Date of Award

Summer 2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Biomedical Engineering - (Ph.D.)

Department

Biomedical Engineering

First Advisor

Cheul H. Cho

Second Advisor

Bryan J. Pfister

Third Advisor

Eun Jung Lee

Fourth Advisor

George Collins

Fifth Advisor

Pranela Rameshwar

Sixth Advisor

Diego Fraidenraich

Abstract

Anti-angiogenic drugs have failed to show significant extended mortality, except when co-administered with chemotherapy drugs in clinical trials. This should be predicted by in vitro models, and yet 2D in vitro models of liver cancer co-administered with these two types of drugs show increased cell viability, contradicting clinical trials. In vitro models should mimic clinical trials in order to accurately predict drug outcomes. 2D in vitro models fail because they lack features of the cancer environment such as presence of stromal cells and a vasculature.

In order to achieve a biomimetic and vascularized in vitro model that would better recapitulate the cancer environment, a vascularized 3D in vitro liver cancer model is proposed in which: 1) heterospheroids, agglomerations of liver cancer HepG2 and stromal cells, are fabricated and encapsulated in collagen gel; 2) heparin crosslinked, wet-spun chitosan microfiber/electrospun chitosan mat tube are coated with endothelial cells; and 3) spheroids and endothelial coated electrospun chitosan mat tube are embedded together on Matrigel.

Using the hanging drop method, spheroids are formed and encapsulated in collagen before exposure to the anti-cancer drug doxorubicin. Cell viability, bile canaliculi, and cytochrome p450 activity are measured afterwards, showing greater maintenance of liver cell function for heterospheroids in collagen gel.

Blood vessel constructs are developed using chitosan microfibers/tubes. Chitosan is modified with heparin and is confirmed by Toluidine blue staining and FTIR (Fourier Transform Infrared Spectroscopy). Fibronectin and VEGF are then adsorbed showing greater cell adhesion. Microfibers/electrospun mat tubes are incubated with endothelial cells and embedded on Matrigel resulting in vascular sprouting.

Triculture spheroids, heterospheroids which also contain endothelial cells, and cell coated chitosan electrospun mat tube are combined on Matrigel to form a vascularized model. Triculture spheroids on Matrigel are exposed to anti-cancer drugs, and vascular sprouts are measured showing expected decrease in length. Spheroids and cell coated tube together show anastomosis and migration, and fluorescent compounds injected into the model show their presence in nascent lumen.

Overall, a vascularized model is made which exhibits similar qualities to cancer in vivo and therefore can be used as a platform for anti-cancer drug testing.

Share

COinS