Rapid detection of low‐level HeLa cell contamination in cell culture using nested PCR

Abstract HeLa cells are a commonly used cell line in many biological research areas. They are not picky for culture medium and proliferate rapidly. HeLa cells are a notorious source of cell cross‐contamination and have been found to be able to contaminate a wide range of cell lines in cell culture. In this study, we reported a simple and efficient method for detecting the presence of HeLa cell contamination in cell culture. HPV‐18 was used as a biomarker. The cell culture supernatant was used directly as the template for nested PCR without extracting nucleic acid. By PCR amplification of the cell culture supernatant with the designed primers, we were able to detect the presence of HeLa cells in the culture. The sensitivity of this method can reach 1%, which is 10‐fold higher than Short tandem repeat sequence (STR) profiling. This simple, rapid, and “noninvasive” quality checking method should find applications in routine cell culture practice.

results, more and more scientific journals require the authors to submit a proof of cell purity before paper submission. 14 There are many methods to detect cross-contamination of cell lines, including isoenzymes zymogram analysis, 15 human leucocyte antigen typing (HLA typing), 16,17 DNA fingerprinting, 18 and short tandem repeat sequence profiling (STRs). 17 Isoenzymes, commonly found in cells of higher organisms, are a group of enzymes that have the same catalytic activities, but differ in composition, physicochemical properties, and structure. Cells from different origins have different isozyme distributions. Analysis of gel electrophoresis banding patterns and relative migration distances for the individual isoforms of intracellular enzymes can be used to detect cross-contamination of cells in cell banks. [19][20][21][22] However, studies have shown that the proportion of contaminated cells needs to have at least 10% of the total cell mass in order for the isoenzymes to be reliably differentiated. 20 Human leucocyte antigen (HLA) complex is a major histocompatibility complex (MHC) in humans. There are quite a few differences in bases among HLA genes in different individuals, resulting in different numbers of restriction endonucleases recognition sites. After amplification of the target gene fragment by PCR, various restriction enzymes can be used to digest the amplified product to generate different digested products, and then the electrophoresis pattern is used for identification. It is also possible to carry out the analysis by hybridizing a probe to the amplification product. 23,24 Recently, the major HLA typing resolution is achieved by the Sequence-Based Typing (SBT) method through direct DNA sequencing. 24 For DNA fingerprinting, the variable numbers of tandem repeats (VNTRs) were amplified first to obtain the DNA profiles. Image analysis was then performed to determine the size of each amplicon of a locus on the agarose gel. Finally, the DNA profiles of all the samples were compared among each other to determine the difference. 25 DNA fingerprinting is commonly used in the identification of human stem cell lines. 26,27 In recent years, STR profiling has been suggested as a golden method for authenticating human cell lines. 5,[28][29][30][31] STRs are tandemly repeated short DNA sequences, which are highly polymorphic in the human genome. The repeat sequence is usually 2-6 bp in length. 32 In the analysis of STR, the genomic DNA of target cell samples is extracted first, and then fluorophore-labelled primers are used for PCR. The target STR sites can be amplified and the amplicons are marked with different colours of fluorescence. The amplified products were then separated by capillary electrophoresis to generate a multicolour fluorescence-time profile based on differences in fluorescence colour and fragment size. After comparing the obtained STR profile with the reference in the database, it is possible to detect the existence of cell cross-contamination. 17,33 Through the STR profiling, cross-contamination of human cell lines can be identified. 5 However, STR profiling method requires expansive equipment, the process is relatively complex, the cost more expensive, and the sensitivity low. Furthermore, STR is unable to detect cell contamination when the portion of contaminating cells was less than 10% of the total cell mixtures. 17 Human papilloma virus (HPV) is a group of small, nonenveloped and double-stranded DNA viruses belonging to the papillomaviridae family. 34 HPV infections can lead to diseases such as cervical, anogenital, and head and neck cancer. 35 In 1997, Walboomers 36 confirmed that almost all cervical malignancies demonstrated oncogenic strains have the HPV gene, and HPV infection is considered to be an important cause of cervical cancer. 37 In clinical testing for HPV, DNA or mRNA is usually extracted from the cervical specimen first. After amplification by PCR with specific primers, the presence of the HPV sequence can be identified by a probe to determine whether the sample is infected with HPV. 38 There are approximately 200 HPV genotypes. 34  The HPV genome has a circular double-stranded DNA structure, which including nonstructural proteins (E1, E2, E4, E5, E6, and E7), structural proteins (L1, L2), and a transcriptional control region. 40 E1 and E2 participate in the initiation of viral DNA replication. E6 and E7 modulate the cell cycle control and contribute to viral genome maintenance. Both L1 and L2 are capsid proteins. [41][42][43][44] The human papillomavirus virions first penetrate the damaged area of the epithelium and infect the basal cells. 45 Following viral entry and uncoating, HPV genomic DNA is maintained at a low-copy number in the nuclei of basal cells. 46 After leaving the basal membrane, HPV begins to replicate with the differentiation of the infected cells. 47 Following the amplification of the genome, the synthesis of the capsid protein is triggered and the assembly of the virus is completed. Eventually, the progenitor virions are released externally with peeled keratinocytes. 40 In HeLa cells, partial copy of the HPV-18 genome is integrated at chromosome 8q24. 1

