imi l a r ly ,

G - 6 - P D H i n c r e a s e d w i t h t h e r a p i d i t y o f g r o w t h ,

a n d L D H d i d n o t v a r y s i g n if ic a n tl y b e t w e e n t h e

d i f fe re n t he pa t oma s . The s i mi l a r i t y be t we e n t he

re su l ts f rom s t e a dy-s t a t e c on t inuous c u l t u re s a nd

t he mu c h mo re b io log ic al ly he t e roge ne ous mi n i -

m a l d e v i a t io n h e p a t o m a s , s u g g es ts t h a t t h e

f o r m e r m a y b e u s e d a s a m o d e l s y s t e m t o e x -

a mi ne me t a bo l i c sh i f t s t ha t oc c ur a s t i s sue s

c ha nge t he i r g row t h ra t e .

PROTEIN SYNTI IESIS AND GROWTH RATE

Wi t h c e r t a i n e xc e p t i ons , t he e nz yme s we ha ve

me a sure d t e n d t o i nc re a se as t he c e ll s g row m ore

s l owly , a nd so t oo , doe s the t o t a l p ro t e i n c on t e n t

of t he L S c el ls (F i g . 1 ) . Th i s i s t rue a l so o f H e L a

c e l l s g rowi ng i n suspe ns i on i n se rum-suppl e -

m e n t e d m e d i u m , a n d i s i n s h a r p c o n t r a s t t o t h e

r i bonuc le i c a c id pe r c e ll , o r p e r un i t DN A, whi c h

i s s e e n t o doubl e be t we e n s l owl y growi ng a nd

ra p i d l y g rowi ng c e l l s (28) . Th i s i s shown e ve n

more d ra ma t i c a l l y whe n c a l c u l a t e d a s t he ra t e

o f R N A a c c u m u l a t io n p e r u n i t D N A ( t h e

s t e a d y - s t a t e R N A / D N A t im e s th e d i lu t io n r a t e ) ,

whi c h i nc re a se s 10- fo l d wi t h i n t he ra nge me a -

sured. The rapidly growing ce l l s a re , of course ,

p r o d u c in g m o r e p r o t e in p e r u n i t t i m e t h a n s lo w l y

growi ng c el ls , a nd t h i s r e l a t ionsh i p m a y be qu a n-

t i fied by the e ff ic iency of pro te in synthes i s , a

p a r a m e t e r t h a t h a s b e e n r e l a t e d t o g r o w t h r a t e

i n a num be r o f ba c t e r i a l sys t e ms (23 , 24) . The

e f f ic i e ncy of p ro t e i n syn t he s i s i s de fi ne d a s t he

ou t p u t o f p ro t e i n (a s a r a t e ) r e l a t i ve t o t he

s t e a d y - s t a t e c o n c e n t r a t i o n o f R N A , a n d i s c a n

c u l a t e d a s t he d i l u t i on ra t e t i me s p ro t e i n / R NA.

As se e n i n F i g . 5 , t h i s qua n t i t y re ma i ns re l a t ive l y

constant a t higher di lut ion ra tes , but fa l l s off

ma rke d l y a t l ow d i l u t i on ra t e s . To i nve s t i ga t e

thi s loss of eff ic iency, we ha ve exam ined the cyto -

plasmic r ibosomal prof i les of the ce l l s a t di f fe r-

e n t g rowt h ra t e s , a nd f i nd t ha t r a p i d l y g rowi ng

c el ls ha ve a g re a t e r p ropo r t i on o f po l ysome s , a nd

a g re a t e r numbe r o f r i bosoma l subuni t s r e l a t i ve

t o m o n o m e r i c r i b o s o m e s ( T a b l e 1 ) . I t m a y b e

n o t e d t h a t t h e p r o p o r t i o n o f p o l y so m e s a p p e a r s

t o i nc re a se p ropor t i ona t e l y wi t h g rowt h ra t e ,

and does not re f lec t the di scont inui ty i l lus t ra ted

in the e ff ic iency of the prote in-synthes iz ing sys-

t e m re fe r re d t o e a r li e r. Th i s sys t e m i s, o f c ourse ,

h i gh l y c ompl e x a nd i s und e r a va r i e t y o f c on t ro ls ,

o f whi c h t he a va i l a b i l i t y o f r i bosoma l subuni t s

a nd t he fo rm a t i on of po l ysoma l c ompl e xe s i s on l y

one a spe c t , a nd c e r t a i n l y t he da t a p re se n t e d he re

are suff ic ient only to emphasize an inte res t ing

a re a fo r fu r t h e r i nve s t i ga ti on .

