All ETDs from UAB

Advisory Committee Chair

Palaniappan Sethu

Advisory Committee Members

Ho-Wook Jun

Ramaswamy Kannappan

Lufang Zhou

Ganesh V Halade

Document Type

Dissertation

Date of Award

2018

Degree Name by School

Doctor of Philosophy (PhD) School of Engineering

Abstract

Leukocytes carry critical information regarding the immediate immune and inflammation status of the patients. Analysis of leukocytes requires isolation from blood since the leukocytes only consist <1% of all blood cells. Commonly used isolation processes exploit physical or biochemical differences between different cell types to enable separations. Current isolation methods inevitably subject cells to physical or biochemical stress that can activate leukocytes and change their natural state. Thus, information contained within isolated leukocytes is a combination of the natural state of the cell and an artifact of the isolation process. Microfluidics which takes advantage of scaling effects within microscale structures can ensure rapid and gentle alternatives to conventional isolation methods and is ideal for sorting, and analysis of cells and particles. The precise control of flow/particles and scaling effects of microfluidics have been applied for blood cells sorting and isolation. However, the limited processing capability and throughput as well as complex fabrication and operations have hampered widespread adoption of these approaches for replacement of conventional methods. My goal was to develop a simple yet powerful microfluidic tool that enables rapid separation of leukocytes while ensuring sufficient throughput, and minimizing isolation process induced activation without the need any sample pre-processing. This project is divided into two specific aims: i) to miniaturize the conventional density gradient centrifugation using centrifugal microfluidics for PBMC isolation and evaluate separation efficiency and activation status of isolated cells in comparison to conventional techniques; ii) develop new approach for PBMC sorting using microfluidic phase partitioning to enable separation of cells based on differences in cell surface energy. For specific aim 1, PBMCs were isolated based on differences in densities using microfluidic density gradient centrifugation. We show that PBMCs can be isolated from 100µl whole blood within 5 minutes. Evaluation of leukocyte activation via profiling of expression of surface integrins and chemokine receptors shows that our microfluidic approach significantly reduced PBMC activation in comparison to conventional isolation approaches. In specific aim 2, we sought to isolate cells based on differenced in surface energy using two-phase partitioning of dextran (DEX) and polyethylene glycol (PEG). Proof of concept demonstrations was accomplished using with polystyrene and silicon dioxide microbeads which represent a mixture of white blood cells and red blood cells. The DEX and PEG mixtures were introduced into a circular channel and phase separated under centrifugal force into unique DEX and PEG phases. The bead mixture was isolated via differences in their affinity to the two phases.

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