Carrel 1912 Indefinite Life of Tissues Outside the Body

8/6/2019 Carrel 1912 Indefinite Life of Tissues Outside the Body

1/15

O N T H E P E R M A N E N T L I F E O F T I S S U E S O U T S I D EO F T H E O R G A N I S M . *BY ALEXIS CARREL, M.D.

(From the Laboratories of The Rockefeller Institute for Medical Research,New York.)PLATES 75 AND 76.I N T R O D U C T I O N .

I t i s known tha t the l i f e of a t i ssue cul t iva ted in vitro is veryshor t ? G e ne r al l y , i n t h re e to f i f te e n da ys a f t e r t he p re pa r a t i on o fthe cul tures the i r growth becomes less r apid and eventua l ly s topscomple te ly . The t i ssues then die and the ce ll s d is integra te .

The purpose of the exper iments descr ibed in th is a r t ic le was tode te rmine the condi t ions under which the ac t ive l i f e of a t i ssueouts ide of the organism could be prolong ed indef inite ly . I t mightbe supposed tha t seni l i ty and dea th of the cul tures , ins tead of be ingnecessary, r esul ted mere ly f rom preventable occur rences ; such asa c c umula t i on o f c a t a bo li c subs ta nc es a nd e xha us t i on o f t he me d ium.The suppress ion of these causes should br ing about the regenera t ionof old cul tures and prevent the i r dea th . I t i s even conce ivable tha tthe length of the l i f e of a t i ssue outs ide of the organism couldexceed grea t ly i t s normal dura t ion in the body, because e lementa ldea th migh t be pos tponed indef ini tely by a prop er a r t i f icia l nut r i t ion.

In the fol low ing exper im ents I soug ht not a comple te solut ion ofthe prob lem of p rolong ing indef ini te ly the l i f e of t i ssues in vitro,bu t t he f a c t s t ha t w ou ld show in w hic h d i r e c t i on w e shou ld p r o -ceed in order to secure a comple te solut ion, i f such a solut ion bepossible.

In September , I9I I , I t r ied to increase the dura t ion of the l i f e ofcul tures of co nnec t ive ti ssue. ~ Th e cul tures we re wash ed for

*Received for publication, March I5, I9~2.1Harrison, Harvey Lectures, Philadelphia and London, I9II-I 912 ; .four.Exper. Zoi~l., I9IO, ix, 787; Carrel and Burrows, Jour. E~rper. Med., I9II, xiii,387.*Carrel, Jour. Am. Med. Assn., I9II, lvii, I6II.

516

Published May 1, 1912


2/15

Alexis Carrel. 51 7

s e v e r a l m i n u t e s i n R i n g e r ' s s o l u t i o n a n d w e r e t h e n p l a c e d i n a n e wm e d i u m . A s a c on ,s e q u e n c e , t h e g r o w t h w a s o b s e r v e d t o b e c o m em o r e ra p id . B y r e p e a t e d w a s h i n g s a n d p a s s a g es th e o c c u r r en c e o fs e n i li t y w a s p r e v e n t e d , a n d t h e l e n g t h o f l i f e w a s v e r y m u c h i n -c r e a s e d . S o m e c u l tu r e s c o u l d b e m a i n t a i n e d i n a c t i v e l i f e f o r f i f ty ,f i ft y - fi v e , a n d e v e n f o r s ix t y d a y s ? T h e s e re s u l t s s h o w e d t h a t t h ee a r l y d e a t h o f t i ss u e s c u l t iv a t e d in vitro w a s p r e v e n t a b l e , a n d , t h e r e -f o r e , t h a t t h e i r p e r m a n e n t l i f e w a s n o t i m p o s s ib l e .

S u b s e q u e n t l y , I d e v e l o p e d o t h e r t e c h n i q u e s b y w h i c h i t m i g h tb e p o s si b le to o b t a i n p e r m a n e n t li f e o f ti s su e s . T h e i d e a l m e t h o dw o u l d h a v e b e e n t o g i v e t o t h e c u l t u r e s a n a r t i f i c i a l c i r c u l a t i o n b yw h i c h t h e y m i g h t h a v e s e c u r e d t h e i r n u t r it i o n a n d e l i m i n a t e d t h e irw a s t e p r o d u c t s . I t w a s a t t e m p t e d t o p la c e t h e t is s u e s i n a t h i n l a y e rc o n s i s t in g o f c e l lu l o s e a n d c o a g u l a t e d p l a s m a , a n d t h r o u g h t h i s as l o w s t r e a m o f s e r u m f l o w e d . B u t i t a p p e a r e d s o o n t h a t b e t t e rr e s u l t s c o u l d b e o b t a i n e d b y a s im p l e r a n d m o r e i n d i r e c t m e t h o d .I n s t e a d o f e s t a b li s h in g a c ir c u l a t io n o f t h e m e d i u m t h r o u g h t h et i s s u e s , i t w a s e a s i e r t o h a v e t h e c u l t u r e s p a s s f r o m m e d i u mt o m e d i u m . T h e m e d i a u s u a ll y em p l o y e d c o n si st e d o f R i n g e r ' ss o l u t i o n a n d h y p o t o n i c p l a s m a . I t w a s f o u n d t h a t t h e r e s is t -a n c e o f a t i ss u e p l a c ed i n a n o n - n u t r i t i v e , b u t p r e s e r v a t i v e m e d i u mw a s v e r y m u c h s t r o n g e r if t h e t e m p e r a t u r e w a s lo w . T h e c u l tu r e sw e r e , t h e r e f o r e , p l a c e d u n d e r c o n d i t i o n s h a v i n g t w o a l t e r n a t ep h a s e s : t h e r e w a s a p h a s e o f a c t i v e l i f e a t 3 8 C . d u r i n g w h i c h t h et i s s u e g r e w , u s e d i t s m e d i u m , a n d w a s s u r r o u n d e d b y i t s c a t a b o l i cp r o d u c t s ; a n d a p h a s e o f l a t e n t l i f e a t o C . d u r i n g w h i c h i t w a sw a s h e d f r e e o f i t s p r o d u c t s , t o b e s u b s e q u e n t l y p l a c e d i n a n e wm e d i u m .

I n F e b r u a r y , I 9 1 2 , t h i s t e c h n i q u e w a s m o d i f i e d a n d t h e p h a s e o fl a t e n t l i f e i n R i n g e r ' s s o l u t i o n i n c o l d s t o r a g e w a s r e p l a c e d b y ap h a s e o f a c t iv e li f e i n s e r u m i n t h e i n c u b a t o r . I n s t e a d o f b e i n gi n t e r r u p t e d , t h e l i f e o f t h e t i s s u e s w a s c o n t i n u o u s a n d t h e i r g r o w t hm o r e r a p i d .

M E T H O D S ,T h e m e t h o d s c h ie f ly e m p l o y e d w e r e t h re e . T h e y a r e a s f o l lo w s :Technique/.--The o r i g i n a l p ie c e o f t i s su e a n d t h e s u r r o u n d i n g8 P o z z i , Bul l . de l 'Acad , de todd . , I 9 1 2 , l x v i i , 2 6 .



