Cell culture contamination: sources, consequences, prevention, and elimination.

The subject of the chapter is cell culture contamination. Contamination may enter the cell culture system as a physical, chemical, and/or biological component of the environment. The potential sources and consequences of cell culture contamination are unique to the cell culture system and the contaminant. A basic understanding of cell culture contamination is necessary to appreciate the need to develop and practice standardized cell culture procedures. General sources, consequences, and preventative measures are discussed for physical and chemical contamination based on current technology. Mycoplasmal contamination is the focus of the discussion on biological contamination and its impact on cell cultures. The introduction of other biological contaminants should be controlled by the institution of cell culture management procedures needed to minimize the incidence of mycoplasmal contamination. The need to eliminate the routine use of antibiotics in cell culture systems and institute routine testing to detect contamination is emphasized. More rapid detection of contamination should reduce the incidence of cross-contamination and minimize the consequences of any contamination event.

Inhibition of DNA synthesis in senescent-proliferating human cybrids is mediated by endogenous proteins.

Cytoplasts were prepared from senescent human diploid fibroblasts. Brief treatments of the senescent cells with cycloheximide or puromycin prior to or after enucleation eliminated the ability of senescent cytoplasts to block initiation of DNA synthesis in senescent-young cybrids. Senescent cells treated with cycloheximide, enucleated and allowed to recover in complete medium without cycloheximide, regained the ability to block initiation of DNA synthesis in senescent-young cybrids. These results support the hypothesis that senescent cells synthesize an inhibitor of DNA synthesis which is either a protein(s) or its activity is mediated by a protein(s) found in the cytoplasm of the senescent cell.

Use of temperature-sensitive mutants to study the morphogenesis of poliovirus.

Three temperature-sensitive (ts) mutants of poliovirus (type 1 Mahoney) were isolated after nitrous acid treatment and characterized as phenotypically RNA+. When cells were infected at 37 degrees with two of the three RNA+ ts mutants (ts109 and ts739), reduced levels of 14 S particles were synthesized. One RNA+ mutant (ts520) synthesized significant amounts of viral 14 S particle subunits. All of the mutants synthesized reduced amounts of procapsids and virions at 37 degrees. At 39.5 degrees, with all three ts mutants, the production of all virus-related particles in infected cells was markedly suppressed. Isoelectric focusing of the viral-related particles produced at 37 degrees by the ts mutants and electrophoretic analysis of their structural polypeptides revealed the following: (i) ts739 synthesized an altered VP0 polypeptide and produced 14 S particles with an altered isoelectric point; (ii) ts109 produced 14 S particles with a normal pI but containing what appeared to be an altered VP1; (iii) ts520 produced normal 14 S particles as demonstrated by their pI, the electrophoretic behavior of their constituent structural polypeptides in SDS-PAGE, their ability to self-assemble, and their ability to form procapsid-like structures when incubated in extracts from wild-type (wt) virus-infected cells. However, ts520-infected cells contained few, if any, procapsids and extracts made therefrom were unable to assemble ts520 or wt 14 S particles into detectable amounts of pI 6.8 empty capsids. These and other findings are consistent with ts739 (and probably ts109) possessing an altered structural protein and ts520 being mutant in its morphopoietic factor.

Inhibition of DNA synthesis in proliferating human diploid fibroblasts by fusion with senescent cytoplasts.

Cytoplasts were prepared from senescent human diploid fibroblasts. The cytoplasts were fused to young human diploid fibroblasts and DNA synthesis was analyzed in the fusion products. DNA synthesis was inhibited (greater than or equal to 40%) in the senescent cytoplast fusion products when compared to unfused young cells or young cytoplasts fused with young cells. These results are consistent with previous experiments that have shown the blockage of DNA synthesis in both nuclei of heterokaryons from fusions of senescent and young human diploid fibroblast cells. Furthermore, these results support the postulate that senescent cells synthesize a specific substance(s), which is present in the cytoplasm of the senescent cell that inhibits DNA synthesis.