Download Ethan Frome - proteomics.dk

Contributions to Early
Cell Biology
Persons and Events
- A Danish Perspective
1. Introduction
2. Cultivation of microbes
3. Cultivation of cells from
multicellular organisms
4. Cultivation of protozoa
5. The films
6. Concluding remarks
7. Notes
8. References
Leif Rasmussen
Institute of Medical Biology
Odense University, Denmark
Private address:
Torpvej 18
5260 Odense S
Denmark
2
1. Introduction
Cell biology integrates knowledge
and viewpoints from biology,
biochemistry, cytology, genetics,
physiology, medicine, microbiology
etc. The primary goal of this subject
is to discuss how the cell is built,
maintains its life and multiplies. One
result of such studies is that one gets
an understanding of the interactions
between cells, either in cultures or in
multicellular bodies.
Cell cultivation is a central part of
cell biology. We grow all types of
cells: bacteria, unicellular plants and
animals and cells from multicellular
organisms. These cultures give us
possibilities to grow economically
and scientifically interesting species
and to test hypotheses on living
cells.
The history of cell cultivation may be
divided in several parts. First it dealt
with very basic problems. Which cells
were easy to grow? Which
techniques will give the best results?
How should one quantitate growth of
cells? Later, more complicated
problems were addressed: we were
led to the concepts of nutritional
requirements, of the cell cycle, of
regulation of gene activities, and of
communication between cells. Little
by little we got insight into
controlling mechanisms of the cells.
That meant that we began to
understand the outlines of very basal
biological and medical problems. Due
to the human curiosity, fantasy and
intuition, people at all times
pondered on possible connections
and explanations, correct as well as
incorrect ones.
Utilisation of cells in culture goes far
back. Firstly, people used microorganisms for production of bread
and alcohol for millennia. In the 19th
century physicians studied bacteria,
e.g. first by staining methods. Later,
they developed techniques to grow
them in pure cultures. Soon after
physicians began growing cells from
multicellular organisms in pure
cultures, and later again
physiologists, virologists and
biochemists took part in this work.
Cultivation of bacteria began in
Germany. Cultivation of cells from
multicellular organisms began in the
USA. In both cases people from
other countries were quickly drawn
into the work. Danes were active in
cell biology from 1920 and onwards.
One day in 1972 Knud Max Møller,
the Biological Institute of the
Carlsberg Foundation, 16 Tagensvej,
Copenhagen, Denmark, found three
films in the attics of the Institute.
One was recorded at the Rockefeller
Institute in Copenhagen shortly after
1928, and two others at the
Biological Institute, one in 1932 and
another in 1936. They had been put
away, were forgotten, but had not
suffered any damage, in spite of the
fact that they were made on
explosive celluloid film. Max Møller
got them transferred to “Safety film”.
They all show how people worked
with problems in cell biology in the
1930’ies.
2. Cultivation of microbes
This part of the story begins in the
middle of the 19th century. Louis
Pasteur (1822-1895) grew microbes
in order to obtain arguments against
theories of “self-creation”. In 1865
he found two which produced
diseases in silkworms, one a
bacterium and the other a protozoon.
He proposed that microbes also could
give rise to diseases in man. There
was a cholera epidemic in Marseilles,
France, the same year. He wanted to
find the guilty bacterium. He and his
collaborator, Claude Bernard (18131878), did not succeed, because they
had their attentions on infective
agents in the air and on the dust
particles in the hospital wards. Had
they looked for the bacteria in the
stools of the patients, they might
have sped up development by 20
years. Many colleagues saw their
negative results as an indication that
Pasteur’s ideas were wrong. There
was one famous exception: John
Lister (1827-1912) from Scotland,
3
known for introducing aseptic
techniques in operations from 1867.
Gerhard Armauer Hansen (18411912) from Norway, was the first
one who noticed a connection
between bacteria and diseases in
man. He found the leprosy bacterium
in his patients and saw that it could
be communicated (note 1). But it
was the German physician Robert
Koch (1843-1910) who best
connected bacteria and diseases
from the middle of the 1870’ies. He
worked with anthrax bacteria on
solid substrates and followed the
bacteria to the spore forms and from
spore forms back to the bacteria. He
also showed that inoculation of these
bacteria into animals produced the
disease. And from sick animals he
could re-isolate the anthrax bacteria.
