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The Horrid History of Cell Culture

The Horrid History of Cell Culture and its Future

Cell Culture in the Present Day

Cell culture refers to the growth of eukaryotic cells: most commonly mammalian (human and animal) and insect. Just to be clear: this is not the culture of an entire animal in a Petri dish but the culture of specific cells that were once isolated from a biopsy of an insect, cow or human etc.

Cell culture is a widespread tool used in the fields of oncology (the study of cancer), virology, immunology, microbiology, and pharmacology. Arguably, oncology has benefited most as cancer cells, by their very nature of uncontrolled growth, are amenable to culture in vitro. Virologists too have benefited from the ability to propagate viruses in cell culture. Viruses are not capable of self-replication and must infect a host to hijack its molecular machinery to produce progeny. If there were no cells to infect in vitro (in a glass Petri dish in the olden days or nowadays a disposable plastic version), there would be no viable viruses to study. Interestingly, due to certain high profile problems surrounding cell culture over the last few decades some pharmacologists (scientists who study the action of a drug on the body and the action of the body onn the drug) have tended to avoid cell culture in favour of biochemical assays followed by in vivo testing. The mantra being “you can’t trust those whacky cell lines”. This may certainly have been a valid concern for the discipline (or lack thereof in times gone past) but these days it’s never been easier to conduct high quality work in cell culture.

Origins

Cell culture’s origins go back quite a long way: 1882. Sidney Ringer FRS – a clinician, physiologist and pharmacologist. His many achievements are too numerous to list here but he is chiefly remembered for his invention of Ringer’s Solution – a variation on normal saline solution. Normal saline is an isotonic salt solution used in hospitals in intravenous drips. Ringer's Solution includes potassium chloride, calcium chloride and sodium bicarbonate as a buffer to maintain a physiological pH. This bicarb buffer system is used in most of the classical cell culture media: Eagle's Minimal Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM), Ham’s F12 (often mixed 50:50 with DMEM), Roswell Park Memorial Institute formulation 1640 (RPMI-1640), McCoy’s 5A. These are considered the classical growth media invented in the mid-1900s. They usually require supplementation with bovine serum and/or a hormone such as insulin to actually support the growth of cells but once this supplementation is carried out the comlete medium has a lifespan of a few weeks at most due to the instability of serum and hormones. The classical media are stable for about a year hence their separate supply and they are still the most widely used media today.

The Bicarb Buffer System

Because of the bicarb buffer system introduced by Sidney Ringer a CO2 incubator will broadly maintain pH7-7.4 until too much lactic acid is produced by the cultured cells. Lactic acid production is one of the main reasons for regular feeding (replacing the growth medium) or subculture (putting the cells into new culture vessels which are lager or more numerous thus allowing total cell nmber to expand). It is also the reason phenol red is included in media formulations for mammalian cells – it is a pH indicator. A vibrant red colour indicates pH7-7.4 – this is good as almost all mammalian cells are very sensitive to pH.

A purple tinge indicates a higher pH: usually seen in nearly empty media bottles in the fridge because CO2 has come out of solution and increased the pH, like a nearly empty bottle of fizzy drink. Such media should be discarded. Cells which grow rapidly and/or are infrequently fed/subcultured will produce enough lactic acid to reduce the pH and turn the media yellow. This latter scenario is something to be aware of as I often see yellow cultures in various labs and those cells will be under significant stress. Instead of pain being the indicator, it's the colour of the phenol red. There is a risk that the culture will not survive unless immediate action is taken.

We experience the same phenomenon: if we run faster than our metabolism can cope with, the lack of oxygen activates a non-oxyen based method of generating energy. This alternative method of energy production (called fermentation but we're not making beer!) produces lactic acid in the muscles which is painful and felt as a burning sensation. If you stop exercising, breathe heavily for a minute or so and rest - the lactic acid is no longer produced and is cleared by the liver and the burn goes. Lactic acid may also be responsible for getting a stitch - the liver in pain due to high levels of lactic acid.

There are always exceptions to these rules! Leibovitz’s formulation 15 (L-15) is a classical media but it contains no sodium bicarbonate and as such putting L-15 in a CO2 environment will drop the pH significantly. So it’s compulsory to use a non-CO2 incubator.

The origins of the potassium chloride and calcium chloride in Ringer’s Solution are a little more interesting. He originally intended to make a normal saline solution to allow a frog heart to continue beating in a Petri dish for an extended period of time. He used London tap water to make his saline and realised that it contained “contaminants” and repeated the experiment with distilled water and sodium chloride. The heart did not beat as well or as long so he realised the physiological importance of contaminating potassium and calcium ions. As is often the case serendipity played its part in science. Ringer’s solution is still found ubiquitously in hospitals today as an adjunct to normal saline.

