Levine R F, Fedorko M E
J Cell Biol. 1976 Apr;69(1):159-72. doi: 10.1083/jcb.69.1.159.
Methods have been devised to harvest megakaryocytes from guinea pig femoral marrow and to isolate them in high yield. When marrow tissue was disaggregated the megakaryocytes underwent degenerative changes characterized by the loss of cytoplasmic granules and alterations in membrane topography, similar to the changes seen in aggregating platelets. These morphologic changes were interpreted to mean that megakaryocytes possessed functional attributes of platelets. The use of agents which inhibit platelt aggregation (0.38% sodium citrate. 10(-3) M adenosine, and 2 x 10(-3) M theophylline) in a medium free of bivalent cations prevented these changes. This solution resulted in both an excellent morphologic preservation and a significantly increased recovery of megakaryocytes from marrow tissue. A two-step purification of the intact megakaryocytes was carried out on the basis of their low density and large size, with equilibrium density gradient centrifugation followed by velocity sedimentation. This sequence gave approximately a 100-fold enrichment of megakaryocytes, significantly better than that achieved with either method alone. These techniques for harvesting and concentrating megakaryocytes make it possible for the first time to study megakaryocytes in vitro.
已设计出从豚鼠股骨骨髓中收获巨核细胞并高产分离它们的方法。当骨髓组织被分散时，巨核细胞会发生退行性变化，其特征是细胞质颗粒丢失和膜拓扑结构改变，类似于聚集血小板中所见的变化。这些形态学变化被解释为意味着巨核细胞具有血小板的功能特性。在不含二价阳离子的培养基中使用抑制血小板聚集的试剂（0.38%柠檬酸钠、10⁻³ M腺苷和2×10⁻³ M茶碱）可防止这些变化。这种溶液既实现了出色的形态学保存，又显著提高了从骨髓组织中回收巨核细胞的效率。基于其低密度和大尺寸，通过平衡密度梯度离心随后进行速度沉降对完整的巨核细胞进行两步纯化。该序列使巨核细胞富集约100倍，明显优于单独使用任何一种方法所达到的效果。这些收获和浓缩巨核细胞的技术首次使得在体外研究巨核细胞成为可能。
