RECURSOS DISPONIBLES

In this thesis, we developed a novel human liver model that closely mimics the architecture and function of a liver sinusoid where most liver activities take place. The liver sinusoid is a blood-carrying microfluidic system lined with LSECs. The hepatocytes are separated from LSECs by a small extracellular-matrix-protein-enriched Space of

Disse. Bile is secreted from the hepatocytes and transported to the intestines through bile

ducts. In order to develop in vitro liver model to mimic the liver sinusoid, we executed a progressional study from a macroscale culture system to a microfluidic culture system (Figure 6-1). First, PRHs were co-cultured with endothelial cells in layers using a transwell plate without flow. Second, PRHs were co-cultured with endothelial cells in a dual PDMS microchannel with or without continuous perfusion that simulates the blood sinusoid and a lower channel for the removal of the secreted factors from hepatocytes. Third, as our ultimate goal, we developed an in vitro human liver on a chip using a dual microfluidic channel to mimic the liver sinusoid based on the reliable results obtained from rat liver sinusoid on a chip. Morphological and biochemical analyses data support that our liver models mimics the liver sinusoid and maintain the viability and functionality of hepatocytes for a long-term. Extensive experimental studies were conducted to optimize the culture conditions and design parameters. In addition, we demonstrated the utility of our human liver model via HBV replication study. We found an interesting observation ‘hanging monoculture’ where only hepatocytes were cultured on the bottom of a transwell membrane, while we were developing the in vitro liver

model. The morphology, and differentiation of PRHs cultured in hanging configuration were maintained a little longer compared to hepatocytes in normal monoculture.

Thus, we believe that our in vitro human liver model closely mimics the in vivo liver sinusoid and could be extended to diverse liver biology studies and liver-related disease research such as drug induced liver toxicology, and drug study. In addition, our hanging monoculture system has the potential to be a viable culture method to culture primary hepatocytes without the help of other cell types.

Each study in this thesis was directed towards testing and optimizing cell culture conditions in each culture system with the goal of the development of ‘Engineering the liver sinusoid’. The results and conclusions for each progressional study were summarized in this chapter as following:

6.1.1. A layered long term co-culture on a transwell membrane

The first goal was to develop a novel in vitro liver model system using commercial standard 6-well and 6-transwell tissue culture plates under static condition. For this goal, primary liver cells were co-cultured with endothelial cells on the 6-well and 6-transwell plates under static condition. We verified that the cultured liver cells retained the phenotypes and functions for at least 30 days like in vivo through the study of cell morphology, viability, liver-specific functions and liver de-differentiation, when PRHs and endothelial cells were co-cultured on the opposite side of membrane.

The second goal was to develop the liver sinusoid on a chip using the PDMS microfluidic platform. To achieve this goal, we combined the layered co-culture of PRHs and endothelial cells with a single and dual microchannel system that is fabricated using microfabrication and microfluidic technology. The primary rat liver cells were co- cultured with endothelial cells in the microchannel under static or perfusion condition. When PRHs and BAECs were co-cultured on the opposite sides of a microporous membrane in a dual microchannel platform, along with the addition of continuous perfusion, the hepatocytes maintained their normal morphology and hepatocyte-like functions for at least 30 days. In addition to standard function tests, we also demonstrated the utility of our liver model by detecting successfully the HBV secreted from PRHs in the microfluidic channel.

6.1.3. The human liver model for HBV replication study

The third goal was to develop the human liver on a chip using a dual microfluidic channel to mimic the liver sinusoid based on the reliable results obtained from the rat liver sinusoid on a chip. Primary human hepatocytes co-cultured with BAECs in a dual microfluidic microchannel platform retained their viability for up to 40 days. The

developed human liver model was provided as an effective platform for HBV replication study with primary hepatocytes. Primary human hepatocytes cultured in liver on a chip were infected with HBV. HBcAg from the infected primary human hepatocytes was detected through an immunofluorescence assay. The HBV secreted from the infected cells was successfully detected by PCR. Thus, we demonstrated the utility of our human liver model via HBV replication study.

6.1.4. The hanging culture system for primary hepatocytes only culture

The fourth goal was to investigate, if indeed, hanging culture configuration of primary hepatocytes only can support a long-term primary liver-cell culture. In order to achieve this goal, we had compared hanging culture and normal culture for up to 21 days in terms of visible cell morphology, differentiation, and function. PRHs in hanging culture retained their morphology, function, and differentiation longer than PRHs in the normal monoculture configuration without the help of any other cell type. Furthermore, we investigated into the factors that play critical roles in the long term maintenance of cell phenotype and function in the hanging monoculture of primary hepatocytes. First, the cortical localized and depolymerized actin distribution was observed in the investigation of actin distribution of primary hepatocytes in hanging culture. Second, primary hepatocytes cultured in the increased gap showed improved morphology than the original gap in the investigation for gap difference between two compartments of hanging culture.

Figure 6-1. Summary of liver on a chip: Engineering the liver sinusoid.