Identification of structural DNA variations in human cell cultures after long-term passage

Abstract

Random amplified polymorphic DNA (RAPD) analysis was adapted for genomic identification of cell cultures and evaluation of DNA stability in cells of different origin at different culture passages. DNA stability was observed in cultures after no more than 5 passages. Adipose-derived stromal cells demonstrated increased DNA instability. RAPD fragments from different cell lines after different number of passages were cloned and sequenced. The chromosomal localization of these fragments was identified and single-nucleotide variations in RAPD fragments isolated from cell lines after 8-12 passages were revealed. Some of them had permanent localization, while most variations demonstrated random distribution and can be considered as de novo mutations.

Keywords: Cell cultures; DNA stability in cells; Random amplified polymorphic DNA (RAPD); different number of passages; mutations.

Figure 1.

RAPD analysis of DNA from cell cultures at different passages using the P29 primer. A 100 bp Ladder+ and a 1 kb Ladder (Fermentas) were used as molecular weight markers. Culture nos. 1–5, adipose-derived stromal cells; MSCs, mesenchymal stem cells; nos. 11, 14, 16, 18–21, and 23, skin fibroblasts; lanes 1–20, different passages; ‘*’ indicates that the cell line was cultured on different medium; nos.1–23, cell cultures obtained from different donors.

Figure 2.

RAPD analysis of DNA from cell cultures at different passages using the R45 primer. For designations, see Figure 1.

Figure 3.

RAPD analysis of DNA from cell cultures at different passages using the P447 primer. For designations, see Figure 1.

Figure 4.

RAPD analysis of DNA isolated from different human cell cultures at different passages: variations observed for the P447 primer. A 100 bp Ladder+ and a 1 kb Ladder (Fermentas) were used as molecular weight markers. Culture nos. 2 and 3, adipose cells; nos. 11 and 20, skin fibroblasts; lanes 1–12, different passages.

Figure 5.

RAPD analysis of DNA isolated from different human cell cultures at different passages: variations were observed for the P447 primer. A 100 bp Ladder+ and a 1 kb Ladder (Fermentas) were used as molecular weight markers. Culture nos. 2, 3, and 5, adipose-derived stromal cells; no. 11, skin fibroblasts; lanes 1–12, different passages. RAPD bands used in further experiments are marked by white frame.