Micropatterned cell-cell interactions enable functional encapsulation of primary hepatocytes in hydrogel microtissues.
ABSTRACT: Drug-induced liver injury is a major cause of drug development failures and postmarket withdrawals. In vitro models that incorporate primary hepatocytes have been shown to be more predictive than model systems which rely on liver microsomes or hepatocellular carcinoma cell lines. Methods to phenotypically stabilize primary hepatocytes ex vivo often rely on mimicry of hepatic microenvironmental cues such as cell-cell interactions and cell-matrix interactions. In this work, we sought to incorporate phenotypically stable hepatocytes into three-dimensional (3D) microtissues, which, in turn, could be deployed in drug-screening platforms such as multiwell plates and diverse organ-on-a-chip devices. We first utilize micropatterning on collagen I to specify cell-cell interactions in two-dimensions, followed by collagenase digestion to produce well-controlled aggregates for 3D encapsulation in polyethylene glycol (PEG) diacrylate. Using this approach, we examined the influence of homotypic hepatocyte interactions and composition of the encapsulating hydrogel, and achieved the maintenance of liver-specific function for over 50 days. Optimally preaggregated structures were subsequently encapsulated using a microfluidic droplet-generator to produce 3D microtissues. Interactions of engineered hepatic microtissues with drugs was characterized by flow cytometry, and yielded both induction of P450 enzymes in response to prototypic small molecules and drug-drug interactions that give rise to hepatotoxicity. Collectively, this study establishes a pipeline for the manufacturing of 3D hepatic microtissues that exhibit stabilized liver-specific functions and can be incorporated into a wide array of emerging drug development platforms.
Project description:Three-dimensional (3D) tissue models replicating liver architectures and functions are increasingly being needed for regenerative medicine. However, traditional studies are focused on establishing 2D environments for hepatocytes culture since it is challenging to recreate biodegradable 3D tissue-like architecture at a micro scale by using hydrogels. In this paper, we utilized a gelatin methacryloyl (GelMA) hydrogel as a matrix to construct 3D lobule-like microtissues for co-culture of hepatocytes and fibroblasts. GelMA hydrogel with high cytocompatibility and high structural fidelity was determined to fabricate hepatocytes encapsulated micromodules with central radial-type hole by photo-crosslinking through a digital micromirror device (DMD)-based microfluidic channel. The cellular micromodules were assembled through non-contact pick-up strategy relying on local fluid-based micromanipulation. Then the assembled micromodules were coated with fibroblast-laden GelMA, subsequently irradiated by ultraviolet for integration of the 3D lobule-like microtissues encapsulating multiple cell types. With long-term co-culture, the 3D lobule-like microtissues encapsulating hepatocytes and fibroblasts maintained over 90% cell viability. The liver function of albumin secretion was enhanced for the co-cultured 3D microtissues compared to the 3D microtissues encapsulating only hepatocytes. Experimental results demonstrated that 3D lobule-like microtissues fabricated by GelMA hydrogels capable of multicellular co-culture with high cell viability and liver function, which have huge potential for liver tissue engineering and regenerative medicine applications.
Project description:3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures.Our paper combines an interesting new way for making capsules with cultivation of difficult-to-maintain primary epithelial cells (hepatocytes). The microcapsules described here will enable high density suspension culture of hepatocytes or other cells and may be used as building blocks for engineering tissues.
Project description:Engineered tissue models comprise a variety of multiplexed ensembles in which combinations of epithelial, stromal, and immune cells give rise to physiologic function. Engineering spatiotemporal control of cell-cell and cell-matrix interactions within these 3D multicellular tissues would represent a significant advance for tissue engineering. In this work, a new method, entitled CAMEO (Controlled Apoptosis in Multicellular tissues for Engineered Organogenesis) enables the non-invasive triggering of controlled apoptosis to eliminate genetically-engineered cells from a pre-established culture. Using this approach, the contribution of stromal cells to the phenotypic stability of primary human hepatocytes is examined. 3D hepatic microtissues, in which fibroblasts can enhance phenotypic stability and accelerate aggregation into spheroids, were found to rely only transiently on fibroblast interaction to support multiple axes of liver function, such as protein secretion and drug detoxification. Due to its modularity, CAMEO has the promise to be readily extendable to other applications that are tied to the complexity of 3D tissue biology, from understanding <i>in vitro</i> organoid models to building artificial tissue grafts.
Project description:Current 2-dimensional hepatic model systems often fail to predict chemically induced hepatotoxicity due to the loss of a hepatocyte-specific phenotype in culture. For more predictive in vitro models, hepatocytes have to be maintained in a 3-dimensional environment that allows for polarization and cell-cell contacts. Preferably, the model will reflect an in vivo-like multi-cell type environment necessary for liver-like responses. Here, we report the characterization of a multi-cell type microtissue model, generated from