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 threedimensional (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.

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

“…For hepatocytes, this challenge is typically addressed with a multi-step process where cells are first formed into aggregates and then encapsulated. [39, 40] Herein, we describe an alternative one step strategy combining capsule fabrication, hepatocyte encapsulation and cell aggregate formation. Microfluidic devices with coaxial flow focusing were used to create polymer droplets, composed of cross-linkable PEG4Mal and inert PEG, in mineral oil.…”

“…Therefore, entrapment of hepatocytes in hydrogels typically requires multiple steps, wherein aggregates are first assembled from single cells and are encapsulated only after cell-cell adhesion has been established (typically after 1–2 days). [4, 12, 13] Such multi-step encapsulation approaches are somewhat limited in terms of the throughput and precision of the aggregate formation.…”

Engineering a perfusable 3D human liver platform from iPS cells

“…We phenotypically stabilized primary human hepatocytes by aggregating them with 3T3-J2 fibroblasts in pyramidal microwells 58, 59 . Aggregates were encapsulated into small polyethylene glycol (PEG) microtissues 60, 61 , which maintained inducible CYP activity. By trapping microtissues in a microfluidic device we were able to provide sufficient transport of oxygen and nutrients to establish albumin secretion for more than 28 days.…”

Novel strategies for liver therapy using stem cells

“…To achieve full maturity may therefore require current blocks to be overcome through further manipulation of cell intrinsic, cell extrinsic or both types of signalling pathways. Many investigators are currently focusing their efforts on these investigations to try and make ‘mature’ hepatocytes in vitro with some success 14 15. It is important to concurrently consider alternative explanations and solutions, however.…”