3/15

518 Permanent L ife of T issues Ou tside of the O rganism.n e w c e ll s w e r e e x t i r p a t e d w i t h a c a t ar a c t k n i f e f r o m t h e f r a g m e n t o fc o a g u l a t e d p l a s m a t h a t c o n t a i n ed th e m . T h e y w e r e w a s h e d f o rs e v e r a l m i n u t e s i n R i n g e r ' s s o l u t i o n a t t h e te m p e r a t u r e o f t h el a b o r a t o r y , a n d w e r e t h e n p l a c e d i n a n e w m e d i u m c o m p o s e d o f o n eo r t w o p a r t s o f d i s ti ll e d w a t e r a n d o f t h r e e p a r t s o f n o r m a l p l a s m a ,t o w h i c h a d r o p o f em b r y o n a l o r m u s c l e e x t r a c t w a s a d d e d . T h ew a s h i n g s a n d p a s s a g e w e r e m a d e e i t h e r b e f o r e t h e a p p e a r a n c e i nt h e c u l t u r e o f t h e s y m p t o m s o f s e n i l i t y , o r w h e n t h e s e w e r e s t i l l v e r ys li g ht . T h e p a s s a g e w a s r e p e a te d m o r e o r l es s f r e q u e n t l y a c c o rd -i n g t o t h e r a t e o f t h e g r o w t h a n d t o t h e c o n d i t i o n s o f t h e c e l l s .

Technique 2 . - - I n t h e s e c o n d m e t h o d t h e w a s h i n g o f t h e c u l t u r e sw a s m o r e p r o l o n g e d a n d w a s p e r f o r m e d w h i l e t h e t i s s u e s w e r e i nc o l d s t o r ag e . T h e l i f e o f t h e c u l t u r es w a s m a i n t a in e d u n d e r c o n -d i t i o n s i n w h i c h t w o p h a s e s a l t e r n a t e d : a p h a s e o f a c t i v e l i fe i n ap l a s m a t i c m e d i u m i n t h e i n c u b a t o r , a n d a p h a s e o f l a t e n t l i fe i nR i n g e r ' s s o l u t i o n i n t h e r e f r i g e r a t o r .

Technique 3 . - - I n t h e th i r d m e t h o d t h e c u l tu r e s w e r e m a i n t ai n e di n a c o n d i t io n o f c o n t i n u o u s l i fe b y a l t e r n a t in g p a s s a g e s f r o mp l a s m a to s e r u m a t t h e t e m p e r a t u r e o f t h e o r g a n i sm . T h e c u l t u r e sw e r e p r e p a r e d i n t h e f o l l o w i n g m a n n e r : A p i e c e o f s i lk v ei l a b o u to n e c e n t im e t e r s q u a r e w a s p l a c e d o n a c o v e r g la s s a n d m o i s t e n e dw i t h a d r o p o f p la s m a . A f r a g m e n t o f t is s u e w a s d e p o s i t ed in th ec e n t r e o f th e s q u a r e a n d c o v e r e d b y a f e w d r o p s o f p l a sm a . A f t e r -w a r d s t h e c u l t u r e w a s s e a l e d o n a h o l l o w s l i d e , p u t i n t h e i n c u b a t o r ,a n d a l l o w e d t o g r o w . A f t e r a f e w d a y s th e si lk v e il t o g e t h e r w i t ht h e c o a g u l a t e d p l a s m a a n d t i s s u e , w a s d e t a c h e d f r o m t h e c o v e r -g l a s s w i t h a k n i fe . A s t h e s i l k v e i l a c t e d a s a s k e l e t o n f o r t h ep l a s m a t i c j e l ly , t h e o r i g in a l f r a g m e n t , a n d t h e n e w t i s s u e c e ll s , t h ec u l t u r e c o u l d b e h a n d l e d e a s i l y w i t h o u t f o l d i n g a n d r e t r a c t i o n o ft h e m e d i u m , a n d w i t h o u t d e f o r m a t i o n o f th e ce ll s. T h e v e i l a n dt h e c u l t u r e a t t a c h e d t o i t w e r e p l a c e d i n a t u b e c o n t a i n i n g h o m o -g e n i e s e r u m , w h i c h w a s k e p t a t i n c u b a t o r te m p e r a t u r e . A f t e r af e w d a y s i t w a s p u t a g a i n i n p l as m a , a n d s u b s e q u e n t l y th e t w op h a s e s a l t e r n a t e d r e g u l a r l y .

Exper imen t s and Resu l t s . - -A g r e a t m a n y e x p e r i m e n t s w e r e p e r -f o r m e d . I n m o s t o f t h e cu l t u re s w e r e u s e d c o n n e c t iv e t is s u e o f



4/15

Alexis Carrel. 519

blood vessels, heart, skin, muscles, peritoneum, and spleen, of four-teen to twenty day old chick fetuses. In othe r cultures were em-ployed fragments of the Rous sarcoma, and in a few cases, tissuesof adult dogs. Many cultures survived six to fourteen or fifteenpassages in a condition of inter rupted or continuous growth. Afew cultures were transplanted seventeen, eighteen, and even twentyor more times. Some are still living and have reached the begin-ning of the third month of their life in vitro. It was possible,therefore, to study the morphological and dynamic characters oftissues cultivated for more than two months outside of theorganism.

MORPHOLOGICAL CHARACTERS.The appearance of the cultures grown by technique I was almost

identical with that o f the cultures grown by technique 2. Thefragment of tissue was surrounded rapidly by cells spreading in athin layer through the medium. Aft er section of the plasma, thenew tissue retracted around the original fragment forming anopaque crown. During the period of washing there was no modi-fication of the culture, but as soon as the culture was put in a newmedium in the incubator, elongated cells began to grow from theedges of the old plasma and to spread into the new medium. Af te ra few passages the original fragment appeared diminished in sizeand surrounded by a dense envelope of a greyish tissue from whichradiated a great many elongated cells, living and dead.The original fragment was often encircled by several concentricrings, each represent ing a period of active life. Soon the ringswere united in a homogeneous tissue. Whe n they remained dis-tinct, however, they might indicate the number of passages under-gone by the culture, as the age of a tree can be read on a section ofits trunk. Progressively the original fragmen t disappeared andwas replaced by an amorphous yellowish substance. The for m ofthe culture was also modified. At the beginning of the growththe cu lture appeared like a thin plate, but it grew progressively moreand more like a flattened sphere in the center of which were ac-cumulated a few foreign bodies and pieces of old plasma (f igure I ),which were encysted by the living cells. Af te r about forty days the



5/15

5 2 0 Permanent Li fe of Tissues Outside of the Organism.c u l t u r e s o f c o n n e c t i v e t i s s u e g e n e r a l l y t o o k t h e ty p i c a l a p p e a r a n c er e p r e s e n t e d i n t h e d r a w i n g s ( f i g u r e s 2 a n d 3 ) o f a c u l t u r e f i f t y d a y so l d .

