5. HeLa Cultures and Contamination: The Intersection of Biology, Race, Class, and Gender

Cell Culture and Cell Banks¶

Today, thousands of cell lines have been established using refined methods of tissue culture. The first human cell lines, much like HeLa in 1951, were established by placing freshly removed human tissue in a growth medium containing animal cells, or human fetal cells. These “feeder” cells provide the newly transplanted cells with the appropriate growth factors and extracellular matrix (ECM) to establish the environmental conditions necessary to sustain growth for this new cell lineage, or cell line.o promote cell viability, cell lines must be incubated at the right temperature, humidity, and CO2 levels. The cell lines are maintained by human researchers who “split the cells” daily. Splitting involves washing the cells and reducing their numbers by distributing (splitting them up) them over numerous Petri dishes. This process of splitting removes waste, dead cells, excess cells, and old culture media and provides cells with fresh media containing growth factors and adequate space to ensure continued cell division (Slide 29: What is Cell Culture).

Because researchers split the cells daily, great care must be taken to ensure a sterile environment. In order to limit contamination by cells either shed from the body of the person performing cell culture or present in the environment a tissue culture hood is used. The tissue culture hood uses air flow to pull air up and out away from the cultures, keeping the working environment sterile. Laboratories using human cell lines are designated as Biosafety level 2 (BSL2) and require special ventilation and air flow rates, as well as sterile biosafety cabinets and sterilizing equipment. Thus, human tissue culture experimentation is a costly endeavor, but one that can produce a wealth of knowledge and a range of biomedical products. Cells can be cultured short-term to serve as sites of experimentation for screening toxins and environmental chemicals, or for the development and testing of drugs and vaccines. They can also be used as biological factories to create humanized proteins for therapeutic outcomes. Cell lines can also be frozen at -80°C for long-term storage and thawed out when needed, allowing researchers to start and stop time with respect to the life of the cell line. A short video produced by EuroStemCell presents a “walk- through” of a cell culture lab, highlighting some of the most important aspects of cell culture. Duguid, 2010

Video: Duguid, C. 2010. Video: Cell Culture. EuroStemCell. (Producer Kate Doherty) (6 minutes). Link

In the story of The Immortal Life of Henrietta Lacks, written by Rebecca Skloot, the notion that time can be frozen and a cell line resurrected is revisited throughout the narrative. Because cells can be thawed, placed in in the optimal growth conditions, and divide, it means that cell lines “live” past the typical human life span. In the case of the Lacks family, this caused some family members to pause and consider the spiritual and cultural ramifications of such manipulations (Skloot, 2010).

As the title of Cynthia Verspaget’s artwork suggests, such cells are exhibiting a type of “anarchy,” either refusing to stay connected to the individual from whom they originated or, perhaps, reaffirming this human connection. In this work, Verspaget fused her own cells with HeLa cells to create what she calls a new “artistic cell line.” Her work questions the notion of ownership and cell memory that can be rendered obsolete through laboratory manipulation, and poses philosophical questions regarding kinship and ontology (Verspaget, 2004). (Slide 41: Anarchy Cell Line). Helen Wilson-Roe seeks to redress this loss of familial connection through a collective work, “A Brush With Immortality,” in which she painted portraits of individual Lacks family members and exhibited these alongside photographs of HeLa cells caught in various stages of cell division (Wilson-Roe, 2013). Adele Senior, has written about other bioartists who have used HeLa cells and the narrative of their derivation to highlight the human connection, and RadioLab conducted an extra segment on the evening of the premiere of the film adaptation of Skloot’s book (Senior, 2011; Radiolab; Wolfe, 2017)

Anthropologist Hannah Landecker explores further what the ontology of cell culture means to society, and writes in the introduction to her provocative book Culturing Life: How Cells Became Technologies:

Despite its relative novelty in historical terms, this state of life has quickly become normal, imbuing scientific objects such as cell lines with the aura of inevitability or, ironically enough, with an air of natural existence. How is it that life, once seated firmly in the interior of the bodies of animals and plants, came to be located in the laboratory? At what point did living matter get extracted from and stripped of the individual forms of organisms? Further, why did the cells of humans become incorporated in the research biomass along with those of other organisms, and how do the lives of such human-derived objects affect the concept of the human subject? How did life, including human life, take this contemporary disembodied, distributed, continuous form? The question of where tissue culture came from is not only one of origins but also of conditions - of what makes it possible for these biotechnical things to exist in these detached, and transformed ways. (Landecker, p. 4, 2007).

