Cryogenic Preservation of Monocytes from Human Blood and Plateletpheresis Cellular Residues.

Abstract : A separation procedure was devised for the isolation of human monocytes from buffy coat of whole blood and the cellular residues in platelet bags after plateletpheresis of donors with the Haemonetics Model 30 Cell Processor. The mononuclear cells were isolated using Ficoll-Isopaque (Lymphoprep) and monocytes were then separated from lymphocytes by centrifugal cytography using two separation chambers in a Beckman JE-6 rotor. About 30,000,000 monocytes were obtained from 100 ml of whole blood. These were 93% myeloperoxidase positive (MPO), while 98% showed intact membranes in the fluorescein diacetate (FDA)eithidium bromide (EB) test. Eight-seven percent ingested 1-5 or more opsonized latex particles and 93% ingested 1 or more ethidium-treated zymosan particles. In three procedures 1,000,000,000 monocytes were obtained from cellular residues in platelet bags. Monocytes were frozen using 5% dimethylsulfoxide (DMSO), 6% hydroxyethyl starch (HES), 4% albumin, and 56 mM glucose in Normosol-R, pH 7.1. Two ml aliquots containing 6.7 x 10 to the sixth power - 7.2 x 10 to the sixth power monocytes were cooled at 4 C per minute to -80 C, transferred and stored for 1 to 3 months in liquid nitrogen. After storage for periods up to 3 months, they were thawed. In some studies the monocytes were diluted to reduce the DMSO to 1.25 percent and in others the monocytes were washed with a 4-fold volume of the solution used to freeze them, but without DMSO. Ninety-eight percent of monocytes frozen were recovered morphologically intact, 90 percent were MPO positive, and 87 percent were FDA positive.