Cultivation of Connective Tissue (Experiment 715).--A s h o r t s e g m e n t o ft h e i li a c v e s s e l s o f a t w e n t y - o n e d a y o l d c h i c k f e t u s w a s e x t i r p a t e d o n N o v e m -b e r I , 1 9 1 1 , a n d p l a c e d i n h y p o t o n i c c h i c k e n p l a s m a a n d m u s c l e e x t r a c t . Al a r g e g r o w t h o f c o n n e c t i v e t i s s u e c el l s d e v e l o p e d i n t h e n e x t f e w d a y s .

O n N o v e m b e r 3 t h e p l as m a w a s c u t a n d t h e c u l t u r e w a s p l a c e d i n R i n g e r ' ss o l u t i o n i n c o l d s t o r a g e f r o m 9 : 5 2 A . iV [. t o I O : 3 5 A . M . A f t e r w a r d s , t h e f i rs tp a s s a g e i n t o a n .e w m e d i u m w a s m a d e . A l a r g e g r o w t h r e s u l t e d .

O n N o v e m b e r 6 t h e s e c on d p a s s ag e w a s m a d e a f t e r w a s h i n g i n R i n g e r 's s o lu -t i o n a n d k e e p i n g i n c o l d s t o r a g e f r o m 9 : 5 5 A . M . t o I O : 5 8 A . M . A t 2 : 3 0 P . M .f u s i f o r m c e l l s h a d a l r e a d y i n v a d e d t h e n e w m e d i u m .

O n N o v e m b e r 9 t h e th i r d p a s sa g e w a s m a d e a f t e r w a s h i n g f o r s i x m i n u t e si n R i n g e r ' s s o lu t io n , a n d o n N o v e m b e r 1 3 t h e f o u r t h p a s s a g e w a s m a d e a f t e rw a s h i n g i n R i n g e r ' s s o l u t i o n a n d k e e p i n g i n c o l d s t o r a g e f r o m 9 : 5 o A . M . t ol O : 4 2 A . I V [. T h e i l i a c v e s s e l s w e r e b e c o m i n g p r o g r e s s i v e l y s m a l l e r , w h i l e at h i c k c r o w n o f c e l l s i n c o n c e n t r i c l i n e s s u r r o u n d e d t h e o r i g i n a l f r a g m e n t .

O n N o v e m b e r 1 6 t h e c u l t u r e w a s w a s h e d i n R i n g e r ' s s o l u t i o n a n d i n c o lds t o r a g e f r o m 9 : 2 0 A . M . t o l O :1 2, A . M . , a n d t h e n p l a c e d i n a m e d i u m c o m p o s e do f p l a s m a an d m u s c l e e x t r a c t . T h e r e w a s a l a r g e g r o w t h ( f i f t h p a s s a g e ) .

O n N o v e m b e r 2 0 t h e c u l t u r e w a s w a s h e d i n R i n g e r ' s s o l u t i o n i n c o lds t o r a g e f r o m 9 A . M . t o I I : 12 A . M . , a n d t h e n t r a n s f e r r e d t o p l a s m a a n d m u s c l ee x t r a c t ( s ,i x th p a s s a g e ) .

O n N o v e m b e r 2 4 t h e c u l t u re w a s p l a c e d in a m e d i u m c o m p o s e d o f h y p o t o ni cp l a s m a a n d e m b r y o n i c e x t r a c t ( s e v e n t h p a s s a g e ) , a f t e r h a v i n g b e e n w a s h e d inc o l d s t o r a g e a n d i n R i n g e r ' s s o l u t i o n f r o m 9 A . M . t o l O : 1 8 A . M .

O n N o ~ e m b e r 2 5 a l a r g e g r o w t h o f c e l ls w a s s e e n in t h e m e d i u m . T h eo r i g i n a l f r a g m e n t h a d a l m o s t c o m p l e t e l y d i s a p p e a r e d , a n d t h e c u l t u r e h a s at e n d e n c y t o t a k e a s p h e r i c a l f o r m .O n N o v e m b e r 2 8 t h e c u l t u r e w a s p l a c ed i n R i n g e r ' s s o l u t i o n a n d i n c o l ds t o r a g e f r o m 9 : 3 0 A . M . t o 1 2 :0 2 P . M . a n d a f t e r w a r d s i n h y p o t o n i c p l a s m a a n de m b r y o n a l e x t r a c t ( e i g h t h p a ss a g e ) , a n d o n N o v e m b e r 2 9 a l ar g e g r o w t h w a so b s e r v e d . T h e c u l t u r e a s s u m e d t h e f o r m o f a f la t t e n e d s ph e r e , t h e c e n t e r o fw h i c h w a s c l e a r a n d a m o r p h o u s .

F r o m D e c e m b e r 2 t o D e c e m b e r 1 2 t h e c u l t u r e u n d e r w e n t i ts n i n th , t e n t h ,e l e v e n t h , a n d tw e l f t h p a s sa g e s . B e f o r e e a c h p a s s a g e i t w a s w a s h e d f o r f o u r o rf i v e m i n u t e s i n R i n g e r ' s s o l u t i o n a t t h e t e m p e r a t u r e o f t h e l a b o r a t o r y .

O n D e c e m b e r 1 4 t h e c u l tu r e w a s c o m p o s e d o f a c e n t r a l a m o r p h o u s p a r t o f at h i c k r i n g o f n e w t i s s u e , a n d o f a p e r i p h e r a l c r o w n o f e l o n g a t e d c e l l s r a d i a t i n gt h r o u g h t h e c u l t u r e m e d i u m .

O n D e c e m b e r 1 5 t h e c u l tu r e w a s p l a c e d in t o a n e w m e d i u m a f t e r h a v i n g b e e nw a s h e d f o r f o u r m i n u t e s , a n d o n D e c e m b e r 1 6 a l a r g e g r o w t h t h r o u g h t h e n e wm e d i u m w a s se e n . T h e r a t e o f g r o w t h h a d m a r k e d l y i n c r e a s e d .

O n D e c e m b e r 1 8 t h e cu l t u r e w a s w a s h e d f o r t h r e e m i n u t e s a n d u n d e r w e n ti t s f o u r t e e n t h p a s s a g e , i n w h i c h a r a p i d g r o w t h o f e l o n g a t e d t i s s u e c e l l s w a so b s e r v e d .



6/15

Alexis Carrel. 521O n D e c e m b e r 2 0 t h e c u l t u r e w a s f i x e d in . f o r m a l i n a n d s t a i n e d w i t h h e m a -

t o x y l i n . T w o d r a w i n g s o f th e s t a i n e d s p e c i m e n w e r e m a d e ( f i g u r e s 2 a n d 3 ) ,s h o w i n g t h e a m o r p h o u s c e n t e r a n d i t s c r o w n o f a c t i v e l y g r o w i n g t i ss u e .

E v e n a f t e r s e v e n t y - f iv e o r e i g h t y d a y s , c u l t u r e s o f c o n n e c t i v et i ss u e m a d e b y t h e f i r st t w o m e t h o d s h a d a b o u t t h e s a m e a p p ea r .a n c e , al l o f t h e m r e s e m b l i n g t h e l a t e r s t a g e s o f t h e c u l t u r e o f t h ei l i ac ves se l f i r s t desc r ibed .

