Identification of a novel cell type in peripheral lymphoid organs of mice. V. Purification of spleen dendritic cells, new surface markers, and maintenance in vitro.

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Abstract
Dendritic cells (DC) were purified from mouse spleen in good yield. Spleen cell suspensions were floated on dense bovine plasma albumin (BPA) columns, and the low density fraction was adhered to glass. The adherent cells consisted of DC and immature macrophages most of which eluted in a viable state from the culture dish after overnight incubation. The macrophages were then removed by selective rosetting with opsonized erythrocytes and recentrifugation on dense BPA. A purified DC fraction was obtained containing 1-3 .times. 105 DC/spleen, which was homogeneous and distinctive in its properties. All cells exhibited the phase contrast and transmission electron microscopy cytologic features that were previously described for freshly isolated adherent DCs. By scanning electron microscopy, most purified DC exhibited a remarkable array of bulbous protrusions of varying length and shape, unlike any other lymphoid cell. All DC expressed surface Ia [immune response-associated] and other major histocompatibility complex (MHC)-linked alloantigens. DC lacked surface Ig [immunoglobulin] and T[thymus-derived]-cell antigens, and did not bind or interiorize opsonized erythrocytes. Purified DC were maintained in vitro for 3 days. Recovery of cultured purified cells was 70% or more of starting cell numbers. When [3H]uridine-tagged DC were mixed with nonlabeled heterogeneous spleen cells, 70-80% of the labeled DC were recovered as viable cells 2-3 days later. Purified DC did not readhere to tissue culture surfaces and did not proliferate, even when cultured with mitogenic doses of concanavalin A and lipopolysaccharide. DC did not change their cytologic or surface properties after 3 days of culture. DC are a novel cell type and provide useful properties and techniques for their further study.