She goes on to investigate the ways in which the freezing of cell lines plays an important role in creating a new temporality and commodity. By freezing and cataloguing cell lines, researchers can store, ship, and exchange cell lines with one another.

As cell culture techniques expanded, a new industry emerged, supplying researchers with reagents, media, and materials as well as the cell lines themselves. Thus, although George Gey, with his firm belief in the free exchange of scientific knowledge, supplied researchers worldwide with the HeLa cell line at no cost, much like Leonard Hayflick did with WI-38, present practices often involve the purchase of cell lines through national or international clearinghouses.

As cell line expert John Masters of University College London puts it, “Much of what we know today and much of what we do tomorrow depends on the supply of HeLa…” HeLa was used to study infectious agents, including polio, making it possible for Jonas Salk to develop the life-saving polio vaccine in 1952 (Masters, 2002). Salk, like Gey and Hayflick, operated on the unwritten rule that if scientific results are published, scientists are obliged to share their work freely with other scientists to promote scientific knowledge and to advance human health. To that end, HeLa cells were grown in space to test the effects of zero gravity and radiation on human tissue, and were exposed to countless drugs and toxins on earth for drug development purposes. Even in today’s modern research, HeLa is still the most widely used human cancer cell line. To trace its provenance and use, Walbaum created an infographic for a short article authored by Erin Biba for Wired Magazine (Biba, 2010). (Slide 42: HeLa Everlasting)

To produce the vast quantities of cell lines needed to conduct this work, scale-up techniques were developed. Walter Nelson Rees, the researcher who discovered through use of genetic markers that HeLa cells had contaminated other cell lines, co-authored an historical perspective entitled “Henrietta Lacks, HeLa Cells, and Cell Culture Contamination” published in the Archives of Pathology and Laboratory Medicine Online just before his death in 2009 (Lucey, 2009). The perspective outlines a rich history, and describes the inventions, such as the bioreactor, that produced the enormous quantities of cells needed for such clearinghouses. Two major repositories are the ATCC and the European Collection of Animal Cell Cultures (ECACC), which provide international access to a variety of cell lines that have been tested and had their histories documented. This standardization assists experimental reproducibility and regulatory approval procedures. Using the ATCC online database, one can review information about the creation of the cell line, the genetic and biological characteristics of the cells, and any additional genetic characteristics unique to the cell line such as HPV 18 DNA and the presence of genomic instability M regions (ATCC CCL2 accession number). Both of these genetic characteristics contribute to the immortality phenotype.

A closer look at the genomic instability information reveals a common phenomenon in cell culture, which is the power of evolution. Cells in a Petri dish are competing for resources such as growth media and space. Any genetic changes that would allow a cell to utilize resources more efficiently or quickly, will be at an advantage. Cancer cells often have DNA mutations in genes that code for proteins that control mutation rate, or DNA repair. Thus, they tend to accumulate mutations at a faster rate than non-cancerous cells, and often these mutations take the shape of large DNA rearrangements. So not surprisingly, many cell lines are established from cancerous tissues because these cells have genetic mutations that allow them to create more mutations. These frequent DNA rearrangements are described as genomic instability, because entire regions of chromosomes are fragmented, translocated to other chromosomes, deleted, or duplicated. Thus, as cells compete for limited space and resources in the Petri dish, they undergo rapid cell divisions due to continuous exposure to growth factors and accumulate genetic mistakes or mutations at a quick rate. The HeLa cell line, being one of the oldest cell lines in culture, can contain cells in its Petri dish with up to 80 chromosomes, rather than the typical 46 that appear in our bodies. Given this rather dramatic difference in genetic content, some researchers have proposed that the HeLa cell line represents a species distinct from human. Evolutionary biologist Leigh Van Valen and his colleague Virginia Maiorana suggest renaming HeLa as Helacyton gartleri; cyton, from the Greek cytos, reflecting the neutral gender of the cells; and gartleri after geneticist Stanley Gartler, the researcher who documented the cells’ evolutionary fitness advantage over other cell lines (Oliwenstein, 1992).