B y t h e s a m e m e t h o d a f e w f r a g m e n t s o f a d u l t d o g t i ss u es , s u c h a st h y r o i d a n d p e r i o s t e u m , w e r e c u l t i v a t e d , a n d s e v e r a l g e n e r a t i o n s o fc o n n e c t i v e t i s s u e c e l l s w e r e o b t a i n e d .

F r e q u e n t l y t h e c u l t u re s o f R o u s s a r c o m a d ie d a f t e r a f e w d a y s.T h e p l a s m a b e c a m e li q u e fi e d a n d t h e t i ss u e d i s i n t e g r a te d . N e v e r -t h e l e s s , a s m a l l f r a g m e n t o f i t c o u l d b e k e p t i n a c t i v e l i f e f o r f o r t y -s i x d a y s .

Culture of the Rous Sarcoma.--On J a n u a r y 2 4 , 1 9 1 2 , a s m a l l p i e c e o f a R o u ss a r c o m a w a s c u l t i v a t e d i n h y p o t o n i c p l a s m a . T h e g r o w t h w a s r a p i d a n da b u n d a n t . O n J a n u a r y 2 6 t h e f r a g m e n t w a s s u r r o u n d e d b y a n im m e n s e n u m b e ro f r a d i a t i n g f u s i f o r m c e l ls a n d b y a p e r i p h e r a l c r o w n o f r o u n d a n d p o l y g o n a lc e ll s. O n J a n u a r y 2 6 a n d 2 9 t h e c u l tu r e w a s w a s h e d f o r t h r e e o r f o u r m i n u t e si n R i n g e r ' s s ol u t i on a n d p u t i n a n e w m e d i u m . O n J a n u a r y 3 o f u s i f o r m c e l lso n l y c o u l d b e s e e n in t h e c u l t u r e . O n J a n u a r y 3 o a n d o n F e b r u a r y I , 3, 5 , a n d7 t h e c u l t u r e u n d e r w e n t i t s t h i r d , f o u r t h , f if t h , s i x t h a n d s e v e n t h p a s s a g e s . T h eg r o w t h w a s n o t v e r y a c t iv e . O n F e b r u a r y 7 t h e c u l t ur e w a s d i v i d e d i nt o t w op a r t s. O n e o f th e s e w a s w a s h e d a n d u n d e r w e n t i t s e i g h t h p a s s a g e o n F e b r u a r y9 . A f t e r t h e n i n t h a n d t e n t h p a s s a g e s o n F e b r u a r y I 0 a n d 1 2 , a m i e ro b i a t~ c o l o n ya p p e a r e d i n t h e m e d i u m . N e v e r t h e l e s s t h e a c ti v e p a r t o f t h e c u l t u r e c o u ld b ee x t i r p at e d w i t h o u t b e i n g c o n t a m i n a t e d a n d u n d e r w e n t i t s e l e v e n t h p a s s a g e o nF e b r u a r y 1 5 .

A f t e r w a r d s t h e g r o w t h w a s s m a l l b u t a s ep t ic . A f t e r t h e t w e l f t h p a s s a g e o nF e b r u a r y 1 7 t h e t is s u e p r o d u c e d a p a r t i a l l i q u e fa c t i o n o f t h e m e d i u m w i t hd i v i s i o n o r d i s s o c i a t i o n o f t h e n e w t i s s ue . N e v e r t h e l e s s , a f t e r t h e t h i r t e e n t hp a s s a g e o n F e b r u a r y 2 0 a n d t h e f o u r t e e n t h p a s s a g e o n F e b r u a r y 2 2, t h e t i s s u eb e g a n t o g r o w m o r e a b u n d a n t l y a n d r e g u l a r l y w i t h o u t l i q u e f a c t io n o f t h em e d i u m . F r o m F e b r u a r y 2 4 t o M a r c h I t h e c u l t u r e u n d e r w e n t i ts fi f te e n t h ,s i x t ee n t h , se v e n t e e n th , a n d e i g h t ee n t h p a s sa g e s. T h e g r o w t h w a s a b u n d a n t a n dt h e c u l t u re i n c r e a s e d v e r y m u c h i n s iz e. O n M a r c h 5 a m a r k e d r e t r a c ti o n o ft h e p la s m a o c c u r re d . A f t e r t h e n in e t e e n t h a n d t w e n t i e t h p a s s a g e s th e g r o w t hb e c a m e v e r y s m a l l a n d d e a t h o c c u r r e d w i t h o u t a n y a p p a r e n t c a u se .

W h e n t h e t i s s u e s w e r e c u l t i v a t e d a c c o r d i n g t o t h e t h i r d m e t h o dt h e a p p e a r a n c e o f t h e c u l t u r e s w a s d i f f e r e n t . I n s t e a d o f b e i n g in -t e r r u p t e d , t h e g r o w t h w a s c o n t i n u o u s , a n d a s t h e r e w a s n o s e c t i o n -i n g o f t h e p l a s m a , t h e r e w a s , a s a c o n s e q u e n c e , n o r e t r a c t i o n .



7/15

522 Permanent Life of Tissues Outside of the Organism.

Moreover , as the plasma was suppor ted by i t s ske le ton of s i lk , thecul ture could not take a spher ica l form, and the t i ssue grew in athin layer ; tha t i s , under the bes t condi t ions for a t i ssue depr ived ofc i rculat ion. The plasmat ic je l ly was adh erent to the s ilk ve il , andthe t i ssue imbedded in the plasma grew as in an ordinary cul ture .The ce l l s could use the s i lk threads as a suppor t , but they se ldomdid. Th e purpose of the s i lk was mere ly to give such a cons is tencyto t he me d ium tha t i t w ou ld no t f o ld ove r o r be c ome sphe r ic a ldur ing the passages in plasma and in se rum.

No rm al hear t was cul t iva ted accordin g to th is technique . I t s ap-pearance a f te r a f ew passages was the same as a f te r a f ew days .T he s iz e o f t he g r ow th a lone w a s modi f ie d .

B y t h e s a m e p r o c e d u r e m a n y f r a g m e n t s o f t h e R o u s s a r c o m awere a lso cul t iva ted. Th ey could be kept in a condi t ion of act ivea nd c on t inuous de ve lopme nt f o r mor e t ha n f i f t ee n da ys . Bu t t heme d ium w a s p r ogr e s s ively ove r c r ow de d w i th de a d c e ll s a nd t hegr ow th o f t he t i s sue be c a me mor e i r r e gu l a r a nd l e s s a bunda n t .T he f r a gme nt s c ou ld no t a da p t t he mse lve s t o t he i r ne w c ond i t i onsof l i f e and w ere less r es i s tant than norm al connec t ive t issue .

