Liver-derived extracellular matrix hydrogels augment the survival and function of primary rat liver sinusoidal endothelial cells

Background: Liver sinusoidal endothelial cells (LSECs) have great potential as target cells for novel therapeutic strategies aimed at preventing or reversing metabolic dysfunction-associated steatotic liver disease (MASLD). However, isolating and culturing primary LSECs is notoriously difficult as they rapidly dedifferentiate, losing their specific phenotype and function. The extracellular matrix (ECM), including its composition, 3-dimensional structure, and mechanical properties, likely plays a crucial role in maintaining the fate and function of LSECs. In traditional 2D culture models, LSECs dedifferentiate into vascular-type endothelial cells, losing their specific functions quickly. <br/>Aim: to explore the effects of liver-derived ECM (L-ECM) hydrogels on LSECs and develop culture conditions that maintain the differentiated function of LSECs for prolonged periods in vitro. <br/>Methods: The L-ECM was isolated from porcine liver and made into hydrogels to culture primary rat LSECs in vitro. Biochemical and mechanical properties of L-ECM hydrogels were measured. Ultrastructure of L-ECM hydrogels was performed by scanning electron microscopy (SEM). Primary rat LSECs were isolated from male Wistar rats. The viability of LSECs was analyzed by the uptake of fluorescein diacetate/propidium iodide. Gene expression was determined by qPCR and protein expression was determined by immunofluorescence and the determination of fenestrae and sieve plates by SEM. <br/>Results: L-ECM hydrogels showed a protein content of 34.9% and the presence of glycosaminoglycans (0.045%) with a low residual DNA level (41.2 ng/mg). The proteomic analysis and molecular weight distribution of proteins of solubilized L-ECM hydrogels revealed ECM core matrisome components, abundant presence of collagens and, unexpectedly, liver-specific function proteins. L-ECM hydrogels had a stiffness of 2.1 ± 1.1 kPa and 87.9% stress relaxation after 100 seconds. The hydrogels reduced cell death of LSECs. LSECs cultured on L-ECM hydrogels presented higher expression of the LSEC-specific mRNAs Stab-2, Nos3, LYVE1, lower expression of Mmp9, CD31, and more abundant fenestrae compared to non-coated tissue-culture polystyrene (TCPS) and collagen type I coated TCPS.<br/>Conclusion: L-ECM hydrogels enhanced the cell viability of LSECs, and slowed down the dedifferentiation of LSECs compared to traditional culture models. Our results suggest that L-ECM hydrogels create a better environment for sustaining differentiated LSEC function, offering the potential for therapeutic interventions against metabolic liver diseases.