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8/10/2019 J Exp Med-1912-Carrel-516-28
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 E
O 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.
INTRODUCTION
I t i s known tha t the l i fe o f a t i ssue cu l t iva ted
in vitro
i s very
shor t ? Genera l ly , in th ree to f i f teen days af te r the prepara t ion of
the cu l tu res the i r g rowth becomes less rap id and eventua l ly s tops
completely. Th e t issues then die and the cells disintegrate .
The purpose of the exper iments descr ibed in th is a r t ic le was to
determine the condi t ions under which the ac t ive l i fe o f a t i ssue
outs ide of the organ ism could be pro longed indef in ite ly . I t might
be supposed tha t sen i l i ty and dea th of the cu l tu res , ins tead of be ing
necessary , resu l ted mere ly f rom preventab le occur rences; such as
accumula t ion of ca tabol ic substances and exhaust ion o f the medium.
The suppression of these causes should br ing about the regenera t ion
of o ld cu l tu res and prevent the i r dea th . I t i s even conceivab le tha t
the leng th of the l i fe o f a t i ssue ou ts ide of the organ ism could
exceed grea t ly i t s normal dura t ion in the body , because e lementa l
deat h migh t be postpone d indefinitely by a prop er ar t if icial nutr i t ion.
In the fo l lowing exper ime nts I sought no t a comple te so lu t ion of
the prob lem of p ro longing indef in i te ly the l i fe o f t i ssues
in vitro
but the fac ts tha t would show in which d i rec t ion we should pro-
ceed in order to secure a complete solution, if such a solution be
possible.
In September , I9 I I , I t r ied to increase the dura t ion of the l i fe o f
cu l tu res o f connect ive t issue . ~ The cu l tu res were washe d for
Received for publication, March I5, I9~2.
1Harrison, Harvey Lectures, Philadelphia and London, I9I I-I912 ; .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
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Alexis Carrel. 517
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 in a n e w
m e d i u m . As 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 e
m o r e r a 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 e s t h e o c c u r r e n c e o f
s e n i l i 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 t u r e s c o u l d be 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 if t y ,
f i ft y - fi v e , a n d e v e n f o r s i x t y d a y s ? T h e s e r e s u lt s s h o w e d t h a t t h e
e 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 ab 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 si b le .
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 t
b e p o ss i bl e to o b t a in p e r m a n e n t l i f e o f ti s su e s . T h e i d e a l m e t h o d
wo 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 y
w 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 i t i o n a n d e l i m i n a t e d t h e i r
wa s t e p ro d u c t s . I t wa s a t t e m p t e d t o p l a c e t h e t is s u e s i n a t h i n l a y e r
c o n s i s ti n g o f c e l l ul o se a n d c o a g u l a t e d p l a sm a , a n d t h r o u g h t h i s a
s l ow s t r e a m o f s e r u m f lo w e d . B u t i t a p p e a r e d s o o n th a t b e t t e r
r e s u l ts c o u l d b e o b t a in e d b y a s i m 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 l is h i n 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 e
t 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 m
t o m e d i u m . T h e m e d i a u su a l l y e m p l o y e d c o n s i st e d o f R i n g e r s
s o l u t io n a n d h y p o t o n i c p l a sm a . I t w a s f o u n d t h a t t h e re s i st -
a n c e o f a t i s s u e p la c e d i n a n o n - n u t r i t iv e , b u t p r e s e r v a t i v e m e d i u m
w a s v e r y m u c h s t r o n g e r i f t h e t e m p e r a t u r e w a s l o w . T h e c u l t u r e s
w 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 e
p h a s e s : t h e r e wa 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 e
t 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 c
p 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 s
w a s h e d f r e e o f i ts p r o d u c t s , t o b e s u b se q u e n t l y p l a c e d in a n e w
m e d i u m .
I n F e b r u a r y , I 9 1 2 , th 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 f
l 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 a
p h a s e o f a c ti v e l i 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 g
i 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 h
m 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 i e fl y e m p l o y e d w e r e t h r e e . T h e y a r e a s f o l l o w s :
Technique/. The o r i g i n a l p i e c e o f t i s s u e a n d t h e s u r r o u n d i n g
8 P o z z i
B ul l . de l A c ad , de t odd .,
I 9 1 2 l x v i i 2 6 .