S i mi l a r r e l a t i onsh i ps be t we e n t he po l ysoma l

c o n t e n t a n d t h e r a t e o f g r o w t h h a v e b e e n r e -

por t e d i n o t he r e xpe r i me nt a l s i t ua t i ons , fo r e x-

am ple , in asc i tes ce ll s t ran sfe rred to enr iched

me di um (25) , a nd i n He La c e l l s r e l e a se d f rom

p u r o m y c i n t r e a t m e n t ( 2 6 ). M i c r o o rg a n i s m s

grown i n c he m os t a t c ondi ti ons a l so i nc re a se t he i r

prote in-synthes iz ing e ff ic iency by an increased

nu mb e r o f po l ysom e s a t h i gh grow t h ra t e s (23 ,

24) . The re duc e d ra t e o f p ro t e i n syn t he s i s a t low

grow t h ra t e s m e a ns t ha t , i n t he se ce ll s, a g re a t e r

p r o p o r t i o n o f t h e R N A p r e s e n t i s u n u s e d , a n d

t h is h a s b e e n r e f e r re d t o a s e x t r a R N A b y

Koch (27) , who has extensive ly di scussed i t s role

i n t he l i f e h i s t o ry o f

Escher ichia col i

The i n forma t i on d i sc usse d t o t h i s po i n t ha s

be e n ob t a i ne d wi t hou t t he use o f r a d i o i so t ope s .

I t r e qu i re s on l y b r i e f r e f l e c t i on t o a ppre c i a t e

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C E L L B E H A V I O R I N S T E A D Y - ST A T E C U L T U R E 3 0 3

S.O

4.0

3.0

2.0

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lhG. 5 . Th e e ff ic iency (D • pro te in /R N A , by we ight ) of the protein-synthes i~ . ing sys-

t e m i n H e L a c el ls g rown i n Ea g l e ' s m i n i m um e s sen t ia l me d i um p l us 10% c a l f s e rum, a t

va r i ous d i l u t i on r a t i o s . Di f fe re n t symbo l s r e p re se n t s e pa ra t e e xpe r ime n t s .

T A B L E 1

COMPARATIVE PROPORTIONS OF RIBOSOMES, AS

POLYSOMES, MONORIBOSOMES~ AND RIBOSOMAL

SUBUNITS IN

HEL

CELLS AT VARIOUS DILU-

TION RATES

Dilution

Rate

(day L

0.12

0 .25

0 .48

0.57

Polysomes

%

25.7

25 .9

35 .6

40 .5

Monosomes

%

74.3

52 .9

4 3 . 9

33 .0

Ribosomal

Subunits

%

0

21 .2

20 .5

26 .5

Polysome

Monosome

0 .35

0 .50

0.81

1 2 2

t h a t t h e a d d i t i o n o f s u c h p r o b e s t o a c o n t i n u o u s

c u l t u r e s y s t e m g e n e r a t e s a c o m p l e t e l y n e w s e t

o f p rob l e ms . The se a r i s e i n i t i a l l y a s p ra c t i c a l

d e c i s i o n s w h i c h m u s t b e m a d e , a n d q u i c k l y e s -

c a l a t e i n t o t h e o r e t i c a l p r o b l e m s d e m a n d i n g k i -

n e t ie a n a l y si s , w h i c h h a s n o t y e t b e e n m a d e . E x -

p e r i m e n t a l l y , t h e a d d i t i o n o f a r a d i o a c t i v e

c o m p o u n d a n d i t s c o l d c a r r i e r , u n l e s s c o m p l e t e l y

i ne r t a s a nu t r i e n t , w i l l a f f e c t t he de l i c a t e l y ba l -

a n c e d s t e a d y - s t a t e c o n d i t i o n s i n t h e c h e m o s t a t .

F u r t h e r , t h e p e r m u t a t i o n s o f p u l s e a n d ch a s e

b e c o m e e n d l e s s . I s t h e l a b e l e d c o m p o u n d a d d e d

t o t h e g r o w t h v e s s e l o n l y ? I f s o , i s t h e d i l u t i o n

c o n t i n u e d o r s t o p p e d ? I s t h e r e s e r v o i r s u p p l e -

m e n t e d w i t h a n e q u a l c o n c e n t r a t i o n o f c o l d c o m -

p o u n d ? I s t h e l a b e l e d c o m p o u n d a d d e d t o t h e

r e s e r v o i r o n l y , a n d i t s c o n c e n t r a t i o n a l l o w e d t o

