Services and Techniques

Authentication of Cultured Cell Lines (per NOT-OD-08-017)

Authentication and quality assurance (AQA) of cell lines is a service offered by The University of Pittsburgh Cancer Institute Cell Culture and Cytogenetics Facility. “All cell lines examined in this study will undergo authentication and quality assurance (AQA) 1) upon development or receipt of the cell cultures, 2) at the beginning of the study, 3) at the end of the study, 4) and every three months the cells are in culture as required by the NIH. AQA, as recommended by American Type Culture Collection (ATCC), includes at least DNA fingerprinting, but may also include karyotyping to rule out cross-contamination, Mycoplasma testing, and species identification, which will be carried out in the UPCI Cell Culture and Cytogenetics Facility.”

1) DNA fingerprinting

  • The minimum authentication will include DNA fingerprinting, which is done by examining microsatellite loci using a multiplex PCR reaction. The results will be compared to the fingerprint of the earliest passage of the cell line or original tumor or fibroblast DNA from the patient. Sixteen STR loci are studied using the AmpFℓSTR® Identifiler®. Loci include: Amelogenin, CSFIPO, D13S317, D16S539, D18S51, D19S433, D21S11, D2S1338, D3S1358, D5S818, D7S820, D8S1179, FGA, TH01, TPOX, and V WA. Cell lines with fingerprints that differ from the original should be retested using an earlier passage or a new cell line obtained and tested for consistency prior to continuation of a study.

  • Sample requirements: 10µl DNA in a 1.5 ml microfuge tube.

2) Karyotyping to rule out cross-contamination

  • Karyotyping should be carried out upon thawing cell cultures. The earliest baseline karyotype available on a cell line should be compared with the karyotype of the current experimental cultures.

  • Sample requirements: One T-25 flask or two to three ≥35 mm wells or culture dishes of actively dividing cells.

3) Species identification

  • If indicated, identification of the species of a cell culture can be carried out on metaphase spreads without preparation of a complete karyotype. This procedure can be beneficial for ruling out cross-species cell culture contamination, validating the species of origin of cells grown on feeder layers, or verification of the species of origin of cell cultures after xenografting.

  • Sample requirements: One T-25 flask two to three ≥35 mm wells or culture dishes of actively dividing cells.

Cytogenetic Services

  • Classical karyotyping of human and mammalian cell cultures

  • Monitoring of established cell lines using classical and/or molecular cytogenetic techniques to rule out interspecies culture contamination.

  • Special studies using a variety of banding techniques, including G-, C-, G11-, Q-, R-, DA/DAPI-, and AgNOR-banding.

  • Molecular cytogenetic analysis, including:

    1. chromosomal fluorescence in situ hybridization (FISH) for mapping cancer-related genes, transgenes, viral integration sites, and oncogene amplifications and to identify marker chromosomes, the origin of which is unidentifiable by classical cytogenetic methods, and

    2. interphase FISH for examining the molecular karyotypes of archived (frozen, paraffin-embedded) or otherwise nondividing cells.

  • Primary cell culture of human and mammalian cells and other cell culture as required for the analyses listed above.

  • Quality assurance (QA) of cell lines in conjunction with the BGPF. The Cytogenetics Facility carries out two parts of the three-part UPCI Cell Line QA Standard Operating Procedure:

    1. Karyotyping to rule out interspecies cross-contamination and karyotypic evolution;

    2. Mycoplasma testing by two methods, as recommended by the ATCC (PCR-based detection and direct visualization by staining); and

    3. The BGPF carries out microsatellite analysis for identity testing to compare with the original cell line and/or repository standards.

Cytogenetic Techniques

  • Chromosome harvesting (directly from research biopsies, blood samples, bone marrow aspirations, or following monolayer or suspension cell culture), slidemaking, and standard trypsin-Giemsa banding of mitotic tumor cells, embryonic stem cells, or cell lines.

  • Classical chromosome analysis to identify modal chromosome number, chromosome pattern, and designate karyotype signature.

  • Use of special stains and banding techniques, including C-, G11-, Q-, R-, DA/DAPI-, and AgNOR-banding to identify marker chromosomes and/or the species of origin of the specimen, the origin of which may be unidentifiable by routine cytogenetic methods.

  • Chromosomal instability assays for aneuploidy, chromosome breakage, etc.

  • Chromosomal FISH using gene-, region-, virus- or chromosome-specific DNA probes to map genes, transgene or viral insertion sites, or oncogene amplifications or to identify marker chromosomes that cannot be identified readily by classical cytogenetic methods

  • Interphase FISH for examining the molecular karyotypes of archived or otherwise nondividing cells, after preparation of nuclei from paraffin blocks or fresh or frozen tissues

  • Comparative genomic hybridization to identify regions characterized by genomic gain or loss.

  • Microscopic analysis, photomicrography, and/or digital imaging requisite for documenting the results of these studies.

Cell Culture and Cytogenetics Facility
University of Pittsburgh
Department of Human Genetics
3060 Parran Hall Lab Annex-5C
130 De Soto Street
Pittsburgh PA, 15261

412-624-5358 (Laboratory)
412-624-5390 (Dr. Gollin's Office)