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518
Permanent Life 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 a r a c t k n i f e f r o m t h e f r a g m e n t o f
c o a g u l a t e d p l a s m a th a t c o n t a i n e d t h e m . T h e y w e r e w a s h e d f o r
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 io 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 , 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 e
o r t w o p a r t s o f d i s t il le 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 e m 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 e
w 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 th e a p p e a r a n c e i n
t 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 il it y , o r w h e n t h e s e w e r e s ti ll v e r y
s li g ht . T h e p a s s a g e w a s r e p e a t e d m o r e o r l e ss f r e q u e n t l y a c c o r d -
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 s
w 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 n
c o l d s t o r a g e . T h e l i f e o f t h e c u l t u r e s w a s m a i n t a i n 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 f e i n a
p 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 f e in
R 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 t h i rd m e t h o d t h e c u lt u r e s w e r e m a i n t a i n e d
i 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 m
p l a s m a t o 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 s m . T h e c u l t u r e s
w 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 t
o n e c e n t i m 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 d
w i t h a d r o p o f pl a 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 e d i n t h e
c 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 s m 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 i l t o g e t h e r w i t h
t 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 f e . A s t h e s i lk v e il a c t e d a s a s k e l e t o n f o r t h e
p l a s m a t i c j e l ly , th e o r i g i n 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 e
c u l t u r e c o u l d be h a n d le d e a s i ly 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 f
t 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 d
t 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 a
f e w d a y s i t w a s p u t a g a i n i n p l a s m a , a n d s u b s e q u e n t l y t h e t w o
p 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 Res u l t s . A g r e a t m a n y e x p e r im 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 c u lt u r e 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
Published May 1, 1912
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blood vessels, heart, skin, muscles, peritoneum, and spleen, of four-
teen to twenty day old chick fetuses. In other cultures were em-
ployed fragments of the Rous sarcoma, and in a few cases, tissues
of adult dogs. Many cultures survived six to fourteen or fifteen
passages in a condition of interrupted or continuous growth. A
few cultures were transplanted seventeen, eighteen, and even twenty
or 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 of
tissues cultivated for more than two months outside of the
organism.
MORPHOLOGICAL CHARACTERS
The appearance of the cultures grown by technique I was almost
identical with that of the cultures grown by technique 2. The
fragment of tissue was surrounded rapidly by cells spreading in a
thin layer through the medium. Afte r section of the plasma, the
new tissue retracted around the original fragment forming an
opaque crown. During the period of washing there was no modi-
fication of the culture, but as soon as the culture was put in a new
medium in the incubator, elongated cells began to grow from the
edges of the old plasma and to spread into the new medium. Aft er
a few passages the original fragment appeared diminished in size
and surrounded by a dense envelope of a greyish tissue from which
radiated a great many elongated cells, living and dead.
The original fragment was often encircled by several concentric
rings, each representing a period of active life. Soon the rings
were 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 of
its trunk. Progressively the original fragment disappeared and
was replaced by an amorphous yellowish substance. The form of
the culture was also modified. At the beginning of the growt h
the cu lture appeared like a thin plate, but it grew progressively more
and more like a flattened sphere in the center of which were ac-
cumulated a few fore ign bodies and pieces of old plasma figure I ),
which were encysted by the living cells. Aft er about fort y days the
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5 2
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 e
r 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 s
o l d .
Cultivation of Connective Tissue Experiment 715). --A s h o r t s e g m e n t o f
t 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 . A
l 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 a s 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 s
s 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 t
p 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 th e s e c o n d p a ss a g 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 s
i n R i n g e r s s o l u 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 r
w 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 ld s t o r a g e f r o m 9 : 5 o A . M . t o
l 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 a
t 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 l d
s 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 d
o f p l a s m a a n 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 l d
s 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 e
e 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 c
p 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 en w a s h e d i n
c 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 e
o 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 a
t 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 th 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 s s o l u t i o n a n d i n c o l d
s 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 d
e 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 s
o 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 f
w 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 to 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 r
f 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 a
t 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 g
t 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 n
w 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 rg e g r o w t h t h r o u g h t h e n e w
m e d i u m w a s s ee 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 in 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 t
i 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 s
o b s e r v e d .
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521
O 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 ty - f i ve 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 ve
t i s s u e m a d e b y t h e f i r s t 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 e a r .
a n c e a l l o f t h e m r e s e m b l i n g th e l a t e r s t ag e s o f t h e c u l t u r e o f t h e
i l i ac vesse l f i r s t descr ibed .
