**5. Future perspectives**

Hybrid cell lines require new methods to ensure that cell-cell fusion is detected and such cultures can be authenticated to demonstrate their validity as research models. Detection of spontaneous cell-cell fusion is particularly important. Somatic cell hybridization may arise when using feeder layers in vitro or when working with patient-derived xenograft models in vivo. In many cases, cell–cell fusion is associated with increased tumorigenicity or the development of malignant behavior in adjacent cell populations [21, 22]. This has the potential to alter the behavior of patient-derived xenograft models, which may be used as the final step before a novel drug proceeds to clinical evaluation [64].

Current authentication methods are not always effective to detect hybrid cell lines. The advantages and limitations of the available methods are displayed in **Table 4**. Common to all methods is the fact that the chromosomal contributions from each parental cell are not possible to predict in advance. This means that chromosomal makeup and number and corresponding genetic information (e.g., surface antigens, HLA types, enzyme isoforms, alleles at STR loci) will be unique to each fusion cell. In hybrid cells, STR profiles become more

(be it parental chromosome(s), species-specific isoenzyme bands or hybrid bands, loss of heterozygosity in SNP or STR profiles, loss of surface staining characteristic of one of the parental cells, etc.). On the other hand, gains in chromosome counts, addition of new isoenzyme bands, appearance of new alleles in SNP or STR profiles, or increases in DNA content with time in culture would not be expected, and such would be considered a red flag during authentication. For instance, such a result might indicate the presence of a cross-contaminating cell type.

• Fusion of two different cell types to create an inter-species or intra-species hybrid results in an unpredictable assortment of genetic material derived from one or the other parental

• The outcome of the fusion process in terms of genetic content contributed by the two parental cells will impact the results of the methodologies typically used for determining

• Isoenzyme analysis may indicate the presence of electrophoretic bands migrating as expected for one parental cell, the other parental cell, or bands migrating differently than

• Hybrid cells may retain surface antigens characteristic of one parental cell or the other, or

• Chromosome number (total, and number derived from one parental cell or the other) and

• Human chromosomes found in inter-species hybrid cells tend to be unstable, and are often

• Regardless of the authentication method to be used, it is recommended that a baseline evaluation be performed as soon as possible after the fusion process used to create the hybrid cell, and that the result be used as a reference against which future authentication

Hybrid cell lines require new methods to ensure that cell-cell fusion is detected and such cultures can be authenticated to demonstrate their validity as research models. Detection of spontaneous cell-cell fusion is particularly important. Somatic cell hybridization may arise when using feeder layers in vitro or when working with patient-derived xenograft models in vivo. In many cases, cell–cell fusion is associated with increased tumorigenicity or the development of malignant behavior in adjacent cell populations [21, 22]. This has the potential to alter the behavior of patient-derived xenograft models, which may be used as the final

**4. Executive summary**

162 Cell Culture

cell into the hybrid.

cell line authenticity.

of both.

expected for either parental cell.

lost over time in culture.

results may be compared.

**5. Future perspectives**

total DNA content will vary from hybrid to hybrid.

step before a novel drug proceeds to clinical evaluation [64].


**Table 4.** Limitations and advantages of methods for authenticating hybrid cells.

complex and difficult to interpret, while mitochondrial-based methods may not be effective for species detection if mitochondria from one species are retained preferentially (as is true for the mouse). However, many methods can be optimized to allow for detection of cellcell hybrids. For example, SNP genotyping is increasingly used for cell line authentication and has been used as a test method for patient-derived xenograft models [64]. SNP panels could be modified to include species-specific marker sets, or human and mouse SNP panels could be run in parallel to confirm species and strain identifications and search for additional markers. Although this type of comprehensive assessment is not usually performed, it could be incorporated into testing pipelines if laboratories are aware of the spontaneous (unintentional) cell fusion issue and look specifically for such markers of cell fusion. A role has been suggested for cell-cell fusion in cancer, stem cell plasticity, and trans-differentiation [17–20, 54, 59, 65–67]. A better set of tools is needed to explore hybrid cell lines and the role of somatic cell hybridization in health and disease.

**Author details**

Raymond W. Nims<sup>1</sup>

\*, Amanda Capes-Davis<sup>2</sup>

\*Address all correspondence to: rnims@rmcpharma.com

Westmead, New South Wales, Australia

Cellular Medicine. 2012;**1**:75-87

fusion. FEBS Letters. 1981;**133**:169-174

Biology. 1982;**67**:165-182

Biochimica et Biophysica Acta. 1982;**694**:227-277

4 ATCC, Manassas, VA, USA

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Authenticating Hybrid Cell Lines

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