Evaluation of methods for preparing and thawing cryopreserved CD34+ and CD34− cell lines for use as reagents in flow cytometry of hematopoietic progenitor cells

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Abstract
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.

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