b u i l d u p i n t h e g r o w t h v e s s e l ? I s t h e l a b e l e d

c o m p o u n d a d d e d t o b o t h g r o w t h v e s s e l a n d r e s -

e r v o i r ? H o w t h e n c a n i t b e c h a s e d i f t h i s is d e -

s i r e d? E a c h o f t he se s i t ua t i ons r e su l t s i n d i ff e r -

e n t k i n e t ic s o f t h e p a s s a g e o f t h e r a d i o i s o t o p e

t h r o u g h t h e o r g a n i s m , a n d o f c o u r s e w i l l v a r y

e n o r m o u s l y w i t h t h e i s o t o p e o f i n t e r e st a n d t h e

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3 0 4 S I N C L A I R

r a d i o a c t i v e c o m p o u n d i n t o w h i c h i t i s i n c o r p o -

r a t e d .

T h e s e p r o b l e m s a r e n o t a s a c u t e i n s e m i c o n -

t i n u o u s c u l t u r e , a n d i n o r d e r t o i n v e s t i g a t e f u r -

t he r t he l o s s i n r i bosom a l e f f ic i e ncy , we h a ve

p r o c e e d e d , u n d a u n t e d , t o a d d t r i t i a t e d u r i d i n e ,

a n d t o m e a s u r e i t s i n c o r p o r a t i o n i n t o R N A i n

ra p i d l y a nd i n s l owl y g row i ng c el ls 28 ) . Af t e r a

4 5 - m i n p u l s e , r i b o s o m e s a n d r i b o s o m a l R N A

w e r e i s o la t e d b y s t a n d a r d d e t e r g e n t - w a s h i n g a n d

p h e n o l e x t r a c t i o n m e t h o d s . I t w a s o b s e r v e d t h a t

t h e m a j o r i n c o r p o r a t i o n w a s i n t o t h e m o n o s o m e

a n d s u b u n i t p e a k , o r m o r e s p e c if ic a ll y , in t o t h e

c y t o p l a sm i c 1 8S R N A o f t h e r a p i d l y g r o w i n g

c e l l s . B y c ompa r i son , t he i nc o rpo ra t i on o f l a be l

i n t o r R N A i n s l o w l y g r o w i n g c ells i n 4 5 r a in w a s

i ns i gn if i c an t . Th e a dd i t i on o f l a be l fo r 12 h r r e -

s u l t e d i n 2 8 S a n d 1 8 S R N A o f e q u al s p e ci fi c a c-

t i v i t y i n r a p i d l y g row i ng c el ls bu t , e ve n a f t e r

t h a t t i m e , i n s l o w l y g r o w i n g c e l l s , t h e 1 8 S R N A

h a d a l o w e r s p e c i f i c a c t i v i t y t h a n 2 8 S R N A

T a b l e 2 ) . T h u s , t h e r e a p p e a r s t o b e a d i f f e r -

e n t i a l r a t e o f p r o c e s si n g o f t h e t w o R N A s p e ci es ,

be t we e n fa s t a nd s l ow c e l l s . Ev i de nc e ye t t o be

c o n f i r m e d s u g g e s t s t h a t t h e n u c l e o p l a s m o f t h e

r a p i d l y g r o w i n g c e l l c o n t a i n s a s m a l l p r o p o r t i o n

o f 18S R NA o f h i gh spe c i f i c a c t i v i t y , i n c on t ra s t

t o t he nuc l e op l a sm o f t he s l owe r c e l l s , wh i c h c on -

t a i n m o r e 1 8 S R N A o f m u c h l o w e r s p e c i f i c a c -

t i v i t y . T h u s , c o n t r o l o f t h e a v a i l a b i l i t y o f n e w

c y t o p l a s m i c r i b o s o m e s m a y b e t h r o u g h t h e

s m a l l e r s u b u n i t , c o n t r o l l e d i n t u r n b y r a t e o f

m o v e m e n t , o r m o d i f i ca t io n o f t h e 1 8S R N A , i n

t he nuc l e o p l a sm o f t he c e ll .

T h e s e , th e n , a r e t e n t a t i v e e x a m p l e s o f th e

p o t e n t i a l u s e o f t h e c o n t i n u o u s c u l t u r e m e t h o d ,

t h r o u g h i t s c a p a c i t y t o g e n e r a t e b i o c h e m i e a l l y

use fu l a moun t s o f c e l l s wi t h va ry i ng phys i o l og i c a l

p ro f il e s, a nd i l l u s t r a t e t he pos s i b i l i ty o f i nve s t i -

g a t i n g f u n d a m e n t a l b i o c h e m i c a l a n d b i o l o g i c a l

processes .

