Autocrine-Controlled Formation and Function of Tissue-Like Aggregates by Primary Hepatocytes in Micropatterned Hydrogel Arrays

Abstract

The liver carries out a variety of essential functions regulated in part by autocrine signaling, including hepatocyteproduced growth factors and extracellular matrix (ECM). The local concentrations of autocrine factors are governed by a balance between receptor-mediated binding at the cell surface and diffusion into the local matrix and are thus expected to be influenced by the dimensionality of the cell culture environment. To investigate the role of growth factor and ECM-modulated autocrine signaling in maintaining appropriate primary hepatocyte survival, metabolic functions, and polarity, we created three-dimensional cultures of defined geometry using micropatterned semisynthetic polyethylene glycol-fibrinogen hydrogels to provide a mechanically compliant, nonadhesive material platform that could be modified by cell-secreted factors. We found that in the absence of exogenous peptide growth factors or ECM, hepatocytes retain the epidermal growth factor (EGF) receptor ligands (EGF and transforming growth factor-a) and the proto-oncogenic mesenchymal epithelial transition factor (c-MET) ligand hepatocyte growth factor (HGF), along with fibronectin. Further, hepatocytes cultured in this three-dimensional microenvironment maintained high levels of liver-specific functions over the 10-day culture period. Function-blocking inhibitors of a5b1 or EGF receptor dramatically reduced cell viability and function, suggesting that signaling by both these receptors is needed for in vitro survival and function of hepatocytes in the absence of other exogenous signals.

One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids

“…[4–7] It has been suggested that enhanced cell-cell contact and the accumulation of autocrine factors, such as epidermal growth factor receptor (EGFR) ligands and deposited ECM, may be at play in enhancing hepatic phenotype in spheroid cultures. [6] Encapsulating spheroids in engineered 3D hydrogels has also been shown to enhance formation of organotypic structures in vitro ,[8–10] and can improve hepatocyte survival upon transplantation. [11]…”

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays

“…Spheroids harvested from hanging drop arrays on Day 7 were stained with Alexafluor-488 conjugated phalloidin (green) and counter stained with nuclear stain, DAPI (blue). Cortical actin staining using phalloidin is commonly used to demonstrate cell–cell interactions and cell aggregates in 3D culture systems, including spheroids [19–23]. Conjugated phalloidin stained the actin cytoskeletons of cells within spheroids.…”

“…Three-dimensional presence and cell–cell interaction was further confirmed using confocal microscopy upon cytoskeletal actin and nuclei staining. Cortical actin staining using phalloidin is commonly used to demonstrate cell aggregates in 3D culture systems, including spheroids [19–23]. The observance of cortical actin staining, as opposed to diffuse cytoplasmic staining and stress fiber staining of cells grown on monolayers, indicated the three-dimensional morphology of cells grown in attachment-free culture.…”

Bioreactor technologies to support liver function in vitro

“…For example, the dramatic improvement in function of primary rat hepatocyte co-cultures with fibroblasts on collagen-modified polydimethylsiloxane (PDMS, silicon rubber) substrates as a means to enhance oxygenation [62] may also arise in part due to more physiological presentation of the adhesion matrix or to partitioning of lipid-soluble hormones and nutrients into PDMS [63,64] providing a local depot regulating gene expression in a favorable manner. Similarly, culture in recessed wells may allow enhanced accumulation of autocrine factors that favorably regulate cell function [65]. …”