**2.1. Isoenzyme analysis**

(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

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

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.

by fusion of mouse splenic cells and mouse myeloma cells [34].

vaccines [14, 15].

152 Cell Culture

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.

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, 36–38, 41, 42].

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 species (**Figure 1**).

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

**Figure 1.** Isoenzyme analysis of (A) lactate dehydrogenase; and (B) 6-phosphogluconate dehydrogenase in parental cells, a cell mixture, and a hybrid cell. Lane 1: parental rat-SV40 cell; lane 2: hybrid H3 (rat-SV40 × mouse 3T3 TK−); lane 3: mixture of rat-SV40 and mouse 3T3 TK−; lane 4: parental mouse 3T3 TK− (from [31]). The black lines in each lane indicate the origins (the slots in the wells into which the protein is loaded).

In the case of an inter-species cell mixture, immunostaining reagents directed against conserved surface antigens of each parental species would each be expected to demonstrate reactivity. In the case of inter-species hybrids, the result that might be obtained is not so easily predicted in advance. For instance, as shown in **Table 1**, the surface antigens that are actually detected in a set of hybrids may be derived from only one or the other of the parental species

Malate dehydrogenase Human Bovine Human Bovine + Bovine + Lactate dehydrogenase Human Bovine Human +<sup>c</sup> Bovine + Bovine + Nucleoside phosphorylase Human Bovine Human Bovine Bovine

Total DNA content 171.1 ± 5.3 166.0 ± 4.9<sup>d</sup> 254.7 ± 6.8 297.7 ± 10.3 288.6 ± 7.5

Total chromosomes includes human, bovine, and unidentified chromosomes.

**Table 1.** Authentication results for three hybrid (human × bovine) cell lines (data from [32]).

Bands expected for bovine were observed, along with extra bands.

Bands expected for human were observed, along with extra bands. <sup>d</sup>Mean ± standard deviation, units are relative DNA content.

It is also possible, depending upon the species-specific antisera employed and the chromosomal make-up of the hybrid cell, for an inter-species hybrid cell to display surface staining for antigens of both parental species. For instance, Kano et al. [43] reported that all human × mouse hybrids evaluated in their study displayed mouse surface antigens, while most but not all also displayed human surface antigens. Surface antigens characteristic of both parental species were displayed by all human × hamster and human × rat hybrid cells evaluated. Gallagher et al. [44] reported similar results in their analysis of the surface antigens in human [HeLa] × mouse hybrid [3T3.4E] cells. Surface antigens characteristic of both parental cells

It is also possible that by the staining of interspecies hybrid cells, one may detect a surface antigen that is not expressed by either parental cell. For instance, van Someren et al. [41]

(**Figure 2**).

were displayed by the hybrid cells.

**Method Parental** 

Immunofluorescent staining for surface

Number of human chromosomes

Number of bovine chromosomes

Total number of chromosomesa

*Isoenzyme analysis* Glucose-6-phosphate dehydrogenase

*Flow cytometry*

a

b

c

antigen

*Karyotyping*

**293-Puro**

**Parental MDBK-Neo** **Hybrid cell line**

Human Bovine Human Bovine Bovine

62 0 71 27 19

0 60 4 48 47

62 60 97 113 103

Human Bovine Human Bovine +<sup>b</sup> Bovine +

**BHH2C BHH3 BHH8**

Authenticating Hybrid Cell Lines

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

authentication by isoenzyme analysis, allowance must be made for loss of parental chromosomes during extended culture, as this may result in loss of electrophoretic bands associated with certain enzyme isoforms over time. Reagents for performing isoenzyme analysis are no longer commercially available, so other methods to be described below are now more commonly being used for hybrid cell authentication.

#### **2.2. Immunostaining for surface antigens**

The species-level identification of cells through use of antisera directed against species-specific cell surface markers has also been applied to the authentication of inter-species cell hybrids [32].


b Bands expected for bovine were observed, along with extra bands.

c Bands expected for human were observed, along with extra bands.