B y t h e sa 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 ti s su e s s u c h a s
t h y r o i d a n d p e r io s t e u m w e r e c u l ti v a t ed a n d s e v e r a l g e n e r a t i o n s o f
c o n n e c t i v e t i s s u e c e l l s we re o b t a i n e d .
F r e q u e n t l y th e c u l t u r e s o f R o u s s a r c o m a di e 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 l iq u e fi e d a n d t h e t i ss u e d is 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 fo r fo 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 s
s 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 d
a 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 r
o 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 l
c 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 s
i n R i n g e r s s o l u t io n 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 ll s
o 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 d
7 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 e
g 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 o
p 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 y
9 . 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 y
a 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 e
e 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 n
F 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 n
F 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 h
d 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 h
p 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 e
b 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 e
m 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 t s f i ft e e n th ,
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 d
t h e c u l t u r e i n c r e a s e d v e r y m u c h i n s i ze . O n M a r c h 5 a m a r k e d r e t r a c t i o n o f
t 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 h
b 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 s e .
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 d
t 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 if 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 in u o u s a n d a s t h e r e w a s n o s e c t io 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 on s e q u e n c e n o r e tr a c t i o n .
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522 Permanent Life of Tissues Outside of the Organism
Moreove r as the p lasma was suppor ted by it s skele ton of s i lk the
cu l tu re cou ld no t take a spherica l fo rm and the ti ssue g rew in a
th in layer ; tha t i s unde r the best condit ions fo r a t issue depr ived of
c i rcu lat ion . The p lasmat ic je lly was adheren t to the si lk ve il and
the t i ssue imbedded in the p lasma grew as in an ord inary cu l tu re .
The ce lls cou ld use the s ilk th reads as a suppor t bu t they se ldom
did. The purpose o f the s i lk was mere ly to give such a consis tency
to the medi um tha t i t would no t fo ld over o r become spher ical
dur ing the passages in p lasma an d in serum.
Nor mal hear t was cu l t iva ted accord ing to th is techn ique . I t s ap-
pearance af te r a few passages was the same as a f te r a few days.
The s ize o f the g rowth a lone was modif ied .
B y th e sam e p r o ced u r e m an y f r ag m en t s o f t h e R o u s sa r co m a
were a lso cu lt iva ted . Th ey cou ld be kep t in a condi t ion of ac t ive
and con t inuous development fo r more than f i f teen days . But the
m ed iu m was p r o g re ss iv ely o v e r c r o wd ed wi th d ead ce ll s an d th e
growth of the t i ssue became more i r regu lar and less abundant .
The f ragments cou ld no t adap t themselves to the i r new condi t ions
of l i fe and were less res is tan t than norma l connect ive t issue.
The appearance of the ce l l s g rowing in cu l tu res o f normal con-
nect ive t i ssue o f ten var ied per iodica l ly . Du r ing the f i rs t few days
of g rowth the fusi fo rm ce l ls were long and th in and con ta ined
clear cy top lasm. Af ter four o r five days they became larger and
the i r cy top lasm was darker and f i l led wi th la rge fa t ty g ranu la t ions .
At the same t ime the ra te o f g row th d iminished . Af ter be ing
wash ed an d t r an s f e r r ed to a n ew m ed iu m th e ap p ea ran ce o f th e
ce lls was again modif ied . Th ey regained the i r o r ig ina l appearance
and the ra te o f g row th was acce lerated . Ins tead of be ing per iod-
ical the morpholog ica l modif ica t ions might be con t inuous. For in -
s tance in the cu l tu res o f sp leen the o r ig ina l f ragm ent was sur -
rounded dur ing the f i r s t phases o f ac t ive l i fe by an envelope of
ameboid ce lls. A f te r a few days long chains o f fu si fo rm cel ls
appeared arou nd the f ragment whi le the ameboid ce lls d ied and
disappeared . Ul t im ate ly the fusi fo rm cel ls were rep laced by small
po lygonal ce l l s which spread around the f ragment in a con t inuous
layer. In the cu l tu re o f the Rous sa rcoma the round ce lls d isap-
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Alexis Carrel 528
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 te d c e ll s w e r e s ti ll 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 la r g e a m e b o i d ce ll s
w a n d e r e d o u t f r o m t h e c e n t r 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 i n g o f t h e t h i r d m o n t h o f i t s l i f e t h e c u l t u 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 o l d p l a sm a . T h e y
w 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 d
cel ls .
