EFFECTS OF THE LIVER MICROENVIRONMENT ON HUMAN LIVER SINUSOIDAL ENDOTHELIAL CELLS DURING IN VITRO CULTURE

Liver Sinusoidal Endothelial Cells (LSECs) are specialized endothelial cells that play an important role in the metabolic and xenobiotic clearance functions of the liver. LSECs are distinguished by their hallmark characteristics of lacking a distinct basement membrane and containing open pores called fenestrae. As such, LSECs are an important cell type for bioengineering of functional liver tissue for clinical applications. When LSECs are removed from their native liver microenvironment they often change their phenotype to a diseased-like phenotype and are extremely difficult to expand in culture. The overall goal of this thesis was expand and maintain healthy human LSECs (hLSECs) in vitro for eventual repopulation of the liver sinusoidal endothelium of a clinically relevant liver scaffold. We examined the effects of three specific aspects of the liver microenvironment on expansion and maintenance of hLSECs in vitro: (1.) extracellular matrix (ECM) composition, (2.) cellular composition, and (3.) oxygen (O2) concentration. This is a first step for future repopulation of the liver sinusoidal endothelium of clinically relevant sized bioengineered livers. We hypothesized that providing hLSECs with ECM proteins found in the native liver sinusoidal endothelium, coculturing hLSECs with other hepatic cells and by culturing hLSECs in physiologically normal O2 levels would enhance proliferation of healthy hLSECs. The first aim of this thesis looked at culturing hLSECs on different ECM surfaces (fibronectin, collagen I, and laminin). hLSECs had higher proliferation rate and maintained their native phenotype when cultured on fibronectin. The second aim of this thesis evaluated the proliferation and phenotype of hLSECs in the presence of primary human hepatocytes, hepatic stellate cells (HSCS) (quiescent and activated), and hepatocyte growth factor (HGF). We observed that the ECM composition still plays a critical role in the maintenance of hLSECs native phenotype and their proliferation regardless of the presence of hepatocytes, HSCs or HGF. There was increased proliferation of hLSECs cultured on fibronectin in the presence of hepatocytes, HSCs, and HGF than when hLSECs were cultured alone on fibronectin. Finally, the third aim of this thesis studied the effect of O2 on hLSECs proliferation and phenotype. In vitro, hLSECs required O2 at levels higher than physiological O2 (3-12%) levels for proliferation and maintenance of native hLSEC phenotype. Overall, the findings of this project demonstrated the importance of ECM composition, the presence of other hepatic cells, and hypertonic levels of O2 on the expansion and maintenace of hLSECs phenotype in vitro for liver bioengineering and regenerative medicine purposes.

Grantor dc:publisher

Wake Forest University

Year dc:date.issued

2017

Author dc:creator

• Dhal, Abritee

• Extracellular Matrix

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10339/82193

OAI identifier oai:identifier

oai:wakespace.lib.wfu.edu:10339/82193