<sup>d</sup>Mean ± standard deviation, units are relative DNA content.

authentication by isoenzyme analysis, allowance must be made for loss of parental chromosomes during extended culture, as this may result in loss of electrophoretic bands associated with certain enzyme isoforms over time. Reagents for performing isoenzyme analysis are no longer commercially available, so other methods to be described below are now more com-

**Figure 1.** Isoenzyme analysis of (A) lactate dehydrogenase; and (B) 6-phosphogluconate dehydrogenase in parental cells, a cell mixture, and a hybrid cell. Lane 1: parental rat-SV40 cell; lane 2: hybrid H3 (rat-SV40 × mouse 3T3 TK−); lane 3: mixture of rat-SV40 and mouse 3T3 TK−; lane 4: parental mouse 3T3 TK− (from [31]). The black lines in each lane indicate

The species-level identification of cells through use of antisera directed against species-specific cell surface markers has also been applied to the authentication of inter-species cell hybrids [32].

monly being used for hybrid cell authentication.

the origins (the slots in the wells into which the protein is loaded).

154 Cell Culture

**2.2. Immunostaining for surface antigens**

**Table 1.** Authentication results for three hybrid (human × bovine) cell lines (data from [32]).

In the case of an inter-species cell mixture, immunostaining reagents directed against conserved surface antigens of each parental species would each be expected to demonstrate reactivity. In the case of inter-species hybrids, the result that might be obtained is not so easily predicted in advance. For instance, as shown in **Table 1**, the surface antigens that are actually detected in a set of hybrids may be derived from only one or the other of the parental species (**Figure 2**).

It is also possible, depending upon the species-specific antisera employed and the chromosomal make-up of the hybrid cell, for an inter-species hybrid cell to display surface staining for antigens of both parental species. For instance, Kano et al. [43] reported that all human × mouse hybrids evaluated in their study displayed mouse surface antigens, while most but not all also displayed human surface antigens. Surface antigens characteristic of both parental species were displayed by all human × hamster and human × rat hybrid cells evaluated. Gallagher et al. [44] reported similar results in their analysis of the surface antigens in human [HeLa] × mouse hybrid [3T3.4E] cells. Surface antigens characteristic of both parental cells were displayed by the hybrid cells.

It is also possible that by the staining of interspecies hybrid cells, one may detect a surface antigen that is not expressed by either parental cell. For instance, van Someren et al. [41]


**2.3. Karyotypic (cytogenetic) analysis**

parental species or the other [26, 32, 36, 46, 47].

Karyotypic analysis of inter-species hybrid cells enables an investigator to visualize the rearrangement and addition/deletion of chromosomes that are typically (but not always) observed in such hybrids as a result of the fusion of the two parental cells (**Tables 1** and **2**). The analysis may amount to determination of modal chromosome number and/or the range of chromosomal numbers observed in a set of metaphase spreads [38, 45]. The chromosomes comprising the karyotype may also be analyzed for morphology (telocentricity, acrocentricity, and banding pattern analysis, see **Scheme 1**), enabling assignment of chromosomes to one

**Parameter Parental cell lines Hybrid cell line**

Chromosomes (mean ± standard deviation) 38 ± 3 38 ± 2 84 ± 7 HLA type A2 A1,2 A1,2

**Table 2.** Authentication of a human × human hybridoma cell line using HLA typing (data from [46]).

**GM15006TGOB ECEBV GMEC-101**

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B18 B5,17 B17 C7 C6,7 C7 Bw6 Bw4 Bw4,6 DR5 DR6 DR5,6

Jacobsen and co-workers [22] used fluorescence in situ hybridization (FISH) to demonstrate inter-species cell hybrids between human breast cancer and mouse stromal cells in patientderived xenografts. The authors labeled human and mouse Cot-1 DNA (enriched in repetitive DNA sequences) with different fluorophores and used these as FISH probes. They were able to highlight the origin of individual nuclei in formalin-fixed, paraffin-embedded tissue

**Scheme 1.** Part 1. Schema for chromosomal structure. Part 2. Human × mouse karyotype from [36]. The arrow indicates

the single human submetacentric chromosome among numerous telocentric mouse chromosomes.

