Engineering Three-dimensional Cell Culture Microenvironment Based On Vascular Networks In Vitro

Reproducing three-dimensional (3D) physical microenvironment for the cells growth in vitro and investigating the morphological and functional changes of cells under controlled physiological conditions are of great importance in the biomedical study. However, traditional cell culture technique could only provide static and2D microenvironment which is very different from the3D physical microenvironment in vivo. It is also difficult to pinpoint the impacts of specific factors on the cell functions with the traditional cell culture method. The vascular networks in the circulatory system play critical roles in complex mass transport including delivery of oxygen and nutrient to, and removal of metabolic by products from tissues. Therefore, engineering3D vascular networks in vitro to simulate the in vivo cell growth microenvironment, is an essential approach for various biomedical research such as angiogenesis and tumor metastasis.In recent years, the microfluidic technology has been developed as powerful and versatile tools to mimic artificial organs in tissue engineering along with the advance of micro fabrication technology. With the advantages of biological compatibility, optical properties and permeability, hydrogels could be ideal candidates for use as microfluidic biomaterials for cell culture. In the hydrogel microfluidic chip, the3D dynamic gradient of factors could be generated and the cell growth microenvironment could be mimicked in vitro, and the changes of cell behaviors under the hemodynamics and chemical gradients could be observed intuitively. It could provide a good guiding significance in the disease diagnosis and therapy such as blood disease and tumor. Therefore, the following researches were carried out in this thesis:1. We describe a vascular-like structure based on a3D cellulose hydrogel chip using glass capillary as the template. The cellulose hydrogel was obtained by chemical crosslinking with the epichlorohydrin as crosslinking agent. The diffusion of fluorescent molecules through the cellulose hydrogel and the cell viability of human umbilical vein endothelial cells (HUVECs) cultured on the hydrogel are also studied. The experiment results showed that the cellulose hydrogel with collagen filling exhibited excellent biocompatibility and good structure replication, and it could be used as a microfluidic chip materials for the reproducing of3D cell growth microenvironment. Furthermore, porous cellulose tubular hydrogel was obtained by physical crosslinking with the polyethylene oxide (PEO) as pore-foaming agent. To better simulate the structures and functions of blood vessel in vivo, collagen with different concentration were stuffed into the3D scaffold of cellulose tubes for mimicking the vascular cell biology community. The transparent, porous and elastic artificial blood vessels are obtained by constructing polysaccharide cellulose-based microtubes using chitosan sacrificial template, and the porosity, lumen diameter and wall thickness could be well controlled. The cellulose/collagen microtubes with good transparency possess excellent cytocompatibility, permeability, and mechanical characteristics and could be used as scaffold biomaterial in the blood vessel tissue engineering.2. The cellulose/collagen microtubes were then fully implanted into collagen matrix to reconstruct the3D microsystem for modeling invasion of tumor cells. Well-defined simulated vascular lumens were obtained by proliferation of the HUVECs lining the artificial blood vessels, which enable us to reproduce structures and functions of blood vessel and replicate various hemodynamic parameters. Based on this model, the adhesion and transvascular migration (including intravasation and extravasation) of tumor cells across the artificial blood vessel have been well reproduced under the normal hemodynamic conditions.3. We engineer interconnected3D microfluidic vascular networks in hydrogels using molded sodium alginate lattice as sacrificial templates. The sacrificial templates were rapidly replicated in polydimethylsiloxane (PDMS) microfluidic chips via Ca2+-crosslinking and then fully encapsulated into hydrogels. Interconnected channels with well controlled size and morphology were obtained by dissolving the monolayer or multilayer templates with EDTA solution. The HUVECs were cultured on the channels linings and proliferated to form vascular lumens. The strong cell adhesion capability and adaptive response to shear stress demonstrate the excellent cytocompatibility of both the template and template-sacrificing process. Furthermore, the barrier function of endothelial layer was characterized and the results showed that a confluent endothelial monolayer was fully developed. Taken together, we develop a facile and rapid approach to engineer vascular model that could be potentially used in physiological studies of vascular functions and vascular tissue engineering.