Engineering In Vitro Models of Pregnancy-Induced Liver Changes

PROJECT SUMMARY / ABSTRACT Alterations in hormone levels during pregnancy significantly affect liver physiology, including organ size, lower insulin sensitivity, increased serum concentrations of several proteins/metabolites, and alterations in drug metabolism enzymes. While 80% of pregnant females take at least one medication (not including dietary supplements), most drugs used in obstetrics patients lack population-specific dosing information due to an incomplete mechanistic understanding of functional alterations in the pregnant liver and model systems for drug testing. Mice are useful for elucidating pregnancy-induced changes in the liver due to some morphological and molecular similarities with humans; however, species-specific differences necessitate complementing mouse studies with human-relevant models. We have utilized high-throughput droplet microfluidics to develop liver microtissues (~300µm) in which primary hepatocytes (mouse or human) are embedded within microscale extracellular matrix (ECM) protein hydrogels, and liver sinusoidal endothelial cells (LSECs) are coated on the surface of the hydrogels to create sinusoidal compartmentalization between hepatocytes and LSECs. Microtissues functionally outperform self-assembled hepatic spheroids and hepatocytes within bulk (macro) gels, and they can be further augmented with other liver cell types, such as hepatic stellate cells (HSCs) and Kupffer cells (KCs). Here, we will test the novel hypothesis that microtissues containing multiple primary mouse liver cells isolated from pregnant and non-pregnant livers can be used to elucidate the role of paracrine (blood-borne) factors and preconditioning in vivo on phenotypic changes in liver cells during pregnancy. In aim 1, we will fabricate non-pregnant mouse liver microtissues using the four liver cell types above and liver decellularized ECM (dECM). We will compare the responses of mouse liver microtissues and human liver microtissues to non- pregnant and pregnant mouse sera from different gestation days to determine how each species responds to the same pregnancy blood-borne factors. The functional and gene expression changes over prolonged mouse liver microtissue culture will be benchmarked to fresh cells before culture. In aim 2, we will fabricate mouse liver microtissues from liver cells and liver dECM isolated from pregnant mice at different gestation days. We will then elucidate the effects of non-pregnant and pregnant sera on phenotypic changes in the pregnant mouse liver microtissues. We will engineer the first long-term, in vitro 3D mouse liver model of pregnancy and elucidate how blood-borne factors and in vivo preconditioning affect the phenotypes of liver cell types during pregnancy.