T he a ppe a r a nc e o f t he c e l l s g r ow ing i n c u l t u r e s o f no r ma l c on-nec t ive t i ssue of ten var ied per iodical ly . Du r ing the f i r st f ew daysof g r ow th t he f us i f o r m c e l l s w e r e l ong a nd t h in a nd c on t a ine dc lear cytoplasm. Af te r fou r or f ive days they became la rger andthe i r cytoplasm was darker and f i l led wi th la rge fa t ty granula t ions .A t t he s a me t ime t he r a t e o f g r ow th d imini she d. A f t e r be ingw a she d a nd t r a ns f e r r e d t o a ne w me d ium, t he a ppe a r a nc e o f t hece ll s was aga in modif ied. Th ey rega ined the i r or igina l appearancea nd t he r a t e o f g r ow th w a s a cc e l er a te d . I ns t e a d o f be ing pe riod -ical , the morph ologica l modif ica t ions migh t be cont inuous . Fo r in-s tance , in the cul tures of spleen the or igina l f r agment was sur -rounded dur ing the f i r s t phases of ac t ive l i f e by an enve lope ofameboid ce ll s. A f te r a f ew days long cha ins of fu s i fo rm ce ll sappeared a round the f ragment , whi le the ameboid ce l l s d ied anddisappeared. Ul t im ate ly the fus i fo rm ce l ls were replaced by smal lpo lygona l c e l l s w h ic h sp r e a d a r ound t he f r a gme nt i n a c on t inuouslayer . In the cul ture of the Rou s sa rcom a the round ce l ls d isap-



8/15

Alexis Carrel . 52 8

p e a r e d a f t e r a f e w d a y s , w h i l e t h e e l o n g a t e d c e l l s w e r e s t i l l g r o w -i n g a f t e r f o r t y d a y s . I n a c u l t u r e o f h e a r t v e r y l a r g e a m e b o i d ce ll sw a n d e r e d o u t f r o m t h e c e n tr a l f r a g m e n t f o r m o r e t h a n t w o m o n t h s.A t t h e b e g i n n in g o f t h e t h i r d m o n t h o f i ts l i fe t h e c u l tu r e w a s c o m -p o s e d o f e l o n g a t e d c e ll s r a d i a t i n g t h r o u g h t h e ol d p l a sm a . T h e yw e r e a p p a r e n t l y t h e r e s u l t o f t h e d e v e l o p m e n t o f t h e l a r g e a m e b o i dcel ls .

DYNAMIC CHARACTERS.

T h e r a t e o f g r o w t h c o u ld b e a p p r e ci a te d b y t h e r a p i d it y w i t hw h i c h t h e c el ls , a f t e r a p a s s ag e , a p p e a r e d a n d s p r e a d i n t h e n e wm e d i u m . T h e n a t u r e o f th e m e d i u m , i ts o s m o t ic t e ns io n , t h e w a yi n w h i c h t h e p la s m a w a s c u t , t h e a m o u n t o f o l d p la s m a l e f t a r o u n dt h e c e ll s, t h e f o r m o f t h e c u l t u r e , a n d t h e f r e q u e n c e o f t h e p a s s a g e s ,o f t e n h a d a m a r k e d i n f l u en c e o n t h e r at e o f th e g r o w t h . T h eo s c i ll a ti o n s i n t h e r a t e s e e m e d g e n e r a l l y to b e t h e r e s u l t o f a c c i d e n t a lc a u s e s , b u t i t w a s v e r y d i f f ic u l t t o e x c l u d e t h e p o s s i b i l i t y t h a t t h e ym i g h t b e d u e t o c a u s e s i n h e r e n t i n th e c o n d i t i o n o f t is s u e s l i b e r at e df r o m t h e c o n t r o l o f it s o r g a n i s m . G e n e r a l l y , a f t e r t h e f ir s t p a s s a g et h e ce ll s a p p e a r e d i n t h e m e d i u m w i t h o u t a l a t e n t p e r i o d . T h i s w a st r u e e v e n in t h e c u l t u r e o f p e r i o s t e u m a n d o f o t h e r t i ss u e s o f a d u l td o g s. T w o o r t h r e e h o u r s a f t e r a p a ss a g e m a n y e l o n g a t e d c el lsw e r e o b s e r v e d g r o w i n g o u t f r o m t h e e d g e s o f t h e o l d p la s m a i n tot h e n e w m e d i u m , a n d t h e y s o o n s u r r o u n d e d t h e c e n t r a l p i e c e b y ad e n s e l a y e r. T h e r a p i d i ty o f th e i r g r o w t h o f t e n d im i n i s h e d a f t e ra f e w p a s s a g es , b u t a f t e r w a r d s in c r e a s e d p r o g r e s s i v e ly . T h e r a t eo f g r o w t h o f a c u l t u r e f o r t y o r f o r ty - f iv e d a y s o l d w a s m o r e r a p i dt h a n w h e n it w a s t e n o r f i f t e e n d a y s o l d . I t s e e m e d t h a t t h e o l d e ra c u l tu r e w a s , t h e q u i c k e r i t g r e w . T h e r a t e o f g r o w t h o f a f i ft y -f o u r d a y o l d c u l t u r e o f c o n n e c t i v e t i ss u e f r o m t h e p o r t a l v e i n w a sm o r e r a p i d t h a n a t t h e b e g i n n i n g o f i t s l i f e in v i t ro . T h e r a t e o fg r o w t h o f a c u l t u re o f h e a r t w a s g r e a t e r a t t h e b e g i n n i n g o f t h et h i r d m o n t h o f i ts l if e o u t si d e o f t h e o r g a n i s m t h a n a t a n y o t h e rp e r i o d .

T h e s e a c c e l e r a t i o n s o f t h e g r o w t h w e r e p o s s i b ly f u n c t i o n s o f t h ea g e o f t h e c ul tu r e . B u t t h e n u m b e r o f t h e e x p e r i m e n t s w a s n o tl a r g e e n o u g h t o e x c l u d e t h e p o s s i b il it y t h a t t h e y w e r e d u e t o o t h e rc a u s e s .



9/15

5 2 4 P e r m a n e n t L i f e o f T i s s u e s O u t s i d e o f t h e O r g a n i s m .

T h e si z e o f t h e c u l t u r e s u n d e r w e n t a l s o m a n y m o d i f i c a t i o n s . F o rl o n g p e r i o d s o f t i m e , t h e c u l t u r e s i n c r e a s e d i n s i ze , d i m i n i s h e d , o rw e r e n o t m o d i f i e d .

T h e d i m i n u t i o n s i n si z e w e r e d u e c h i e f ly t o a c c i d e n t al c a u s e s ,s u c h a s d e s t r u c t i o n o f c e ll s d u r i n g t h e h a n d l i n g o f t h e c u l t u r e s ,f o l d i n g o f t he p l a s m a , a n d c o n c e n t r a t i o n o f t h e m e d i u m , b u t e s p e -c i a ll y t o m i c r o b i a n i n f e c t io n .

