Introduction:
The need to create physiologically realistic in vitro liver models has resulted in the emergence of technologies such as organ on a chip, 3D printing and rotational culture methods, yielding protocols to create improved human preclinical models.[1]However, 3D cellular aggregates present certain drawbacks. First, there is no vascular network formation therefore nutrient and waste transport limits the maximum size of 3D tissues grown in vitro. [2] Second, in vivo niche-based cues such as ECM components are missing, which slows initial aggregation and cell activation resulting in loss of function and phenotype in long term cultures.[3] Unlike other organs, the liver has a unique makeup with a proportionately small ECM in relation to its volume, mainly consisting of collagen, fibronectin, and laminin.[4] Collagen represents 60% of human liver ECM molecules and constitutes mostly fibrillar collagens such as type I and III collagen,[5] providing tensile strength to the organ. Whereas non-collagenous proteins such as fibronectin and laminin are vital to maintaining basement membrane and functional integrity.[6] Fibronectin is a multifunctional adhesive glycoprotein that originally synthesized by liver cells and abundantly present in liver tissue. This protein is directly involved in regulating cellular behavior such as cell survival and proliferation.[7] Laminin is another major non-collagenous adhesive glycoprotein presents in the hepatic perisinusoidal space (space of Disse). It includes specific combinations of α, β, and γ chains giving rise to functional diversity within a common structural framework. Within this family the distribution and expression of α5, β1 and β2 laminin chains in mammalian liver have been widely reported.[8,9]
ECM-based molecules (peptides or whole proteins) have been widely incorporated into biomaterials to promote the formation and function of various types of spheroids and organoids.[10,11]For example, human intestinal organoids were generated from pluripotent stem cells in a synthetic hydrogel based on a four-armed, maleimide-terminated poly(ethylene glycol) macromer functionalized with arginine-glycine-aspartate (RGD) adhesive peptides.[12] The authors showed that organoids encapsulated in this scaffold show high viability as well as expression levels of pluripotency, endoderm, and epithelial junction markers compared to non-modified gels. Similarly, Lin et al.[13] prepared a hydrogel based on poly (ethylene glycol)-tetra-norbornene (PEG4NB) functionalized by bioactive peptides (e.g., fibronectin-derived arginine-glycine-aspartate-serotonin (RGDS)) to improve cell–matrix interactions. They observed elevated urea secretion and Cytochrome P450 3A4 (CYP3A4) enzymatic activities, as well as upregulated mRNA levels of multiple hepatocyte genes (e.g., CYP3A4, bile salt export pump (BESP) and sodium-taurocholate cotransporting polypeptide (NTCP)) of two human hepatoma-derived cell lines (Huh7 and HepG2) encapsulated in these gels.
Despite these reports, new methodologies are needed for synthetic cellular microenvironments to reduce mass transport limitations, especially for metabolically active cell types/tissues such as the liver. Various methodologies have been formulated to promote oxygen transport and maintain viability as well as the function of cells in 3D culture.[14,15] In our recent work, we created PFC-MPs to overcome common limitations of spheroids such as inadequate oxygen supply and ultimate loss of cell/organ specific functions over long-term cultures.[16] Our data suggested that PFC- conjugated MPs offer a simple, affordable, and direct approach for improving mass transport of nutrients within spheroids and other engineered tissues. In the present study, we extended our PFC-MP approach to yield improved hepatic 3D cell culture models using ECM components. First, we examined if PFC- MPs play a role in driving oxygen into spheroidal aggregates using optical sensing. Then, we covalently tethered ECM proteins including laminin-111, laminin-511, laminin-521, and fibronectin on the surface of PFC- MPs followed by co-culturing these particles with two immortalized human liver cells including human hepatoma (HepG2) and hepatic stellate cells (HSCs). We characterized liver-specific synthetic functions and cell adhesion patterns, which allowed us to collect evidence that different ECM proteins presented from PFC-MPs have distinctive roles in the physiological regulation of liver cells in vitro 3D culture.