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Cell culture technology: it all started with frog nerve fibres

The history of cell culture technology is closely connected with cell biology and the discoveries made in the field of cell biology. Ever since the study of science became a professional activity, researchers have been trying to keep cells and tissues alive outside organisms for purposes of investigation.

In the early days, cell culture was carried out with embryonic frog nerve fibres. The American zoologist Ross Granville Harrison from Yale University is credited as being the first scientist to work successfully with artificial tissue culture. In 1907, he was the first to successfully grow animal tissue outside the body. In 1885, Wilhelm Roux successfully kept embryonic chicken cells in a saline solution for several days, thereby establishing the principle of tissue culture.

Penicillin counteracts bacterial contaminations

Alexis Carrel, surgeon and Nobel Laureate (1912), is regarded as the father of tissue culture to whom the scientific community owes the dogma that cells kept in culture are able to divide indefinitely. Leonard Hayflick and Paul Moorhead overthrew this dogma in 1961 after discovering that normal cells did have a finite replication capacity after all.

With the discovery in 1928 of penicillin, an antibiotic exuded by the fungus “Penicillium notatum”, Alexander Fleming laid the foundations for effective cell culture methods that had previously been hampered by bacterial contaminations. In the late 1940s, Wilton Earle and his colleagues initiated the use of protein-free chemically defined cell culture media, and so an “art” became a “science” and such media continue to be the standard media for the cultivation of primary cultures and established cell lines.

Broad beans and immortal HeLa cells

Nature creates the most beautiful shapes: dendritic cells à la Silker Brüderlein, a cytogenetist at Ulm University © S. Brüderlein

In 1951, Alma Howard and Stephen Pelc discovered an important cell cycle phenomenon in broad beans (Vicia alba) - they found that the cell goes through many discrete phases before and after cell division. In 1952, the first human cell line was derived from cervical cancer cells taken from Henrietta Lacks, hence the name HeLa cell line. HeLa was the first “immortal” cell line that nowadays is propagated in many laboratories around the world. It is one of the most commonly used cell lines used in scientific investigations. HeLa cells are also known to contaminate other cell cultures in the same laboratory. In 1962, Hayflick developed the first diploid cell strain WI-38 from the lung tissue of a three-month old female embryo. These cells are still used today for the manufacture of vaccines.

Over the following decades, cell type-specific media were discovered and developed. With the development of the HAT (hypoxanthine, aminopterin, thymidine) selection medium for mammalian cell culture, John W. Littlefield succeeded in culturing and isolating somatic cell hybrids in 1964, and Richard Ham introduced a serum-free culture medium in 1965.

In 1974, MacFarlane Burnet coined the term “Hayflick limit”, which refers to the number of times a normal cell population divides before it stops; cancer cells, on the other hand, can replicate infinitely. In 1975, George F. Köhler and César Milstein were the first to establish the continuous culture of fused cell hybrids (lymphocytes fused with cancer cells). These fused cell hybrids became the first hybridoma cell line to be used for the production of antibodies. In the same year, the biological clock of cells, which Hayflick termed replicometer (a cell division counting mechanism), was localised in the cell nucleus. Elizabeth Blackburn discovered in Paramecium that the telomeres consisted of simple repeated sequences, and ten years later, Elizabeth Blackburn and her doctoral student Carol Greider co-discovered telomerase, a reverse transcriptase that maintains the length of chromosomes and is critical for the survival of all living cells.

In 1981, Hayflick managed to turn a normal human cell population into an immortal cell line using a chemical carcinogen and irradiation. In 1998, Woodring Wright and the US company Geron showed that the chromosome ends, i.e. the telomeres, are what Hayflick previously referred to as replicometers. They demonstrated that telomeres shorten as the cells divide and that cells die when the telomeres become short enough. In 2000, Jerry Shay and Woodwring Wright showed in a Scid mouse (Scid = severe combined immunodeficiency) how hTERT (human telomerase reverse transcriptase)-immortalised cells can be used to develop tissue culture methods.

