ABSTRACT
The yield of hematopoietic progenitor cells recovered from bone marrow and peripheral blood varied widely between different clinical centers when Percoll was used (44-48). Across these different studies, the recovery of colonyforming units (CFU) varied between 35 and 90% for bone marrow and 9 and 90% for peripheral blood. A more recent study showed that the median recov ery of CD34 cells from the blood of patients with non-Hodgkin’s lymphoma mobilized with recombinant human granulocyte colony-stimulating factor (rHuG-CSF) was 81% (range, 10-100%) (39). This illustrates not only the variability among patient samples, but also the inconsistency in reproducibility in cell separation that is the most fundamental hurdle in density gradient separation. In our laboratory the biggest challenge in performing reproducible cell separations was related to the methods of formulation, the storage condi tions of silanized colloidal silica, and the accuracy of recovering cells from the interface after centrifugation. Colloidal silica is provided by the manufacturer as a raw material that requires further dilution to obtain a desired density, pH, and osmolality. The formulation is performed using in-house protocols that are not standardized between clinical centers and rarely are adequately quality controlled. Furthermore, the manufacturer and literature instruct the user to formulate Percoll solutions by measuring the raw materials on the basis of volumes rather than weight. In our experience, formulation of small (100 mL)- to medium (1 L)-scale batches of colloidal silica based on volumetric mea sures, using standard laboratory equipment such as pipettes and regular fluid transfer pipettes, results in significant batch-to-batch variations. Variations in density as great as 0.001 g/mL were observed between different batches formulated by volumetric means. This variation is undesirable for the separa tion of particular cell types, especially hematopoietic progenitor cells, whose density varies within a 0.001 g/mL range, as will be discussed. Especially for small to medium batches, it is advisable to formulate colloidal silica solutions based on weight measurements and to control the physicochemical specifica tions of the final product. Another feature of the “home-brew” approach that is often underestimated is the condition and the choice of container used for storage of the final product. Many laboratories store Percoll solutions for prolonged periods in wide-mouth flasks with a large headspace of air. The amount of water that can evaporate in this space is sufficient to change the specifications of the final material. This is especially important for the separation of cell types with a narrow density distribution, such as hematopoi
etic progenitor cells. In this context, it is advisable to store premade solutions in narrow-mouth containers in volumes that are rapidly used. Under these conditions, isotonic solutions of silanized colloidal silica were stored in our facility for more than a year without observing changes in density ( < 0.0005 g/mL), osmolality (<10 mOsm), or pH (< 0.5).
