Culture Collections

Authenticating hybrid cell lines

STR profiling

A lesson in cell line authentication

Authentication of cell lines is important in scientific research to ensure that you are not working with cells that are misidentified, cross contaminated, or that differ genetically from the original stocks.

It is a fundamental part of good cell culture practice and is increasingly required to secure funding for research and for publication in scientific journals.

At ECACC, we authenticate all our Human cell lines by Short Tandem Repeat (STR) profiling and our animal cell lines via DNA barcoding. 

 

Hybrid cell lines

Hybrid cell lines arise through fusion of somatic cells having different origins. They can be difficult to identity as the authentication results may not be as expected for the two parental cell types; they may display characteristics of one of the parental cell types or both (refer to article ‘Authenticating Hybrid Cell Lines’ by Nims et al: DOI: 10.5772/intechopen.80669).

ECACC has a series of four human electrofused hybrid cell lines. A researcher contacted us with concerns that the human cell line 1.1B4 also contained rat IL-22 cDNA sequences (a receptor involved in insulin response). This initiated an investigation into these electrofused cell lines to confirm their authenticity. Please note 1.1B4 and 1.1E7 were withdrawn from distribution at the start of this investigation.

Background

Three human insulin secreting cell lines were deposited into the ECACC catalogue in 2010:

  • 1.1B4
  • 1.1E7
  • 1.4E7

These cell lines were generated by the electrofusion of primary human pancreatic Βeta cells and with the immortal human epithelial PANC-1 cells. The original publication (McClusky et al, The Journal of Biological Chemistry, Vol 286, pp.21982-21992) noted the resulting cells retained the immortal phenotype of the PANC-1 cell lines but were responsive to glucose and secreted insulin.

In addition to the above three cell lines, another human electrofused cell line was deposited (1.2B4). This was formed by the electrofusion of primary human pancreatic Βeta cells with the human pancreatic carcinoma cell line HuP-T3.

Finally, a further three electrofused cell lines, the BRIN series, were deposited. These were generated by the electrofusion of primary rat pancreatic Βeta cells with cell line RINm5F, derived from a NEDH rat insulinoma:

  • BRIN BD11
  • BRIN BG5
  • BRIN BG7 

 

History of Quality Control (QC)

On receipt of these cell lines ECACC performed full QC including STR profiling and DNA barcoding.

The human electrofused cell lines 1.1B4, 1.1E7, 1.4E7 and 1.2B4 generated human STR profiles for all four cell lines. For 1.1B4, 1.1E7 and 1.4E7 the profiles matched that for parental cell line cell line PANC-1 and for 1.2B4 the STR profile matched that for parental cell line cell line HuPT3.

Isoenzyme analysis and subsequent DNA barcoding of the mitochondrial Cox-1 gene confirmed 1.1B4, 1.1E7, 1.4E7 and 1.2B4 to be of human origin, and the BRIN series cell lines were confirmed to be of rat origin.

 

Summary of investigation

  • Interspecies testing confirmed the presence of:
    • human and rat nuclear DNA sequences in 1.1B4 and 1.1E7, but only human cytoplasmic sequences
    • only human DNA in 1.4E7 and 1.2B4
    • only rat DNA in the BRIN series
  • Rat STR profiling generated profiles for 1.1B4 and 1.1E7; the profiles were the same in both
  • Rat STR profiling generated profiles for the BRIN series; they were almost identical but were different to those generated for 1.1B4 and 1.1E7 and therefore the BRIN cell lines are not the cause of the presence of rat DNA in 1.1B4 or 1.1E7.

 

Conclusions

STR profiling found that the 1.1B4 cell line contained human nuclear DNA; this test will detect human nuclear DNA but will not detect the presence of contaminating DNA from a different species. The DNA barcode also yielded a clean trace for human DNA.

Electrofusion of cell lines is supposed to produce a heterokaryotic line, that is nuclear DNA representing both the recipient and donor cell line. A mixed cytoplasm may also be expected.

This would have been expected to be picked up on the DNA barcoding as a mixed electropherogram. However, it has been reported in the literature that hybrid cells resulting from the fusion of parental cells of two different species will initially contain cytoplasm and mitochondria from both species but that the mitochondria from one species will quickly dominate.

 

Recommendations

DNA barcoding, an assay which amplifies mitochondrial DNA sequences to determine the species of the cell line, cannot be used to assess if a cell line has mitochondrial DNA from two different species.

Interspecies testing, an assay which will test for the presence of human, mouse, rat, Chinese hamster and African green monkey nuclear DNA sequences in a cell line, will be introduced into the ECACC QC procedures as part of the authentication of hybrid cell lines.

 

References

1. Raymond W. Nims, Amanda Capes-Davis, Christopher Korsch and Yvonne A. Reid. Authenticating Hybrid Cell Lines. DOI: 10.5772/intechopen.80669.

2. Barbara Attardi and Giuseppe Attardi. Fate of Mitochondrial DNA in Human-Mouse Somatic Cell Hybrids. Proc. Nat. Acad. Sci USA. Vol 69, No1, pp. 129-133, January 1972.

 

Written by Edward Burnett, Culture Collections Scientific Development Group Project Manager 

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