258
should have become known as Ebeling's
The myth of cell immorality
Jan Witkowski
In 1978, one of the Citation Classics
series in Current Contents Life Sciences 1
was devoted to the paper by L. Hayrick
and P. S. Moorhead that described the
growth of fetal human diploid cells in
culture2. One of the main features of
this work was the suggestion that the
death of such cells after some 50 sub-
cultures was a phenomenon related to
ageing at the cellular level. This paper
was cited over 1100 times between 1961
and 1978, and established the field of
cytogerontology, one of the most active
areas of research on ageing.
It may therefore surprise many to
know that before 1961, the accepted
dogma was that cells in tissue culture
were potentially immortal, and in one
case this was believed to have been
proved beyond doubt. This was the so-
called 'immortal' strain of chick heart
fibroblasts established by Alexis Carrel
in 1912 and grown for 34 years. The
growth behaviour of these 'immortal'
cells had a major influence on earlier
views on senescence but it is clearly at
variance with current views on cell
ageing.
Who was Carrel, and what were his
'immortal' cells? Why were they import-
ant and how can they be reconciled
with modem research?
Alexis Carrel
Alexis Carrel 3'4 was born in Lyon,
France, in 1873, and studied medicine at
the University of Lyon, completing his
MD thesis in 1902. From an early stage
in his career he was interested in
vascular surgery and organ transplan-
tation 5, and in 1902 published his tri-
angulation method for the end-to-end
joining of blood vessels. In 1904 he
emigrated to North America, first to
Montreal and then to Chicago, where
he and Charles Guthrie carried out
pioneering work on organ transplan-
tation. Simon Flexner 'head-hunted'
him for the newly-established Rockefeller
Institute, and he moved there in 1906 as
head of experimental surgery. In 1912
he was awarded the Nobel Prize for his
contributions to surgery.
Origins of tissue culture
Carrel, at this time, was interested in
nerve regeneration and wound healing,
J. Witkowski is at the Imperial Cancer Research
Fund Laboratories, Dominion House, Bartholomew
Close, London EC1A 7BE, UK.
1985, Elsevier Science Pubfishers B.V., Anlslerdam 0376 - 5067/85?$02.00
and in 1908 he attended the Harvey
Lecture given by Ross G. Harrison who
described his work ~growing amphibian
nerve cells in vitro ° . The tremendous
potential of this technique was clear to
Carrel and he despatched his assistant,
Montrose T. Burrows, to Yale to learn
the method. Burrows made several
technical innovations and was able to
grow chicken tissue 7. On his return to
the Rockefeller Institute, he and Carrel
began to grow as many different k i n d s
of tissue as they could, and between 22
October and 12 November 1910, they
presented no fewer than seven papers to
the Soci~t6 de Biologie (Paris).
It was an unfortunate occasion to
present such revolutionary work. Carrel
had left France under a cloud after
disagreement with the medical hierarchy,
and had compared French science un-
favourably with its American counter-
part. His tissue culture results were
bittedy attacked.
The immortal cells
Carrel's principal critic was Jolly, an
eminent scientist who had himself
attempted in vitro culture of leucocytes
as early as 1903. Jolly's criticism was
that Carrel had not demonstrated cell
growth in vitro, and Jolly suggested that
they were merely dying slowlyS! Carrel
resolved to answer this criticism by
keeping chick cells growing for long
periods of time; indeed he undertook to
determine 'the conditions under which
the active life of a tissue outside of
the organism could be prolonged in-
definitely'9.
On 17 January 1912, he established a
series of chick heart fibroblast cultures,
one of which was destined to become
the 'immortal' cell strain9'1°. Small frag-
ments of chick heart were embedded in
clots of chick plasma and grown in slide
chambers. They were subcultured by
cutting out a growing portion of the
culture and transferring it to another
chamber with fresh plasma. There were,
of course, no antibiotics and Carrel's
success undoubtedly owed much to his
skills in working aseptically, acquired
during his surgical work.
The first paper on these cells was
published in March 1912 by which time
they had been subcultured 19 times. On
1 June 1912 they became the responsi-
bility of Albert Ebeling who looked
after them for the next 34 years. (They
TIBS - July 1985
immortal cells, but all the glory went
to Carrel.) In 1913 Ebeling gave a de-
scription of the state of the cells and
presented a detailed account of their
culture 11. In September 1912 only a
single culture was alive, but this was
saved and by February 1913 the cells
had been subcultured 138 times since
they were first established.
In the same year, Carrel reported that
extracts of chick embryo could promote
cell growth 12, and chick embryo extract
(CEE) became a standard constituent of
the culture medium. (This work caused
a sensation in the popular press, coming
as it did shortly after his Nobel Prize.)
By now the cells had become the
standard material for much research in
Carrel's laboratory.