Feminist scholar and scientist Lisa Weasel points to the irony of demoting HeLa to a microbial species, despite its ability to survive in multiple environments. She considers the inconsistency to be rooted in both sexist and racist thinking. She explains how race, class, sexuality, and gender intersect in this attempt to reclassify HeLa cells as a new species because of their inability to breed with humans.

“The route from human carcinoma to novel microbe was not one uniformly accepted in evolutionary biology, calling into question the researchers’ designation of HeLa cells within the kingdom Protista, which somehow implied that evolution could take place backwards, retroactively transforming a complex metazoan into a primitive protist … And so the story comes full circle, the madly proliferating cells, now verging on becoming a separate and inferior species, linked at least in some readers’ minds to the unbridled, infectious sexuality of a black woman from Baltimore. Is this mere coincidence? After all, the HeLa cell line could just as easily have been derived from a lung carcinoma from Herbert Langston, a middle-class bank teller from suburban New Jersey, or from the prostate cancer of Henrik Larson, a Scandinavian immigrant living in the Midwest, or from any other number of individuals whose first names began with the letters “He” and last names with “La” and were host to a pernicious cancer proliferating wildly within their confines. Then we might read the story differently, or might not tell it at all (Weasel, p 187, 190, 2004).

Though Weasel explains that the HeLa cells’ proliferative power is a result of infection with the sexually transmitted HPV 18, she does not unravel how the very nature of viral infection is caught up in the intersection of biology, race, class, and gender. Henrietta Lacks, unlike the laundry list of theoretical male cancer patients she brings forth, was an African-American woman who did not access healthcare early on due to her social and built environment. In the 1950s, John Hopkins Hospital served as a Charity Hospital and was one of the only hospitals serving African-Americans during this time. Given her responsibilities to raise children in a patriarchal community that had a deep mistrust of the medical establishment, due to centuries of practice during which African-American slaves were used as experimental subjects, she postponed what might have been perceived by others as routine care. Because of her race, class, and gender she arrived at John Hopkins Hospital with late-stage cervical cancer.

The aggressive nature of Henrietta’s cancer was partially due to this delay to seek medical care as well as a compromised immune system. Being infected with multiple sexually transmitted diseases taxed her immune system. Thus, immune cells that address virally infected cells and clear them from the body,were not efficient, resulting in multiple copies of HPV18 genomic DNA being inserted in more of her cervical cells’ genomes. Had the cancer been detected in its earliest stage, the number of insertions would be far less, leading to a less aggressive cancer phenotype. In 1951, the Pap smear was not yet available in the United States, making cervical cancer a challenge to diagnose and prevent. Cervical cancer screening via the Pap commenced in the late 1950s, increased rapidly after 1960, and grew steadily until 1973, when it stabilized. These statistics are applicable to Caucasian women who had access to healthcare, but does not address the African American community that remained marginalized and fearful of the medical establishment. It should be noted that the unethical Tuskegee Syphilis Trial conducted on African American men was not terminated until 1972.

As Rebecca Kumar states in her Open Letter to the faculty at universities and colleges, to teach the story of Henrietta Lacks without giving close attention to the social and political context in which these cells were brought into the biomedical arena disregards the systemic injustice that has occurred in the African-American community, and does nothing to remedy the health disparities that continue to challenge marginalized societies today (Slide: 43: Open Letter). Kumar writes:

“ I confess that I too have given blood for HPV vaccine research so I could get $75 to help supplement my grad school stipend; I did it so that perhaps one day less women will suffer from HPV, but I admit, shamelessly, I did it for the money as well. When I went to give my blood, the office was populated primarily by African-American women, Latina women, and students. Skloot’s narrative is therefore part of a much longer history that intimately ties quantifiable “knowledge” to eugenics and the female body....Thus, I am of the opinion that the “immortality” to which the title of the book refers is as much a reference to the racism behind the first cut that took Lacks’ cells as it is to Lacks’ cells themselves. (Kumar, 2012)

Charnell Covert, artist, actor, activist, and health justice scholar, echoes Kumar’s view in her theatrical production “They called me HeLa,” which provides viewers with a counter narrative about the provenance of the HeLa cell line.

