Date

2018

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Bioengineering

First Adviser

Liu, Yaling Liu

Abstract

Circulating tumor cell (CTC) plays a significant role to lead tumor become life-threatening. The appearance of CTC in the circulating system of tumor patients is deemed as the start of metastasis. To obtain CTC from blood is critical for vast biomedical applications, such as using CTC for DNA sequencing to reveal the gene difference between CTC and tumor cells at original site, creating in vitro tumor models based on CTC, and develop new and effective therapeutic schemes, etc. Based on this, the focus of the dissertation mainly on the research towards CTC. In brief, the dissertation demonstrates several techniques on how to isolate them from patient blood samples, how to use them as seed to form 3D tumor models, and how to use these 3D tumor models for highly efficient anti-tumor drug screening.Firstly, a wavy-herringbone (wavy-HB) structured microfluidic device is developed to effectively and selectively capture and release circulating tumor cells (CTCs) by using immunoaffinity and magnetic force. The device is designed to create passive turbulence and increase the possibility of tumor cells colliding onto the device wall. Under an external magnetic field, magnetic particles (MPs) coated with anti-EpCAM against tumor cell surface protein (EpCAM) are immobilized over the wavy-HB surface to capture tumor cells. After removing the magnetic field, the captured cells with surplus MPs are released from the device and collected, thus cells can be re-cultured for further analysis. On optimized conditions, the capture efficiency of tumor cells can be as high as 92%±2.8%. Capture experiments are also performed on whole blood samples and the capture efficiency is in a high range of 81%-95%, based on different tumor cell concentrations.Next, to isolate CTC clusters (CTCC), which has been shown to have higher invasiveness than CTC, a spiral channeled microfluidic device is introduced. By the centrifugal force created by spiral channels in microfluidic conditions, the device can isolate three types of cells, i.e. white blood cell (WBC), CTC, and CTCC. Due to the size difference among these cells, when flowing into the microfluidic device, the different centrifugal force they experience is different, and this difference enables them to exit from different outlets of the device. At lower flow rate, WBC could be firstly isolated, while CTC and CTCC could be isolated at higher flow rate. This device is able to isolate rare CTC and CTCC from massive WBC, so this device with this method can be potentially used for isolation of CTC and CTCC from patient blood samples.To use the CTC and CTCC obtained from the first two techniques, a facile method for generation of tumor spheroids in large quantity with controllable size and high uniformity is presented. HCT116 cells are used as the model cell line. Individual tumor cells are sparsely seeded onto petri-dishes. After a few days of growth, separated cellular islets are formed and then detached by dispase while maintaining their sheet shape. These detached cell sheets are transferred to dispase-doped media under orbital shaking conditions. Assisted by the shear flow under shaking and inhibition of cell-to-extracellular matrix junctions by dispase, the cell sheets curl up and eventually tumor spheroids are formed. The average size of the spheroids can be controlled by tuning the cell sheet culturing period and spheroid shaking period. The uniformity can be controlled by a set of sieves which were home-made using stainless steel meshes. Since this method is based on simple petri-dish cell culturing and shaking, it is rather facile for forming tumor spheroids with no theoretical quantity limit. This method has been used to form HeLa, A431 and U87 MG tumor spheroids and application of the formed tumor spheroids in drug screening is also demonstrated. The viability, 3D structure, and necrosis of the spheroids are characterized.Finally, to more closely mimicking the microenvironment of in vivo tumor, a bi-layer microfluidic device is presented to facilitate anti-tumor drug screening. The bi-layer microfluidic device consists of two PDMS pieces with channels and the two pieces are separated by a semi-permeable membrane to allow water, oxygen and nutrition supply but prevent cell migration. The two channels on the two PDMS pieces have a long overlapping to ensure a larger exchange area to mimic the blood vessel-tumor model. High concentration of EC is firstly seeded onto the membrane through the apical channel, and after two-day culture to ensure a confluent EC monolayer forming, tumor spheroids laden Matrigel is seeded into the basal channel. After the Matrigel is cured, the device is ready for drug test. Confocal and ImageJ are used to assess the efficacy of different concentration of drugs and combination of drugs therapies. Optical coherence tomography is employed to determine the tumor shrinkage after drug treatment.

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