In Vitro Studies of Cryopreserved Granulocytes.

Abstract : Granulocytes, isolated from the buffy coat of baboon blood by counterflow centrifugation, were frozen in polypropylene tubes in 2.0 ml volumes containing 1 x 10 to the 7th power granulocytes. The medium consisted of 5% DMSO, 6% HES, 4% human serum albumin, and 6 mM glucose in Normosol-R, pH 7.1. The best cooling rate for granulocytes was found to be 4 C for 30 minutes, then 4 C per minute to -80 C and stored for 1-3 weeks in liquid nitrogen at -197 C. Tubes were thawed manually with swirling for 130 seconds at 42 C in a water bath. The yield after thawing was 98 + or - 14%. Granulocytes isolated from fresh blood all fluoresced green when incubated with fluorescein diacetate (FDA). Less than 1% of cells showed nuclear uptake of ethidium bromide (EB). After freezing and thawing, an average of 79 + or - 8% granulocytes fluoresced green with FDA and 21 + or - 8% fluoresced red with EB. About 98 + or - 2% of the granulocytes isolated from fresh blood exhibited a serum dependent capacity for ingestion of latex and yeast. Thawed granulocytes were diluted 1:4 dropwise with a medium containing 7% HES, 4% albumin, and 6 mM glucose in Normosol-R, incubated with fluorescent latex or fluorescent zymosan at 37 C for 30 minutes and then washed twice. After this, 75 + or - 9% of the granulocytes fluoresced green with FDA, 76 + or - 5% demonstrated serum-dependent latex ingestion and 67 + or - 8% ingested zymosan.