Culture of Porcine Embryonic Germ Cells in Serum-Supplemented and Serum-Free Conditions: The Effects of Serum and Growth Factors on Primary and Long-Term Culture

Abstract

Fetal bovine serum (FBS) is a commonly used medium supplement with variable and undefined composition, which presents problems in culture of pluripotent stem cells. The purpose of this study was to determine if FBS can be replaced with Knockout Serum Replacement (KSR), a defined medium supplement, and to examine the effects of FBS and growth factors on short- and long-term culture of pig embryonic germ cells (EGC). No significant differences were observed in total and mean colony areas in primary cultures between FBS- and KSR-supplemented medium (421 x 10(3) mum(2) vs. 395 x 10(3) microm(2), p = 0.68, n = 11, and 6375 microm(2) vs. 6407 microm(2), p = 0.885, respectively). Total and mean colony areas were significantly larger in KSR-supplemented medium compared with medium supplemented with KSR and growth factors (505 x 10(3) microm(2) vs. 396 x 10(3) microm(2), p = 0.016, n = 12, and 8769 microm(2) vs. 6513 microm(2), p = 0.003, respectively). The cultures proliferated for significantly higher numbers of passages in FBS-supplemented medium and in medium supplemented with KSR and growth factors compared with medium containing KSR alone (31.1 vs. 21.9, p = 0.004, n = 10, and 35.5 vs. 21.6, p = 002, n = 10, respectively). Porcine EGC maintained in serum-free conditions were positive for pluripotent stem cell markers, maintained stable karyotypes for up to 54 passages, and were capable of differentiating in vitro into cells from the three primary germ layers. These results will help improve and standardize culture of pluripotent stem cells in the pig.