Thus he had unequivocally
established the relation between
bacteria and disease. This led to a
lasting interest in the cultivation of
bacteria all over the world, and there
was an explosion in techniques and
knowledge in microbiology during the
following years.
3. Cultivation of cells from
multicellular organisms
Many attempts must have been
made to grow cells from whole
organisms in culture after the
success with bacteria. Sometimes
the attempts went well, but most
often the difficulties were so
overwhelming that it was not
possible to reproduce the results,
and therefore they could not be
used. It was Ross Granville Harrison
(1870-1959) from USA who
developed the first techniques which
succeeded and which could be
repeated. They were published in
1907 (ref. 1). He was a comparative
anatomist and worked with
embryological problems. He was
especially interested in the
development of the nerve cells. He
managed to isolate ganglia from
tadpoles and place them on a piece
of coagulated lymph in a hanging
drop in a depression slide under
sterile conditions. He sealed his
preparations and this kept his cells
alive for more than a week. In the
course of this time the nerve cells
made the same type of extensions,
as they would have done in the living
frog (note 2). Thus he had shown
how nerve cells are formed. He was
also the first one to keep cells alive
outside the body, founding the
techniques of cell cultivation.
Ross Harrison came to the Yale
Medical School, New Haven, CT, in
1907, when his results were
published. His student Montrose T.
Burrows improved the technique by
using coagulated blood plasma,
which contains more growth factors
than lymph – we know now. He
introduced this technique to Alexis
Carrel (1873-1944) at the
Rockefeller Institute in New York.
Carrel was a surgeon and got the
Nobel Prize in Medicine or Physiology
in 1912 for his work with sutures of
blood vessels and transplantation of
blood vessels and organs. Burrows
and Carrel collaborated on cultivation
of cells and quickly expanded their
techniques to other cell types than
nerve cells. The Rockefeller Institute
became a centre for this type of
investigations for many years.
In Denmark Albert Fischer (18911956) studied medicine. While he
was studying he published results on
quantification of growth of bacteria
by means of respiration
measurements (note 3). In 1916 he
visited the Haukeland Hospital in
Bergen, Norway. Here he met
Magnus Haaland (1876-1935) who
showed him a strain of mice with a
50 per cent chance of developing
cancer. He also met Olof
Hammersten (1841-1932) and John
Runnström (note 4), both from
Sweden. Especially the latter turned
Fischer’s interest away from bacteria
and towards proper cell biology.
They were all determining the
direction of Fischer’s future life.
Back in Copenhagen Fischer tried in
vain to grow cells in culture. The
difficulties with respect to nutrient
4
medium and sterility were, however,
too big. He graduated in 1919 and
had for some time corresponded with
the well-known pathologist and
microbiologist Simon Flexner (18631946), Director of the Rockefeller
Institute. Fischer wanted to get
permission to go to New York as a
guest in Carrel’s laboratory. There
were no guest programmes available
and Carrel was not interested in
more collaborators. Anyway, Fischer
went to New York and after some
time he got a grant and a working
laboratory. During this stay he learnt
the techniques and grew – as the
first one - epithelial cells in culture.
(The cells were from the iris of a cat.
The advantage with these cells was
that their blue pigment identified
them.) He went back to the Institute
of Pathology in Copenhagen in 1922,
and wrote his thesis: “Tissue Culture.
Studies in Experimental Morphology
and General Physiology of Tissue
Cells in Vitro. A Textbook”. It
became the handbook in cell
cultivation and appeared in two
editions in German in the course of
three years.
His thesis had many consequences:
Otto Warburg (1883-1970) (note 5)
from the Kaiser Wilhelm Institut für
Zellphysiologie (note 6) in Berlin,
Germany, had just seen that cancer
cells had a higher rate of glycolysis
than normal cells. He used sections
of tumours for these studies, but
wanted to be able to subcultivate
these cells. He read Fischer’s thesis
and invited him to join him in Berlin.
Fischer stayed there from 1926 to
1932.
