Many researchers in the biosciences work with cell cultures, but not all of them seem to understand them at a deeper level. The biologist Silke Brüderlein from the Institute of Pathology in Ulm is a specialist in cell culture and is well aware of the problems that might arise when working with cell cultures.
Based on the Ulm University Campus, Brüderlein establishes cell lines from tissue samples of rare tumours for her research colleagues. The cell culture specialist was recently awarded a 10,000-dollar research prize by the American Chordoma Foundation, in recognition of the significant contribution her development of the new chordoma cell line makes to the field of chordoma research.Chordoma is a rare bone tumour (prevalence of 0.5: 1,000,000) that arises from bone in the skull base and along the spine. It accounts for 0.2 per cent of all central nervous system tumours and for two to four per cent of all primary neoplasms of the bone. Once the disease has been diagnosed, the median survival time of chordoma sufferers is seven years.
The impression one gets is that establishing a cell line is really rather difficult. Major problems can arise related to cross-contaminations and erroneous identification of clones. However, researchers shy away from talking and writing about drawbacks, in order to avoid potentially negative headlines and because they feel under pressure to come up with successful research outcomes. Specialists believe that no less than one third of all tumour cell lines originate from tissues and even species other than the one indicated.Findings that have been published in renowned scientific journals and attracted a lot of attention are frequently impossible to repeat due to the fact that the cell lines used are contaminated or reagents have been improperly handled. A recent example, one of many, involves researchers who claimed to have found a link between a mouse retrovirus (XMRV) in people who have chronic fatigue syndrome (Science, 326, 585), and later had to refute their findings following evidence that the virus accidentally originated during mouse lab experiments.
Cancer researchers use immortalized tumour cell lines to investigate the biology of tumours, from which they glean potentially important preclinical knowledge about potential therapeutic targets. This is why it is extremely important to work with the correct cell lines. Although this appears to be stating the obvious, current practice shows that the use of the correct cell lines is not that easy. Silke Brüderlein is very well aware that it is quite easy to think that one is working with the correct cell line, when in fact one is not.In cooperation with colleagues from the USA, Silke Brüderlein investigated all five of the chordoma cell lines that are currently available. They used an array of technologies to characterise the molecular and genetic features of these cell lines and came up with rather depressing results: three of the five cell lines turned out not to be chordoma cell lines at all, one of them lacked the typical chordoma biomarkers despite being derived from chordoma cells, and one cell line was derived from a mouse rather than from human tissue. The researchers found that only their own cell lines were suitable as a valid tool for the international biomedical research community.
So the question arises as to what makes work with cell cultures so difficult. Why does it seem to be so problematic and so tricky to establish a sought-after cell line? Silke Brüderlein struggles to find answers to this question as, for her, establishing a cell line is a relatively easy task. “The trick is quite simple: you have to closely monitor the cells and recognise when something goes wrong and when it is necessary to interfere. But in order to do this, researchers need to know what the cells look like when they are on top form.”Brüderlein reports that many researchers find it difficult to differentiate normal cells from tumour cells. She firmly believes that this lack of expertise leads to researchers adding excess medium when they leave for the weekend. The cells then consume too much nutrients, stop producing growth factors and stop growing.
For Silke Brüderlein, it is easy to list the typical errors that might occur during the establishment of a cell culture: in Europe, many researchers still believe that foetal calf serum (FCS), which is commonly added to the culture medium, needs to be inactivated at 56°C. However, this habit leads to the rapid death of cells, including cells that are as robust and rapidly growing as liposarcoma cells. High temperatures lead to the destruction of many cellular growth factors. The international research community originally worked with the serum of adult cattle, whose immune system (complement system) had to be switched off. Embryos do not yet have a complement system.
Silke Brüderlein believes that the real deadly sin is when researchers fail to add glutamine to the medium, or do not add enough. Tumour cells require up to four times more glutamine than healthy/normal cells. The lack of glutamine kills the tumour cells within 24 hours. She advises researchers who work on the establishment of new cell lines to avoid commercially available stabilised glutamine. Although commercially available glutamine has a much longer half-life than normal, the majority of cells do not tolerate it, said Brüderlein shaking her head.
Silke Brüderlein’s chordoma cells do not need a special nutrient cocktail to grow. She points out that it takes a long time to develop a special cocktail when a “fresh tumour is delivered”. The researcher from Ulm developed “her” culture medium many years ago while she was working at the DKFZ in Heidelberg, and she tested it for use with colon carcinoma and neuroblastoma cells. The culture medium is made up of Iscove's RPMI and FCS (4:1) that contains penicillin, streptomycine and glutamine.
Brüderlein established her first (the only one in the world at the time) valid chordoma cell line in 2003. This was more or less a matter of chance, as Brüderlein had come across and was able to help a young trainee doctor who was looking for a relatively unknown field of research. Back in 2003, very little was known about chordoma.
Brüderlein’s second valid chordoma cell line – a third one is currently being developed – provided extra income for the Ulm-based institute which does not normally have any extra funding available. The new cell line will provide the international chordoma research community with new opportunities for studying chordoma. Since 2008, the Chordoma Foundation has distributed the first cell line to over 30 different laboratories and in the past month the new cell line has been distributed to 12 laboratories.Silke Brüderlein is not interested in common tumours because they are catered for by cell collections such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms and Cell Cultures (DSMZ) and the European Collection of Cell Cultures (ECACC). Brüderlein receives the biomaterial she uses from hospitals in Ulm as well as other hospitals around the world, including a hospital in Stanmore in England, with which contact was recently established.
