<i>In Vitro</i> Studies of Cryopreserved Baboon Granulocytes

Abstract

Granulocytes were isolated from the buffy coat of baboon blood by counterflow centrifugation. They were frozen in polypropylene tubes in 2.0 ml volumes containing 1 × 10 7 granulocytes. The medium consisted of 5% DMSO, 6% HES, 4% human serum albumin, and 6 mM glucose in Normosol‐R, pH 7.1. Granulocytes were cooled to 4 C for 30 minutes, then cooled at 4 C per minute to –80 C and stored for one to three weeks in liquid nitrogen at –197 C. Cooling rates of 1 C and 10 C per minute were less efficacious. Tubes were thawed manually with swirling for 130 seconds at 42 C in a water bath. The yield after thawing was 98 ± 14 per cent. Granulocytes isolated from fresh blood all fluoresced green when incubated with fluorescein diacetate (FDA). Less than one per cent of cells showed nuclear uptake of ethidium bromide (EB). After freezing and thawing, an average of 79 ± 8 per cent granulocytes fluoresced green with FDA and 21 ± 8 per cent fluoresced red with EB. About 98 ± 2 per cent 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 ± 5 per cent demonstrated serum‐dependent latex ingestion and 67 ± 8 per cent ingested zymosan.