An episode sheds light on the
“common” attitude towards the work
with tissue cells at this time. The
chair in pathology was vacant in
Copenhagen in 1928. Albert Fischer
had been working here for several
years and applied for this position,
but he had to retract his application.
He was told “that it was difficult to
understand that the study of dying
cells (!) could be so interesting in
connection with medicine that you
needed an expert in that field to
chair pathology”.
Two events in Copenhagen in 1929
and 1930 had far-reaching effects on
cell biology:a) Firstly, Professor Valdemar
Henriques (1864-1936) chairman
of the Board of the Carlsberg
Foundation began negotiations
between the Danish State, the
Rockefeller and the Carlsberg
Foundations. They resulted in the
building of the Biological Institute
financed by the Carlsberg
Foundation. The Rockefeller
Foundation covered daily
expenses and Albert Fischer
became its Director. His main
interest at this time was to
improve conditions of cultivation.
The cells were still grown on
coagulated plasma and he
wanted to design a chemically
defined medium for them. This
was indeed a pioneering job. The
last essential amino acid,
threonine, would be found as late
as 1935 by W.C. Rose, and many
vitamins still remained unknown.
b) Secondly, Einar Lundsgaard
(1899-1968), assistant of
Valdemar Henriques, found
around 1929 evidence for the
view that Otto Meyerhof’s (18841951) prevailing ideas on the
mechanism of muscle contraction
were wrong (note 7). The young
Dane described his experiments
in a letter to Meyerhof in
Heidelberg. Here they were
confirmed by Fritz Lipmann
(1899-1986), Meyerhof’s
assistant. Then Lundsgaard was
invited to Heidelberg for
discussions of his results. During
this stay Lundsgaard and
Lipmann became excellent
friends. Afterwards, Lundsgaard
returned to Copenhagen, and
Lipmann went to Berlin where he
worked at Otto Warburg’s Institut
für Zellphysiologie. Here he met
Albert Fischer who invited him to
join his staff at the new Biological
Institute of the Carlsberg
Foundation in Copenhagen.
5
Lipmann accepted – maybe
because of his friendship with
Einar Lundsgaard. This friendship
was extended to include Herman
Kalckar (1908-1991), Einar
Lundsgaard’s assistant.
Fritz Lipmann described in his
autobiography an important
experiment made in Copenhagen. He
used acetone extracts of bacteria to
provide enzymes to catabolise
glucose. He used routinely a
phosphate buffer for this purpose.
One day he had run out of buffer and
he used a bicarbonate buffer instead.
The yield was only 1 per cent of the
expected amount, he therefore he
quickly made a new phosphate
solution and now obtained results
100-fold greater. He concluded that
phosphate from the buffer
(unexpectedly!) enters the reaction,
phosphate has not only buffering
effects. Lipmann was a Jew, and he
was urged by friends to leave Europe
and go to the USA in 1939. Here he
met again with Herman Kalckar and
together they presented the first
account of energy metabolism in
cells. Herman Kalckar had discovered
oxidative phosphorylation in
Copenhagen just before WW2 and
had arrived to the USA for further
studies (ref. 3, for further details).
Fritz Lipmann received (together with
Hans Krebs (1900-1981)) the Nobel
Prize in Physiology or Medicine for
1953 (note 8).
4. Cultivation of Protozoa
Parallel steps to grow single cell
eukaryotes were taken early in the
20th century. Flagellates were the
first to succeed, followed by ciliates
and soil amoebae. It became
possible to combine techniques from
cultivation of bacteria with cultivation
of cells with nutritional requirements
and regulatory mechanisms like
those of mammalian cells. They
could even be grown like bacteria in
test tubes. André Lwoff (1902-1994)
(note 9) from France grew the first
ciliate, Tetrahymena, in pure culture
in 1923 and George W. Kidder
(1902-1996) from USA grew from
1951 the same cell in a chemically
defined nutrient medium (note 10).
Tetrahymena was thus the first
animal cell to be grown in a medium
in which all the components were
known. R. Jack Neff from USA grew
small soil amoebae – Acanthamoeba
sp. – in pure cultures from the
middle of the 1950’ies.
