Contamination of Primary Human Corneal Epithelial Cells With an SV40-Transformed Human Corneal Epithelial Cell Line: A Lesson for Cell Biologists in Good Laboratory Practice

We recently identified a unique way cultured cells can become contaminated by other cells. Initially, we thought we should not publish these observations out of shear embarrassment as our laboratory prides itself on good laboratory practice and scientific rigor, but on reflecting on this, we decided to put our story together, hoping that it will serve a valuable lesson and reminder for ocular and nonocular researchers performing cell culture about the risks of contamination and therefore the importance of authenticating cells prior to publication. In brief, we discovered that a human corneal epithelial cell (HCEC) line remains viable in growth media in the laboratory refrigerator without gaseous exchange for more than 3 weeks at 2°C and that this was the most likely mode in which our primary human corneal epithelia became contaminated. 

Our investigation began several years ago when eye banks across Australia moved from cold storage toward preserving donor human corneas in organ culture. For the uninitiated, there are two principal methodologies used to support corneas ex vivo prior to grafting. They include hypothermic storage at 4°C^1,2 and organ culture preservation at temperatures ranging from 30°C to 37°C,^3,4 with both approaches delivering comparable clinical results.^5,6 Although this is reassuring from a medical end point, we discovered that the corneal epithelium in these long-term organ culture stored specimens is far from intact (i.e., it typically detaches from Bowman's layer). Given these findings, and realizing that these storage conditions may favor cell survival, we propositioned that sloughed corneal epithelia accumulate as viable cells in the Eye Bank Organ Culture Media (EBOCM) and that the submerged organ culture arrangement (Fig. 1A) would render these cells less differentiated, allowing them to be propagated for several generations for basic biochemical and functional analyses or perhaps for grafting purposes if they contained a healthy population of stem cells. 

Figure 1

 

The effect of organ culture on corneal epithelial integrity. The Lions NSW Eye Bank (Sydney) currently preserves donor human corneas long term by suspending specimens in EBOCM (A). Phase contrast microscopy of cells derived from sample 1 after 15 days (B) in primary culture (P1). The hatched line demarcates a proliferative colony comprising smaller cells. The EBOCM-derived cells were detached from primary culture at different passages (P), collected, and dispensed at low density into tissue culture plates. Colonies were expanded, plates were stained with 1% rhodamine blue, and images were taken by conventional photography (C, D). A colony that formed at P33 was photographed under phase contrast microscopy (E). Immunofluorescence was performed on cytospin cell preparations using an anti-human pan-cytokeratin (F) (clone MNF116; Dako Corporation, Carpinteria, CA, USA) to determine epithelial cell content, a polyclonal antibody against the proliferation marker Ki-67 (F) (clone RB-1510-P1, Ab-4; Neomarkers, Fremont, CA, USA), a monoclonal Ab to the putative limbal epithelial SC marker K15 (G) (clone LHK15, Ab-1; Neomarkers), and appropriate isotype control Abs (G, inset). Some cells (>P23) were analyzed by RT-PCR for stem, corneal, and conjunctival marker mRNA expression (H) as previously described.^9–11 PCR products were run on agarose gels next to a 100-bp HyperLadder (E). The RT-PCR assay controls included template with no primers (−Pr) and reactions with no template (−Tm). Flow cytometry (I–K) was performed on paraformaldehyde-fixed cells derived from sample 1 at >P29 using Abs to TLR2 (I) (clone TL2.1; eBiosciences, San Diego, CA, USA), TLR4 (J) (clone HTA125; eBiosciences), and HLA-DR (K) (clone L243; BD Biosciences) with appropriate isotype control Abs (IgG₁, Dako; or IgG₂, eBiosciences).

Therefore, EBOCM was collected and centrifuged, and after resuspending the pellet, Trypan blue exclusion disclosed that many cells were indeed viable (Table). These cells were subsequently deposited into a single well of a six-well plate with commercial media (CnT50; CellnTec, Bern, Switzerland) formulated for the expansion of corneal progenitor cells. For the majority of samples (n = 12/17), cells adhered to the culture vessel and readily expanded, but generally not beyond the first generation. However, in two independent specimens derived from samples 1 and 6 (Table) that were acquired 10 days apart, clusters of small cells with high nuclear to cytoplasmic content were detected amongst larger differentiated cells (Fig. 1B). These cells had high proliferative activity and formed colonies that rapidly expanded (Figs. 1C, 1D). On closer inspection, most colonies were circular with defined smooth edges, were densely packed, and contained a central region of stratification (Fig. 1E), consistent with the formation of holoclones.^7,8 In all other samples that contained viable cells, replicative senescence was reached either before or soon after subcultivation from the initial seeding plate. 