DYNAMIC CHARACTERS
T h e r a t e o f g r o w t h c o u l d be a p p r e c i a t e d b y t h e r a p i d it y w i t h
w h i c h t h e c e ll s a f t e r a p a s s a ge a p p e a r e d a n d s p r e a d i n t h e n e w
m e d i u m . T h e n a t u r e o f t h e m e d i u m i ts o s m o t i c t e n s io n t h e w a y
i 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 l a s m a l e f t a r o u n d
t h e c el ls t h e f o r m o f t h e c u lt 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 ss a g e s
o f t e n h a d a m a r k e d i n f l u e n c e o n t h e r a t e o f t h e g r o w t h . T h e
o s c i l l a t i o n s i n t h e r a t e s e e m e d g e n e ra l l y t o b e t h e r e s u l t o f a c c i d e n t a l
c a u s e s b u t i t wa s v e ry d i ff i c u lt t o e x c l u d e t h e p o s s i b i li t y t h a t t h e y
m i g h t b e d u e t o c a u s e s i n h e re n t i n t h e c o n d i t i o n o f t i s s u e s l i b e ra t e d
f r o m t h e c o n t ro 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 i rs t p a s s a g e
t 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 te n t p e ri o d . T h i s w a s
t r u e e v e n i n 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 is s u e s o f a d u l t
d o g s . T w o o r t h r e e h o u r s a f t e r a pa s s a g e m a n y e l o n g a t e d c e ll s
w 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 l a s m a i n t o
t 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 ce b y a
d e n s e l a y e r . T h e r a p i d i t y o f th e i r g r o w t h o f t e n d i m i n i s h e d a f t e r
a f e w p a s s ag e s 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 si v e l y . T h e r a t e
o 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 t y - f iv e d a y s o l d w a s m o r e r a p i d
t h a n w h e n it w a s te n o r f i f te 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 r
a c u l t u 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 f t 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 s
m 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 f
g r o w t h o f a c u l t u r e 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 in g o f t h e
t h i r d m o n t h o f i t s l i f e o u t s i 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 r
p e r i o d .
T h e s e a c c e l e r a t io 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 e
a g e o f t h e c ul t u re . B u t t h e n u m b e r o f th e e x p e r i m e n t s w a s n o t
l 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 ib i li ty t h a t t h e y w e r e d u e t o o t h e r
causes .
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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 s iz e o f t h e c u l tu 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 ca t i o n s. F o r
l 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 iz e , d im i n i s h e d , o r
w 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 l y t o a c c i d e n t a l 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 th e 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 l ly t o m i c r o b i a n i n f e c t i o 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 si ze 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 tu 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 d
a 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 t ly .
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 i v e a n d a f t e r t h e f if t ie t h d a y t h e
v o l u m e o f t h e t i s s u e i n c r e a s 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 e
t is s u 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 ti o n o f g r o w t h a n d a m a r k e d i n c r ea s e i n s iz e w a s o b s e r v e d
a 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 an c e a c u l t u re o f h e a r t b e c a m e s o
l 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 b e d i v i d e d i n to 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 f
t 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 e
t 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 , th e
o 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 t e d s u s p e n s i o n o f c e l ls 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 a s e i n th e v o l u m e o f t h e t i s s u e c o u l d t a k e
p l a c e .
Culture of Cells Suspended in R inger s Solution.--On February I , I912 a
nine day old chick embryo was cut into very small pieces, and to the pieces an
equal vo lume o f Ringer s so lut ion was added . The suspension was c en tr i f -
ugalized fo r five minutes, and a dro p of the super natant fluid was then added
to a culture medium. On Fe bru ary 2 ma ny isolated cells wer e seen in the
per ipheral par t o f the med ium. On Fe bruary 5 the numbe r o f these cel ls had
increased very much . The pa r t o f the p lasma where they had g rown was
resected, washed, and put in a new medium. Th e culture u nderw ent its second,
third, fourth, and fifth passages on Fe brua ry 2, IO, 13, and 16. The cells had
then increased so muc h in n.umber that the y form ed a real t issue, and had the
appearan ce o f an o rd inary cu l tu re . The l i fe o f these cells af terwards was l ike
that of other cultures.
S i n c e a f e w s c a t t e r e d c e ll 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 e
c u l t u r e o f f r a gm e n 4 c s o f t i s s u e , i t i s 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 ls
m u l t i p l y a n d t h a t t h e y d o n ot m e r e l y w a n d e r f r o m t h e c e n t ra l
p i e c e .