5. The films
The first film from the inauguration
of the Biological Institute of the
Carlsberg Foundation shows the date
“19. October” (but not the year
which was 1932). We are informed
that the Foundation carried the costs
of the building, the Rockefeller
Foundation the daily expenses and
the Danish State granted the ground
on which it was built. The building is
shown from the outside together
with its architect, Christen Borch
(1883-1972). Guests arrive for the
festivities, the chairman of the
Foundation, A.B. Drachmann (18601935), the Danish Prime Minister Th.
Stauning (1873-1941), the American
and German Ambassadors etc. We
see parts of the speeches by the
chairman and the new Director,
Albert Fischer. Then everybody is
shown around: we see rooms with
autoclaves, daily work with the tissue
cells on depression slides covered by
Petri dishes, and Carrel flasks with
coagulated plasma. The personnel
are in black coats and we see Fritz
Lipmann measuring respiration (with
a van Slyke apparatus?). In the end
the camera pans over guests
gathered on the balcony of the
Institute.
The second film from 1936 shows
again the building from the outside
and daily life. Fritz Lipmann is
measuring respiration with a
Warburg apparatus – the
manometers are shaken, stopped
and read. A sequence shows
(unclearly) Colonel Charles
Lindbergh’s (1902-1974) (note 11)
“artificial heart”, a pump circulating
nutrient fluid through an isolated
thyroid gland. (This pump was
developed in the USA in collaboration
6
with Alexis Carrel and it arrived in
Denmark on the occasion of the
Fourth International Congress of
Cytologists in Copenhagen in August
of 1936.) Finally, Albert Fischer is
shown engaged in preparing cells for
recordings with a camera. We see a
rheostat controlling the duration of
the intervals between single
exposures, “time lapse recordings”. A
text informs us that these intervals
can be set to vary between 10
milliseconds and 10 hours.
The third film is from 1928 in the
Rockefeller Institute in Copenhagen.
What the films have in common are
aspects of early cell biology: studies
of cell cultivation and energy
metabolism. We see Einar
Lundsgaard isolating a muscle, fix it
in a stand, and attach it to a turning
chymograph to record its
contractions in time. After addition of
mono-iodo-acetic acid (blocking
glycolysis) the contractions continue
until the muscle goes into a state of
cramp. We are possibly shown the
measuring of the pH in a
homogenate of the muscle - the
reaction was alkaline, which was
incompatible with Meyerhof’s idea
that lactic acid was the compound
responsible for muscle contraction.
(We know now that lactic acid is
rather a “by-product” of the
contraction.) These sequences last
for just 2 minutes.
6. Concluding remarks
The important problem for cell
culturists is, was and will always be
the hazard of infection. At the
Biological Institute in the 1930’ies
the walls of the operation rooms
were cleaned with water from a
garden hose to make them sterile.
Transitions between ceilings and
walls and between walls and floors
were rounded to let the water run
away easily into an outlet in the
corner of the room. Windows were
provided with a set of double panes
in metal frames, and installations for
compressed air, water and gas were
sunk deep into the walls and covered
by watertight doors. No wonder it
was difficult to keep cultures sterile!
The personnel wore black lab coats.
These coats and the fact that the
tiles in Copenhagen were of the
same type that was used by Carrel in
New York testify that you did not
deviate at any point from the way
that the Master from New York had
shown.
Two important events took place in
cell biology after WW2. Firstly, we
got access to antibiotics, penicillin
and streptomycin. The danger of
infections could now be drastically
reduced and therefore it was much
easier to work with tissue cultures.
Secondly, John Franklin Enders
(1897-) and collaborators
(re-)discovered around 1950 that
virus particles can multiply in tissue
cultures. They received the Nobel
Prize for their discovery and it meant
that researchers no longer needed to
infect animals in order to propagate
the virus. These two features made
cell cultivation very interesting for
virologists and knowledge of the
techniques spread among them like
an explosion. Today, we see new
applications of the techniques in
connection with biotechnology, and
we see a new explosion in the
interest for the procedures.
It is characteristic for active scientific
institutions that their members often
influence each other in such a way
that the positive consequences can
be seen over long periods of time,
maybe over whole life spans. So,
they form unforeseen networks of
great importance for the future. A
good example of this is the
Rockefeller Institute in Copenhagen
with many connections to the
Rockefeller University in New York.