I n a f e w e x p e r i m e n t s m a r k e d i n c r e a s e i n s iz e w a s o b s e r v e d .T h e t i s s u e o f a c u l t u r e o f p o r t a l v e i n h a d b e e n c o n s i d e r a b l y r e d u c e da f t e r i n f e c t i o n , a n d f o r s e v e r a l g e n e r a t i o n s i t i n c r e a s e d b u t s l i g h tl y .T h e n t h e g r o w t h b e c a m e m o r e a c t iv e a n d a f t e r t h e f if t ie t h d a y t h ev o l u m e o f th e ti s s ue i n c r e as e d r a p i d l y . I n a c u l t u r e o f c o n n e c t i v et i s su e w h i c h h a d r e m a i n e d v e r y s m a l l f o r t w o m o n t h s , a g r e a t a c -c e l e r a t i o n o f g r o w t h a n d a m a r k e d i n c r e a s e i n s i ze w a s o b s e r v e da f t e r s i x t y d a y s . I n a n o t h e r i n s t a n c e a c u l t u r e o f h e a r t b e c a m e sol a r g e a f t e r s i x t y - t w o d a y s t h a t i t h a d t o be d i v i d e d i n t o s e v e r a l p a r t s .

I t i s g e n e r a l l y d i ff i cu l t t o a s c e r t a i n w h e t h e r o r n o t t h e m a s s o ft h e t i s s u e a c t u a l l y i n c r e a s e s , b e c a u s e c h a n g e s i n t h e d e n s i t y o f t h et i s s u e c a n b e m i s t a k e n f o r a c h a n g e i n i ts m a s s . N e v e r t h e l e s s , t h eo b s e r v a t i o n o f a c u l t u r e o f a d i l u te d s u s p e n s i o n o f c e ll s d e m o n -s t r a t e d t h a t a r e a l i n c r e as e i n t h e v o l u m e o f t h e t i ss u e c o u l d t a k ep l a c e .

Culture of Cells Suspended in Ringer's So lution.--On February I , I912 anine day old chick embryo was cut into very small pieces , and to the pieces anequal volum e of Ringer 's solution was added. The suspension was centrif-ugalized fo r five minutes, and a d rop of the supern atant fluid was then addedto a culture medium. On F ebru ary 2 ma ny isolated cells were seen in theper iphera l pa r t o f the medium. On F ebruary 5 the number o f these ce lls hadincreased ve ry much . The p a r t o f the p lasma where they had g rown wasresected, washed, and put in a new medium. Th e culture un derwen t its second,third, fou rth, and fifth passages on Fe bru ary 2, IO, 13, and 16. Th e cells hadthen increased so much in n.umber that th ey for me d a real tissue, and had theappearance o f an o rd ina ry cu ltu re . The l i fe o f these ce lls a f te rwards was l ikethat of other cultures .

S i n c e a f e w s c a t t e r e d c e l l s c o u l d g e n e r a t e a c u l t u r e s i m i l a r t o t h ec u l t u r e o f f r ag m e n 4 c s o f t i s s u e , i t is a b s o l u t e l y c e r t a i n t h a t t h e c e l lsm u l t i p l y a n d t h a t th e y d o n o t m e r e l y w a n d e r f r o m t h e c e n tr a lp i e c e .



10/15

Alex i s Carre l . 525

G e n e r a l l y , t h e c u l t u r e s r e m a i n e d v e r y s m a l l in s p i te o f t h e i r c o n -s t a n t g r o w t h , a n d o f t e n t h e y d i d n o t i n c r e a s e a t a ll in s iz e . I t i sp r o b a b l e t h a t t h e l i m i t a t i o n o f g r o w t h i s d u e t o c e r t a i n m e c h a n i c a lc o n d i t io n s . C u l t u r e s h a v e a t e n d e n c y t o b e c o m e sp h e r ic a l . T h en u t r i m e n t t h a t r e a c h e s t h e c e n t e r o f s u c h c u l t u r e s i s in s u ff i ci e n t, b u tt h a t w h i c h is s u p p l ie d to t h e p e r i p h e r y is a b u n d a n t . H e n c e , th ec e l ls a t t h e p e r i p h e r y m u l t i p l y c e a s e le s s l y . B u t a s s o o n a s c e ll s a r ei s o la t e d f r o m t h e m e d i u m b y a s u f f ic i en t t h i c k n e s s o f n e w t is s u e ,t h e i r n u t r i t i o n p r o b a b l y b e c o m e s d e f i ci en 't a n d t h e y d i s i n t e g r a te .W h i l e t h e c u l t u r e i n c r e a s e s c o n s t a n t l y b y i t s p e r i p h e r y , i t d i m i n i s h e si n it s c e n t e r a n d t h e r e f o r e i t s v o l u m e d o e s n o t i n c r e a se .

A t i s s u e d e p r i v e d o f c i r c u l a t i o n p r o b a b l y c a n n o t g r o w i n a n u t r i -t iv e m e d i u m b e y o n d a c e r t a i n v o lu m e w h i c h i s a f u n c t i o n o f i tsp e r m e a b i l it y to t h e m e d i u m a n d c o n s e q u e n t ly o f t h e t h i c k n es s o ft h e c u l tu r e . I f t h e c u l t u r e b e s p h e r i c a l i ts v o l u m e w i l l r e m a i n v e r ys m a l l , s i n c e t h e n u t r i t i o n o f i t s c e n t e r is i n s u f f ic i e n t . F r o m a t h e o -r e t i c a l s t a n d p o i n t , o n t h e o t h e r h a n d , a t i s s u e g r o w i n g i n a v e r yt h i n l a y e r c o u l d i n c r e a s e i n d e f i n i t e l y . I t i s f o r t h i s r e a s o n t h a t t h et i s s u e s w e r e c u l t i v a t e d o n a s i l k v e i l , f o r t h e v e i l p r e v e n t e d t h ep l a s m a o f t h e c u l t u r e f r o m r e t r a c t i n g a n d b e c o m i n g s p he r ic a l.

T h e a t t e m p t w a s a l so m a d e t o a s c e r t a in w h e t h e r t is s u e s a f t e r al o n g p e r i o d o f l i f e i n v i t ro c o u l d r e t a i n t h e i r n o r m a l f u n c t i o n s .T h e e x p e r i m e n t s w e r e c a r ri e d o u t w i t h f r a g m e n t s o f c h ic k f e t u sh e a r t , w h i c h , a s i s w e l l k n o w n , c a n g r o w a n d p u l s a t e f o r s e v e r a ld a y s w h e n k ep t a t a p r o p e r t e m p e r a t u r e i n p l as m a . I n D e c e m b e r ,1 91 I , i t w a s o b s e r v e d t h a t w h e n t h e p u l s a t i o n s o f a c u l t u r e o f h e a r ts t o pp e d , t h e y b e g a n a g a i n a f t e r t h e t is s u e h a d b e e n w a s h e d a n dt r a n s p la n t e d i n to a n e w m e d i u m . T h e n i n J a n u a r y , 1 9 12 , t h e a t-t e m p t w a s m a d e t o d e t e r m i n e t h e in t e r v a l d u r i n g w h i c h t h e c a r d i a cm u s c l e w o u l d r e t a i n i ts p r o p e r t y o f c o n t r a c t i n g r h y t h m i c a l l y .T h r e e e x p e r i m e n t s g a v e p o s it i v e a n d i d e n t i c al r e s u lt s . O n e o ft h e s e w i l l b e d e s c r i b e d .

