A Perfusion Bioreactor for Longitudinal Monitoring of Bioengineered Liver Constructs

Abstract

In the field of in vitro liver disease models, decellularised organ scaffolds maintain the original biomechanical and biological properties of the extracellular matrix and are established supports for in vitro cell culture. However, tissue engineering approaches based on whole organ decellularized scaffolds are hampered by the scarcity of appropriate bioreactors that provide controlled 3D culture conditions. Novel specific bioreactors are needed to support long-term culture of bioengineered constructs allowing non-invasive longitudinal monitoring. Here, we designed and validated a specific bioreactor for long-term 3D culture of whole liver constructs. Whole liver scaffolds were generated by perfusion decellularisation of rat livers. Scaffolds were seeded with Luc+HepG2 and primary human hepatocytes and cultured in static or dynamic conditions using the custom-made bioreactor. The bioreactor included a syringe pump, for continuous unidirectional flow, and a circuit built to allow non-invasive monitoring of culture parameters and media sampling. The bioreactor allowed non-invasive analysis of cell viability, distribution, and function of Luc+HepG2-bioengineered livers cultured for up to 11 days. Constructs cultured in dynamic conditions in the bioreactor showed significantly higher cell viability, measured with bioluminescence, distribution, and functionality (determined by albumin production and expression of CYP enzymes) in comparison to static culture conditions. Finally, our bioreactor supports primary human hepatocyte viability and function for up to 30 days, when seeded in the whole liver scaffolds. Overall, our novel bioreactor is capable of supporting cell survival and metabolism and is suitable for liver tissue engineering for the development of 3D liver disease models.

Recent Advances in Liver Engineering With Decellularized Scaffold

“…Compared to acids, alcohols are more routinely used to sterilize decellularized matrix materials. Ethanol (4%) and peracetic acid (0.1%) are frequently used to sterilize decellularized scaffolds in liver engineering ( Xiang et al, 2016 ; Devalliere et al, 2018 ; Nishiguchi and Taguchi, 2021 ; Sassi et al, 2021 ).…”

“…Therefore, obtaining sufficient viable hepatocytes is the biggest impediment to using primary hepatocytes for recellularization. The primary hepatocytes in a decellularized liver scaffold, cultured in a dynamic perfusion system, have higher viability, function, and can most crucially, proliferate, when compared to those cultured under static conditions ( Kojima et al, 2018 ; Yang et al, 2018 ; Debnath et al, 2020 ; Sassi et al, 2021 ). Nonetheless, the prolonged culturing of primary hepatocytes results in the onset of apoptosis and a persistent decline in the function of certain cells.…”

“…Additionally, residual DNA may activate platelets and exert significant pro-inflammatory and pro-thrombotic effects in the extracellular space, hence promoting the development of immunothrombosis ( Fuchs et al, 2010 ; Shi et al, 2020 ). The resultant thrombosis contributes to an inadequate supply of nutrients and oxygen to the implanted cells, which impedes the long-term survival of the neo-liver ( Sassi et al, 2021 ). Thus, re-establishing blood flow to the new liver entails inhibiting thrombosis and constructing a new endothelial layer.…”

Physiologically relevant microsystems to study viral infection in the human liver

“…Perfused decellularized scaffolds Sassi et al (2021) recently published a platform to perfuse a recellularized rat liver dECM in a bioreactor. The authors cultured individual lobes of the reseeded liver under syringe pump perfusion and showed significant upregulation of hepatocyte maturation markers in the perfused culture over the course of 11 days (Sassi et al, 2021). The use of decellularized scaffolds for perfusion is a novel technique that may be an optimal method for sustaining a differentiated hepatocyte phenotype.…”

“… Sassi et al (2021) recently published a platform to perfuse a recellularized rat liver dECM in a bioreactor. The authors cultured individual lobes of the reseeded liver under syringe pump perfusion and showed significant upregulation of hepatocyte maturation markers in the perfused culture over the course of 11 days ( Sassi et al, 2021 ). The use of decellularized scaffolds for perfusion is a novel technique that may be an optimal method for sustaining a differentiated hepatocyte phenotype.…”