Z2 - Investigation of Fontan-associated Thrombosis and Fibrotic Remodeling in a Bioengineered Model of a Liver Sinusoid

Introduction: : The most common birth defect in the US are congenital heart defects (CHD), affecting about 1% of births annually. Single-ventricle disease, a type of CHD in which the infant is born with a severely underdeveloped ventricle, requires a series of three invasive surgeries to restore proper blood oxygenation levels. The final surgery is called the Fontan procedure, which provides an alternative circulation path that bypasses the malfunctioning ventricle, as well as a reliance on systemic increased central venous pressure to drive blood flow to the lungs. The surgery provides an excellent option to critically ill patients, however the effect of systemic increased pressure on the liver and how this leads to the development of Fontan-associated liver disease (FALD) is largely unknown. All Fontan patients experience some degree of congestion and fibrosis in the liver, but for some of these patients it develops into cirrhosis and FALD. Current methods for diagnosis and monitoring of Fontan patients for FALD include biomarker blood tests, medical imaging for liver nodules, and liver biopsy, however these are not specific enough or too invasive for routine monitoring. There is therefore a critical need to better understand the pathophysiology of the liver sinusoid under these systemic increased pressure conditions, to discover alternative methods for disease diagnosis and monitoring.

Materials and

Methods: : Microphysiological systems provide an excellent way to test controlled pressure and flow differences on liver cells. Through the utilization of freeform-reversible embedding of suspended hydrogels (FRESH) 3D-printing, we engineered a collagen-based high-resolution internally perfusable scaffolds (CHIPS) liver tissue with Immortalized Human Hepatic Sinusoidal Endothelial Cells (ABM T0056) and Immortalized Human Hepatocytes (ABM T0063), with an extracellular matrix composed of 23 mg/mL Lifeink 240 Acidic Type I Collagen Bioink. The liver cell lines were grown to confluency with a count of at least 10 million cells total before FRESH printing. After FRESH printing, some liver CHIPS were analyzed with a Live / Dead assay for cell viability, then fixed with 4% PFA and kept in the fridge for further immunofluorescent staining. The rest of the liver CHIPS were analyzed in culture over several days. The liver CHIP design works in conjunction with our vascular and perfusion organ-on-a-chip reactor (VAPOR). Pressure and flow can be modulated separately by controlling channel dimensions of the liver CHIPS, to determine which mechanical factors are significant in causing fibrosis and congestion. The liver tissue was left to grow under static and perfused conditions over several days in the VAPOR chamber. After seven days, the CHIPS were analyzed with a Live / Dead assay for cell viability and then fixed with 4% PFA before immunofluorescent staining. After fixing, the CHIPS were stained with NucSpot, Phalloidin, CD-31, and CK-8 and imaged on the Leica Stellaris-8 confocal microscope.

Results, Conclusions, and Discussions:: The liver CHIPS are printed with the multi-material printer (Figure 1A) inside a biosafety cabinet, with sterile collagen I and the hepatocyte and liver sinusoidal endothelial cells, at the ratio of 70% hepatocytes and 30% endothelial cells. The CHIPS are printed with 10 mm in length, 9 mm in width, and 2 mm in height, with ~300 um thickness in the liver cell layer (Figure 1B). To perfuse media through the liver CHIPS over several days, the CHIPS are placed inside the VAPOR chambers and connected to a peristaltic pump, sterile tubing, and a bubble trap (Figure 1C), all inside an incubator. The engineered liver tissue integrates into the VAPOR perfusion bioreactors to modulate flow and pressure for healthy (10-15 mmHg) and Fontan (> 15 mmHg) circulations. The liver CHIPS demonstrate specificity in layer multi-material printing (Figure 1D), with clear separation of Collagen-I and Liver Bioink materials. The liver cells demonstrate maturation of liver cell health markers (Figure 1E) for hepatocytes with CK-8 and LSECs for CD-31. Comparing the liver CHIPS on Day 1 to Day 7, there’s a significant difference in cell alignment and proliferation (Figure 1F). On Day 1, the liver cells remain in the diagonal 3D printed pattern design, but by Day 7, the liver cells have proliferated and remodeled the tissue. After seven days in culture, the 3D printed liver tissues show endothelial cells and hepatocytes expressing CD-31 and CK-8 respectively, and the cellular layer (~300 um) shows proliferation and growth into the collagen tissue structure. From this preliminary research, we have a working model of a perfusable bioengineered liver tissue with the ability to remodel and proliferate under culture. With a working liver tissue model for FALD, there’s incredible potential for biomarker discovery for better diagnosis and monitoring.

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