**1. Introduction**

Fusion of cells occurs normally in vivo, such as of muscle cells, bone cells, macrophages, during fertilization of germ cells, and in placenta formation [1]. Somatic cell hybrid cell lines (or more simply hybrid cell lines) are cell lines that arise through intentional or nonintentional fusion of somatic cells having different origins [2]. Intra-species and inter-species (cross-species) cell fusions have been described since the 1950s [3] and can occur either spontaneously or can be mediated by human oncogenic viruses (such as Sendai virus, Epstein-Barr virus, human papilloma viruses, hepatitis B and C viruses, human T-cell lymphotropic virus type 1

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

(HTLV-1), herpesviruses-8/Kaposi sarcoma herpesvirus (HHV-8/KSHV)) [4, 5], polyethylene glycol [6], or electrical pulses (electrofusion) [7–12], resulting in viable syncytial cells (giant cells or polykaryotes) with hybrid genotypes, namely heterokaryons. Mouse-human heterohybridoma technology has advanced significantly with the use of electrofusion technology [13]. Currently, electrically induced cell fusion is also being used to develop cancer cells with increased immunogenicity by fusion with dendritic cells for development of anti-tumor vaccines [14, 15].

**2. Methodologies for authenticating hybrid cell lines**

Isoenzyme analysis was one of the first methods to be used (as early as 1970s) for determining the species-level identity of cell lines. This method is still being used [39], despite the fact that reagents for performance of the method are not commercially available. There is a considerable amount of historical data, in the public domain, for non-hybrid and hybrid cell lines, therefore, discussion of these results has relevance in deciphering hybrid cell line identities.

Authenticating Hybrid Cell Lines

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In isoenzyme analysis, the gel electrophoresis banding patterns and relative migration distances of intracellular enzyme isoforms are used to confirm the expected animal species of origin for test cells. Normalized migration distances obtained for the set of enzymes evaluated are compared to a set of tabular values for various animal species, and through a process of elimination, the most likely animal species of origin for the test cell is determined. Although the results of isoenzyme analysis historically have been used to confirm species-level (intraspecies) identity of a cell line, the method also can be used to demonstrate the existence of an inter-species cell mixture [40] or to authenticate inter-species hybrid cell lines [27, 31–32,

When evaluating inter-species cell mixtures using isoenzyme analysis, bands migrating as expected for each of the parental species comprising the mixture are observed, provided that a sufficient percentage of cells of both species are present in the mixture [31, 38–41]. In the case of inter-species hybrid cell lines, however, a variety of possible outcomes may be obtained when authenticating using isoenzyme analysis. These outcomes might include, for instance, bands for certain enzymes that migrate as expected for both parental species or for only one of the two parental species, or bands that migrate differently than expected for either parental

As is evident from **Figure 1**, interpretation of an isoenzyme analysis electropherogram for a hybrid cell is not as straightforward as it is for a cell mixture. The chromosomes contributed to the hybrid cell by the two parental cells determine the outcome of the isoenzyme analysis results for any given enzyme, as the genes encoding the enzymes evaluated in this method are scattered among the various chromosomes of the various animal species [37]. In fact, isoenzyme analysis was performed commonly in early gene mapping studies because linkage between genes encoding an isoenzyme and a gene of interest could be used to assign the chromosomal location for the gene of interest. Due to uncertainty of the assortment of parental chromosomes (and encoded enzyme genes) into a hybrid cell, it is not possible to predict in advance the phenotype and, therefore, the electrophoretic characteristics of enzymes being evaluated using isoenzyme analysis. This is depicted well by the results of authentication of a

series of human × bovine hybrid cell lines (**Table 1**) by van Olphen and Mittal [32].

Authenticating an intentionally created hybrid cell line using isoenzyme analysis, therefore, entails evaluation of the hybrid as soon as possible after fusion of the parental cells. The migration patterns displayed by the enzymes evaluated are then considered to be the reference pattern to be expected for the hybrid cell during subsequent authentication assays. This is similar to the case for DNA fingerprinting. When reviewing historical data of cell line

**2.1. Isoenzyme analysis**

36–38, 41, 42].

species (**Figure 1**).

Cell fusion can be important in the establishment and evolution of cell lines (e.g., [16]) and can lead to cancer progression and metastasis via genetic instability [17–20]. Hybrid cell lines also can arise spontaneously. Numerous examples have been documented [21], including a case where a patient-derived xenograft model underwent spontaneous fusion with normal mouse stromal cells, forming a hybrid cell that was more tumorigenic than the parental lines [22]. Spontaneous cell-cell fusion can act as a mechanism for DNA exchange between malignant and non-malignant cells and for horizontal transmission of malignancy [21, 23]. Spontaneous cell-cell fusion can be challenging to detect. Some cases are only detected incidentally—for example, when unexpected chromosomes are detected during cytogenetic analysis [24].

Intentionally created hybrid cell lines have been used for a variety of purposes, including monoclonal antibody production by mouse × mouse and mouse × human hybridomas [25], gene mapping studies [26], studies of gene expression [27], study of cancer initiation, progression, and metastasis [21, 23, 24, 28, 29], evaluation of drug resistance mechanisms [30]; as well as in the field of virology [31, 32]. Perhaps the most commonly employed inter-species hybrid cell lines, currently, are mouse × human somatic cell hybrids. Examples of intra-species cell hybrids might include mouse × mouse (inter-strain) hybrid cells [33] or hybridomas created by fusion of mouse splenic cells and mouse myeloma cells [34].

Hybrid cell lines can be challenging to authenticate and to confirm that they are valid research models. This paper reviews historical and more recent technologies that have played a role in the authentication of inter-species and intra-species hybrid cell lines. As part of cell line authentication, the identity of a cell line is expected to be established to the species level, or if possible, to the individual donor level. There are a variety of approaches that may be used for this purpose. Over the years, these have included isoenzyme, cytogenetic, and immunological analyses, and more recently, a variety of molecular methods such as restriction length fragment polymorphism (RFLP), nested PCR analysis of mitochondrial genes, short tandem repeat (STR) profiling, single nucleotide polymorphism (SNP) profiling, sequence-based human leukocyte antigen (HLA) typing, and next generation sequencing. Each of these approaches may also be applicable to the authentication of intra-species or inter-species hybrid cell lines, although the results to be expected and, therefore, the interpretation of such results in arriving at the identification of the cell line may differ from those for non-hybrid cell lines. Interspecies hybrid cell lines have a propensity to lose chromosomes during continued passage of the cultures [18]. Such loss occurs especially in the case of hybrids of human and rodent cells, such as human × mouse or human × rat hybrids. In these cases, the human chromosomes tend to be lost with continued passage of the cultures. The results expected to be obtained with several of the authentication techniques mentioned below tend, therefore, to evolve over time as the hybrid cells are cultured [35–38]. This is especially true in the case of karyotyping and total DNA content, but also may impact isoenzyme analysis and molecular-based methods.
