Abstract
Microphysiological systems (MPS), comprising human cell cultured in formats that capture features of the three-dimensional (3D) microenvironments of native human organs under microperfusion, are promising tools for biomedical research. Here we report the development of a mesoscale physiological system (MePS) enabling the long-term 3D perfused culture of primary human hepatocytes at scales of over 106 cells per MPS. A central feature of the MePS, which employs a commercially-available multiwell bioreactor for perfusion, is a novel scaffold comprising a dense network of nano- and micro-porous polymer channels, designed to provide appropriate convective and diffusive mass transfer of oxygen and other nutrients while maintaining physiological values of shear stress. The scaffold design is realized by a high resolution stereolithography fabrication process employing a novel resin. This new culture system sustains mesoscopic hepatic tissue-like cultures with greater hepatic functionality (assessed by albumin and urea synthesis, and CYP3A4 activity) and lower inflammation markers compared to comparable cultures on the commercial polystyrene scaffold. To illustrate applications to disease modeling, we established an insulin-resistant phenotype by exposing liver cells to hyperglycemic and hyperinsulinemic media. Future applications of the MePS include the co-culture of hepatocytes with resident immune cells and the integration with multiple organs to model complex liver-associated diseases
Highlights
Over the past few decades, integration of engineering and life sciences have led to the development of three-dimensional (3D) culturing methods allowing for the creation of organ-like systems, known as organ-on-chip (OOC) or microphysiological systems (MPS) [1]
Recognizing that in vitro liver models are desirable for a range of applications from assessing metabolism and toxicity to disease modeling [27, 30, 52,53,54,55,56], we used a variety of metrics to assess the functional performance of primary human hepatocytes (PHH) cultured in the mesoscale physiological system (MePS) compared to the standard scaffold over a culture period of 3 weeks. Metrics include both standard functional metabolic assays as well as a demonstration of disease modeling after characterizing the enabled lower inflammation profile in our MePS compared to standard scaffold. For the latter, motivated by the billions of individuals worldwide affected by non-alcoholic fatty liver disease [57] and type 2 diabetes [18, 58], we report here as a proof-of-concept the use of the MePS to replicate certain clinical features of a phenomenon common to both of these diseases, hepatic insulin resistance
The inherent tradeoff between resolution and throughput guided the design of the MePS so that flow distribution remained unaffected by small defects of fabrication, allowing less stringent criteria for rapid fabrication of the scaffolds
Summary
Over the past few decades, integration of engineering and life sciences have led to the development of three-dimensional (3D) culturing methods allowing for the creation of organ-like systems, known as organ-on-chip (OOC) or microphysiological systems (MPS) [1]. While biomedical research has made groundbreaking advances using traditional static two-dimensional (2D) culture methods, which offer benefits such as simplicity, throughput and reproducibility, researchers have been confronted with mounting evidence that 2D culture on hard plastics lacks physiological translatability when comparing in vitro outcomes with in vivo data [2] These observations have fueled the efforts to create ‘MicroPhysiological Systems’ (MPS) that better capture complex 3D features of human tissues [3, 4] for applications in toxicology, pharmacokinetics, and disease modeling [4]. While some optical or molecular assays are compatible with 103–105 cells, assays ranging from detailed transcriptional analyses to mass spectrometry or phosphoproteomics may require 106–108 cells [15, 19] Disease modeling such as tumor metastases in a host tissue may require substantial cell mass to replicate phenomena adequately [15, 20, 21]. These observations motivate the need to design physiological systems at mesoscopic scale between micrometric MPS and macrometric animal models, which would help bridge the gap between the two fields and provide more physiologically relevant in vitro models
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