Heroes

Sidney Ringer’s place as one of the earliest pioneers in cell culture is well cemented. He contributed the bicarb buffer system and realised the physiological importance of potassium, calcium and chloride ions in beating hearts. Another scientist is worthy of mention: Ross Granville Harrison grew frog neurons using an unusual method: he suspended a drop of frog lymph from the underside of a microscope slide and the biopsied neurons successfully grew. This was no mean feat, neurons are challenging to grow in vitro. The so-called hanging drop method of culture he developed became a footnote in the history of science but has made a comeback recently in the culture of stem cells. It seems to be well suited to the culture of delicate cells.

I recently discovered at the Pirbright Institute that tick cell lines were cultured in this way in the past. They represent a real challenge as the cell lines can take a decade to be established but they are vectors for several BSL-3 and BSL-4 pathogens that are of concern such as Crimean-Congo Haemorrhagic Fever.

Villains

No good story would be complete without villains and cell culture has its share. Probably the most noteworthy were Alexis Carrel and his collaborator Charles Lindbergh. Carrel had achieved fame in the early 1900s as a Nobel Prize Laureate for pioneering work in heart surgery. Lindbergh was the aviator who first flew solo across the Atlantic from the US to France. Also his child was tragically abducted and later found dead in one of the FBI’s earliest famous cases.

Lindbergh used his aviation fame to seek out Carrel as his sister had suffered rheumatic heart fever. It was possible to repair the damage surgically but in the absence of heart bypass the operation was not feasible. Lindbergh was an avid inventor and approached the elusive and eccentric Carrel. Remarkably they got on The pair designed a heart bypass machine in the 1930s and were featured on the cover of Time Magazine for it. Heart bypass wouldn’t take place until 1960 so they were ahead of the times.

Their interest in cell culture was the achievement of immortality. Carrel had apparently kept a sliver of chicken heart alive for over 30 years – well beyond the lifespan of the average chicken. Although the experiment was flawed and unrepeatable it captured the public’s imagination. If a piece of chicken could have its lifespan extended perhaps the same could be done with an entire chicken. And if chickens, why not humans?

And therein lay their villainy: they were proponents of eugenics and wanted immortality for only the right kind of people. There is a photo of Lindbergh with Herman Göring and Carrel would likely have been imprisoned as a Nazi sympathiser had he not died at the end of WWII.

Nonetheless we owe the monolayer culture and trypsin subculture methodology to Carrel - a century old technique still heavily relied on in dug discovery. Needless to say immortality remains a pipe dream.

The Problems with Cell Lines

Simply put, the majority of cell line problems come from people sharing cells: the black market of cell biology. Engaging in this practice would seem to save time and money but cells lose provenance (this is the history of the cell line consisting of records of the origin and propagation of the line, and can include assays for authentication and contamination), cell lines risk becoming cross contaminated and are invariably much older than those available from biorepositories leading to genotypic and phenotypic artifacts. The life science literature is a litany of false interpretation in cell culture for this reason. Unreproducible results are rife.

Misidentified or cross-contaminated cells have been known about since 1966 when Stanley Gartler reported that most of the 18 cell lines he had studied (the majority in those days) were in fact all contaminated with HeLa cells, the first immortalized human cell line. He proved this by examining whether the cell lines had isoenzymes and genetic polymorphisms specific to their origins. This technique is still used today to test for cross-contamination or misidentification, which remains highly prevalent.

The first immortal human cell line HeLa was isolated 15 years prior from Henrietta Lacks’ cervical adenocarcinoma. So aggressive was the tumour that it killed her within 8 months of her first presentation at Johns Hopkins Hospital. By the mid 1950s HeLa was ubiquitous in biology labs across the world. The man whose lab first isolated it, George Gey, toured the world sharing the cells, reagents and methods for culturing his cell line. It grew fast: killing Henrietta Lacks remarkably quickly and with a doubling time of 24 hours in culture. Whilst the cell line was a boon to the emerging field of cell biology it contaminated putative cell lines very effectively without anyone noticing for 15 years. HeLa contaminants are still used and published on today: WISH was supposed to be normal amniotic cells and Chang was supposed to be normal liver. They were both identified as HeLa by Gartler in 1966. It is now known nearly 500 other cell lines suffer the same problem with HeLa being the most frequent contaminant closely followed by T24 bladder cancer cells. Estimates of the amount of money wasted in basic oncological research are in the tens or hundreds of millions, let alone the human cost.

In the 1970s Walter Nelson-Rees ran the premier cancer cell bank for the National Cancer Institute (NCI) part of the NIH. Nixon wanted to be remembered as the president who cured cancer so there was an abundance of funding. Nelson-Rees used this to develop more CLA methods, e.g. karyology, again still used today. History turned out differently for Tricky Dicky, but also for Nelson-Rees. Nelson-Rees discovered the misidentification problem extended far beyond the original 18 cell lines that Gartler had reported on in 1966 at a conference. However, Nelson-Rees went a step further than a presentation at a single conference. He named and shamed anyone using misidentified or contaminated cell lines in prominent journals such as Nature & Science (there are 3 good examples in Science: 1974, 1976 & 1981) and for his efforts he was fired in 1981 and became an art dealer. He was certainly a poor diplomat and did not play politics but crucially he was right.

