Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells

Abstract

Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a “gold standard” in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.

Bioinspired tuning of glycol chitosan for 3D cell culture

“…Recently, non-cell adherent polymers have been utilized for 3D spheroid formation and culture. [7][8][9] In this case, cells may not adhere to the substrate but could form spheroids on the non-adherent substrate because cell-cell interactions are favored over cell-substrate interactions. In this regard, chitosan, a deacetylated chitin, has been widely studied as a non-adherent polymer to promote the formation of self-assembled 3D cellular spheroids.…”

Engineering 3D Hydrogels for Personalized In Vitro Human Tissue Models

“…The simplest example is Matrigel layer, which will induce small adherent hepatocyte aggregates. Other weakly or nonadhesive materials include alginate, heparin/heparin sulfate/dermatan sulfate‐coated substrate, poly(2‐hydroxyethyl methacrylate) coatings, agarose, or PEG . In addition to direct seeding of hepatocytes on a low‐adhesive hydrogel layer, there are a variety of 3D manipulations which induce spheroid formation of hepatocytes with enhanced liver‐specific function.…”

Microfluidic device‐assisted etching of p‐HEMA for cell or protein patterning

“…p-HEMA was chosen as the nonadhesive substrate for our material due to its successful use for the study of rat hepatocyte spheroids, flexibility in use for techniques, such as optical tweezing, and the fact that it is inexpensive, easily available, and biocompatible. [10][11][12]16 In our study, we first coated glass coverslips or a six-well cell culture plate with a thin layer of p-HEMA (Figure 1). This created an initial nonadhesive substrate to prevent protein or cell adhesion.…”

“…Historically, poly(2‐hydroxyethyl methacrylate) (p‐HEMA) has been used to create regions of decreased protein and cell adhesion of spheroid cultures, and in specific, rat hepatocytes . The fabrication of p‐HEMA‐coated regions with microfluidic devices has been used for creation of walls and molds that allow for single cell isolation of cells and other cell types .…”

“…Historically, poly(2-hydroxyethyl methacrylate) (p-HEMA) has been used to create regions of decreased protein and cell adhesion of spheroid cultures, and in specific, rat hepatocytes. [10][11][12] The fabrication of p-HEMA-coated regions with microfluidic devices has been used for creation of walls and Correspondence concerning this article should be addressed to Bonnie L. Firestein at Firestein@biology.rutgers.edu molds that allow for single cell isolation of cells and other cell types. 13,14 While very useful for studies of single cells, this fabrication type has several limitations.…”