Cryopreservation of Canine Peripheral Blood Mononuclear Cells in Untreated and Ficoll-Hypaque Treated Buffy Coats

摘要

Peripheral blood mononuclear cells (MNC) were obtained from the buffy coats collected during 21 platelet apheresis procedures using the Haemonetics 30 Blood Processor. The mononuclear cells in the buffy coat were divided into two equal portions. One portion was treated with ficoll-hypaque to purify the mononuclear cells by removing the granulocytes and red cells, the other poriton was not treated. Dimethylsulfoxide (Me2SO) in McCoy's medium was added to the untreated buffy coats and to the ficoll-hypaque treated buffy coats rapidly in1 to 2 minutes or slowly over 15 to 20 minutes. The cell suspensions were frozen in polyolefin plastic bags at 2-3 C/min by placing the plastic bag in a 080 C freezer, or at 1 C/min by use of a graded freezing apparatus. The percentage of viable MNC's was determined after thawing and washing by measurement of uptake of fluorescein diacetate and ethidijm bromide. There was no significant difference in the percentage of viable mononuclear cells recovered between the untreated buffy coat and the ficoll-hypaque treated buffy coat depleted of contaminting red blood cells and granulocytes regardless of the rate of addition of the Me2SO-McCoy's solution and the rate of freezing. There was a significant difference in the number of viable MNC'S recovered when comparing untreated buffy coat and ficoll-hypaque treated buffy coat due to 22% loss of MNC's during ficoll-hypaquetreatment. (Author)