SEPARATION OF FELINE BONE MARROW CELLS BY COUNTERFLOW CENTRIFUGAL ELUTRIATION: IDENTIFICATION AND ISOLATION OF PRESUMPTIVE EARLY AND LATE MYELOID/ERYTHROID PROGENITORS
SEPARATION OF FELINE BONE MARROW CELLS BY COUNTERFLOW CENTRIFUGAL ELUTRIATION: IDENTIFICATION AND ISOLATION OF PRESUMPTIVE EARLY AND LATE MYELOID/ERYTHROID PROGENITORS
复制标题
通过逆流离心淘洗分离猫骨髓细胞：鉴定和分离推定的早期和晚期骨髓/红系祖细胞
作者：
N. Gengozian;A. Legendre
逆流离心淘洗（CCE）已用于基于大小从哺乳动物骨髓中分离有核细胞，从而分离处于不同谱系发育阶段的造血细胞。我们研究了从猫骨髓中鉴定和分离这种细胞的可行性。使用Beckman J6 MI离心机和Sanderson室进行CCE，使用3000 rpm的固定转子速度，并以（1）16-、（2）21-、（3）25-、（4）32 ml/min和（5）转子关闭级分收集细胞。在四个重复实验中，总输入细胞的回收率平均为86%，在级分4中回收的细胞数量最多。通过流式细胞术和单克隆抗体的分析揭示了级分1和2中的单核细胞以及级分2至5中的早期和晚期分化的髓系/红系细胞。在混合淋巴细胞反应（MLR）中的T淋巴细胞和同种异体反应性仅限于部分1和2;通过免疫磁性耗竭去除T细胞和MLR活性。克隆形成细胞的体外培养物显示在级分2至5中的CFU-GM和BFU-E集落，其中级分4含有最大绝对数量的髓系集落，级分3和4含有大部分红系集落。更重要的是，在检查不同级分中的克隆形成细胞的接种效率时，发现当培养时间从7天延长至14天时，在级分2和3中这显著增加;相反，级分4和5在7天内达到其最大接种效率，在第14天没有进一步增加。我们解释这些发现，以表明在大细胞大小的部分4和5中存在晚期分化的祖细胞，而部分2和3中的较小的单核细胞代表了更早，更原始的造血细胞群体，需要在培养中延长时间才能完全集落发育。
Counterflow centrifugal elutriation (CCE) has been used to separate nucleated cells from mammalian bone marrow on the basis of size with the resultant isolation of hematopoietic cells in varying stages of lineage development. We examined the feasibility of identifying and isolating such cells from feline bone marrow. CCE was performed with a Beckman J6MI centrifuge and a Sanderson chamber, using a fixed rotor speed of 3000 rpm and collection of cells at (1) 16–, (2) 21–, (3) 25–, (4) 32 ml/min, and (5) a rotor off fraction. Recovery of the total input cells in four replicate experiments averaged 86%, with the maximum number of recovered cells in fraction 4. Analysis by flow cytometry and monoclonal antibodies revealed mononuclear cells in fractions 1 and 2 and early and late differentiating myeloid/erythroid cells in fractions 2 through 5. T lymphocytes and alloreactivity in a mixed lymphocyte reaction (MLR) were restricted to fractions 1 and 2; removal of T cells and MLR activity was accomplished by immunomagnetic depletion. In vitro cultures for clonogenic cells revealed CFU-GM and BFU-E colonies in fractions 2 through 5, with fraction 4 containing the greatest absolute number of myeloid colonies and fractions 3 and 4 the majority of the erythroid colonies. More important, in examining the plating efficiency for clonogenic cells in the different fractions it was found that this increased significantly in fractions 2 and 3 when the culture time was extended from 7 to 14 days; in contrast, fractions 4 and 5 reached their maximum plating efficiency within 7 days with no further increase on day 14. We interpret these findings to indicate the presence of late differentiating progenitors in the large-cell size fractions 4 and 5, while the smaller mononuclear cells in fractions 2 and 3 represent an earlier, more primitive population of hematopoietic cells requiring an extended time in culture for full colony development.