As bone marrow becomes a more utilized source of cells for novel cell therapies and regenerative medicine, approaches to cell processing may require modification to accommodate smaller volume samples. The need for modification may be most obvious for immunomagnetic selection of specific subpopulations of cells. Immunomagnetic selection devices were designed for processing apheresis products and higher hematocrit values have been shown to be a hindrance to their function [10
]. A minimum volume of product is required as well; while this requirement may be addressed by simply diluting the starting material with a buffer solution, dilution would not address the possible need for a minimum or optimal target cell count.
The manufacturer recommends that a maximum of 8 × 1010 nucleated cells be loaded onto the instrument. There is no maximum or minimum target (CD34+) cell number recommendation. However, as with all antigen-antibody reactions, there is an optimal relative antigen: antibody concentration. The optimal proportion of antigen: antibody would allow for the most efficient interaction, and, hence, cell selection. The Isolex® Anti-CD34 Monoclonal Antibody vials contain 2.5 mL (1.0 mg/mL) in sterile, non-pyrogenic phosphate buffered saline solution, and the Dynabeads® are packaged in vials containing 4 × 109 beads suspended in 10 mL of phosphate buffered saline with 0.1% human serum albumin.
The Isolex® Stem Cell Reagent Kit is optimized for use with larger mononuclear cell apheresis collections. We suspected that selections using smaller volume marrow samples would not be as efficient, and we initiated this study to further define the efficiency of such selections. Our results suggest that smaller volume marrow processing is as efficient or more efficient when compared to the processing of larger volume products.
Several investigators have shown consistency in mean CD34+
cell recovery with mobilized peripheral blood apheresis collections and the Isolex®
system with Hildebrandt et al. [10
] reporting 53.6%, Rowley et al. [12
] reporting 58.4%, and Gryn et al. [13
] reporting 55%. Final product purity in these three studies was consistent as well at 85.36%, 90.8%, and 91.7%, respectively [10
]. However, CD34+
cell selection using the Isolex®
and larger volume marrow has been shown to be less efficient when compared to mobilized peripheral blood [11
]. Kasow et al. reported 29.93% recovery and 92.02% purity with primary bone marrow grafts (n=20). They hypothesized that greater hematocrit/red blood cell volume in the marrow collections led to these less efficient results. To minimize the negative effect of red cells on selection efficiency, Kasow et al. [11
] performed an inverted centrifugation step (400 g for 10 minutes) and attained a red cell mean volume of 21.5 mL (range: 12.0 – 28.1 mL). We, however, did not attempt to remove the red cells prior to selection and initiated processing with a mean volume of 34.5 mL (range: 16.7 – 45.0 mL).
Because the Isolex® reagents are packaged for optimal selection of larger products, we included a comparison of full vial reagent versus half vial reagent in Phase I of the study. Four CD34+ cell selections were performed with half of the typical amount of Isolex® Anti-CD34 Monoclonal Antibody and half of the Dynabeads®. Qualitatively, there was no significant difference noted with the post- Isolex® selection mean CD34+ cell recovery being 115.6% and 88.7% for full vial and half vial, respectively. The post- Isolex® selection purity results were almost identical with mean values of 84.7% and 82.8% for full vial and half vial, respectively. We suspect this lack of difference was due to antibody/bead saturation with smaller volume marrows even when only a half vial was used during processing. Use of lesser amounts of reagents may be an option if the manufacturer is willing to consider alternatives to current packaging of kits.
The final products in this small study had relatively few cells, and this raises a few technical considerations. Of course, optimal cell doses for most (if not all) regenerative medicine/cellular therapies are not known, and, perhaps, few cells are needed for certain clinical applications. However, this study emphasizes the need for high quality starting material. The largest final doses were generally obtained from bone marrow samples with the highest total nucleated cell counts. Successful marrow aspiration is dependent upon technique and several small aspirations following re-positioning of the aspirate needle are necessary. Finally, when dose is limited, final QC testing should be performed on as few cells as possible or even the negative fraction (i.e., when technically feasible and if the FDA agrees with the plan).
In this study, CD34+
cell recoveries were widely variable and occasionally greater than 100%. The variability is likely to be at least in part, attributable to the small ‘n’ of the study. Recoveries greater than 100% have been reported previously with hematopoietic stem cells [14
], and we believe that the initial results (i.e., pre-selection) for CD34%/count may have been underestimations of “rare” events. An under-estimate at that stage would cause the perception of >100% recovery with presumably more accurate analysis of the post-processing/post-shipping samples. Likewise, the lower overall recoveries in phase II may have been secondary to overestimation of “rare” events with the starting material. Certainly a larger study with a focus on intra-and inter-laboratory standardization of flow cytometry would be a logical next step.
Inter-laboratory standardization of quality control testing, including flow cytometry, was intentionally not considered in this exercise. Despite the inherent differences among each of the individual samples, reanalysis of Phase II data at Pittsburgh showed consistency in evaluation of the unmanipulated samples. Other studies have shown that standardization of CD34+
cell enumeration is possible if attention is paid to the nuances of flow cytometry gating strategy [15
In summary, the primary charge of the PACT group is to provide translational support and clinical production of cell therapy products for institutions engaged in clinical research involving cellular therapies and regenerative medicine. The success of this study demonstrates the feasibility of regional cell processing of one such product. Smaller volume samples of bone marrow were efficiently processed using an immunomagnetic selection device designed for apheresis products. Further, satisfactory shipping conditions were maintained throughout each shipment, and stability of the final product was shown by good post-shipping cell recovery, viability, CFU-GM, and negative sterility testing.