Silke Brüderlein recently attended a conference in the USA to give a lecture about the development of the chordoma cell line. She presented the recipe for the culture medium she uses and the steps that are necessary for developing such a cell line. Although it sounds quite simple, the establishment of a valid cell line nevertheless requires in-depth experience, patience and many successful individual steps.All beginnings are difficult, even more so when it comes to the development of cell lines. Biopsied tissue material always behaves differently. For the development of her second cell line, Silke Brüderlein cut the tissue into a Petri dish using a sterile pair of scissors, put the tissue sections into a tube containing collagen R solution and placed the tube into an incubator where the tumour cells were able to detach from the tissue chunks.
The handling of biopsied tissue material is an art in itself. Brüderlein has already developed cell lines from 100 cells. In principle, researchers who attempt to establish cell lines want to have as many cells as possible available as this helps them to find which one is able to optimally adapt to the plastics material. “The establishment of a cell line usually involves the selection of the fittest cell, i.e. the selection of the most malignant tumour cell.”
Once the biopsied material has softened, it only takes a few hours for the sample to disintegrate and the enzyme collagenase to separate the cells. Harder tissue has to be left in the enzyme solution for longer, usually overnight, without applying mechanical pressure. In the case of chordoma cells, Brüderlein did not use trypsin, an enzyme that is commonly used to detach adherent cells, as it tends to destroy these types of cells.The turbid culture broth is washed and centrifuged several times to remove cell debris; the pellet, which in the optimal case contains the living (tumour) cell mass, is resuspended and transferred to a new culture flask. This primary cell culture is then used to establish a cell line.
Depending on the size of the tumour, numerous flasks are prepared to collect the biopsied material. It is recommended that the supernatant be delivered to the laboratory on the day after collection, centrifuged and a new flask prepared, said Silke Brüderlein describing the procedure that she meticulously documents in a laboratory logbook. This seemingly complex procedure is necessary because the supernatant might contain dead cell material as well as tumour cells that have not yet adhered to the flasks. This might happen if the tumour cells are located between two normal cells. “You never know which flask is the best one, even after four weeks.”Therefore, Brüderlein has a number of different strategies she relies on. She never discards anything because, as she tells us, this increases the chances of obtaining the sought-after cells in a form that is as pure as possible and of being able to set up a primary culture. Constant subcultivation enables the researchers to change the ratio between tumour cells and other cells, thereby giving the tumour cells an advantage. “As far as chordoma cells are concerned, we succeeded after the third or fourth passage, when we had only tumour cells in the culture.” When do the cells need to be supplied with fresh medium? Brüderlein: “I believe that is a matter of experience, you just have to learn to recognise when it is necessary to add more medium.”When is the primary culture ready for developing subcultures? Chordoma cells are “quite unproblematic tumour cells” as they tend to grow quite well, said Brüderlein. However, as in the human organism, they tend to grow rather slowly and they take more than five days to divide. Sometimes it can take three months or more before the first subculture can be established. Usually, it takes two months before the first subculture can be established and another month before the second can be established. In other words, Brüderlein took virtually a whole year to establish her second chordoma cell line. As far as the culture of colon cancer cells are concerned, it takes much longer. About 70 per cent of the colon cancer cells die every day.
Cells need to adapt to life in artificial media. “Once we have successfully handled the first passages, i.e. managed to keep the cells growing and living under cultured conditions, we have won the battle, in particular as far as tumour cells, and to a greater extent, chordoma cells, are concerned,” said Brüderlein explaining that a few passages are required for the cells to undergo the necessary epigenetic modifications (methylation, acetylation, histone code) that enable them to grow faster.Once the first subcultures have been established, a single subculture is frozen in liquid nitrogen in order to prevent an infection from occurring. Brüderlein’s colleagues at the Royal National Orthopaedic Hospital in Stanmore then take over where she has finished; they characterise the cell line before sending it to the Chordoma Foundation.
The researchers can characterise a cell line when several passages lead to no further phenotypical changes. This means, for example, that the cells do not increase in size or display any signs of senescence. On the chromosomal level, this means that the tumour cells need to have more than 46 chromosomes. On average, chordoma cells need to have 56 chromosomes characterised by numerous translocations. In addition, comparative genomic hybridisation (a molecular-cytogenetic method for analysing copy number changes in the DNA content) needs to generate more or less constant results. In the majority of cases, though not all, the chordoma cells need to express the brachyury gene (transcription factor), which has been identified as a definitive diagnostic marker of chordoma.
Brüderlein is currently growing her third chordoma cell line in a packed incubator. She is extremely enthusiastic when she talks about different cell types such her U-CDS cell lines, and fluff-shaped dendritic cells that look like spiders under the microscope. And we are gradually realising what the biologist meant when she said she had green fingers. As she said with a laugh: “I am good at growing plants too. In fact, I can grow anything. If something doesn’t grow with my help, it will not grow anywhere.” The biologist entered the realm of cell culture by pure chance. During her studies, she worked as a student assistant in the Department of Human Genetics at the University Hospital in Erlangen in order to earn some extra money. And the cultivation of cells has remained one of her favourite activities ever since. And all this work has given her a great deal of comprehensive experience in this field, although she simply says “I have learned quite a bit over the last years”. It’s clear that her experience has become a true passion from which her oncology research colleagues now stand to benefit.
Silke Brüderlein, Joshua B. Sommer, Michael Kelley et al.: Molecular Characterization of Putative Chordoma Cell Lines, Hindawi Publishing Corporation, Sarcoma, Volume 2010, Article ID 630129, 14 pages, doi:10.1155/2010/630129.
List of false leukaemia and lymphoma cell lines:https://www.gesundheitsindustrie-bw.dewww.dsmz.de/human_and_animal_cell_lines/main.php?contentleft_id=97