Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics

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Abstract
We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.