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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 i n s p it e o f t h e i r c o n -
s t a n t g ro wt h , a n d o f t e n t h e y d i d n o t in c re a s e a t al l i n s iz e . I t i s
p 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 l
c o n d i t i o 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 s p h e r i c a l. T h e
n 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 i n s u ff i c ie n t , b u t
t h a t w h i c h is s u p p li e d t o t h e p e r i p h e r y i s a b u n d a n t . H e n c e , t h e
c e l ls a t t h e p e r i p h e ry m u l t i p l y c e a s el e s s ly . B u t a s s o o n a s c e ll s a r e
i s o la t e d f r o m t h e m e d i u m b y a s u ff ic i en t t h i c k n es 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 ro b a b l y b e c o m e s d e f ic i e n t a n d t h e y d i s i n t e g ra t e .
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 ly b y i t s p e r ip h e r y , i t d i m i n i s h e s
i n i t s c e n t e r a n d t h e re fo re i t s v o l u m e d o e s n o t i n c re a s e .
A t i ss 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 ly c a n n o t g r o w i n a n u t r i -
t i v e m e d i u m b e y o n d a c e r t a i n v o l u m e w h i c h i s a f u n c t i o n o f i ts
p e r m e a b i l i t y t o t h e m e d i u m a n d c o n s e q u e n t l y o f t h e t h i c k n e s s o f
t h e c u l tu r e . I f t h e c u l t u r e b e sp 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 y
s 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 i s i n s u f fi c i e nt . F ro m a th 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 y
t h i n l a y e r c o u l d i n c re a s e i n d e f i n i te l y . I t i s f o r t h is r e a s o n t h a t t h e
t i s s u e s we re 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 e
p 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 h e 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 i n w h e t h e r t i ss u e s a f t e r a
l o n g p e r i o d o f l i f e i n v i t r o 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 r i 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 s
h 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 l
d a y s w h e n k e p t a t a p r o p e r t e m p e r a t u r e in p l a sm 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 io n s o f a c u l t u r e o f h e a r t
s t op p 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 i s s u e h a d b e e n w a s h e d a n d
t r a n s p l a 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 i n 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 c
m 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 ve a n d i d e n ti c a l r e s u lt s . O n e o f
t h e s e w i l l b e d e s c r i b e d .
Cultiva tion o f the Hea rt Expe rime n t 72o- 1) . - -0n Janu ary I7 1912 a small
fragment of the heart of an eighteen day old chick fetus was cultivated in
hypotonac plasma. The f ragme nt pulsated regularly fo r a few days and grew
extensively. Afte r the first washing and passage on January 24 the culture
grew again very extensively but there were no rhythmical contractions. On
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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 Febr uar y I 3 6 9 12 I5 I7~ 20 24 a nd 28 the culture under-
went e leven wash ings and passages. I t became sur rounded by fus i fo rm ce l ls
and many dead ce l ls . The re w ere no pu l sa tions . A~f te r the twe l f th passage the
cu l tu re d id no t g row a t a l l. The n the t i ssue was d i s sec ted and the o ld p lasma
was comple te ly ex t i rpated . A small cen t ra l f r agm ent was removed washed
and pu t in a new medium. On M arch I i t was pu l sa t ing a t a r a te tha t va r ied
be tween 60 and 84 pe r minu te . On March e the pu l sa t ions w ere lO4 a t 4 I C.
and on M arch 3 8o a t 40 C . bu t o n M arch 4 the pu l sa t ions were ve ry weak and
s topped a l toge the r a t 2 P . M . On March 5 the cu ltu re underwent i t s four teen th
passage and the pulsat ions reappe ared immediately. Th ey becam e wea k again on
March 2 . On M arch 8 the f i ft een th passage was made . On M arch 9 the pu lsa -
t ions were aga in 80 to 82 pe r minu te a t 40 C . a nd on M arch 12 they were
6o pe r minu te . They then became s lower and weaker . Af te r the s ix teen th
passage on March I2 the pu l sa t ions were ir regu la r and the f ragm ent bea t fo r a
series of 3 to 4 pulsat ions and then stopped fo r about 20 seconds. Af ter the
seven teen th passage on March 16 regu la r pu l sa t ions a t 52 bea t s pe r minu te reap-
peared and the t i s sue g rew abundan t ly . Af t e r the e igh teen th passage on March
19 the pulsat ions were irregular .
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 d
s ti ll p u l s a t e 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 ts
l 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 t h a t a f r a g -
m e n t o f ti s s u e l iv i n g
i n v i t r o
c o u l d r e t a i n it s n o r m a l f u n c t i o n f o r a
l o n g t i m e .
