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.