Human liver models that are three-dimensional (3D) in architecture are indispensable for compound metabolism/toxicity screening, to model liver diseases for drug discovery, and for cell-based therapies; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months. Therefore, here we determined how microscale 3D collagen I presentation and fibroblast interaction affect the longevity of PHHs. High-throughput droplet microfluidics was utilized to generate reproducibly sized (∼300-μm diameter) microtissues containing PHHs encapsulated in collagen I ± supportive fibroblasts, namely, 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as controls. Hepatic functions and gene expression were subsequently measured for up to 6 weeks. We found that microtissues placed within multiwell plates rescued PHH functions at 2- to 30-fold higher levels than spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Importantly, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically relevant manner to drug-mediated cytochrome P450 induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display high hepatic functions for 6+ weeks and are useful for assessing drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find utility for modeling liver diseases and as building blocks for cell-based therapies.
We explore the possibility of re-engineering mitochondrial genes and expressing them from the nucleus as an approach to rescue defects arising from mitochondrial DNA mutations. We have used a patient cybrid cell line with a single point mutation in the overlap region of the ATP8 and ATP6 genes of the human mitochondrial genome. These cells are null for the ATP8 protein, have significantly lowered ATP6 protein levels and no Complex V function. Nuclear expression of only the ATP8 gene with the ATP5G1 mitochondrial targeting sequence appended restored viability on Krebs cycle substrates and ATP synthesis capabilities but, failed to restore ATP hydrolysis and was insensitive to various inhibitors of oxidative phosphorylation. Co-expressing both ATP8 and ATP6 genes under similar conditions resulted in stable protein expression leading to successful integration into Complex V of the oxidative phosphorylation machinery. Tests for ATP hydrolysis / synthesis, oxygen consumption, glycolytic metabolism and viability all indicate a significant functional rescue of the mutant phenotype (including re-assembly of Complex V) following stable co-expression of ATP8 and ATP6. Thus, we report the stable allotopic expression, import and function of two mitochondria encoded genes, ATP8 and ATP6, resulting in simultaneous rescue of the loss of both mitochondrial proteins.
Changes to the cellular microenvironment are an integral characteristic of numerous pathologies, including cancer, fibrosis, and autoimmune disease. Current in vitro methodologies available to study three-dimensional tissue remodeling are ill-suited for high-throughput studies as they are not scalable for large-scale experiments. Combining droplet microfluidics and patterned low-adhesion culture surfaces, we have engineered a workflow to incorporate cell-extracellular matrix (ECM) interactions in a versatile and high-throughput platform that is compatible with existing high-throughput liquid handling systems, enables long-term experiments (>1 month), and is well suited for traditional and novel biological measurements. With our platform, we demonstrate the feasibility of high-throughput ECM remodeling studies with collagen microtissues as one application of a tissue-level function. In this study, we use our workflow to examine ECM remodeling at the tissue, cell, and subcellular levels, leveraging assays ranging from immunohistochemistry and live cell imaging, to proliferation and contraction assays. With our unique culture system, we can track individual constructs over time and evaluate remodeling on several scales for large populations. Finally, we demonstrate the ability to cryopreserve our microtissues while retaining high viability and cell function, an invaluable method that could allow for dissemination and freezing of microtissues after mass production. Using these methods, our ECM-based system becomes a viable platform for modeling diseases characterized by tissue reorganization as well as a scalable method to conduct in vitro cell-based assays for drug screening and high-throughput biological discovery.
The objective of this study was to create a method for studying cell–matrix interactions in a physiologically relevant 3D protein-based tissue construct that could be scaled up to perform large-scale screens, study cell–matrix interactions on a population basis, or be remodeled by cells to build larger tissues. We have developed an easy-to-use method to miniaturize protein-based tissue constructs that maintains the 3D in vitro environment, while alleviating several obstacles associated with larger avascular tissue constructs. In this study, we demonstrate that (i) cells can interact with the 3D environment both while encapsulated or while interacting only with the surface of the microtissues, (ii) encapsulated cells are highly viable and, for the first time, (iii) microtissues on this size scale (~200 μm) can be used to quantify cell contractility. This versatile platform should facilitate large-scale screens in 3D in vitro culture conditions for drug development and high throughput mechanistic biology.
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