*2.5.2. STR profiling*

sections, and the origin of individual chromosomes in metaphase spreads, and were able to

At the time of creation, hybrid cells contain a complement of chromosomes, a portion of which are attributable to one of either parental cells, while some may be of unknown origin (**Table 1**). Inter-species chromosomal rearrangements may also occur in somatic cell hybrids [26]. In many, but not all cases, loss of chromosomes attributed to one or the other parental

Measurement of total or nuclear DNA content is used in characterization of somatic cell hybrids, but is not necessarily intended as an identity test for authenticating such cells. The excess number of chromosomes present in certain cell hybrids relative to the parental cells (discussed above) is also reflected in an increase in DNA content in the hybrid cells. This increase may be detected using microspectrophotometric analysis for nuclear DNA [38] or flow cytometric analysis of propidium iodide-stained cells for total DNA [32]. For instance, while authenticating a series of human × bovine hybrid cell lines, van Olphen and Mittal [32] found that the total DNA content of the hybrid cells was 51–77% greater than the average

Levels of nuclear DNA in bovine × mouse hybrid cells corresponded to the relative increases in chromosome count for the hybrids [38]. A hybrid with mean chromosomal count of 53 (vs. parental values of 44 and 44) was found to have nuclear DNA content similar to the parental mouse cell, while a hybrid with mean chromosome count of 89 displayed a bimodal nuclear DNA content, with one peak similar to that of the parental mouse cell and another peak at

Both total DNA and chromosome count for a hybrid cell may evolve with continued passage of a culture, due to the propensity for loss of chromosomes derived from one or both parental

The ability to detect intra-species and inter-species cell hybrids has been greatly facilitated by the development of nucleic acid sequencing methods, such as DNA barcoding (PCR- or sequencebased approaches targeting mitochondrial genes), STR analysis, and next generation sequencing.

Ono et al. [48] used a nested PCR targeting the cytochrome b gene of 7 animal species (human, mouse, rat, rabbit, cat, cow, and pig) to authenticate two inter-species hybrid cell lines. These included the 4G12 hybridoma cell line (human B lymphocyte × mouse myeloma) and the N18-RE-105 hybridoma cell line (mouse glioma × rat neural retina). Cytoplasmic isoenzymes from the two hybridoma cell lines were found to display human- or rat-specific migration patterns in isoenzyme analysis, but yielded a result expected for mouse in the nested PCR (**Figure 3**). The authors concluded that the preferential retention of mouse mitochondria in

detect hybrid chromosomes consisting of both human and mouse DNA.

cell is experienced as the hybrid cells are cultured [26, 31, 32, 35, 36, 46].

**2.4. DNA content**

158 Cell Culture

value for the parental cells (**Table 1**).

cells, as mentioned above.

*2.5.1. DNA barcoding*

around twice the parental cell peak value [38].

**2.5. Nucleic acid sequence-based methods**

Chan et al. [49] used STR profiling to identify presumed intra-species human hybrid cell lines comprised of HeLa × EBV-negative NPC (Epstein-Barr virus-negative nasopharyngeal carcinoma) cells. Four EBV-negative NPC cell lines (CNE-1, CNE-2, HNE-1, and HNE-2) were found to have STR profiles similar to each other, and also shared at least one allele with HeLa across 16 STR loci, as well as additional alleles at several of the STR loci that were attributed to an unknown EBV-negative NPC cell. High-throughput RNA sequencing by Strong et al. [50] resulted in similar conclusions and raised similar concerns for three other EBV-negative NPC cell lines (HONE-1, AdAH, and NPC-KT).

Because these particular EBV-negative NPC cell lines have additional alleles, they do not satisfy the usual match criteria for human cell line authentication [51]. However, it is important to consider all available evidence when deciding if cross-contamination has occurred. The data from Chan et al. [49] showed that CNE-1, CNE-2, HNE-1, and HNE-2 carry an allelic variant (D13S317 13.3) that is characteristic of HeLa derivatives [51]. Strong et al. [50] showed that CNE-1, CNE-2, HONE-1, AdAH, and NPC-KT carry human papillomavirus 18 (HPV18), which is an unexpected finding for NPC cell lines, and display viral and cellular genomic rearrangements that are consistent with HeLa. Looking at all the evidence, it is reasonable to conclude that these EBV-negative NPC cell lines represent not simply cross-contamination with HeLa, but rather somatic cell hybridization with HeLa.