Video Slide Show: Covert, C., Chamany, K. and Elie, C. 2013. They Called Me HeLa Educational Slide Show. Stem Cells Across the Curriculum/Media. Link

Ruha Benjamin, a sociologist interested in scientific innovation and equity, challenges us to be more forward thinking and to learn from our mistakes of the past. She challenges the the biomedical community to be critical of approaches that result in study designs that are ill matched to community context and needs. In a TedX Talk she discusses the derivation of a profitable cell line in the face of health and economic inequities (Benjamin, 2014).

Cell Line Contamination¶

Undergirding the exciting discoveries and innovation that accompanied the development of cell culture techniques is one of the most challenging issues plaguing biologists over the past 50 years, namely cell line contamination. Because a cell line is defined as a collection of cultured cells derived from one specific tissue in a single donor, its provenance is of importance. While the ability to divide indefinitely is certainly the most essential quality of a cell line, the specificity of the cells’ origin from a single tissue type and from a single donor is a crucial aspect of the research conducted using this tool. For example, a potential stomach cancer drug would need to be tested specifically on human stomach cancer cells, not lung or other tissue origins. Similarly, for stem cell transplant therapies, most often the donor and the recipient should be one and the same, to reduce graft rejection due to immune incompatibility. In these instances, cross-contamination of cell lines (where a more aggressive cell line take over the culture) becomes an issue, as the validity of the study or the efficacy of transplant therapy depends on knowing the very specific origin of the cells (Masters, 2002).

After HeLa was developed in 1951, scientists were able to culture other human cancers using Gey’s technique, and soon after the issue of contamination emerged. As scientists started culturing non-cancerous human cells, they noticed that their cells “spontaneously transformed” in culture to divide rapidly and continuously like cancer. Not long after, scientists began finding evidence of interspecies contamination among cell lines. As early as 1957, researchers were using immunological and karyotyping techniques (observation of chromosomes) to determine species of origin and detect cross-contamination between lines of different species. The use of fluorescent antibody detection in the early 1960s furthered such discoveries. This led scientists to wonder whether cross-contamination was occurring for cultured cells within a species as well.

In 1967, Stanley Gartler made an astonishing discovery that was deeply disturbing to biologists working on human cell lines (Culliton, 1974). Using isoenzyme analysis (a technique comparing enzymes that vary within a species), Gartler showed that two separate human enzymes, G6PD and PGM, which were known to vary within the human population, were identical for each cell line in his survey of 18 human lines sampled from the ATCC. The statistical odds of all 18 cell lines sharing identical phenotypes for these two proteins are nearly impossible. There were only two explanations: either all cell lines reverted to this phenotype in culture, or the cell lines were identical, a result of contaminating overgrowth by an aggressive cell line (Oliwenstein, 1992; Masters, 2002).

In the 1950s, there were a limited number of cell lines and most were established from tissues obtained from Caucasians. Thus, the frequency distribution of two variants of the gene coding for the G6PH enzyme was utilized as a means of identifying the HeLa cells line as Black. The “A” variant is the result of a genetic mutation that reduces the efficiency of the enzyme, but is believed to provide a protective effect against malaria, reducing infection rates by 46-58% (Ruwende et al., 1995). This variant is more common in individuals of African descent given their continued environmental exposure to mosquitoes harboring the protozoa Plasmodium falciparum responsible for malaria. Over time, African populations living in endemic malaria zones evolved to carry this gene variant as it improved survival.

