Stromal cells are critical regulators of bone marrow hematopoietic niches, but assessment of their regulatory roles has been impeded by difficult and ineffective dissociation methods. Here, we methodically address bone marrow stromal cell dissociation. Yield of bone marrow CD45⁻/Ter119⁻/CD31⁺/CD202b⁺ endothelial cells (ECs) and CD45⁻/Ter119⁻/CD44⁻/PDGFR⁺ mesenchymal stromal cells (MSCs) were determined by flow cytometry. Liberase DL, Collagenase D, and Dispase II (all supplemented with DNase) enhanced EC and MSC yields, with Dispase II + DNase proving most effective. Combinations of these enzymes did not exhibit additive benefits, nor did the addition of Elastase, TrypLE, Hyaluronidase, or Accutase. Similarly, common mechanical dissociation approaches also proved ineffective. However, the combination of gentle Dispase II + DNase dissociation with magnetic sorting dramatically enriched both ECs and MSCs. This work methodically addressed common approaches for bone marrow stromal dissociation and established an effective approach for enrichment.

基质细胞是骨髓造血壁ches的关键调节剂，但是其调节作用的评估受到困难和无效解离方法的阻碍。在这里，我们有条不紊地解决了骨髓基质细胞的分离。骨髓CD45的产量^- / TER119 ^- / CD31 ⁺ / CD202b ⁺内皮细胞（EC）和CD45 ^- / TER119 ^- / CD44 ^- / PDGFR ⁺间充质基质细胞（MSCs）通过流式细胞仪确定。Liberase DL，胶原酶D和Dispase II（均添加DNase）可提高EC和MSC产量，其中Dispase II + DNase最有效。这些酶的组合没有表现出加和作用，也没有添加弹性蛋白酶，TrypLE，透明质酸酶或Accutase。同样，常见的机械分解方法也被证明是无效的。但是，柔和的Dispase II + DNase解离与磁性分选相结合，极大地丰富了EC和MSC。这项工作有条不紊地解决了骨髓基质分离的常见方法，并建立了有效的富集方法。