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re ve a l t he be ha v i o r o f popu l a t i ons o f c e l l s , a nd

t h e i r e x t e n s i v e p r o p e r t i e s u n d e r c h o s e n c o n d i -

t io n s . T h e y m a y a ls o b e u s e d t o i n v e s ti g a t e r e g u -

l a t i o n i n t h e i n d i v i d u a l c e l l b y m o d i f y i n g t h e

s y s t e m t o g e n e r a t e s y n c h r o n i z e d c e l l s . T h i s m a y

b e a c h i e v e d b y r e g u l a ti n g t h e l im i t i n g s u b s t r a t e .

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t h e l i m i t i n g s u b s t r a t e i s a d d e d t o t h e c h e m o s t a t

i n a s e ri e s o f pu l s e d a dd i t i ons , t he c el l po pu l a t i on

w i l l b e c o m e e n t r a i n e d , o r s y n c h r o n i z e d i n s o m e

T A B L E 2

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f a s h i o n r e la t e d t e m p o r a l l y t o t h e p u l s e d a d d i t i o n .

Thus , i f a c ons t a n t g rowt h r a t e i s e s t a b l i she d ,

a n d t h e l i m i t i n g s u b s t r a t e i s p r o v i d e d i n m e a -

s u r e d a m o u n t s a t i n t e r v a l s e q u a l t o t h e d o u b l i n g

t i m e o f t h a t s t e a d y - s t a t e p o p u l a t i o n , t h e n a f t e r

s i x t o e i gh t a dd i t i ons , t he c e l l s shou l d be syn -

c h ron i z e d , d i v i d i ng onc e e a c h i n t e rva l . The r e su l t

i s a sy s t e m wh i c h ge ne ra t e s sync h ron i z e d c e l l s

wi t h a c on t ro l l e d d i v i s i on t i me , a nd wh i c h o f fe r s

t h e p r o s p e c t o f a n a l y z i n g t h e e v e n t s o f t h e c el l

c y c l e a n d t h e i r i n t er r e la t io n s h i p s , w i t h m u c h

gre a t e r p re c i s i on t ha n ha s be e n pos s i b l e h i t he r t o .

A l t h o u g h t h e t e c h n i q u e h a s b e e n a p p l i ed s u c ce s s-

fu l l y t o ye a s t c el ls 29 , 31 ) , i t ha s no t ye t be e n

u s e d w i t h m a m m a l i a n c e l l s , o n e r e a s o n b e i n g

t h a t t h e l i m i t i n g s u b s t r a t e h a s n o t b e f o r e b e e n

de f i ne d i n ma mma l i a n c e l l c he mos t a t e xpe r i -

m e n t s .

A n a t t e m p t h a s b e e n m a d e t o s u r v e y t h e c u r -

r e n t s t a t e o f k n o w l e d g e o f t h e b e h a v i o r o f t h e

c e l l i n s t e a dy -s t a t e suspe ns i on c u l t u re , a nd i n

p a r t i c u l a r t h e p h y s i o l o g i c a l c h a n g e s t h a t o c c u r

i n r e sponse t o a l t e ra t i ons i n g ro w t h r a t e , t h i s

r a t e b e i n g c o n t r o l l e d b y t h e i n v e s t i g a t o r . I t h a s

b e e n s h o w n t h a t t h e e n e r g y m e t a b o l i s m i s s u r -

p r i s i n g l y c o n s t a n t a t d i f f e r e n t g r o w t h r a t e s , b u t

t h e p r o t e i n - s y n t h e s i z i n g s y s t e m c a n d e m o n s t r a t e

c o n s i s t e n t s h i f t s t h a t m a y b e u s e d t o s p e c u l a t e

o n r e g u l a t o r y m e c h a n i s m s w i t h i n t h e c e l l . O t h e r

pos s i b l e u se s ha ve be e n d i s c us se d , a t t e mp t i ng t o

s h o w t h a t t h e c h e m o s t a t i s a v e r s a t i l e t o o l f o r

i nve s t i ga t i ng bo t h t he c o l l i ga t i ve a nd i n t r i n s i c

p rop e r t i e s o f t he g row i ng c el l.

R E F E R E N C E S

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c h e m o s t a t f o r t h e g r o w t h o f m a m m a l i a n

7/21/2019 Mamall Cell Steady State

http://slidepdf.com/reader/full/mamall-cell-steady-state 11/11

C E L L B E H A V I O R I N S T E A D Y - S T A T E C U L T U R E 3 05

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