Cult iva t ion o f the Hear t (Exper iment 72o-1) . - -0n January I7, 1912, a smallfragment of the heart of an eighteen day old chick fetus was cultivated inhypotonac plasma. The f ragment pulsated regula rly for a few days and grewextensively. Afte r the first washing and passage on January 24 the culturegrew again very extensively, but there were no rhythmical contractions. On



11/15

5 2 6 P e r m a n e n t L i f e o f T i s s u e s O u t s i d e o f t h e O r g a n i s m .Jan ua ry 29 and Fe bru ary I , 3, 6, 9, 12, I5, I7~ 20, 24, and 28, the cul tu re u nde r-wen t el even washings and pas sages. I t became sur rou nded b y fus i form ce ll sand man y dead ce ll s. Th e re w ere no pul sa tions . A~f te r t he twe l f th pas sage thecul ture d id not grow a t a ll . Th en the t i s sue was d i s sec ted and the o ld p l a smawas comple te ly ext i rpa ted . A sma l l c ent ra l f r agm ent was r emoved , washed,and put i n a new medium. On March I i t was pul sa t ing a t a r a t e tha t va r i edbe tween 60 and 84 pe r minute . On March e the pul sa tions w ere lO4 a t 4 I C.,and o n M arch 3 , 8o a t 40 C. , but on March 4 the p ul sa t ions were v e ry w eak ands t opped a lt oge t he r a t 2, P . M . O n M a r c h 5 t he c u lt u r e unde r w e n t i t s f ou r t ee n t hpassage, and the pul sa t ions r eappea red immedia te ly . Th ey became weak aga in onMarch 2 . On M arch 8 the f if t e enth pas sage was made . On M arch 9 the pul sa -t ions were aga in 80 to 82 pe r minute a t 40 C. , and on March 12 they were6o pe r m i nu t e . T he y t he n be c a m e sl ow e r a nd w e a ke r. A f t e r t he s i x t e e n thpa s s a ge on M a r c h I 2 t he pu ls a ti ons w e r e i r r egu l a r , a nd t he f r a gm e n t be a t f o r ase r i e s of 3 to 4 pul sa tions, and then s topped fo r about 20 seconds . Af t e r t heseventeenth pas sage on March 16 r egula r pul sa t ions a t 52 bea t s pe r minute r eap-pea red, and the t i s sue grew abundant ly . Af t e r t he e ighteenth pas sage on March19 the pul sa t ions were i r r egula r .

I t w a s t h u s d e m o n s t r a t e d t h a t a f r a g m e n t o f c h i c k e n h e a r t c o u l ds ti ll p u l s a te r h y t h m i c a l l y a t t h e b e g i n n i n g o f t h e t h i r d m o n t h o f i tsl if e o u t s i d e o f t h e o r g a n i s m . I t s h o w e d , c o n s e q u e n t l y , th a t a f r a g -m e n t o f t i s su e l i v i n g i n v i t r o c o u l d r e ta i n i ts n o r m a l f u n c t i o n f o r al o n g t im e .

T h e m a x i m u m a g e t h a t ti s su e s l i v i n g i n v i t r o c a n r e a c h i s s t i l lu n d e t e r m i n e d . M a n y c u l t u r e s h a v e d ie d a f t e r l es s t h a n t w om o n t h s , b u t a f e w w e r e v e r y a c t i v e a t t h e b e g i n n i n g o f th e t h i r dm o n t h o f th e i r li f e o u t s i d e o f th e o r g a n i s m . G e n e r a l l y , t h e t i ss u e ss e e m e d , a f t e r a t i m e , t o a d a p t t h e m s e l v e s t o t h e ir n e w c o n d i t io n ,a n d a f t e r t h e f o u r t e e n t h o r f i ft e e n t h p a ss a g e , v e r y f e w c u l t u r es d i e ds p o n t a n e o u s l y . I n t h e h a n d l i n g a n d c h a n g e s r e q u i r e d b y t h ep a s s a g e s , t h e c u l tu r e s w e r e e x p o s e d t o m a n y a c c id e n t s , c h i ef l y t om i c r o b i a n i n f e ct i o n s . I n c a se o f a l o c al i n f e c t i o n t h e p a r t o f t h ec u l t u r e t h a t w a s n o t y e t i n f e c t e d w a s r e s e ct e d w i t h a c a t a r a c t k n i f ea n d p l a ce d i n a n e w m e d i u m . O f t e n t h e c u l t u r e re c o v e r e d a n dp r o d u c e d s e v e r al g e n e r a t i o n s o f c e l ls t h a t w e r e f r e e f r o m m i c r o b i a ni n f e c ti o n . B u t w h e n i n f e c t io n w a s g e n e r a l iz e d , t h e t is s u e s a l w a y sd i e d r a p i d l y . M a n y c u l t u r e s d i e d o f s e p si s . I n t h e d e s c r i p t i o nt h a t f o l l o w s i s a n i l l u s t r a t io n o f t h i s k i n d o f d e a t h .

Culture of Portal Vein (Experiment 483).~0n September 27, I9I I , a f r ag-ment of t he por t a l ve in of a chick f e tus s ix t een days o ld was cul t iva t ed in three



