Using Acoustophoresis Cell Washing In The Immune Cell Radiolabeling Procedure

Introduction: We explore the use of acoustophoresis cell washing as a substitute for centrifugal cell washing in the ⁸⁹Zr-oxine immune cell radiolabeling procedure for the steps which require cell purification. The ⁸⁹Zr-oxine radiolabeling process of immune cells requires a series of manual steps which mandate a highly skilled operator with experience working with cells. This research tests the use of acoustophoresis as a viable technology to be used in the immune cell radio-labeling procedure with the potential to reduce the number of manual steps involved in the process and possibly automate the sequence. The goal is to validate acoustophoresis as a viable technology to be used to design and implement a fully automated radio-labeling device suitable for use in GMP radio-pharmacies.

Methods: To perform an acoustophoresis cell washing (acoustic wash) in ⁸⁹Zr-oxine radiolabeling procedure, replacing the centrifuge used to wash cells (centrifugal wash), the AcouWash acoustophoresis system (Acousort, Lund, Sweden) was incorporated. This new acoustic wash procedure was compared to the standard centrifugal cell washing one in side by side comparison tests. Several radio-labeling metrics were measured by the two procedures allowing evaluation of the efficacy of the acoustophoresis cell washing technology using the standard procedure as a control; the radiolabeled specific activity, the labeling efficiency, cell recovery, % unbound ⁸⁹Zr-oxine remaining in the suspension solution and cell viability. EL-4 murine T cell lymphoma cells (American Tissue Culture Collection, Cat. No. TIB-39) were used in the tests. Cell viability was measured using flow cytometry with annexin V/propidium iodide stained cells to exclude apoptotic cells not detected by the traditional trypan blue exclusion test.

Results: The acoustophoresis cell washing procedure produced radiolabeled cells with similar properties to the centrifugal control method. The average labeling specific activity was 24±2 and 26±2 kBq/million cells, using the acoustophoresis method compared to the centrifugal method, for EL4 cells (P=0.0010), 15±2 and 15±1 kBq/million cells for the T cells (P=0.0722), respectively. The radiolabeling efficiencies were 37%±3% and 43%±2% in EL4 cells (P=0.1762, n=3), 5%±1% and 9%±1% in T cells (P=0.0028, n=3) for the acoustophoresis and centrifugal methods. Cell viability for both cell types were within 4% of the viability of the pre-labeled cells. The T cells labeled using the two methods demonstrated similar IFNγ production after a CD3/CD28 stimulation.

Conclusions: The acoustophoresis cell labeling method showed comparable results to the centrifuge method in the specific activity, labeling efficiency, % unbound ⁸⁹Zr-oxine remaining in the suspension solution, and maintenance of cell viability and function. These results suggest that acoustophoresis cell washing can be used in the design of an automated benchtop, good manufacture practice-qualified acoustophoresis cell radiolabeling device.