The mechanism responsible for somatic cell hybridization in these seven EBV-negative NPC cell lines is not known. Cell-cell fusion may have occurred between HeLa and an unknown NPC cell line through exposure to Sendai virus. NPC-KT, one of the cell lines investigated by Strong et al. [50], was established by using Sendai virus to fuse AdAH and primary NPC cells [52]. NPC-KT also carried EBV, which can cause cell–cell fusion in monolayer cultures [5, 53]. If the originating laboratory unknowingly performed this work on a culture that was cross-contaminated with HeLa, it may have resulted in a HeLa fusion cell line, which may have subsequently cross-contaminated other cell lines used by the NPC research community. HeLa cells also contain the gene for HPV18 viral protein E5, which is fusogenic [54]. The E5 protein of HPV16 is a fusogenic membrane protein and if expressed in two cells, the cells can fuse [55, 56]. So if HeLa and another cell line expressed the HPV18 analogue protein E5, this could have also induced fusion.

*2.5.3. Next generation sequencing*

**3. Discussion**

using one of these analytical techniques.

Inter-species and intra-species cell fusion may be detectable by next generation sequencing techniques because of the extensive amount of DNA sequencing and the unbiased selection of the DNA segments (i.e., not using species-specific primers for PCR or selection of DNA fragments). A difficulty may occur in genomic regions that are highly conserved between species for which only small sequence differences exist (e.g., a single or a few bases). In such cases, it may be difficult to determine whether the observed difference is a single nucleotide variation (SNV) between two species or between two samples from the same species. Also, SNPs/ SNVs are generally transitional sequence changes (i.e., either purine to purine or pyrimidine to pyrimidine) and may not provide sufficient information to determine whether a sample contains cells from two different species or from two different individuals of the same species. To overcome this difficulty, one must sequence DNA segments that are highly variable and unique to different individuals. These might include human, mouse [34], or rat [58, 59] STR and human [60, 61] or mouse SNP arrays [62, 63]. Multiple genomic regions must be evaluated for those cases in which a few or even a single chromosome from one species is retained

Authenticating Hybrid Cell Lines

161

http://dx.doi.org/10.5772/intechopen.80669

Hybrid cell lines represent a special problem for the various approaches that have been utilized for authentication up to now. Firstly, the endpoints that are used in cell authentication assays are ultimately, if not directly, dependent upon the genetic make-up of the cell. Intraspecies and inter-species hybrid cells are difficult to test because they contain an assortment of genetic material conferred from the two parental cell types during the fusion process. Thus, specific isoforms of enzymes, the presence or absence of surface antigens, chromosome count, and total DNA content are each subject to the assortment of genetic material that is present in the hybrid cell line following the fusion process. This difficulty also applies to the molecularbased methods that are so useful for determining the authenticity of cells. Thus, one cannot predict, in advance, the results that will be obtained during authentication of a hybrid cell

Secondly, not all of the genetic material in the hybrid cell is stable, as it is not uncommon for one or more chromosomes to be lost from hybrid cells on continued passage of the culture. This means that the authentication profile of a hybrid cell may evolve over time in culture.

Due to these considerations, a hybrid cell should be evaluated as soon as possible after fusion to get a baseline (reference) profile. Any drift or change in subsequent profiles may imply changes within the chromosome number or expression profiles. The profile of authentication resulting from one or more of these methods can then be used as a sort of fingerprint or reference against which subsequent authentication can be compared (as in authentication of a master cell bank or a working cell bank). Evolution of such a reference pattern may occur as chromosomes are lost, sometimes quite soon following fusion, from a hybrid during continued culture. This evolution typically involves the loss of characteristics of one of the parental cells

by the hybrid cell, as is the case with many hybridoma cell lines.

Yoshino et al. [33] used an STR profiling approach to authenticate a series of mouse cell lines, including an inter-strain hybrid (Balb/c mouse × C3H/He mouse) cell line. In this approach, F1 hybrid cells derived from the two parental mouse strains displayed different alleles at each locus, corresponding to the alleles contributed by the two parental strains. Loss of heterozygosity occurring during extended culture was thought to result in loss of one of the parental alleles at the D5 Mit201.1 locus of the dinucleotide STR marker (**Table 3**).

Almeida et al. [34] described results obtained during authentication of an intra-strain (Balb/c mouse) hybridoma (P3X63Ag8.653 × Balb/c mouse splenic cell). In this case, not unexpectedly, identical alleles were detected for eight of nine STR loci evaluated. At the mouse STR 9-2 locus, the parental P3X63Ag8.653 cell was heterozygous, displaying alleles with 15 and 16 repeats, while the hybrid cell contained only the allele with 15 repeats.

