Immunomagnetic cell separation combines simplicity and low cost of separations based on direct application of physical forces to cells with the sensitivity and specificity afforded by use of immunospecific reagents. Current separation strategies and performance highly rely on the availability and choice of the immunomagnetic labels that magnetize cells selected for separation and induce their motion in the magnetic field. It is thus of great importance to synthesize and quantitatively characterize immunomagnetic labels for cell separation, which significantly increases cost of magnetic separation and limits its use. An alternative is to develop a label-less magnetic cell separation strategy. Due to low intrinsic magnetic susceptibility of biological material, however, the latter depends on the development of sensitive analytical instruments and methods to characterize the intrinsic cell magnetophoresis and the ability to identify cells with high intrinsic magnetic susceptibility that are significant for diagnostic or therapeutic applications.
In this dissertation, different versions of an experimental instrument, referred to as Cell Tracking Velocimetry, CTV, were developed to measure magnetophoretic mobility of a micro-sized cell or synthetic particle, either rendered magnetic by tagging or due to their own intrinsic magnetization. Another analytical apparatus, Inductively Coupled Plasma Mass Spectrometry was also employed to measure the intracellular concentration of magnetic metals that contribute to intrinsic cell magnetic susceptibility. Based on the results of these measurements, candidate “magnetic” cell types were identified and commercial magnetic separators were used for their enrichment from mixed cell samples.
The required high accuracy of the CTV instrument was first tested and confirmed by examining the Brownian motion of monodisperse microspheres. The sensitivity of the CTV analysis to different magnetization mechanisms on microscale was then investigated. The new and improved, variable-field CTV was shown to be sensitive to the type of the microparticle magnetization and capable of distinguishing between motions of magnetically unsaturated species (paramagnetic or diamagnetic) and the magnetically saturated species (superparamagnetic). Furthermore, with the aid of a fluorescent microscope, the increased functionality of the fluorescent CTV was tested and confirmed on cells labeled with fluorescent and magnetic tags. The combination of the regular, visible CTV with fluorescent CTV was shown to be able of evaluating the sensitivity and specificity of immunomagnetic labels important for magnetic cell separation.
In the second part of this dissertation, label-less magnetic cell separation was proposed and studied. Cultured RBCs were successfully separated from hematopoietic stem cell bioreactor cultures after deoxygenation by exploiting the paramagnetic nature of deoxygenated hemoglobin. A label-less magnetic selection of nonapoptotic human spermatozoa for in vitro fertilization was then investigated and its performance was compared with an established method of immunomagnetic separation of apoptotic cells labeled with magnetic colloids. The results suggested that apoptotic spermatozoa have a higher intrinsic magnetic susceptibility than nonapoptotic ones. The link to the known elevated intracellular iron and high-spin ROS in the apoptotic cells has been proposed. Lastly, intrinsic cell magnetophoresis of eight cancer cell lines was examined for potential applications to studies on abnormal intracellular iron metabolism in certain cancers and to circulating tumor cell enrichment and detection. Our results reveal the existence of small subpopulations (1-2%) in K-562 and HeLa cell lines with high intrinsic magnetic susceptibility, further confirmed by their isolation (without labeling) using a commercial magnetic separator. Moreover, the effect of a soluble iron compound addition to cell culture media on the intracellular iron concentration was studied and was found to be different for different cancer cell lines. Such cell metabolism-related soluble iron transfer into intracellular compartment establishes the possibility of an alternative magnetic cell labeling to immunomagnetic labeling. In summary, the result of my studies generated a number of promising and important leads for further investigations of magnetic cell separation in biomedical and biotechnological applications.