176 IMPROVED GROWTH OF HUMAN EMBRYONIC STEM CELLS IN A REDUCED OXYGEN ATMOSPHERE
T. Peura A , A. Bosman A and T. Stojanov AASydney IVF, Sydney, New South Wales 2001, Australia. Email: teija.peura@sivf.com.au
Reproduction, Fertility and Development 17(2) 238-239 https://doi.org/10.1071/RDv17n2Ab176
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
Traditional cell culture conditions entail the use of gas atmosphere consisting of 5–6% CO2 in air. These same conditions have also been used universally for the culture of human embryonic stem cells (hES), despite the natural milieu of the embryos, from which these cells are derived, being slightly hypoxic. The aim of this work was to examine if human embryonic stem cells would benefit from the reduced oxygen culture environment, as used for human pre-implantation embryos. In Experiment 1, the relative growth in high and low oxygen atmosphere was compared by cutting undifferentiared hES-colonies into equal-size pieces and transferring them to two dishes, one into the conventional incubator in 6% CO2 in air (HIGH), the other into K-MINC-1000 (Eight Mile Plains, Queensland) mini-incubator in 5% O2, 6% CO2 and 89% N2 (LOW). After 8 days the colony sizes and differentiation status were measured. In Experiment 2, the absolute growth in high and low oxygen atmosphere was compared by cutting exactly same size fragments from undifferentiated hES-colonies and distributing them to two treatments as described. All colonies were measured and passaged at Day 7 and final measurements taken at Day 14. In Experiment 1, improved growth was observed in reduced oxygen, mean percentage of undifferentiated growth from original colony (assigned as 100%) being 217% for HIGH and 482% for LOW over three replicates. In Experiment 2, the total of 16.9 mm2 of undifferentiated colonies in both treatment groups (24 colonies per group over 3 replicates) had by Day 7 grown to total of 51.1 and 79.5 mm2 (P < 0.001), and by Day 14 to 216.8 and 373.3 mm2 (P < 0.0001) in HIGH and LOW, respectively (see Table 1). In neither experiment were there differences in the differentiation status of the colonies between the treatments (mean 6% and 5% at Day 7, and 9% and 9% at Day 14 in HIGH and LOW, respectively). We conclude that culture in reduced oxygen improves growth of human embryonic stem cells.
|
We like to acknowledge Bresagen Ltd. for the supply of hesBGN-01 cell line.
