In vitro Tissue Engineering of Liver and Primary Lymphoid Tissues with Inverted Colloidal Crystal Scaffolds for Drug Testing Application.

Abstract

Effective early stage drug toxicity testing is imperative to minimize failures in the late clinical stages of the drug development process. 2D cell cultures have been dominantly used, but they cannot adequately estimate actual toxic effects of drug molecules due to the limited capability in restoring original cellular behaviors in 3D tissues. As a potential solution to improve the predictive power of in vitro screening procedures, this dissertation explored a new opportunity of in vitro tissue engineering as a part of the drug development process. Besides the biological significance in functional tissue formation, scaffolds should be transparent and support standardized tissue growth. Inverted colloidal crystal (ICC) hydrogel scaffolds having standardized 3D structure and materials as well as retaining a high analytical capability were developed for this purpose. Uniform size spherical pore arrays prepared with cell repulsive polyacrylamide promoted homogenous HepG2 liver tissue spheroid formation, while the transparent hydrogel matrix allowed convenient characterization of cellular processes. The standardized spheroid culture model was successfully applied to the in vitro toxicity testing of CdTe and Au nanoparticles. Significantly reduced toxic effects were observed compared to the conventional 2D culture attributed by tissue-like morphology and cell phenotypic change in the spheroid culture.

In addition, ICC scaffolds combined with a LBL surface modification technique served as a platform for engineering primary lymphoid tissue, i.e. bone marrow and thymus. Under dynamic culture condition, hematopoietic stem cells (HSCs) could travel deep into the scaffold via interconnecting channels, while they were temporarily entrapped due to limited channel size and number. As a result, HSCs extensively interacted with stromal cells growing along the LBL coated pore surface. Such intimate cell-cell and cell-matrix interaction is the key process in HSCs survival and differentiation that was substantiated by ex vivo expansion and B-/T-cell differentiation of HSCs. Overall this thesis introduces a promising application of in vitro tissue engineering as a practical and valuable early stage toxicity testing tool. ICC scaffolds exhibited unique advantage in preparation of spheroid culture model and lymphoid tissue engineering. Standardized in vitro tissue models substantiate the capability to extend current cellular level cytotoxicity to the tissue level.