Carrel published papers describing
the cells after 16 and 28 months in
culture 13'14. He concluded that the cells,
' . . . like microorganisms, multiplied
indefinitely in the culture medium'.
Ebeling described the cells on their
seventh (1919) and tenth birthdays
(1922) 15'16. They were similar in all
respects to the original cultures, and by
1922 had been subcultured on 1860
occasions. It was about this time that
extravagant examples were given to
illustrate how the cells had grown.
Ebeling, for example, remarked that
the accumulated mass of cells would be
very much greater than that of the
sun 16, and The World wrote that they
would have formed a 'rooster . . . big
enough today to cross the Atlantic in a
stride; . . . so monstrous that when
perched on this mundane sphere, the
world, it would look like a weather-
cock'! 17. Public interest was such that
the New York World Telegram asked
after the health of the cells every
January.
The Rockefeller Institute had a policy
of compulsory retirement at 65 years,
and despite his machinations, Carrel
was unable to evade the ruling. In
protest, he rejected the laboratory space
offered to distinguished retired staff and
returned to France. There he estab-
lished the French Foundation for the
Study of Human Problems dealing with
a range of subjects from biochemistry to
paranormal phenomena. He accepted
help from the Vichy Government and
when Paris was liberated in 1944, he was
accused of being a collaborator. He died
in November 1944, before standing
trial.
The cells outlived him, although the
New York World Telegram published a
premature obituary of the cells in January
TIBS - July 1985 259

1940. Arthur Ebeling had taken them remarkable paper. They wrote that 'the distinction between normal cell strains
with him when he went to work at the failure of human fibroblasts to proliferate with these characteristics, and cell lines
Lederle Laboratories of the American indefinitely in standard cell culture that grew indefinitely, had abnormal
Cyanamid Company, and they were media is a more general phenomenon karyotypes and were frequently tumor-
used in cytotoxicity assays TM. The final than is usually recognized'. They pointed igenic. In the subsequent paper 22,
publication in the series came in Scientific out that cells were dying under identical Hayflick drew up a table contrasting the
American in 1942, when Ebeling de- conditions to those in which other cells characteristics of diploid cell strains and
scribed the cells and the uses to which were flourishing and that the behaviour heteroploid cell lines (Table I).
they had been put. He also dismissed of a cell was a function of 'its age in Clarifying these fundamental dis-
some of the fantastic myths that had vitro'. But this was related to selection tinctions was one of the major achieve-
become associated with the cells: that of the physiologically different cells ments of this work, and one that should
they were kept in a glass jar or on a present in the same culture, resulting in be more widely recognized.
marble column, guarded day and night disturbance of 'symbiotic' relations These findings have been confirmed
by scientists, and that pieces had to be between the cells and death of the for a variety of cells and it is generally
'snipped off' to stop the culture growing culture as a whole. accepted that the death of diploid cells
out of the the laboratory! They were The situation in the late 1940s and in vitro is related to the phenomenon of
finally discarded in 1946. 1950s became even more confused cell ageing in vivo. (For a review see
when it was realized that certain cells, Ref. 23.) What explanations can there
The significance of Carrel's cells mainly from rodents, did seem to grow be for Carrel's cells?
Carrel established these cells to prove indefinitely. But this change in growth
that cells could grow in vitro and were potential was always associated with The myth of cell immortality
not merely surviving. But by the time of other changes in cell behaviour. For It might be that Carrel's cells under-
the first publication9, he was already example, there were chromosomal went spontaneous transformation, and
writing of prolonging the life of tissues changes, alterations in cell morphology the decline to a single culture by
indefinitely and, after 10 years, Ebeling and the cells usually became tumorigenic. September 1912 followed by growth is
concluded that the cells were potentially The process was called transformation. suggestive of a 'crisis' period. However,
immortal 16. It seemed that ageing was a the cells were described throughout
phenomenon restricted to cells in multi- Human diploid cells and cell ageing their existence as being unchanged in
cellular organisms, and analogies were The resolution of this confusion came growth rates and morphology. Until
drawn with the indefinite growth of with the publication in 1961 of the paper very recently24, there have been no
protozoa and bacteria. (It should be by Hayflick and Moorhead, and the authenticated reports of spontaneous
noted that it is now by no means clear subsequent detailed study of cell death transformation of chicken cells in vitro;
that all, if any, protozoa are capable of in vitro as an ageing phenomenon by like human diploid cells, chick cells
indefinite growth.) This had a signifi- Hayflick in 1965 (Ref. 22). Hayflick and seem very stable in culture.