| Nested PCR validation
For the nested PCR assay, four primers HVP-424FW, HVP-747RV, HVP-530FW, and HVP-680RV (Table 1)   After cells were mixed and cultured for 24 hours, the mixed cell culture supernatants were collected for two round PCR. As illustrated in Figure 2, after the cells were mixed and cultured for 24 hours, the size of amplification product is about 150 bp that can be detected in the mixed culture supernatants when the proportion of HeLa was 1%, 10%, and 50%. The size of the amplified product was consistent with the target band of 151 bp ( Figure 2C), indicating that the HPV-18 sequence was successfully amplified. Without any optimization of amplification conditions, we were able to detect this target fragment by nested PCR using PCR kits from commercial suppliers ( Figure 2D).
We could not detect HeLa at low doping rate of 0%, 0.01%, and 0.1% after incubation of 48 hours (Figure 3). Even after 72 hours of incubation, no amplification products could be detected in the mixed cell culture supernatants when the proportion of HeLa was 0%, 0.01%, and 0.1%.
From these results, we estimated that the limit of detection sensitivity was between 0.1% and 1%. We then used seven cell lines: HepG2, AGS, A549, SNU-216, HCT-116, HGC-27, N87 mixed with Hela cells to repeat the assay. The proportion of HeLa was 0%, 1%, 10%, 50%, and 100%. After 24 hours incubation, the mixed cell supernatants were collected for PCR assay. The results of the experiment are shown in Figure 4. These experiments indicated that as long as the number of Hela cells reached to 1%, the 150 bp amplification product can be detected in the mixed culture supernatant after 24 hours of incubation.

| Sanger sequencing validation
We used Sanger sequencing to verify the PCR amplicons. We selected some of the PCR products from mixed cell supernatant at 1% doping rate for sequencing validation. The sequencing results were confirmed using NCBI blast. 50 The alignment showed that the sequencing results perfectly matched the sequence of 530-680 bp of HPV-18, which agreed with the expected PCR-amplified fragment. The section of the Sanger sequencing peak map is shown in

| Microscopic examination results
During the experiment, we randomly photographed Hela cells mixed with other cells at a 1% doping rate. By observing the morphology of the cells, we could not recognize that the cells had been contaminated with Hela cells (Figure 6). With the increase of cell passage times, Hela cells gradually outgrew the originally cells. If the morphology of the two cell type is close, the original cells may be completely displaced without notice and thus could lead to erroneous results.

| STR profiling compared with PCR method
In order to compare the sensitivity and convenience of STR profiling with the nested PCR method used in this study in detecting HeLa  in some experiments.
Compared with STR profiling, the PCR detection method adopted in this study had a much better sensitivity. Also, the process of STR Also, a certain number of cells need to be killed in order to extract genomic DNA, which may not be practical for precious or slow-F I G U R E 8 Workflow of nested PCR and STR for cell authentication growing cells. PCR assay of cell culture medium neatly obviates such need. Compared with traditional assays such as STR profiling, this method is a "noninvasive" type of detection that does not cause any damage to or loss of cells.
We can envisage that the fast, convenient, inexpensive, and easy to implement method we demonstrated in this study will allow scientists to simply collect culture supernatant for PCR amplification at any time during the process of culturing or passaging, and virtually in real-time monitoring cell culture contamination. This simple approach may be applied to other cell culture system in which a specific marker for contaminating cells is available.