Alternative models, researcher tools, production factories and test medium

Today, cell culture technology has become one of the most common methods for replacing animal experiments. It has become an indispensible tool in cell- and biotechnological research. The production of induced pluripotent stem cells, following the worldwide hype surrounding embryonic stem cells, has become one of the fastest growing areas in biomedical research. In 2010, an Austrian stem cell researcher successfully converted mouse skin cells into nerve cells. Some time later, this also became possible with human cell cultures.

The cell cycle – key in cell culture technology

Immunohistochemical image of keratin and desmosomes in the human tumour cell line ACH1P. © DSMZ

Precise knowledge about the cell cycle is key in culture research, production and testing. Cell types such as nerve and liver cells and lymphocytes stop growing when they have reached maturity. This resting phase can take weeks to months, but lymphocytes can come out of the resting phase upon the addition of plant lectin.

The cell cycle is regulated by many internal and external (in particular physiological) parameters. Cells stop growing when their neighbour cells come too close (contact inhibition) or when damaged DNA sends specific signals out of or into the cell. These processes depend on the sufficient supply of nutrients; starving cells stop growing, go on strike and the cell cycle comes to a halt. Internal factors can also affect the cell cycle: cells only divide when they have reached a certain size or when the DNA has been duplicated. These factors are rather like control points; such control points exist for DNA damage or for the formation of the spindle during the M-phase of the metaphase.

The molecular control mechanisms of the cell cycle are a complex interplay of special cell cycle proteins such as cyclins, cyclin-dependent kinases, kinases and phosphatases. Two classes of inhibitors (CIP = CDK inhibitory proteins; Ink4 = inhibitor of kinase 4) are negative regulators of the cell cycle. In addition, the cell cycle is also controlled by tumour suppressor genes and proto-oncogenes.

Programmed, genetically controlled cell death (apoptosis) and the process of necrosis (induced by external influences such as injured tissue) are two forms of cell death to which cell biologists attach the same physiological importance as to reproduction.

As researchers often work with cancer cell cultures, they need to know the differences between normal cells and abnormal cells, i.e. healthy cells. Degenerated cells are characterised by increased proliferation, they have a limited apoptosis capability, they have either no regulatory proteins or inactivated ones, they are genomically unstable, immortal, do not respond to contact inhibition or to growth factors.

In contrast to tumour or transformed cells, experiments need to be carried out to turn primary cells into immortal ones. This can be done through the addition of mutagenic agents, irradiation and the transfer of foreign DNA. Foreign DNA can be introduced into host cells either through transfection (methods used: calcium phosphate precipitation, electroporation and lipid-mediated transfection) or transformation (frequently through viral infection). 

Cell cultures, cell lines and unwanted guests

The “weed” of many cell cultures. This electron microscope image shows a HeLa cell line infected with spaghetti-shaped mycoplasma. © DSMZ

Cell culture collections such as ATTC (American Type Culture Collection), ECACC (European Collection of Cell Cultures), DSMZ (German Collection of Microorganisms and Cell Cultures) or JCRB (Japanese Collection of Research Bioresources) provide cell lines or cell clones of many species for almost any type of research issue. Although the scientific community is gradually working to improve the situation, many cell lines still suffer from contaminations (in the double-figure percentage range) or originate from other tissue or even another species. This is also confirmed by Hans Drexler, Head of the Department of Human and Animal Cell Lines at the DSMZ. His colleague Willi Dirks uses DNA fingerprinting to identify cell lines that are sent to the DSMZ and, in corporation with JCRB and ATTC, he has developed an online identity check that compares human cell lines with those stored in a huge database.

Mortal (finite) cell cultures can be differentiated from immortal (permanent, continuous) cell cultures. Human finite cell cultures are usually derived from cells isolated from body fluids (pleural secretions, amniotic fluid). Cells that only need to be cultured for a limited period of time can also be derived from tissue and organs of humans, animals and plants.

