Label-free processing of stem cell preparations by acoustophoresis

Abstract: The first bone marrow transplant in a human was performed in 1959, providing evidence that cells can be used for transplantation and treatment, revealing that healing capacities lie within the human body that ought to be understood. Since then hematopoietic stem cell transplantation has developed as a standard treatment for many cancers besides other malignancies. Much research is done to understand and utilize the properties of stem cells and their progenies for clinical application and transplantation. A routinely used valuable non-invasive information source in research and clinical applications is whole blood. However, cell processing including isolation or removal of certain components is desirable for many diagnostics, research, and transplantation applications. This thesis aimed to develop and evaluate the use of a microfluidic technology, called acoustophoresis for processing human blood and bone marrow cell preparations. Acoustophoresis utilizes the phenomenon that cells can be manipulated in an ultrasonic standing wave field in microfluidic devices. In the acoustic wave field cells experience an induced movement based on their acoustophysical properties, either to the channel center (pressure node) or towards the channel walls (pressure anti-node). These properties include size, density, and compressibility also in relation to the suspending medium. This can be utilized as biophysical biomarkers in acoustic separations.In the first two articles, it has been demonstrated that neuroblastoma tumor cells (cell line and neuroblastoma patient derived-xenograft cells) could be isolated from peripheral blood and progenitor cell products without the use of labeling antibodies, which is an advantage of acoustophoresis compared to other methods. The clinical relevance for stem cell graft processing was furthermore validated by acoustophoretic removal of transplant-contaminating tumor cells (“purging”) applicable for diagnostic, prognostic as well as potentially therapeutic purposes, with preserved high cell viability and functions. Moreover, bone marrow stromal cells (BM-MSCs) could be acoustically separated based on specific properties of primary, uncultured BM-MSCs as well as cultured MSCs. In article III, proof-of-principle evidence has been provided for the acoustic separation of functionally different subsets of cultured MSCs, which provides a first step towards better characterized and possibly enhanced cell products for cellular therapies in the future. It has further been demonstrated in article IV that primary stromal cells can be enriched from BM preparations based on their distinct biophysical properties. For clinically relevant cell processing, a stable system with relevant throughput is required. Hence, in article V a new chip holder design with an improved air-cooling unit is presented and validated, providing improved heat distribution along with stable multiplexed separation of beads and leukocyte subpopulations. This was realized at increased sample throughput of 500 µL/min and 300 µL/min, respectively. To facilitate the development of acoustofluidic cell separation applications it is crucial to obtain the properties of cell populations of interest to predict separability together with optimal experimental conditions. Therefore, the study in article VI presents a method to statistically estimate cell compressibility based on acoustophoretic separation data. Collectively, the work at hand provided valuable progress towards the validation and implementation of acoustic blood as well as stem cell processing in clinically relevant applications for cell transplantation, diagnostics, and regenerative medicine.