Células 3t3

Published May 1, 1963

QUANTITATIVE

S T U D I E S OF T H E

G R O W T H OF M O U S E E M B R Y O C E L L S I N
CULTURE AND THEIR DEVELOPMENT
INTO ESTABLISHED

GEORGE

LINES

J. T O D A R O

and H O W A R D

GREEN,

M.D.

From the Department of Pathology, New York University School of Medicine, New York

Disaggrcgatcd mouse embryo cells, grown in monolaycrs, undcrwcnt aprogrcssivc dcclinc
in growth ratc upon succcssivc transfer, the rapidity of the decline dcpcnding, among othcr
things, on the inoculation density. Ncvcrthclcss, ncarly all culturcs dcvclopcd into
cstablishcd lincs within 3 months of culture. Thc firstsign of thc emcrgcncc of an established
line was the ability of thc cells to maintain a constant or rising potential growth ratc. This
occurredwhile thc cultures wcrc morphologically unchangcd. Thc growth rate continued
to incrcasc until it cqualcd or cxcccdcd that of the original culturc. The carly cstablishcd
cclls showed an increasing mctabolic autonomy, as indicated by dccrcasing dependence
on ccll-to-ccllfccding. It is suggcstcd that the process of cstablishmcnt involvcs an altcration in ccll pcrmcability propcrtics. Chromosomestudics indicatcd that thc cells rcsponsiblc
for thc upturn in growth rate wcrc diploid, but latcr the population shifted to the tctraploid
range, often very rapidly. Still later, marker chromosomes appcarcd. Different lines acquired diffcrcnt propcrtics, dcpcnding on thc culture conditions cmploycd; one line dcvclopcd which is cxtrcmcly scnsitivc to contact inhibition.
INTRODUCTION
Whenmammalian ceils are placed in culture they
grow rapidly, often at a rate substantially exceeding that in the intact animal. However, this growth
does not continue indefinitely. Most frequently,
after a variable interval, for reasons as yet unclear,
the cells die (1, 2). In some cases, usually thought
to be uncommon, changes in the cell population
are observed to occur, culminating in thedevelopment of an established line having a variety of
properties which distinguish it from the strain of
origin (2). One of these properties is the ability to
produce tumors when injected into suitable hosts
(3-5), so that the mechanism by which the normal
cell is converted into an established cell may have
a bearing on the problem of carcinogenesis.

In the following experiments the growthproperties of mouse embryonic fibroblasts were closely
studied from the time they were placed in culture,
and especially during their conversion to established lines. The changes in growth properties were
related to chromosomal and morphological
changes, and certain criteria were set up for the
established condition.
MATERIALS

AND METHODS

M edia
T he medium used was Dulbccco's modificationof Eagle's medium (6), containing an approximately
fourfold higher concentration of the amino acids

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ABSTRACT

Published May 1, 1963

a n d vitamins described, plus serine a n d glycine, a n d
10 per cent calf s e r u m (Colorado S e r u m Co., Denver).
T h i s m e d i u m h a s a h i g h bicarbonate concentration,
a n d t h ep H was kept at 7.2 by equilibration with
10 per cent CO2 in air. I n the earlier experiments
10 per cent tryptose p h o s p h a t e was s u p p l e m e n t e d .
All cultures were m a i n t a i n e d in 50 m m d i a m e t e r
plastic Petri dishes a n d were transferred by trypsinization in p h o s p h a t e - b u f f e r e d saline (PBS) (7).

Primary Cultures
C ultures of 17 to 19 dayold Swiss m o u s e e m b r y o s
were p r e p a r e d by fine m i n c i n g of t h e whole e m b r y o s

I

detached, in 10 to 15 minutes, aliquots of t h e suspension were t a k e n from each plate for c o u n t i n g in a
h e m o c y t o m e t e r c h a m b e r u n d e r p h a s e microscopy.
At least 250 cells, a n d in most cases 500 to 1000 cells,
were counted. A p p r o p r i a t e...