Development of biomimetic microfluidic adhesive substrates for cell separation

Abstract

Cell separation is important in medical, biological research, clinical therapy, diagnostics and many other areas. The conventional methods of cell sorting have limited applications due to sophisticated equipment settings, high costs, or time-intensive and labor-intensive processing steps. Inspired from natural cell sorting system-cell rolling, a novel microfluidic device design was proposed for point-of-care and point-of-use applications. It relies on interaction of cells with biomimetic adhesive substrates comprising multiple inclined, asymmetric bands of weak adhesive molecules. Such device design allows continuous sorting of cells without irreversible capture of cells. To realize such device, comprehensive studies of how cells settle onto the substrate, how cells capture by the substrate, the effect of substrate parameters on separation potential, and selection of adhesion molecules are needed to optimize device performance. In this thesis, first, how the cells settle and how they are captured by the receptors were studied using HL60 cells as a model leukocyte cell line and P-selectin as a model receptor. Settling distance of HL60 cells under different shear stresses inside microfluidic channels was identified from the study of convection velocity of cells at different position along the channel. The results show that settling distance increases with increasing shear stress. Cell capture was then quantified by characterizing how far settled HL60 cells travelled before they were captured by P-selectin molecules, defined as the attachment distance. Cumulative probabilities of attachment distance of cells at different shear stresses revealed that increasing shear stress results in exponential increase of the attachment distance of cells by receptor molecules. An empirical model was developed to predict capture probability by an inclined receptor band and the prediction value was verified by experimental data from a device. Second, a patterning method involving microcontact printing was developed to create biomimetic adhesive substrates comprising multiple inclined receptor bands of P-selectin molecules. The patterned substrates were then used to study how transport of HL60 cells can be controlled by the substrate parameters including pattern inclination angle with respect to shear flow direction, shear stress magnitude, and P-selectin incubation concentration. The effects of substrate parameters were quantified in terms of the edge tracking length, lateral displacement, and the rolling velocity. The edge inclination angle was identified as the strongest modulator of edge tracking length on a single band for captured cells. To study optimization of the device design, experimental data of cell settling, cell attachment, and edge tracking length were integrated into a model to predict device performance including device capture efficiency and total lateral displacement. General guidelines for microfluidic device design were established based on the results from the model: smaller band width, edge angle of 15-20°, and lower shear stress. Finally, to develop new specific receptor-ligand systems, M13 pVIII and pIll phage libraries were used for selecting peptides with affinity to CD4 proteins. Screened phage from pVIII library was immobilized on the gold surface and capture efficiency of CD4+ cells were characterized. The interaction between selectin phage and CD4+ cells were demonstrated to be CD4-dependent. Moreover, the selected phage from pIII library and the corresponding synthetic peptides were demonstrated to exhibit specificity to CD4 proteins. In summary, this thesis focuses on development of biomimetic adhesive substrates for microfluidic devices involving transient interactions between the cells and the receptor-patterned substrates. How cells flow and get captured by patterned biomimetic substrates inside the microfluidic channels, how substrates parameters affect cell rolling trajectories and device performance, and how to identify new receptor-ligand systems were discussed in this thesis. This study has led to realization of a microfluidic device for separating neutrophils from blood. This microfluidic system provides continuous sorting without irreversible capture of cells, and is believed to be an effective method that can potentially be used in many point-of-care applications. Keywords: microfluidics, cell separation, cell rolling, selectin, biopanning, M13