Cell Culture Quality Control

The Coriell Cell Repositories has developed and maintains preventative and surveillance procedures to guarantee that the NIGMS Human Genetic Cell Repository (HGCR) distributes highly-characterized, viable, and contaminant-free cell cultures.

All cell cultures are grown and frozen in antibiotic-free media to aid in the detection and prevention of contamination. Cultures are tested and found free of mycoplasma, bacteria, and fungi during expansion, at the time of frozen storage, and after recovery of stock for distribution from liquid nitrogen. Lymphoblast cultures are further tested for the presence of Human Immunodeficiency Virus (HIV), and recovered cultures are also examined to determine viability and growth potential and to verify species of origin.

Cytogenetic Analyses

Chromosomal analysis supports the molecular quality control of DNA preparations. Cytogenetic analysis assures that the chromosomal/genomic characteristics of a DNA or cell line sample has been retained. The analysis of chromosomes in human development and disease is accomplished through classical cytogenetic procedures (such as G-banding) combined with advanced molecular techniques, such as FISH and genomic microarray analysis. G-banded metaphases are prepared from aliquots of cells from each large scale expansion of a cell line. Usually, more than 20 good metaphase cells are counted and analyzed using a microscope, and no less than 5 cells are further karyotyped and documented with a karyotype analysis computer software program (e.g. CytoVision). Under certain circumstances (e.g. cases of mosaicism), more cells are scored, analyzed and/or karyotyped according to the Cytogentic Laboratory Director’s discretion.

FISH analysis using whole chromosome paints, centromere probes, or locus specific probes can be performed to either further confirm the findings revealed by G-banding or detect chromosomal abnormalities (e.g. microdeletions and microduplications), which are beyond the detection limit of G-banding. Array-based comparative genomic hybridization (aCGH) is also a powerful tool for chromosomal and genomic studies. The Affymetrix Genome-Wide SNP Array 6.0 is used for routine analysis of cell lines in the NIGMS HGCR. Using this approach, detailed analyses of genome-wide unbalanced chromosomal aberrations, such as copy number variations (CNVs), loss of heterozygosity (LOH) or long contiguous stretches of homozygosity (LCSH), are easily visualized. Coriell’s Cytogenetics Laboratory is also equipped to analyze samples using the same Affymetrix platform, for various tests.

All DNA samples prepared by the Coriell Cell Repositories are characterized for the following:

1. OD260 /OD280 ratio,
2. agarose gel electrophoresis to confirm the integrity and digestibility with Eco R1 and Hind III, and
3. subject identity by microsatellite analysis, as described below.

Gender Assay

The gender of all DNA preparations is determined by TaqMan methodology, utilizing identities and differences between the X and Y chromosome amelogenin alleles.

Microsatellite Fingerprinting

Since 1998, for all DNA prepared from existing cell cultures, a molecular identity is established using a panel of six microsatellite markers. Future DNA isolations from these cell lines are fingerprinted to verify identity with the original culture.

DNA from the NIGMS HGCR's extended families, including the CEPH reference families, is also currently identified by microsatellite profiles and these are used to verify family relationships. Previous to 1998, DNA samples from the NIGMS HGCR's extended families, including the CEPH Reference Families, were characterized as described for cell cultures. For each new submission, a microsatellite identity is determined; this identity is verified each time a cell expansion is ordered for DNA isolation.

Mutation Characterization

The NIGMS HGCR's inherited disorders collection contains DNA from human cells which have known mutations. Where the mutation has been identified by the submitter or by other experts, this is included in the remarks for the cell line. If the mutation is verified by another expert in response to CCR request, this is also listed as a verification. Where the mutation is identified after submission by an expert laboratory, this is noted under remarks which also provide the method of analysis.