Authentication of hybridoma cell lines is difficult, because of the inbred rodent populations that are used for hybridoma generation. Koren et al. [57] proposed a unique solution using degenerate primers to amplify and sequence the variable regions of the monoclonal antibodies produced by their hybridoma cell lines. Because these regions are highly diverse, they can potentially be used to uniquely identify the hybridoma cell line from which a monoclonal antibody is generated. Koren et al. [57] used this approach to resolve a misidentified cell line in their own laboratory, but the method would be useful for any laboratory working with hybridoma cell lines.


**Table 3.** Authentication of an inter-strain hybrid (mouse) cell line by STR profiling (data from [33]).

#### *2.5.3. Next generation sequencing*

The mechanism responsible for somatic cell hybridization in these seven EBV-negative NPC cell lines is not known. Cell-cell fusion may have occurred between HeLa and an unknown NPC cell line through exposure to Sendai virus. NPC-KT, one of the cell lines investigated by Strong et al. [50], was established by using Sendai virus to fuse AdAH and primary NPC cells [52]. NPC-KT also carried EBV, which can cause cell–cell fusion in monolayer cultures [5, 53]. If the originating laboratory unknowingly performed this work on a culture that was cross-contaminated with HeLa, it may have resulted in a HeLa fusion cell line, which may have subsequently cross-contaminated other cell lines used by the NPC research community. HeLa cells also contain the gene for HPV18 viral protein E5, which is fusogenic [54]. The E5 protein of HPV16 is a fusogenic membrane protein and if expressed in two cells, the cells can fuse [55, 56]. So if HeLa and another cell line expressed the HPV18 analogue protein E5, this

Yoshino et al. [33] used an STR profiling approach to authenticate a series of mouse cell lines, including an inter-strain hybrid (Balb/c mouse × C3H/He mouse) cell line. In this approach, F1 hybrid cells derived from the two parental mouse strains displayed different alleles at each locus, corresponding to the alleles contributed by the two parental strains. Loss of heterozygosity occurring during extended culture was thought to result in loss of one of the parental alleles at the D5 Mit201.1 locus of the dinucleotide STR

Almeida et al. [34] described results obtained during authentication of an intra-strain (Balb/c mouse) hybridoma (P3X63Ag8.653 × Balb/c mouse splenic cell). In this case, not unexpectedly, identical alleles were detected for eight of nine STR loci evaluated. At the mouse STR 9-2 locus, the parental P3X63Ag8.653 cell was heterozygous, displaying alleles with 15 and 16

Authentication of hybridoma cell lines is difficult, because of the inbred rodent populations that are used for hybridoma generation. Koren et al. [57] proposed a unique solution using degenerate primers to amplify and sequence the variable regions of the monoclonal antibodies produced by their hybridoma cell lines. Because these regions are highly diverse, they can potentially be used to uniquely identify the hybridoma cell line from which a monoclonal antibody is generated. Koren et al. [57] used this approach to resolve a misidentified cell line in their own laboratory, but the method would be useful for any laboratory working with

Parental Balb/c 141.8 135.5 242.5 94.9 88.3 155.1 Parental C3H/He 185.1 123.8 236.4 92.5 78.4 140.1

**Table 3.** Authentication of an inter-strain hybrid (mouse) cell line by STR profiling (data from [33]).

Hybrid UV.CC3.11.1 142.0, 185.2 135.5, 123.9 242.6, 236.4 94.8 88.4, 78.5 155.2, 140.2

**D1 Mit159.1 D2 Mit395.1 D4 Mit170.1 D5 Mit201.1 D13 Mit256.1 D17 Mit51.1**

repeats, while the hybrid cell contained only the allele with 15 repeats.

**Strain/cell line Size of amplicons for alleles at STR loci**

could have also induced fusion.

marker (**Table 3**).

160 Cell Culture

hybridoma cell lines.

Inter-species and intra-species cell fusion may be detectable by next generation sequencing techniques because of the extensive amount of DNA sequencing and the unbiased selection of the DNA segments (i.e., not using species-specific primers for PCR or selection of DNA fragments). A difficulty may occur in genomic regions that are highly conserved between species for which only small sequence differences exist (e.g., a single or a few bases). In such cases, it may be difficult to determine whether the observed difference is a single nucleotide variation (SNV) between two species or between two samples from the same species. Also, SNPs/ SNVs are generally transitional sequence changes (i.e., either purine to purine or pyrimidine to pyrimidine) and may not provide sufficient information to determine whether a sample contains cells from two different species or from two different individuals of the same species. To overcome this difficulty, one must sequence DNA segments that are highly variable and unique to different individuals. These might include human, mouse [34], or rat [58, 59] STR and human [60, 61] or mouse SNP arrays [62, 63]. Multiple genomic regions must be evaluated for those cases in which a few or even a single chromosome from one species is retained by the hybrid cell, as is the case with many hybridoma cell lines.