Immortality can be achieved in the laboratory

Continuous cell lines, i.e. cell lines with the capacity to perpetually re-divide, have either been derived from tumour cells, transformed or stably transfected (i.e. genetically modified) cells or have been made immortal with the enzyme telomerase which allows the cells to divide virtually forever when kept in suitable media. Many continuous cultures based on cancer cells have retained only a few of their original characteristics: they grow on soft agar and have a larger number of chromosomes.

Researchers who plan to work with a normal, though continuous, cell line, can use cell lines that have been made immortal as a result of having been transfected with telomerase. These h-TERT (human telomerase reverse transcriptase) cell lines enable long-term biochemical and physiological investigations of cell growth to be carried out.

Continuous cell lines are characterised by a “transformed phenotype”, which means that they have a different morphology from primary cells; cells that normally adhere to each other or to the substrate can lose this property as well as the ability to arrest cell growth when they come into contact with each other (contact inhibition) or can grow without being in contact with substrate. Cells in continuous cultures are less dependent on growth factors, they have reduced demands on the serum used, and typically display chromosome aberrations or aneuploidy or both. Such cells do not age and never die as they can divide infinitely and have typically lost the ability to undergo apoptosis. Transformed cells are not necessarily malignant. Just like cancer cells, these cells have the advantage of being constantly available. However, they have the disadvantage of having moved on from their in vivo origin.

Primary cells have undergone the fewest changes and are therefore more representative of the main functional component of the tissue from which they were derived than continuous cell lines. As they are a more representative model of the in vivo state, they are particularly suited to finding answers relating to cell metabolism and cell morphology. They are also very suitable for volunteer and patient studies. Differentiation processes of immune cells can only be investigated in primary cultures. Primary cells are also used for gene expression analysis/cell-based screening as these cells more closely mimic the physiological state of cells in vivo.

Application of cell cultures

Biberach-based Boehringer Ingelheim produces biopharmaceuticals on the basis of cell cultures. Here, the inoculation of cell cultures is shown. This is the first step in industrial-scale cell culture. © Boehringer Ingelheim

Cell cultures can be used to investigate many fundamental processes without needing to sacrifice animals. However, animals are required for the production of specific substances that form part of the culture media (sera) and for the provision of organs. Cell culture systems have proven to be an excellent alternative to animal experiments. Cell cultures have the decisive advantage that they can be meticulously controlled and standardised (temperature, nutrient media).

Cell cultures are not only used for research purposes. Genetically modified cells can also be kept in bioreactors to produce biopharmaceuticals for the treatment of numerous diseases. The production of biopharmaceuticals with cell cultures has been standard for around 20 years now. CHO (Chinese hamster ovary) cells and the baculovirus-insect system are the most common systems used for the production of recombinant proteins.

The increasing integration of cell biology, molecular biology, bioprocess engineering and functional genome analysis and bioinformatics might help cell culture technology to enter the realm of exemplary and theoretical dimensions (systems biology).

Although mammalian cells play the greatest role of all cells in pharmaceutical research, plant cells, bacteria, fungi and yeasts also play a major role in pharmaceutical production (cultivation in bioreactors).

Cell cultures can be used to answer, or at least clarify and investigate, many questions, for example questions relating to intracellular parameters such as the synthesis of DNA in the nucleus, the regulation of proteins or the cellular transport and signalling pathways. On the cellular level, viral infection mechanisms can be elucidated and differentiation processes be induced. Cell cultures can be used to assess the toxic potential of implants, pharmaceutical drug candidates, of substances those that are harmful to the environment and other chemical substances (the EU project “DETECTIVE” - Detection of endpoints and biomarkers of repeated dose toxicity using in vitro systems,  is one example).

Walter Pytlik - July 2011
© BIOPRO Baden-Württemberg

Selcted publications:

Sabine Schmitz, Zellkultur (Experimentator series), Heidelberg  2009.
Olaf Fritsche, Biologie für Einsteiger, Heidelberg 2010.
Reinhard Renneberg, Biotechnologie für Einsteiger, Heidelberg 2006
DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig (www.dsmz.de)
Website address: https://www.gesundheitsindustrie-bw.de/en/article/dossier/cell-culture-technology-it-all-started-with-frog-nerve-fibres