Table

 

Details of Donor Organ Cultured Corneas

 

Sample 1 was the initial specimen received and the first in which this vast cellular expansion was noted. Sample 6 was obtained 10 days later, and cells from this specimen were propagated for 10 generations before a decision was made to cryopreserve all cells arising from this sample because they displayed similar morphologic and functional characteristics to those from sample 1 (Table). Therefore, we undertook the majority of phenotypic and functional assessments on cells derived from sample 1. Because EBOCM-stored corneas include bulbar conjunctiva and endothelium, the identity of the cells being cultivated was next assessed. First, it was noted that, beyond the third passage, the entire culture consisted of proliferative epithelial cells as indicated by coexpression of cytokeratin and Ki-67 (Fig. 1F), of which a large proportion were K15-positive cells (Fig. 1G) and expressed mRNA for this and other putative limbal epithelial SC-associated genes such as ΔNp63α and ABCG2. Finally, these cells expressed the corneal-specific K3 but not the conjunctival-related marker K19 (Fig. 1H). Flow cytometry showed that they lacked antigens responsible for eliciting and immune response such as Toll-like receptors (TLR)-2 and TLR-4 (Figs. 1I, 1J), and did not express the HLA class II activation marker HLA-DR (Fig. 1K). At this stage of the investigation, we were excited about the prospect of having discovered a potentially new source of readily expandable epithelia, but at the same time, we were concerned about microbial transformation as these cells expanded well beyond what is typical for primary human corneal epithelia. Therefore, we screened for epitheliotrophic human papillomavirus-16 and -18, which are known to infect ocular surface epithelia,¹² as well as cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, enterovirus, herpes simplex virus, human immunodeficiency virus, and human T-cell lymphotrophic virus, which all returned negative findings (data not shown). 

One remaining concern was that EBOCM-derived cells were contaminated by another cell line. At the time these experiments were conducted, no other cells were being propagated in our facility. However, the SV40-immortalized HCEC line¹³ had been cultured 10 days prior to us receiving the first EBOC human corneal specimen (sample 1). Therefore, DNA was extracted from the most recent cryopreserved aliquot of this line, as well as from cells derived from sample 1, and after submitting both for DNA fingerprinting, they were found to be genotypically identical at 16 different loci (Figs. 2A–2D; data not shown). To confirm the presence of SV40, immunofluorescence for the SV40 T-antigen was performed. Our results showed SV40 T-antigen expression in the SV40 HCEC line (Figs. 2E, 2F), as well as in cells from sample 1 (Figs. 2H, 2I) and sample 6 (Figs. 2K, 2L). However, SV40 was not detected in freshly prepared primary human limbal epithelial cells (Fig. 2N, 2O). Finally, all cells displayed immunoreactivity to the human nuclei antigen (Figs. 2J, 2M, 2P), confirming their species origin. 

Figure 2

 

Short-tandem repeat sequencing and SV40 T-antigen expression. Histograms of short-tandem repeat sequences at 2 of the 16 loci analyzed by the Powerplex assay: D21S11 (A, B) and vWA (C, D) using DNA from sample 1 (A, C) and SV40 HCECs (B, D). Immunofluorescence was performed using monoclonal Abs to the SV40 T-antigen (E, F, H, I, K, L, N, O) (clone PAb416; Abcam, Cambridge, UK) and the human nuclei (hNu) antigen (J, M, P) (clone 3E1.3; Chemicon, Temecula, CA, USA) on SV40 HCECs (E–G), cells from sample 1 (H–J) and sample 6 (K–M), and primary human limbal epithelial cells (N–P). An Alexa-Fluor 488–conjugated goat anti-mouse secondary Ab (Invitrogen; Carlsbad, CA, USA; 1:300 dilution) was added after washing off the primary Ab. Immuno-labeled cells displayed green fluorescence, and DAPI (blue) was applied as the nuclear counterstain (F, G, I, L, O). Cells incubated with an isotype control IgG Ab were nonreactive (G). Passage number is given in parentheses. Images were taken under oil immersion.

Given that SV40 HCECs were not being used for experimentation at the time our newly discovered EBOCM-derived corneal epithelial cells were being characterized; we propositioned that poor culture technique must have contributed to the contamination. This could only have eventuated if the contaminating cell line was accidently inoculated into the growth media stock bottle, and cells remained viable in cold storage for several weeks. To test this hypothesis, SV40 HCECs were dispensed into multiple CnT50 media aliquots and stored in sealed tissue culture–grade tubes in the laboratory refrigerator for 4 weeks. Primary human corneolimbal epithelial cells (of a finite lifespan) and the transformed human adult low calcium high temperature (HaCaT) skin keratinocytes were assessed in parallel. Our results showed that SV40 HCECs were viable and expanded to form colonies after cold storage at temperatures averaging 2°C for greater than 3 weeks (Figs. 3A–3C), although with decreasing efficiency (Fig. 3D). Our data also revealed that primary human corneolimbal epithelia are more prone to hypothermic damage than their immortalized SV40 HCEC counterparts, but behave in a similar manner to transformed HaCaT keratinocytes in relation to viability and colony formation (Fig. 3D), suggesting that different cell lines have diverse levels of hypothermic tolerance. Notably, other cell lines (e.g., CHO-clone 161 and HEK 293) have been “paused” for 9 days at 4°C and shown to resume exponential growth when reincubated at 37°C.¹⁴ Although we did not conduct mechanistic investigations to explain this phenomenon, it has been suggested that ATP synthesis, membrane permeability,^15,16 and heat shock proteins¹⁷ play a pivotal role. 

