**3. Discussion**

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 using one of these analytical techniques.

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 (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.

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

> Rapid; inexpensive; visual endpoint; useful for interspecies hybrids

Rapid, visual endpoint; useful for interspecies hybrids; HLA immunostaining enables detection of intra-species (human) fusion cells

Visual endpoint; useful for inter-species hybrids; can directly determine chromosomal makeup derived from the parental cells; useful for inter-species

Total DNA content is simple to measure; DNA content is useful in detecting intraas well as inter-species

Rapid; DNA barcoding is used for species of origin confirmation of cells

Rapid; STR profiling can enable authentication of human, dog, or mouse cells to the individual donor level; useful for authenticating mouse hybridoma cells

Detects inter- and intraspecies hybrid cells

hybrids

hybrid cells

Reagents no longer commercially available; results must be compared to a reference result; certain isoforms may be lost with continued passage of the hybrid

Authenticating Hybrid Cell Lines

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Requires species- or HLA type-specific immunostaining reagents and fluorescent microscopy; results must be compared to a reference result; certain surface antigens may be lost with continued passage of the

Preparing and interpreting karyotypes takes expertise; human chromosomes in inter-species hybrids are often lost with

Total DNA content in hybrid cells is often not stable with passage in culture as

cells

hybrid cells

passage in culture

chromosomes may be lost

Nested PCR must be created with species-specific primers for parental cells; preferential retention of mouse mitochondria in inter-species bybrid cells diminishes utility of this method

Not typically useful for inter-species hybrid cells; loss of heterozygosity during extended passage in culture may result in

Multiple genomic regions must be evaluated in hybrids containing few or single chromosomes from one of the

loss of STR alleles

parental cells

**Method Principle Advantages Disadvantages**

Isoenzyme analysis Electrophoretic

Immunostaining Surface antigens of

DNA content Fusion cells may

DNA barcoding Mitochondrial

Karyotyping (cytogenetic analysis)

mobilities of cytosolic enzymes in the fusion cell correspond to one or both parental cell

one or both parental cells may be retained

Chromosomes contributed by two parental cells may be directly observed in the fusion cell

contain more total DNA content than either parental cell

DNA sequences are conserved within

STR loci provide identity to the donor

Agnostic sequencing of variable DNA sequences such as STR or SNP arrays

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

species

STR profiling Multiple (8 or more)

Next generation sequencing

level

isoforms
