Cell Culture

Factors affecting culture growth

DDx low mitotic index

Culture failure rates

Harvesting

DDx poor spreading

DDx overspreading

· Definitions:

· Primary culture:

· Started from cells taken directly from an organism

· Established culture:

· Demonstrates the potential to be passaged indefinitely in vitro

· Cell line:

· Consists of numerous lineages of the cells originally present in the primary culture

· Cloned line:

· A culture arising from a single cell

· Specimen Collection:

· Sterile

· Isotonic saline

· Transport media

· Specimen transport & storage:

· Never frozen

· Refrigeration is ok but not recommended due to risk of freezing

· Culture medium:

· components:

· Amino acids (essential, +/- non-essential)

· Fetal bovine serum

· Antibiotics

1. Penicillin

2. Streptomycin

· Phytohemagglutinin (PHA)

· Sodium heparin

· Media examples:

· Medium 199

1. Low in folate for demonstration of folate-sensitive fragile sites

· Dulbecco’s and Vogt’s modified Eagle’s (D&V)

1. Additional amino acids, vitamins, and non-essential amino acids

· Alpha MEM

1. Nonessential amino acids

· Ham’s F-10 and F-12

1. Support clonal growth of mammalialn cells

· RPMI 1640

1. Defined for suspension (lymphoblast) cultures

· Chang

1. Defined for amniotic fluid cultures

· McCoy’s 5A

1. General purpose medium

· Balanced Salt Solutions (BSS)

· Rinse serum from cells prior to trypsinizaiton

· Other specialized procedures

· Ex. Hanks’s solution, PBS

· Maternal cell contamination:

· Greatest for POC and placental biopsy (Rodgers et al, 1996)

· POC – estimated 24% maternal cell contamination (Rodgers et al, 1996)

· Placental biopsy – estimated 18% maternal cell contamination (Rodgers et al, 1996)

· Skin – estimated 12% maternal cell contamination (Rodgers et al, 1996)

· Blotting samples dry on a gauze decreases the incidence of maternal contamination

·

References:

· Barch MJ, Knutsen T, Spurbeck JL. The AGT Cytogenetics Laboratory Manual. 3rd ed. Lippincott Williams & Wilkins; 1997.

· Rodgers CS, Creasy MR, Fitchett M, et al. Solid tissue culture for cytogenetic analysis: a collaborative survey for the Association of Clinical Cytogeneticists. J Clin Pathol. 1996;49(8):638-41.