Another was the Biological Institute
of the Carlsberg Foundation in
Copenhagen. These two placed
Danish Science centrally in the
development which led to increased
biological insight which later led to
molecular biology and biotechnology.
The Biological Institute played an
important role in creating a research
unit with cell cultivation as the
7
central theme, and many
fundamental ideas originated here. It
is important to be reminded of the
involved persons. In our daily routine
we sometimes consider their
contributions as something which has
always been known.
The decision of the Carlsberg
Foundation to erect an Institute for
cell cultivation was very far-sighted.
These techniques were not
introduced at the University of
Copenhagen until 25 years later.
7. Notes
1. He infected his housemaid with it
and was later sentenced to jail for
unethical practice.
2. Harrison reported that the finest
extensions of the nerve cells showed
so fast amoeboid movements that he
could not trace them with his pencil.
3. These reports carried the
affiliation: “Aus dem PrivatLaboratorium für Chemie” (“From the
Private Laboratory for Chemistry” or
“From the Albert Fischer Laboratory
for Biochemistry and Bacteriology”.
In both cases the fine words covered
the fact that he had done the
experiments in his mother’s kitchen.
4. The Swede John Runnström
worked with the development of the
sea urchin egg. He was a co-founder
of the journal “Experimental Cell
Research” (with Tryggve Gustavson
and Torbjörn Caspersson).
5. He had around 1905 in a report
wondered that living systems
maintain high cellular concentrations
of various ions and tried –
prematurely – to connect this to
production of excess energy in the
organisms.
6. The Kaiser Wilhelm Institutes are
called the Max Planck Institutes
today.
7. Meyerhof found proportionality
between consumption of oxygen and
the metabolism of lactic acid in the
muscle and that led him to think
(erroneously!) that lactic acid was
the controlling factor in contraction.
(He shared the Nobel Prize for 1922
(given out in 1923) with A.R. Hill for
studies on energy metabolism in
cells.) Einar Lundsgaard realised that
Meyerhof’s ideas could not be
maintained in the light of his own
results with mono-iodo-acetic acid.
At this time – around 1930 - both
ATP and creatine phosphate were
known, but it was not until 1934 that
Karl Lohmann (1898-1978) in
Meyerhof’s laboratory found the
enzyme, which phosphorylates ADP.
From this moment the compounds
could be put together the right order.
8. Fritz Lipmann shared in 1953 the
Nobel Prize with Hans Krebs.
Lipmann got it for his discovery of
CoA and its significance for the
intermediary metabolism of glucose.
(Krebs got his for his discovery of
the citric acid cycle.) Both had begun
their careers at the Kaiser Wilhelm
Institut für Biologie in Berlin.
9. André Lwoff (1902-1994) from
France received in 1965 (together
with Jacques Monod (1910-1976)
and Francois Jacob (1920-)) the
Nobel Prize. Lwoff got his share for
his work with phages, distinguishing
between their lytic and lysogenic
pathways.
Monod and Jacob worked
successfully with transcription control
in bacteria (the ‘operon concept’).
They all worked – together - at the
Institute Pasteur in Paris.
10. The requirement was close to
that of mammalian cells.
Tetrahymena, however, does not
need cobalamine (B12), but it does
need thioctic acid (also known as
‘lipoic acid’) and that was the last
missing vitamin to be included in the
synthetic medium. It should be
pointed out that Tetrahymena
requires neither lipids, nor proteins.
8
11. Charles Lindbergh was the first
person to fly across the Atlantic
Ocean alone (May 1927).
8. References
1. Harrison, R.G.: Proc. Soc. Exp.
Biol. Med. 4, 140-144 (1907).
2. Rasmussen, L. & Agnisola, C.:
Early bioenergetics and cell
cultivation: the history behind
two films. Forme 4(2), 23-25
(1993). (‘Forme’ is a publication
from the Italian National
Research Council.)
3. Kalckar, H.M.: Biological
Phosphorylations. Development
of Concepts. Prentice-Hall, Inc.
Englewood Cliffs, New Jersey,
1969.