T h e m a x i m u m a g e t h a t t is s u es l iv i n g
i n v i t r o
c a n r e a c h i s s t i l l
u 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 i ed a f t e r le ss t h a n t w o
m o n t h s b u t a f e w w e r e v e r y a c t iv e a t t h e b e g i n n i n g o f th e t h i r d
m o n t h o f t h e i r l i f e o u t s i d e o f t h e o r g a n i s m . G e n e r a l l y t h e t i s s u e s
s 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 i r n e w c o n d i t i o 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 f t e e n th p a s s ag e v e r y f e w c u l t u re s d i e d
s 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 re q u i r e d b y t h e
p a s s a g e s t h e c u l t u r e s w e r e e x p o s e d t o m a n y a c c i d e n t s c h i e f ly t o
m i c r o b i a n i n f e c t i o n s . I n c as e o f a l o c a l i n f e c t i o n t h e p a r t o f t h e
c 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 c t e d w i t h a c a t a r a c t k n i f e
a n d p l ac e 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 re r e c o v e r e d a n d
p r o d u c e d s e v e r a l g e n e r a t i o n s o f c el ls th a t w e r e f r e e f r o m m i c r o b i a n
i n f e c t i o n . B u t w h e n i n f e c t i o n w a s g e n e r a l i z e d t h e t i s s u e s a l w a y s
d 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 n
t 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 .
Cul ture of Portal Ve in Experiment 48 3) .~ 0n
September 27 I9 I I a f rag-
ment o f the por ta l ve in o f a ch ick fe tus s ix teen days o ld was cu l t iva ted in th ree
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Alexis Carrel 5 2 7
p 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 w
v 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 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 f o r f i v e m i n u t e s a n d p u t ir ~ a n e w m e d i u m . O n
O 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 nt o t w o p ar t s . O n e
p 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 k
p 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 s
w 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 e
a 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 d
p 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 e
a 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 n
O 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 e
w 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 h
p 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 d
w 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 e
c 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 30
a 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 t
a 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 h
w 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 d
a 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 n
c 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 s
i 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 d
s o o n a f t e r .
Nearly all the cultures made in the latter part of I9 II died in
the same manner after one to two months. By slight modifications
of 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 January
17 I912 five were still very active in March I912 and of the five
active ones two he art cultures previously described grew slowly
but pulsated and another heart culture which pulsated from time to
time produced a large growth of ameboid and fixed cells which
covered an extensive area of the medium. In this instance afte r
having been motionless for two months the central part of the
culture manifested strong rhythmical contractions on the sixty-fifth
day of its life i n v i t r o Moreover two cultures of connective tissue
made on January 17 were growing actively at the beginning of
April. The rate of growth and the increase in their volume became
very much greater as they grew older.
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528 Permanent L i fe o f T issues Outs ide o f the Organism.
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 r ie s o f e x p e r i m e n t s m a d e a t t h e e n d o f 1 91 1 a n d a t t h e
b e g i n n i n g o f 1 9 12 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 w
o f i n v e s t i g a ti n g t h 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 el y t h e l i f e o f
t i ss u e s i s ol a te 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 iq u e s a r e f a r f r o m
p e r f e c t a n d w i l l d o u b t l e s s b e m o d i f i e d i n t h e fu t u re . B u t t h e y h a v e
a l r e a d y p e r m i t t e d t h e e s t a b li 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 iv e t is s u e h a v e b e e n k ep t i n v i t ro in a cond i -
t i o n o f a c ti v 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 s
a r e n o w e i g h ty - f iv e d a y s ol 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 is
p ro 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 ll
c o n t i n u e fo r a l o n g t i m e .
I n s o m e c a s e 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 i n d ir e c t
r 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 a te 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 f
t h e t h i r d m o n t h o f t h e i r l i f e in v i t ro .
T h e s e f a c t s 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 e
p e r f e c t t e c h n i qu e s a n d f o l l o w e d o v e r l o n g p e r io d s o f t i m e m a y l e a d
t 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 ir e d b y t i ss u e s
l 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 e
d 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 e
c 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 c
l 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 .
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THE JOURNAL OF EXPER IMENTA L MEDICINE VOL XV PLATE 75
~IG I
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THE JOURNAL OF EXPERI MENTAL MEDICINE VOL XV. PLATE 76.
F I G . 2 .
F I G . 3
Published May 1, 1912