Figure 3

 

Cell viability and activity in cold storage. SV40 HCECs were dispensed into sterile tubes containing CnT50 media and placed in a laboratory refrigerator for 4 weeks. At regular intervals, the contents of each tube were decanted into tissue culture plates and incubated at 37°C to assess their viability and replicative activity. Phase contrast micrographs display cells that were refrigerated for 21 days and imaged at 5, 6, and 7 days after seeding (A, B, C, respectively). The same colony is depicted in panels A–C. Inset in C is an image of cells that were left in cold storage for 28 days and then plated and photographed after 7 days. Under these conditions, only two cells were found to have attached and potentially replicated. The bar graph (D) represents the mean number of colonies formed (±SD) from SV40 HCEC (red), HaCaT (blue), and primary corneolimbal epithelium (green) that was assessed after retrieval from cold storage over a 3-week period.

The rationale for disseminating this report to the wider scientific community is to raise awareness of the potential for misidentification and/or cross-contamination of either primary cells or cell lines. For decades, cases of mistaken identity have infiltrated the cell biologists' “tool box,” and although some investigators have been diligent about ensuring authenticity, the vast majority are in denial about either receiving potentially contaminated aliquots of cells or themselves spoiling batches of cells after receiving pure stocks.^18,19 Perhaps the most infamous contamination story is that of HeLa cells, which were originally established in 1951 from a cervical cancer biopsy taken from a 30-year-old African-American woman named Henrietta Lacks.²⁰ Within a decade or so of their dissemination, they were found to contaminate many other cell lines.^21,22 Using a simple and reasonably inexpensive DNA fingerprinting assay, such as the one performed in our current investigation, Phuchareon et al.²³ recently highlighted the extent of this problem. In their quest to authenticate six human adenoid cystic carcinoma cell lines, they discovered that each had been contaminated; three were determined to be HeLa cells, one was a urinary bladder cancer line, and the remaining two harbored nonhuman cells of mouse and rat origin. 

From our basic scientific workup, we confirmed the species origin of the cells in question, their epithelial lineage and corneal heritage, and that they were not contaminated with HeLa cells (absence of HPV-18 integration). Furthermore, their genetic profile at the amelogenin locus (data not shown) renders them likely to have arisen from a female donor; this and the localization of the SV40 T-antigen are additional features that authenticate these cells as being SV40 HCECs.¹³ 

We devoted significant resources to this project, including valuable time, manpower, and research funds, only to realize that our primary cultures were in fact overgrown by an immortalized HCEC line. In recent times, diligent ophthalmic researchers have come to realize that misidentification is a significant problem and are systematically authenticating their cell lines.²⁴ Moreover, popular ophthalmic and vision science journals including Investigative Ophthalmology and Visual Science have understood the ramification of publishing reports with mischaracterized cells and have introduced specific policy around this issue (ARVO/IOVS terms and policies, retrieved September 16, 2015; http://iovs.arvojournals.org/ss/forauthors.aspx). A prime example is in relation to the use of the rat retinal ganglion cell line (RGC-5)²⁵ that likely never existed beyond the first generation outside the laboratory of origin.²⁶ We will probably never know precisely how such contaminations arose; however, our report offers novel insights. In terms of eradicating the problem, we make the following recommendations: (1) avoid culturing more than one cell type at any given time, and if necessary, work with different cells in separate culture suites with designated tools including class II safety cabinets and CO₂ incubators; (2) dispense media and other reagents in small aliquots, allocating them to the respective cell line; (3) discard any unused materials regularly and do not use reagents owned by colleagues; (4) avoid sharing cells (an endemic problem in research) especially if they are commercially available from reputable banks; and (5) develop appropriate protocols to periodically authenticate cells before proceeding to large-scale time-consuming experiments. Finally, our message to institutional hierarchy and other stakeholders such as funding agencies, reviewers, and editors of scientific journals is to continue to raise awareness of this problem and devise stricter guidelines and policies to change the current culture.^27,28 

Acknowledgments

The authors thank Jenna Iwasenko and William Rawlinson (Department of Virology, Prince of Wales Hospital, Randwick, Sydney, Australia) for assistance with the viral multiplex screening assay and Michael Buckley (Genetics Laboratory, Prince of Wales Hospital) for performing the DNA fingerprinting assay. The authors also thank Rajnesh Devasahayam (Lions NSW Eye Bank, Sydney, Australia) for providing all the organ-cultured and fresh human donor corneal tissue. 

Supported by the University of New South Wales.