The race-based analysis of the HeLa cell line was only possible given the small number of cell lines at the time and the limited population sampling represented in these lines. Because there were only 18 cell lines in existence and all but Henrietta’s were derived from Caucasians, generalizations about the distribution of gene variants among populations allowed the tracing of a cell line’s provenance back to HeLa. Additionally, it was known that cell lines do not revert to this phenotype but maintain the phenotype of the original donor. Collectively these data suggested that HeLa cells had infiltrated the other cell lines. This cross-contamination explained why it was suddenly possible to culture non-cancerous tissue types when they “spontaneously transformed” after a short time in culture. HeLa was the elusive “transformation factor,” and the other lines had been overrun early during the culture process and were actually HeLa (ErinC, 2009).

The notion that cells can be defined as Black goes against research that demonstrates that race is socially constructed and not biologically based. It is of note that population genetics is based on frequencies that describe general trends and that traits are not discontinuous in human populations such that one population carries a genetic variant at the exclusion of all other variants. The distribution of G6PH genetic variants is the result of environmental factors that placed selection pressure on the population and, thus, altered the frequency of the A variant in different human populations; this is similar to the explanation of the emergence of haplotypes that vary in frequency among human populations (Online Mendelian Inheritance of Man). So though the genetic structure of human populations corresponds to stressors specific to geographic locations, this structure does not support essentialist conceptions that racial categories are discrete or informative. A project developed with funding from the National Science Foundation and the American Anthropological Association entitled “Race: Are We so Different?” provides a deeper analysis of race and genetic variation, as does the PBS series “Race: The Power of Illusion.” Given the inclination of society to carry essentialist notions concerning genes and race, it is of import to contextualize the narrative of the HeLa cell line within a larger socio-political context. If given cursory address, the discussions regarding cell contamination and race can reify essentialist beliefs regarding race by suggesting that there is an entitativity (characterizing groupness) dimension in this sort of thinking (Donovan, 2013; Donovan, 2015).

In her book Culturing Life, Landecker remarks on the ways in which the boundary between the scientific community and the lay public became blurred around discussions of cell line contamination by HeLa cells (Landecker, 2010). That cell lineage was being described in race-based language, and associated with terms like “contamination” and “ aggressive” presents a particularly unique challenge to scientific communication. Though “contamination” in the lab refers to lab practices and is a result of poor sterile technique, the term carries with it a negative connotation, not unlike the ways in which “contamination” is referenced in non- scientific communities.The term “contamination” was often used during the eugenics movements of the US and Nazi Germany. The prefix “eu” implies purity and is applied to describe the pure genetic lineage of those considered “fit” enough to reproduce. Thus, those individuals who were considered “unfit” were prevented from “contaminating” the gene pool of these “pure” populations through policies that regulated reproduction as well as elimination of those individuals deemed unfit (Brignell, 2010a; Brignell 2010b). This rationale of maintaining a true-breeding population of humans goes against all scientific evidence. Evolutionary theory is based on the fact that hybridization produces genetic diversity in populations allowing them to survive environmental changes.

Additionally, the notion that a cell line derived from an African-American woman was being referred to as “aggressive,” and that the immortality characteristic was attributed to a sexually transmitted viral infection, led to a conflation of the cell line and the person from whom they were derived. The use of “aggressive” was similarly problematic, given the social history of labeling African American women as “angry black females.”

At times it was difficult to know whether the conversation was about the cell line or Henrietta herself when terms like “promiscuous” were used. For scientists, promiscuity refers to a cell line’s ability to take up DNA from its environment and grow more rapidly. However, for the general public, all of these descriptive terms to linked to societal prejudices (Landecker, 2010). During the height of the Social Darwinism movement, the term “unfit” was often used in conjunction with “promiscuity.” More recently, James Watson, co-discoverer of the structure of DNA has, on more than one occasion, remarked that people of color have an elevated sexual appetite. Watson’s ideas echo those who showcased Sarah Baartman, known as the Hottentot Venus, in public venues as a highly sexualized African woman during the 1800s. So though using words like “contamination” and “promiscuous” may seem benign in the lab, it may unintentionally register differently in the minds of students and, be linked to these other discriminatory remarks that have a deep and troubled history.