cant effect on studies of ageing. As Moorhead showed that cells from a Hayflick suggested that the cultures
Pearl said in 1921, senescence is ' . . . an variety of human fetal tissues grew in may have been accidentally replenished
attribute of the multicellular body as a culture for about 50 subcultures before by living cells present in the chick
whole . . . If this conception of the dying, and that this cessation of growth embryo extract used to feed the
phenomenon of senescence is correct in was not related to alterations in the cultures 22. Certainly there was some
its main features . . . it shows the medium, or to the treatment the cells correlation between the addition of
essential futility of attempting to investi- received during culture; cells at early CEE and the growth of the cells, but it
gate its causes by purely cytological subcultures would flourish in media in is only a weak correlation and may have
methods' 19. which late subculture cells were dying. been due to the growth-promoting
Yet no one else achieved Carrel's When 'old' male cells were mixed with activity of the CEE. Nevertheless,
success at growing cells for such lengths 'young' female cells and the mixed Hayflick has anecdotal evidence for
of time. Cells in culture died, but cultures examined after a further 17 this, from a technician who worked in
because Carrel had shown cells were subcultures, only female cells were Carrel's laboratory between 1927 and
immortal, their death was attributed to found. Under precisely the same con- 1931. However, I feel that it is unlikely
'accidental causes' such as inadequate ditions, the 'young' cells had continued that such regular contamination could
media or infection. For example, Haft to grow while the 'old' cells had died. have occurred over such a long period,
and Swim2° performed a careful study of The cells retained their diploid karyo- particularly when Carrel's laboratory
the growth of rabbit and chick cells and type and did not exhibit any factors became aware of the problem very early
found that 17 of 20 cultures established associated with malignant transformation, on, in 191311. The methods used to
died after 8 to 12 subcultures. 'After this and Hayflick and Moorhead made the prepare CEE were quite harsh and it is
period [of initial growth], the rate of
growth either declined gradually over a Table I
period of several weeks or growth
ceased abruptly'. Alterations in medium Heteroploid cell : Transplantable = Diploid cell : Normal somatic
composition had no effect, and they lines tumours strains tissue
remarked it was a 'perplexing problem' (in vitro) (in vivo) (in vitro) (in vivo)
not confined to chick and rabbit cells. 1 Heteroploid 1 Diploid
Indeed Swim and Parker 2~ later re- 2 Cancer cells 2 Normal cells
ported similar findings with cultures of (histological criteria) (histological criteria)
human foreskin and human fetal tissues. 3 Indefinite growth 3 Finite growth
With the benefit of hindsight, it is a
260
not likebj that cells could have frequently
survived. If Carrel's laboratory was
aware of such contamination, it was
highly reprehensible, to say the least, of
the workers not to take steps to
eliminate it.
Another possible explanation comes
from an anecdote told by Dr R.
Buchsbaum of a visit he paid to Carrel's
laboratory in 1930. In the course of the
visit, a technician told Buchshaum the
'immortal' cells d/d die regularly, and
that fresh cultures were started anew 1°.
This seems plausible in view of the great
importance Carrel's laboratory attached
to these cells. If the laboratory dogma
was that the cells were immortal, then if
they did die, it was not because of the
intrinsic mortality of cells, but simply
the effects of external causes, i.e. tech-
nical accidents. Indeed, on one
occasion, Carrel wrote: 'If we exclude
accidents, connective tissue cells, like
colonies of infusoria, may proliferate
indefinitely'14. The scientific staff of the
laboratory may not necessarily have
been aware of what was happening.
Even Ebeling may not have known, for
he took two of his technicians to
l.ederle and presumably the Rockefeller
procedures for growing cells were
implemented at Lederle.
It should be noted that rumours of
this kind were circulating in New York
tissue culture circles in the 1930s, and
this is not the first occasion when it has
been suggested that there was some-
t h i n ~ suspect about the 'immortal'
cells~ .
Conclusion
The influence of Carrel's 'immortal'
cells reached beyond their death to
affect the reception of the work by
Hayflick and Moorhead. Their original
paper was rejected by Peyton Rons,
editor of the Journal of Experimental
Medicine with the following comments:
'The largest fact to have come out from
tissue culture in the last fifty years is that
cells inherently capable of multiplying
will do so indefinitely if supplied with
the right milieu/n vitro' and 'The infer-
ence that death of the c e l l s . . , is due to
"senescence at the cellular" level seems
notably rash '26. Indeed Carrel's ~vork is
still cited as evidence for the immortality
of cells (Ref. 27 but see Refs 28, 29).
