Abstract:

BACKGROUND: It has not been deeply reported how to obtain high-purity and high-vigor stem cells as well as the standardized operation plan has not been seen, yet.
OBJECTIVE: To explore the best condition for isolating human umbilical cord blood mononuclear cells.
METHODS: Umbilical cord blood red blood cells precipitated by hydroxyethyl starch were observed in different time points (20, 30, 40, 50, 60, 70 minutes). The human umbilical cord blood mononuclear cells were separated in human lymphocyte separating medium (LSM) by using centrifugation method at 800, 700, 600, 500, 400 g/min for 30, 25, and 20 minutes.
RESULTS AND CONCLUSION: The 6% hydroxyethyl starch precipitation for umbilical cord blood red blood cells was best within 60 minutes. Using the human LSM of (1.077 0±0.000 1) g/mL at 4 ℃, centrifugation at 700 g/min for 30 minutes, and rinsing for 3 times the best conditions to harvest human umbilical cord blood mononuclear cells. Under the above-mentioned conditions, not only higher umbilical cord blood mononuclear cells rate and higher mononuclear cells vigor were obtained, but fewer red blood cells and other cell fragments were seen. The best condition for isolating the human umbilical cord blood mononuclear cells using two steps methods (6% hydroxyethyl starch precipitation and LSM) at the optimal time can improve the recovery rate of human umbilical cord blood stem cells.