**Figure 2.** Use of immunostaining against species-specific surface antigens to characterize parental human [293-Puro] and bovine [MDBK-Neo] cells, and three human × bovine hybrid cell lines [BHH2C, BHH3, and BHH8]. Antisera are designed α-human (anti-human), α-bovine (anti-bovine), and α-mouse (anti-mouse). The latter was used as a negative control reagent (from [32]).

examined a large number of human × Chinese hamster hybrid cells using human leukocyte antigen (HLA) typing antisera. The parental human cells exhibited reactivity against HLA typing serum 3 only. Most of the hybrids evaluated retained reactivity against this typing serum, and a subset of these displayed reactivity against one or more additional typing sera (i.e., sera 1, 2, 9, and 10) for which the human parental cell was negative.

Immunostaining for HLA antigens may be used for detecting hybrids of parental human cells with differing HLA types, therefore conferring limited utility of this approach for detecting intra-species (human × human) cell hybrids [44, 45]. The results of HLA typing of a human × human hybridoma cell line [46] are displayed in **Table 2**.

As with isoenzyme analysis, immunostaining for surface antigens should be performed as soon as possible once a hybrid cell is created. The results may not in all cases remain the same throughout management of the cell culture over time. For instance, a loss of one or more of the parental surface antigen reactivities may coincide with loss of chromosomal material, and perhaps function, with time in culture.


**Table 2.** Authentication of a human × human hybridoma cell line using HLA typing (data from [46]).

#### **2.3. Karyotypic (cytogenetic) analysis**

examined a large number of human × Chinese hamster hybrid cells using human leukocyte antigen (HLA) typing antisera. The parental human cells exhibited reactivity against HLA typing serum 3 only. Most of the hybrids evaluated retained reactivity against this typing serum, and a subset of these displayed reactivity against one or more additional typing sera

**Figure 2.** Use of immunostaining against species-specific surface antigens to characterize parental human [293-Puro] and bovine [MDBK-Neo] cells, and three human × bovine hybrid cell lines [BHH2C, BHH3, and BHH8]. Antisera are designed α-human (anti-human), α-bovine (anti-bovine), and α-mouse (anti-mouse). The latter was used as a negative

Immunostaining for HLA antigens may be used for detecting hybrids of parental human cells with differing HLA types, therefore conferring limited utility of this approach for detecting intra-species (human × human) cell hybrids [44, 45]. The results of HLA typing of a human ×

As with isoenzyme analysis, immunostaining for surface antigens should be performed as soon as possible once a hybrid cell is created. The results may not in all cases remain the same throughout management of the cell culture over time. For instance, a loss of one or more of the parental surface antigen reactivities may coincide with loss of chromosomal material, and

(i.e., sera 1, 2, 9, and 10) for which the human parental cell was negative.

human hybridoma cell line [46] are displayed in **Table 2**.

perhaps function, with time in culture.

control reagent (from [32]).

156 Cell Culture

Karyotypic analysis of inter-species hybrid cells enables an investigator to visualize the rearrangement and addition/deletion of chromosomes that are typically (but not always) observed in such hybrids as a result of the fusion of the two parental cells (**Tables 1** and **2**). The analysis may amount to determination of modal chromosome number and/or the range of chromosomal numbers observed in a set of metaphase spreads [38, 45]. The chromosomes comprising the karyotype may also be analyzed for morphology (telocentricity, acrocentricity, and banding pattern analysis, see **Scheme 1**), enabling assignment of chromosomes to one parental species or the other [26, 32, 36, 46, 47].

Jacobsen and co-workers [22] used fluorescence in situ hybridization (FISH) to demonstrate inter-species cell hybrids between human breast cancer and mouse stromal cells in patientderived xenografts. The authors labeled human and mouse Cot-1 DNA (enriched in repetitive DNA sequences) with different fluorophores and used these as FISH probes. They were able to highlight the origin of individual nuclei in formalin-fixed, paraffin-embedded tissue

**Scheme 1.** Part 1. Schema for chromosomal structure. Part 2. Human × mouse karyotype from [36]. The arrow indicates the single human submetacentric chromosome among numerous telocentric mouse chromosomes.

sections, and the origin of individual chromosomes in metaphase spreads, and were able to detect hybrid chromosomes consisting of both human and mouse DNA.

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