Bowman C A, Yu M, Cottler-Fox M
Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, Maryland, USA.
Transfusion. 1996 Nov-Dec;36(11-12):985-8. doi: 10.1046/j.1537-2995.1996.36111297091743.x.
Flow cytometry is used to quantitate CD34+ hematopoietic progenitor cells for transplantation. The present study evaluates methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as flow cytometry reagents.
The human myeloid leukemic cell lines KG 1 a (CD34+) and K562 (CD34-) were grown in culture under standard conditions and then prepared on ficoll gradients of different densities to determine which gave the component that was most reproducible. After ficoll preparation, the cells were frozen in standard cryopreservation media and four methods of thawing were examined. Determination of the method that gave the cell component that was most reproducible was based on viability, percentage of cell recovery, and maintenance of CD34 antigenicity status.
Ficoll gradient preparation improved the ease of flow cytometry analysis when original viability was low, and it produced a more uniform cell population. However, it resulted in significant cell loss for both cell lines. While the cell recovery for K562 cells was not significantly different with any of the densities of ficoll, recovery was significantly better for KG 1 a cells with ficoll at a specific gravity of 1.077. Of the thawing methods examined, all three that involved a rapid thaw at 37 degrees C were statistically equivalent to each other and were better than thawing at 4 degrees C.
With a standardized method of preparing cell lines as reagents for quality control purposes, data comparison among cell processing laboratories may more readily be initiated. Such cell lines could also be useful as a teaching tool for flow cytometry and in proficiency testing.
流式细胞术用于定量分析用于移植的CD34+造血祖细胞。本研究评估了制备和解冻冷冻保存的CD34+和CD34-细胞系以用作流式细胞术试剂的方法。
人髓系白血病细胞系KG 1 a（CD34+）和K562（CD34-）在标准条件下培养，然后在不同密度的菲可梯度上制备，以确定哪种方法能得到最可重复的细胞成分。菲可制备后，将细胞冻存于标准冷冻保存培养基中，并检测了四种解冻方法。基于细胞活力、细胞回收率和CD34抗原性状态的维持，确定能得到最可重复细胞成分的方法。
当原始活力较低时，菲可梯度制备提高了流式细胞术分析的便利性，并产生了更均匀的细胞群体。然而，两种细胞系均出现了显著的细胞损失。虽然K562细胞在任何菲可密度下的细胞回收率均无显著差异，但对于比重为1.077的菲可，KG 1 a细胞的回收率显著更高。在所检测的解冻方法中，所有三种在37℃快速解冻的方法在统计学上彼此等效，且优于在4℃解冻。
采用标准化方法制备细胞系作为质量控制试剂，细胞处理实验室之间的数据比较可能更容易开展。这样的细胞系也可用作流式细胞术的教学工具和能力验证。