Unfortunately the effect was that no-one dared address the issue seriously for about 20 years. In fact a paper was published in The Lancet describing this long standing problem in 2001, anonymously.

The Solution

Fortunately it has never been easier to conduct high quality cell culture. Biorepositories around the world ensure that the cell lines deposited with them and used by different researchers are free of contamination and match their original description. The oldest, largest, and most diverse of these repositories is the American Type Culture Collection aka ATCC. It is virtually impossible to work in cell culture, microbiology or virology without acquiring materials from this bank.

I am often asked, “What is the difference between materials available from commercial biorepositories and the same cell line from a colleague?”, The main difference is the QC and validation carried out by the commercial biorepository. Biorepositories generally ensure cell line identity and purity making it easier for comparable studies to be performed on a cell line even decades after it is initially isolated. Regrettably this is not usually the case if you obtain a cell line from a colleague. Time and money are wasted, papers are retracted and there is a loss of reputation that can impede collaborations, especially with industry.

Today we also have the appropriate tools to verify our cells lines. Even if you acquire your cells from a biorepository like the ATCC, there are times when it is important to check their identity. For example, perhaps a working cell bank has been made by a research institution (an excellent practice), or maybe cells have been in culture for an extended period of time (>10 subcultures or >3 months). Other times cell lines are unavailable from the official biorepositories or have been directly isolated from patients. In these cases, a test known as Short Tandem Repeat (STR) profiling can be used to ID the cell line. The same test is so good that it is used as a forensic technique (aka DNA fingerprinting) that can place suspects at the scene of a crime (every human in the world has a unique STR profile) and can also be used as a paternity test. The test is accurate, precise, robust, and STR profiles are available for all the major cell lines on public databases. Here are two papers urging the use of STR profiling

  • Short tandem repeat profiling provides an international reference standard for human cell lines – PNAS, April 2001, Masters et al
  • Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues - In Vitro Cell Dev Biol Anim, Oct 2010, Barallon et al

The Future

Attitudes are changing but yet more is needed to convince people of some of the basic tenets of responsible cell culture outlined in this article. Now many journals strictly require evidence of cell line provenance and identity. The Nature Publishing Group recently reiterated its strict stance on the matter (15 March 2016). Funding bodies such as the Biotechnology and Biological Sciences Research Council (BBSRC) and Medical Research Council (MRC) require authentic cells to be used and STR profiling to be conducted during projects to ensure the results are reliable. With this two-pronged approach, the many mistakes of the past can be prevented and we can progress faster to desired clinical endpoints.

The Author

Nick Amiss is an Outreach Scientist for consultancy in cell culture in drug discovery and bioprocess. He studied biochemistry at Warwick University, England, graduating in 2001. He has gained extensive cell culture research experience in market leading companies including: biopharmaceutical process development at GSK using bioreactor culture of adherent human cells on microcarriers, researching preclinical drug development and culture process optimisation at AstraZeneca-MedImmune, developing novel therapeutics and luminescent cell lines for xenotransplantation at Antisoma & providing technical training & engineering support for bioreactors at Infors & Applikon.

He now conducts consultancy, seminars and training, examples being this horrid history you have read above. Also looking to the future of life science and its reproducibility there are many inexpensive in vitro methods to be used appropriately which will generate valid data to better inform the decision to proceed to in vivo and clinical trials.

From Petri Dish To Human: Lost in Translation

In an increasingly competitive environment more and more life science research has to be translatable to the therapeutic or diagnostic arenas. The journey from Petri dish to human is a long one and fraught with pitfalls. Even the best ideas have an arduous journey if they are to make it to commercial application. Several high profile studies have recently called into question the reproducibility of life science research which at best undermines the reputation of a lab and at worst can lead to retracted papers and threatens future funding from both academic institutions and industrial collaborators. Additionally avoiding doomed in vivo experiments can save much time and money and has significant ethical benefits. Yet the risk of failure in vivo due to flawed in vitro experiments can be easily reduced by using current best practices and technologies. These range from simple, free to implement practices to the very latest cell biology tools. Such quality control practices and use of standards are commonplace in disciplines such as chemistry and engineering – life science is catching up. This seminar will help you get ahead of the curve.

References and further reading

  • Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 7th Edition ISBN-13: 978-1-118-87365-6
  • http://www.wiley.com/go/freshney/cellculture
  • Immortal Life of Henrietta Lacks by Rebecca Skloot, ISBN-10: 1400052181
  • A Conspiracy of Cells by Michael Gold, ISBN-10: 0887060749

Filed Under: Cell Culture Pharmacology Oncology Virology Immunology Pharmaceutical Drug Discovery Development


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User Type: Tutor  Verified
Name: Nick
Uploaded Date: Jun 01,2016

About The Author

I studied biochemistry at Warwick University, England, graduating in 2001. I gained extensive cell technology experience in market leading companies. I currently consult as an Outreach Scientist (e.g., the Crick Institute and UCL) which involves many things including scientific seminars.... Read More

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