Development Of Continuous Density Gradient Cell Separ.

紧凑的台式离心机可根据密度进行连续细胞分离。该装置装有一个小的分离盘，该分离盘配备有一个被隔膜中断的圆形通道（8 ml容量）。在一端连续引入一组具有不同密度的等渗Percoll介质，并从另一端收集。在离心力场下，引入通道近端部分的细胞悬浮液导致细胞根据其密度连续分离。该装置的性能在分离人血沉棕黄层时得到了证实，该血沉棕黄层含有大量（10，000，000，000）RBC中超过100，000，000的有核细胞。用May Grunwald-Giemsa染色的涂片制备物上的白细胞分类计数显示，嗜碱性粒细胞在密度= 1.075 g/ml的级分中浓缩高达15%。流式细胞仪分析显示，CD 34阳性细胞主要收集在单核细胞和淋巴细胞混合的部分中（密度= 1.060）。该方法已成功地应用于分离培养的细胞（超过1，000，000个细胞），其中人肥大细胞（密度= 1.085）与大的成纤维细胞（密度= 1.065）很好地分离，细胞损伤最小。本发明的方法可以有效地应用于从脐带血富集造血干细胞。

A compact bench-top model of the centrifuge enables continuous cell separation according the density. The apparatus holds a small separation disk equipped with a circular channel (8 ml capacity) interrupted by a septum. A set of isotonic Percoll media with different densities was continuously introduced at one terminal and collected from the other. Under centrifugal force field, cell suspension introduced into the proximal portion of the channel results in continuous separation of cells according to their densities. The performance of the apparatus was demonstrated on separation of human buffy coat containing nucleated cells of more than 100,000,000 among a large population (10,000,000,000) of RBC. The differential leukocyte count on a smear preparation with May Grunwald-Giemsa stain revealed that basophils were concentrated as high as 15% in a fraction at density = 1.075 g/ml. The flow cytometric analysis showed that CD34 positive cells were mainly collected in a fraction (density = 1.060) mixed with monocytes and lymphocytes. The method has been successfully applied for separation of cultured cells (over 1,000,000 cells) where human mast cells (density = 1.085) were well separated from large fibroblasts (density = 1.065) with minimum cell damage. The present method may be efficiently applied for enrichment of hematopoietic stem cells from cord blood.