Collection, Separation, Cryopreservation and Characterization of Peripheral Blood and Bone Marrow Stem Cells and Their Use in Treating Lethally Irradiated Dogs

摘要

Peripheral blood mononuclear cells (MNC) were obtained from buffy coats collected during 26 platelet apheresis procedures in the dog using the Haemonetics 30 Blood Processor. The number of mononuclear cells and platelets were determined for each procedure. After separation of the platelet rich plasma, dimethylsulfoxide (Me2SO) in McCoy's medium was added to the residual buffy coats rapidly in 1 to 2 minutes or slowly over 15 to 20 minutes. The cell suspensions were frozen in polyolefin plastic bags at 2-3 C per minute by placing the plastic bag in a -80 C freezer or at 1 C per minute by use of a graded freezing apparatus. The percentage of viable MNC's recovered was determined after thawing and washing by measurement of uptake of fluorescein deacetate and thidium bromide. The average harvest of platelets from a standardized four pass procedure was 1.26 x 10 to the 11th power cells and of MNC's 1,340,000,000 cells. Viable recovery of frozen, thawed and washed MNC's was similar when frozen at 1 C/minute with slow addition of the cryophylactic agent to those frozen at 2-3 C/minute with rapid addition.