Long Term Cryopreservation of Dog Granulocytes.
Technical rept. 1 Dec 80-30 Nov 81,
CENTER FOR BLOOD RESEARCH BOSTON MA
Pagination or Media Count:
Granulocytes isolated by counterflow centrifugation eluriation CCE from leukapheresed dog blood, frozen in liquid nitrogen at -196 C, were studied. The effects of long term cryopreservation on cell recovery and in vitro function were determined. In eight separate experiments, an average of 1.7 x 10 to the 9th power granulocytes were obtained. The white cell differential count was 91 granulocytes and 9 mononuclear cells. There was less than 5 red cells present and no platelets. The granulocytes were placed in Hemoflex bags and mixed slowly with equal volumes of sterile ice-cold hyperosmolar cryoprotectant buffer to make a final composition of 5 dimethylsulfoxide DMSO, 6 hydroxyethylstarch HES and 4 bovine serum albumin BSA, pH 7.1. A total volume of forty ml was frozen at a cooling rate of 4 C per minute by storage in a mechanical freezer at -80 C. After storage periods of 1, 34, 60, 90 and 132 weeks in liquid nitrogen at -196 C, a bag was thawed at a rate of 190 degrees per minute to 10 C. The recovery of cells at these periods was 95, 105, 100, 100 and 88 respectively and the ethidium bromide exclusion, indicative of viable nuclei, was 91, 81, 94, 89 respectively. Ingestion of opsonized Fluolite particles was measured and virtually all cells ingested particles but the number ingested was approximately one half that of prefreeze values. Thawed cells also demonstrated superoxide anion synthesis at rates approximating those in unfrozen granulocytes. These results indicate that dog granulocytes obtained by leukapheresis may be preserved in liquid nitrogen at 196 C with high cellular recovery and at least 50 phagocytic function. Author
- Medicine and Medical Research