Whatever the explanation of the long
life of Carrel's chick heart fibroblasts, it
is extremely unlikely that they were
immortal as we would define cell
immortality today. We will never know
the 'truth' of the matter without examin-
ing either the immortal cells or members
1985,ElsevierSciencePublishersB.V.,Amsterdam 0376- 5067/85/$02.110
TIBS - July 1985
of Carrel's laboratory. But, as Strehier 12 Carrel, A. (1913) Z Exp. Med. 17, 14--19
put it, ' . . . the ultimate effects of the 13 Carrel, A. (1913) J. Exp. Med. 18, 287-298
14 Carrel, A. (1914) Z Exp. Med. 20, 1-2
ageing process have made it impossible
15 Ebeling, A . H . (1919) J. Exp. Med. 30,
for Carrel to respond in his own 531-537
defence '3°. Unfortunately, the myth of 16 Ebeling, A . H . (1922) J. Exp. Med. 35,
the 'immortal' cell strain is proving to be 755--759
longer-lived than the cells themselves! 17 The Worm (New York), 12 June 1921
18 Ebeling, A. H. (1942) Sci. Am. 166, 22-24
19 Pearl, R. (1921) Sci. Mon. 12, 321-335
References 20 Haft, R. F. and Swim, H. E. (1956) Proc. Soc.
Exp. Biol. Med. 93, 208-204
1 Hayflick, L. (1978) Curr. Contents 26, 12
2 Hayflick, L. and Moorhead, P. S. (1961) Exp. 21 Swim, H. E. and Parker, R. F. (1957) Am. I.
Cell Res. 25, 58.5--621 Hyg. 66, 235-243
3 Edwards, W. S. and Edwards, P. D, (1974) 22 Hayflick, L. (1965) Exp. Cell Res. 37, 614--636
Alexis Carrel, Visionary Surgeon, Charles C. 23 Hayflick, L. (1984) Mech. Aging Dev. 28,
Thomas, Springfield, IL, USA 177-185
4 Witkowski, J . A . (1979) Med. Hist. 23, 24 Ogura, H., Fujiwara, T. and Namba, M.
279-296 (1984) Gann 75, 410--414
25 Medawar, P. B. and Medawar, J. S. (1977)
5 Cow.roe, J. H. (1978) Am. Rev. Respir. DIS.
118, 391-402 The Life Science, pp. 125-126, Wildwood
6 Harrison, R. G. H. (1908) Anat. Rec. 2, House, London
385-410 26 Hayliick, L. (1984) Adv. Cell Cult. 3, 303-316
7 Burrows, M.T. (1910) J. Am. Med. Rec. 27 Najafi, H. (1983) J. Am. Med. Assoc. 250,
Assoc. 55, 2057-2058 1086-1089
8 Jolly, J. (1910) C. R. Soc. Biol. 69, 470--473 28 Witkowski, J. A. (1984) J. Am. Med. Assoc.
9 Carrel, A. (1912) J. Exp. Med. 15, 516--528 252, 44
29 Hayllick, L. (1984) Z Am. Med. Assoc. 252,
10 Witkowski, J.A. (1980) Med. Hist. 24,
129-142 44--45
11 Ebeling, A . H . (1913) J. Exp. Med. 17, 30 Strehler, B. L. (1977) Time, Cells and Aging,
273-285 p. 41, Academic Press
Composition and properties of
connective tissues
David W. L Huldns and Richard M. Aspden
Connective tissues consist of a complex system of interacting macromolecules. The
mechanical, and hence biological, properties of this system can be related to its
chemical composition using ideas which have been developed to analyse the
behaviour of fibre-reinforced composite materials.
Connective tissues perform a variety of is implicated in common rheumatic
mechanical functions in the body - conditions such as osteoarthrosis and
tendon transmits tension from a con- lower back pain.
tracting muscle, articular cartilage forms At its simplest the extracellular
a resilient coating on the ends of the matrix consists of collagen fibrils sur-
bones in synovial joints, etc. These rounded by a highly hydrated gel
mechanical functions depend on the known as ground substance. This
structure of the extracellular matrix ground substance contains giycosamino-
which forms the bulk of the tissues. The glycans - polyanions which attract water
chemical composition of the matrix may by the Oonnan effect 1. Glycosamino-
change, and so affect the properties of glycans include chondroitin 4-sulphate,
the tissue, during normal physiological chondroitin @sulphate, dermatan sul-
and pathological processes. For example, phate, keratan sulphate and hyaluronic
the composition of uterine cervix acid. The sulphated glycosaminoglycans
changes in pregnancy to produce a re- are covalently bound to a rod-shaped
markable difference in mechanical protein to form a proteoglycan. In some
properties which allows it to dilate forms of cartilage the protein core of at
during labour. Less dramatic changes least some of the proteoglycans binds to
occur in most tissues during aging and hyaluronic acid2-4. Furthermore, the
mechanical failure of connective tissues sulphated glycosaminoglycans can inter-
D. W. L. Hukim and R. M. Aspden are at the
act with the surfaces of the collagen
Department of Medical Biophysics, University of fibrils. The kinds of macromolecular
Manchester, Stopford Building, Manchester M13 interaction which can occur in cartilage3'4
9PT, UK. are summarized in Fig. 1.