Interestingly, the scientific community on the whole does not seem ready to address this social implication nor the issue of contaminated cell lines in general. Gartler’s work was extended throughout the 1970s by Walter Nelson-Rees of the Cell Culture Laboratory at UC Berkeley. For more than 10 years, Nelson-Rees collected information from 144 publications identifying cross-contaminated cultures and compiled a list of 279 contaminated lines, more than 40 of which were HeLa. Both Gartler and Nelson-Rees were shunned for exposing cell line contamination. Acknowledging it would devalue years of research, commercially available products, and long-held paradigms about how cells behave in response to environmental toxins, drugs, vaccines, and reprogramming factors. In the 30 years since Gartler and Nelson-Rees presented their findings, little attention has been given to the issue of cell culture contamination and its consequences for biomedical research (Lucey, 2009).

Video: Duguid, C. 2010. Video: Cell Culture. EuroStemCell. (Producer Kate Doherty) (6 minutes). Link

With the advent of molecular techniques for identifying cell lines, the issue of cross-contaminated cell lines has resurfaced. John Masters has published widely about the extent to which cell lines appear to be mixed populations, and despite an entire session devoted to the topic in the 2009 American Society for Cell Biology conference, there has been little response to rectify this situation (Masters, 2002, Ouellete & Nardone, 2009)

The most up-to-date list of cross-contaminated cell lines that has been published includes 360 lines, 106 (9%) of which are HeLa. This list, compiled by Capes-Davis (of CellBank Australia) and a team of researchers from other major international cell banks, was generated through the PubMed database, references within articles relating to the topic, and websites of five major cell banks: ATCC, DSMZ (Germany), ECACC (Europe), JCRB (Japan), and RIKEN (private, Japan) (Slide 44: HeLa Contamination). The authors hope that the list will be included in a new initiative improving access to authentication data in the form of a free database, and more recently the lead author, Amanda Capes-Davis, a physician and researcher, has taken to annotating publications in PubMed that contain incorrect cell line identities (Capes-Davis et al., 2010; NCBI, 2014)

The percentage of contaminated cell lines found in collections has apparently decreased, from the initial 100% contamination of ATCC stocks reported by Gartler in 1967, to present-day estimates for various collections of 18%. However, this decrease may be the result of expansion of collections, and not due to more rigorous methods of derivation and storage. When Gartler found that 100% of lines were contaminated with HeLa, there were very few other lines existing, and methods for immortalizing cells were not yet invented. In Nelson-Rees’s time, more cell lines had been created, but not nearly as many as exist today. The fact that recent studies report significant contamination (10-18%) is disturbing, because we now have highly efficient methods for identifying cell origins (Katsnelson, 2010; Masters, 2012; Del Carpio, 2014; Marrow, 2015; Grens, 2015).

The DSMZ cell bank in Germany has played a large role in exposing the issue of cell line cross-contamination, and has been a pioneer among cell banks in practicing measures to improve their collections. It routinely performs multi-parameter (DNA typing, cytogenetic and immunophenotypic) authentication.

Purchasing cells from cell banks instead of sharing among colleagues would help to reduce the misidentification of cell lines, although this may squelch the collaborative and collegial practice of scientific investigation. Others critics suggest that government and private sponsors should require authentication of cell lines when receiving submissions for grants and contracts. In fact, the FDA has adopted this mandate, as has the National Cancer Institute (Chatterjee, 2007; Nardone, 2008).

Although the cross-contamination issue is one that should be addressed by the cell biology community, stem cell researchers learned early on that verifying newly established stem cell lines is essential. The very nature of stem cell research requires a researcher to be able to demonstrate that a specialized cell is capable of adopting a wide set of cell fates without cell fusion or contamination by other cell lines. This was a particular sticking point when the first putative adult stem cell lines were reported, because they were cultured on embryonic feeder cells. Later studies demonstrated these adult cells were able to adopt multiple cell fates as a consequence of cell fusion between the cell line in question and the mouse embryonic feeders. For this reason, more recent studies have taken great care to keep cell lines pure, and include repeated monitoring of lineage-specific characteristics for that cell line, using molecular techniques to ensure that no cell cross- contamination has occurred (NCBI, 2014; Anonymous, 2015; De Los Angeles et al., 2015)