Contributions to Early Cell Biology:
Persons and Events – A Danish Perspective
Leif Rasmussen, Professor emer.
Institute of Medical Biology, University of Southern Denmark, Denmark
Jesper Dybvad Olesen, C.Sc., Mentors' Classroom, Denmark
February 2015
© Copyright Mentorsclassroom.com
1. Introduction
2. Cultivation of microbes
3. Cultivation of cells from multi-cellular organisms
4. Cultivation of protozoa
5. The films
6. Concluding remarks
7. References
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 multi-cellular bodies.
Cell cultivation is a central part of cell biology. We may grow many types of cells: bacteria, unicellular plants and animals, and cells from multi-cellular 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 measure 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 micro-organisms 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 multi-cellular 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 Dr Knud Max Møller (1922-2004), 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. To view the films click on one of the images below.
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 producing 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 (1813-1878), 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, known for introducing aseptic techniques in hospital operations from 1867.
The Norwegian Gerhard Armauer Hansen (1841-1912) was the first one who noticed a connection between bacteria and diseases in man (1873). He found the leprosy bacterium in his patients and saw that it could be communicated (Box 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.
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Box 1 Gerhard Armauer Hansen infected his housemaid with the leprosy bacterium and was later sentenced to jail for unethical practice. A hundred years ago, Bergen had the largest concentration of leprosy patients in Europe. |
3. Cultivation of cells from multi-cellular 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 successful and repeatable techniques. 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 (Box 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.
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Box 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. Harrison was the first who showed that chicken embryo nerve cells could be grown outside the animal (1914). |
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 received 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 (1891-1956) wanted to become a physician. While still studying he published results on quantification of growth of bacteria by means of respiration measurements (Box 3).
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Box 3 These reports carried the affiliation: "Aus dem Privat-Laboratorium 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. |
In 1916 Fischer 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 (Box 4), both from Sweden. Especially the latter turned Fischer's interest away from bacteria and towards proper cell biology. They were all part of pointing Fischer's future life in the right direction.
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Box 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). |
Back in Copenhagen Fischer tried in vain to grow cells in culture. The difficulties with respect to nutrient 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 (1863-1946), Director of the Rockefeller Institute in New York. Fischer wanted to get permission to go as a guest to 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.
Fischer's thesis had many consequences: Otto Warburg (1883-1970) from the Kaiser Wilhelm Institut für Zellphysiologie (Box 5) 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 sub-cultivate these cells. He read Fischer's thesis and invited him to join him in Berlin. Fischer stayed there from 1926 to 1932.
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Box 5 Today the Kaiser Wilhelm Institutes are called the Max Planck Institutes. There are 83 institutes and research facilities (as of January 1, 2014), 5 institutes and one research facility are situated abroad. On January 1, 2014 the Max Planck Society employed a total of 16,988 staff (previous year: 16,918), of whom 5,516 were scientists (previous year: 5,470). This represents 32.5 % of the total number of employees and an increase of 0.5%. Ref.: The Max Planck Society at mpg.de. |
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 for the appointing committee to see 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:
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 (1887-1985), and many vitamins still remained unknown.
Secondly, Einar Lundsgaard (1899-1968), assistant of Valdemar Henriques, found around 1929 evidence for the view that Otto Meyerhof's (1884-1951) prevailing ideas on the mechanism of muscle contraction were wrong (Box 6). 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. Lipmann accepted – maybe because of his friendship with Einar Lundsgaard. This friendship was extended to include Herman Kalckar (1908-1991), Einar Lundsgaard's assistant.
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Box 6 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 muscle contraction. (He shared the Nobel Prize for 1922 (given out in 1923) with A.V. Hill (1886-1977) for studies on energy metabolism in muscles.) Einar Lundsgaard realised that Meyerhof's ideas could not be maintained in the light of his own (ELs) 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 reactions could be put together the right order.
Meyerhof reported around 1905 that cells maintain high cellular concentration gradients of various ions and tried – correctly, but prematurely – to connect this with the production of energy in excess of that required by mere muscle work in the organisms.
Fritz Lipmann described in his autobiography an important experiment made in Copenhagen (ref. 2). He used acetone extracts of bacteria to provide enzymes to catabolise glucose. Routinely he used 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 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 (Box 7).
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Box 7 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. |
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) (Box 8) 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 (Box 9). Tetrahymena was thus the first animal model 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.
Monod and Jacob worked successfully with transcription control in bacteria (the 'operon concept'). They all worked – together – at the Institute Pasteur in Paris.
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Box 8 André Lwoff (1902-1994) from France received in 1965 (together with Jacques Monod (1910-1976) and Francois Jacob (1920-2013) the Nobel Prize in Medicine or Physiology. André Lwoff got his share for his work with phages, distinguishing between their lytic and lysogenic pathways. In the 1930'ies Lwoff established the view of the universal nature of biochemical compounds in metabolism. If an organism does not require a vitamin, the organism synthesises the vitamin itself. |
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Box 9 Tetrahymena is a single-cell ciliate and it was found that the requirement of nutrients 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 by G. W. Kidder in the synthetic medium around 1950. It should be pointed out that Tetrahymena requires neither lipids, nor proteins. |
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).
View the films at YouTube in a new window by clicking an image below.
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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 (1860-1935), 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 the institute: 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) (Box 10) "artificial heart", a pump circulating nutrient fluid through an isolated thyroid gland. (This pump was developed in the USA in collaboration 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.
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Box 10 Charles Lindbergh was the first person to fly nonstop across the Atlantic Ocean alone on May 20–21, 1927, from the Roosevelt Field in Garden City on New York's Long Island to Le Bourget Field in Paris, France. |
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 (1897-1985) 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 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 (year 1932) was very far-sighted. These techniques were not introduced at the University of Copenhagen until 25 years later.
1. Harrison, R.G.: 1907, Proc. Soc. Exp. Biol. Med. 4, 140-144.
2. Lipmann, F.: Wanderings of a Biochemist. Wiley & Sons, Inc. New York, London, Sidney, Toronto, 1971.
3. Kalckar, H.M.: Biological Phosphorylations. Development of Concepts. Prentice-Hall, Inc. Englewood Cliffs, New Jersey, 1969.
Acknowledgements
We thank Morten Rasmussen, Ph. D., for encouragement, technical help, and advice.