Characterization of Purified Cryopreserved Human Monocyte Function in Assays of Superoxide Production, Accessory Cell Function, Chemotaxis, and in Fluorescent Cell Sorter Analysis

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Abstract
The ability to use highly purified cryopreserved human monocytes in various in vitro assays has a number of practical and theoretical advantages, including convenience and the potential for enhanced reproducibility. Large numbers (up to 1 × 109) human monocytes can be isolated from a single donor in a purified suspension state, by a combination of leukapheresis and counter- current centrifugal elutriation (CCE) technologies. Following short- and long- term periods of cryopreservation, the viability and phagocytic function of these CCE-purified monocytes was unimpaired. Cryopreserved monocytes were similar to fresh cells in their ability to release superoxide anion (O2), although unstimulated and stimulated Of release values tended to increase slightly following weeks to months of cryopreservation. In contrast, even short-term cryopreservation diminished both the random migration and chemotactic responses of human monocytes; however, cryopreserved monocytes could be employed in this assay provided the calculated chemotactic ratio (chemotactic migration/spontaneous migration) was used. Cryopreserved monocytes demonstrate 70% of fresh monocyte accessory cell function in pokeweed mitogen- induced lymphocyte proliferation assays. When the binding of OKT3, OKM1, anti-DR, and fluoresceinated pokeweed mitogen to monocytes was analyzed in the fluorescence activator cell sorter (FACS), cryopreserved and fresh monocytes displayed a similar pattern of membrane reactivity.