University of Pittsburgh Cancer Institute (UPCI)

Services

The Cell Culture and Cytogenetics Facility provides critical cell culture services including cell line authentication, Mycoplasma testing, primary cell culture of mammalian cells, and Epstein-Barr virus (EBV) transformation of human B-lymphocytes, and comprehensive, state-of-the-art cytogenetics services including assisting investigators with study design, performing cell culture, preparing probes, carrying out and interpreting classical and molecular cytogenetic analyses, and assisting in manuscript preparation.

The laboratory includes equipment for cell culture, cryopreservation, cytogenetic harvests, cytogenetic analysis of human and other mammalian cells, fluorescence in situ hybridization (FISH), molecular genetic procedures requisite for FISH, including polymerase chain reaction (PCR) amplification, fluorescence and light microscopy, as well as digital imaging.

Cell Culture Services

  1. Authentication and Quality Assurance (AQA) of Cultured Cell Lines
    The Facility offers cell line authentication and quality assurance (AQA), in accordance with the NIH-issued notice NOT-OD-08-017. It is recommended that these services be performed upon development or receipt of cell cultures, at the beginning of a study, at the end of a study, and every three months the cells are in culture. 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.
    1. DNA fingerprinting
      The minimum authentication includes DNA fingerprinting, which is done by examining microsatellite loci using a multiplex PCR reaction. The results are 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. Microsatellite analysis for identity testing to compare with the patient, original cell line and/or repository standards.

      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. Mycoplasma testing
      Mycoplasma testing is carried out using two methods at the same time point (ATCC recommendation): direct visualization by fluorescent stain (DAPI and/or Hoechst) along with positive and negative controls, and a highly sensitive test done by PCR followed by agarose gel electrophoresis, along with internal controls. All contaminated cultures should be treated or discarded, the latter according to University biosafety procedures.

      Sample requirements: One T-25 flask or two to three ≥35 mm wells or culture dishes of actively dividing cells.
    4. 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, 2-3 ≥35 mm wells, or culture dishes of actively dividing cells.

  2. Primary cell culture
    The CCCF offers primary cell culture of human and mammalian cells, including fibroblast-like cells, lymphocytes, and tumor cells necessary for research studies of investigators. Due to the challenges of primary cell culture, results are not guaranteed, but our skilled faculty and staff will do our best to succeed in developing the cell cultures required for your research studies. This includes availability of one fresh culture and three cryopreserved vials of cells for pickup at the facility when ready.

    Sample requirements: We recommend meeting with the CCCF faculty and staff when planning studies involving primary cell culture to optimize the probability of success and avoid surprises when the critical specimens become available.
  3. EBV Transformation of Human B-Lymphocytes
    The CCCF offers EBV transformation of B-cells as a service to University of Pittsburgh investigators. This includes availability of one fresh culture and three cryopreserved vials of cells for pickup at the facility when ready.

    Sample requirements: We recommend meeting with the CCCF faculty and staff when planning studies involving primary cell culture to optimize the probability of success and avoid surprises when the critical specimens become available. One ~8 ml sodium heparin vacutainer of blood drawn within 24 hours and delivered to the laboratory on a workday before 2pm. Please call the laboratory on the previous workday to schedule receipt of the impending specimen.

Cytogenetics Services

  1. Classical Karyotyping
    The CCCF provides karyotyping of human and other mammalian cells. This usually includes short-term cell culture, specialized cell harvesting after mitotic blockade (cells can be harvested 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, specimens, or cell lines. Classical chromosome analysis can be used to identify modal chromosome number, chromosome pattern, and/or designate karyotype signature in as many as 20 mitotic cells. Special staining and banding techniques, including C-, G11-, Q-, R-, DA/DAPI-, and/or AgNOR-banding can be added to identify marker chromosomes and/or the species of origin of the specimen, the origin of which may be unidentifiable by routine cytogenetic methods. Karyotyping of new cell lines is useful at baseline and then at intervals during experimentation to rule out cross-contamination and to validate the species of origin of cells grown on feeder layers or xenografted. Investigator will be provided with images of at least two metaphase cells or at least two karyotypes per clone, methods, results, and discussion of results, if appropriate.

    Sample requirements: One T-25 flask or two to three ≥35 mm wells or culture dishes of actively dividing cells. We recommend meeting with the CCCF faculty and staff when planning studies involving classical cytogenetic analysis to optimize the study design.
  2. Assessment of Chromosomal Instability
    Chromosomal instability assays are useful in a variety of situations, including testing the effects of environmental chemicals, new therapeutic regimens, cancer research studies, and DNA damage response studies. These assays usually involve cell harvesting after mitotic blockade, slidemaking, and conventional Giemsa staining of chromosomes followed by assessment of chromosome number in 150 cells, chromosomal breakage in 200 cells, or a similar assessment method using molecular cytogenetic analysis, depending on the specific goals of the research study. A report and selected digital images to illustrate the results will be provided to the investigator.

    Sample requirements: One T-25 flask or two to three ≥35 mm wells or culture dishes of actively dividing experimental and control cells (if appropriate). We recommend meeting with the CCCF faculty and staff when planning studies involving chromosomal instability assessment to optimize the study design.
  3. Molecular Cytogenetic Analysis
    The CCCF carries out chromosomal fluorescence in situ hybridization (FISH) using gene-, region-, virus- or chromosome-specific DNA probes 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 interphase FISH for examining the molecular karyotypes of archived (frozen, paraffin-embedded) or otherwise non-dividing cells. At least two images of each analysis will be provided along with a report including methods, results, and discussion of results, if appropriate.

    Sample requirements: We recommend meeting with the CCCF faculty and staff when planning studies involving molecular cytogenetic analysis to optimize the study design.

    See our Image Catalog for examples of some of the services provided.