12/15

Alexis Carrel. 5 2 7p a r t s o f c h i c k e n p l a s m a a n d t w o p a r t s o f d i s t i l l e d w a t e r . T h e t i s s u e g r e wv e r y e x t e n s i v e l y . O n O c t o b e r 2 t h e c u l t u r e w a s d i v i d e d i n t o t h r e e p a r t s . O n ew a s w a s h e d i n R i n g e r ' s s o l u t i o n f o r f i v e m i n u t e s a n d p u t i r~ a n e w m e d i u m . O nO c t o b e r 7 i t h a d g r o w n e x t e n s i v e l y a n d w a s a g a i n d i v i d e d i n to t w o p a r ts . O n ep a r t w a s w a s h e d a n d t r a n s p l a n t e d i n t o a n e w m e d i u m a n d a la r g e g r o w t h f o l -l o w e d . O n O c t o b e r 9 a n d I 2 th e t h ir d a n d f o u r t h w a s h i n g s a n d p a s s a g e s t o o kp l a ce . O n O c t o b e r I 4 a v e r y l a r g e g r o w t h o f l o n g , c l ea r , s l e n d e r f u s i f o r m c e l l sw a s p r e s e n t . O n O c t o b e r I 6 th e g r o w t h h a d s t o p p e d a n d t h e c e l l s w e r e l a r g ea n d c o n t a in e d v e r y l a r g e f a t g r a nu l e s . O n O c t o b e r I 6 t h e f if t h w a s h i n g a n dp a s s a g e t o o k p l ac e , a n d o n O c t o b e r I 7 t h e g r o w t h o f f u s i f o r m c e ll s , w h i c h w e r ea g a i n c l e a r w i t h f e w g r a n u l e s, w a s v e r y a c t i v e. A f t e r t h e s i x t h p a s sa g e o nO c t o b e r 2 0 r e t r a c t i o n o f t h e m e d i u m o c c u r r e d , a n d t h e g r o w t h o f t h e t i s s u ew a s s l i g h t . B y O c t o b e r 2 2 t h e o r i g i n a l f r a g m e n t h a d d i m i n i s h e d g r e a t l y i n s i ze .O n O c t o b e r 2 3 a n d O c t o b e r 2 6 t h e c u l tu r e u n d e r w e n t i t s s e v e n th a n d e i g h t hp a s s a g e s , a n d o n O c t o b e r 2 7 t h e o r i g i n a l f r a g m e n t w a s n o l o n g e r t o b e d i s -t i n gu i s h e d. T h e c u l t u r e w a s c o m p o s e d o f a n a m o r p h o u s c e n t r a l p a r t a r o u n dw h i c h a c r o w n o f a c t i v e t i s s u e h a d d e v e l o p e d . T h e t i s s u e w a s d i s p o s e d in c o n -c e n t r i c c i r c le s a n d f r o m i t s p e r i p h e r a l p a r t e l o n g a t e d c e l l s r a d i a t e d t h r o u g h t h ec u l t u r e m e d i u m ( f i g u r e I ) . A f t e r t h e n i n t h a nd t e n t h p a s s a g es o n O c t o b e r 30a n d N o v e m b e r 4 , a g r e a t i n c r e a s e i n t h e r a t e o f t h e g r o w t h w a s o b s e r v e d, b u ta f t e r t h e e l e v e n t h p a s s a g e o n N o v e m b e r 9 t h e c u l t u r e a s s u m e d a s p h e r i c a l s h a pe ,b u t g r e w e x t e n s i v e l y n e v e r t h e l e s s . O n N o v e m b e r I o , I 3, x 6, a n d I 8 , t h e t w e l f t h ,t h i r t e e n t h , f o u r t e e n t h , a n d f i f t e e n t h p a s s a g e s t o o k p la c e . T h e r a t e o f g r o w t hw a s v e r y r ap i d . O n e h o u r a f t e r t h e f i f t e e n t h p a s s a g e , th e f u s i f o r m c e l ls h a da l r e a d y i n v a d e d t h e n e w c u l t u r e m e d i u m , b u t o n N o v e m b e r ~ 9 a l a r g e m i c r o b i a nc o l o n y a p p e a r e d o n o n e s i d e o f th e c u l t u r e , a n d o n N o v e m b e r 2 0 t h e c u l t u r e w a si n v a d e d b y a g r e a t m a n y b a c t e r i a l c o l o n i e s . T h e d e a t h o f t h e t is s u e o c c u r r e ds o o n a f t e r .Nearly all the cultures made in the latter part o f I9 II died inthe same manner after one to two months. By slight modificationsof the technique, however, infection was almost completely elimi-nated in the experiments made in the beginning of I912.

Of sixteen cultures of heart and blood-vessels made on January17, I912, five were still very active in March, I912, and of the fiveactive ones, two heart cultures previously described grew slowly,but pulsated, and another heart culture, which pulsated from time totime, produced a large growth of ameboid and fixed cells whichcovered an extensive area of the medium. In this instance afterhaving been motionless for two months, the central part of theculture manifested strong rhythmical contractions on the sixty-fifthday of its life i n v i t r o . Moreover, two cultures of connective tissuemade on January 17 were growing actively at the beginning ofApril. The ra te of grow th and the increase in their volume becamevery much greater as they grew older.



13/15

528 Pe rmane n t L i f e o f T i s sue s Ou t s ide o f t he Organ i sm .

S U M M A R Y A N D C O N C L U S I O N .I n t w o s e ri e s o f e x p e r i m e n t s m a d e a t th e e n d o f 1 91 1 a n d a t t h eb e g i n n i n g o f 1 9 1 2 , n e w t e c h n i q u e s w e r e d e v e l o p e d w i t h t h e v i e wo f i n v e s t i g a t i n g th e p r o b t e m o f p r o l o n g i n g i n d e f in i t e ly t h e l i f e o ft i ss u e s i s o la t e d f r o m t h e o r g a n i s m . T h e s e t e c h n i q u e s a r e f a r f r o mp e r f e c t a n d w i ll d o u b t l es s b e m o d i f i e d i n t h e f u t u r e . B u t t h e y h a v ea l r e a d y p e r m i t t e d t h e e s ta b l i s h m e n t o f n e w f a c ts .

F r a g m e n t s o f c o n n e c t i v e t i s s u e h a v e b e e n k e p t i n v i t ro i n a c o n d i -t i o n o f a c t iv e l i fe f o r m o r e t h a n t w o m o n t h s . A s a f e w c u l t u r e sa r e n o w e i g h t y -f i v e d a y s o l d a n d a r e g r o w i n g v e r y a c t i v e ly , i t isp r o b a b l e t h a t , i f n o a c c i d e n t o c c u r s , t h e l i f e o f t h e s e c u l t u r e s w i l lc o n t i n u e f o r a l o n g t im e .

I n s o m e c a se s t h e r a t e o f g r o w t h o f t h e t i ss u e s i n c r e a s e d in d i r e c tr a t i o t o t h e a g e o f t h e c u l t u r e .

F r a g m e n t s o f h e a r t p u l s at e d r h y t h m i c a l l y a t t h e b e g i n n in g o ft h e t h i r d m o n t h o f t h e i r l if e i n v i t ro .

T h e s e f a c ts s h o w t h a t e x p e r i m e n t s m a d e w i t h t h e se o r w i t h m o r ep e r f e c t t e c h n i q u e s a n d f o l l o w e d o v e r l o n g p e r i o d s o f t i m e m a y l e a dt o t h e s o l u t i o n o f t h e p r o b l e m o f p e r m a n e n t l i f e o f t i s s u e s in v i t ro ,a n d g i v e i m p o r t a n t i n f o r m a t i o n o n t h e c h a r a c t e r s a c q u i r e d b y t i ss u e sl i b e r a t e d f r o m t h e c o n t r o l o f t h e o r g a n i s m f r o m w h i c h t h e y w e r ed e r i v e d . EXPLANATION OF PLATES.

PLATE 75.F IG . I . A t h i r t y d a y o l d c u l t u r e o f c o n n e c t i v e t i s s u e . T h e c e n t e r o f t h ec u l t u r e c o n s i s t s o f d e b r i s o f o l d p l a s m a . A r o u n d i t i s a r i n g o f c o n c e n t r i cl a y e r s o f v e r y a c t i v e n e w t i s s u e .

PLATE 76.F IG . 2 . A f i f t y d a y o l d c u l t u r e o f c o n n e c t i v e ti s s u e . A c t i v e t i s s u e i s e n c i r -

c l i n g a p i e c e o f o l d p l a s m a .F IG . 3 . P e r i p h e r a l p a r t o f t h e s a m e c u lt u r e .



14/15

THE JOURNAL OF EXPERI MENTAL MEDICINE VOL. XV. PLATE 75.

~IG. I .



15/15

THE JOURNAL OF EXPERI MENTAL MEDICINE VOL, XV. PLATE 76.

F I G . 2 .

F I G . 3 -


Documents

Carrel 1912 Indefinite Life of Tissues Outside the Body