Micropatterned cell-cell interactions enable functional encapsulation of primary hepatocytes in hydrogel microtissues.

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.

Flow-based pipeline for systematic modulation and analysis of 3D tumor microenvironments.

The cancer microenvironment, which incorporates interactions with stromal cells, extracellular matrix (ECM), and other tumor cells in a 3-dimensional (3D) context, has been implicated in every stage of cancer development, including growth of the primary tumor, metastatic spread, and response to treatment. Our understanding of the tumor microenvironment and our ability to develop new therapies would greatly benefit from tools that allow us to systematically probe microenvironmental cues within a 3D context. Here, we leveraged recent advances in microfluidic technology to develop a platform for high-throughput fabrication of tunable cellular microniches ("microtissues") that allow us to probe tumor cell response to a range of microenvironmental cues, including ECM, soluble factors, and stromal cells, all in 3D. We further combine this tunable microniche platform with rapid, flow-based population level analysis (n > 500), which permits analysis and sorting of microtissue populations both pre- and post-culture by a range of parameters, including proliferation and homotypic or heterotypic cell density. We used this platform to demonstrate differential responses of lung adenocarcinoma cells to a selection of ECM molecules and soluble factors. The cells exhibited enhanced or reduced proliferation when encapsulated in fibronectin- or collagen-1-containing microtissues, respectively, and they showed reduced proliferation in the presence of TGF-ß, an effect that we did not observe in monolayer culture. We also measured tumor cell response to a panel of drug targets and found, in contrast to monolayer culture, specific sensitivity of tumor cells to TGFßR2 inhibitors, implying that TGF-ß has an anti-proliferative affect that is unique to the 3D context and that this effect is mediated by TGFßR2. These findings highlight the importance of the microenvironmental context in therapeutic development and that the platform we present here allows the high-throughput study of tumor response to drugs as well as basic tumor biology in well-defined microenvironmental niches.

DNA-templated assembly of droplet-derived PEG microtissues.

Patterning multiple cell types is a critical step for engineering functional tissues, but few methods provide three-dimensional positioning at the cellular length scale. Here, we present a "bottom-up" approach for fabricating multicellular tissue constructs that utilizes DNA-templated assembly of 3D cell-laden hydrogel microtissues. A flow focusing-generated emulsion of photopolymerizable prepolymer is used to produce 100 µm monodisperse microtissues at a rate of 100 Hz (10(5) h(-1)). Multiple cell types, including suspension and adherently cultured cells, can be encapsulated into the microtissues with high viability (~97%). We then use a DNA coding scheme to self-assemble microtissues "bottom-up" from a template that is defined using "top-down" techniques. The microtissues are derivatized with single-stranded DNA using a biotin-streptavidin linkage to the polymer network, and are assembled by sequence-specific hybridization onto spotted DNA microarrays. Using orthogonal DNA codes, we achieve multiplexed patterning of multiple microtissue types with high binding efficiency and >90% patterning specificity. Finally, we demonstrate the ability to organize multicomponent constructs composed of epithelial and mesenchymal microtissues while preserving each cell type in a 3D microenvironment. The combination of high throughput microtissue generation with scalable surface-templated assembly offers the potential to dissect mechanisms of cell-cell interaction in three dimensions in healthy and diseased states, as well as provides a framework for templated assembly of larger structures for implantation.