Engineering a perfusable 3D human liver platform from iPS cells

Abstract

In vitro models of human tissue are crucial to our ability to study human disease as well as develop safe and effective drug therapies. Models of single organs in static and microfluidic culture have been established and shown utility for modeling some aspects of health and disease; however, these systems lack multi-organ interactions that are critical to some aspects of drug metabolism and toxicity. Thus, as part of a consortium of researchers, we have developed a liver chip that meets the following criteria: (1) employs human iPS cells from a patient of interest, (2) cultures cells in perfusable 3D organoids, and (3) is robust to variations in perfusion rate so as to be compatible in series with other specialized tissue chips (e.g. heart, lung). In order to achieve this, we describe methods to form hepatocyte aggregates from primary and iPS-derived cells, alone and in co-culture with support cells. This necessitated a novel culture protocol for the interrupted differentiation of iPS cells that permits their removal from a plated surface and aggregation while maintaining phenotypic hepatic functions. In order to incorporate these 3D aggregates in a perfusable platform, we next encapsulated the cells in a PEG hydrogel to prevent aggregation and overgrowth once on chip. We adapted a C-trap chip architecture from the literature that enabled robust loading with encapsulated organoids and culture over a range of flow rates. Finally, we characterize the liver functions of this iHep organoid chip under perfusion and demonstrate a lifetime of at least 28 days. We envision that such this strategy can be generalized to other microfluidic tissue models and provides an opportunity to query patient-specific liver responses in vitro.

A Pathway to Personalizing Therapy for Metastases Using Liver-on-a-Chip Platforms

“…The 3D-printed tri-culture liver tissue showed higher gene expression of hepatocyte markers (albumin, HNF4α, transthyretin, CYP3A4, CYP2C9 and CYP2C19) and increased albumin and urea production [62]. Schepers et al expanded the 3D culture system into a perfused liver model where iPS-derived hepatocytes and 3T3-J2 fibroblasts were firstly aggregated in pyramidal microwells using low speed centrifugation [122]. The aggregates were then encapsulated with polyethylene glycol (PEG) hydrogel to prevent uncontrollable growth that would limit oxygen and nutrient diffusion.…”

“…The aggregates were also positive for both HNF4a and HNF1b, markers for hepatocytes and biliary epithelium. Although the maturity of these cells is still unclear as the CYPs activities were inferior to those of primary human hepatocytes and AFP expression is unknown, this study shows that encapsulation allows the aggregates to withstand the sheer stress produced by medium perfusion and this provides the flexibility for multi-organ systems integration [122]. All of these technological advancements may hopefully overcome the obstacle in producing uniform and fully functional hepatocytes for proper liver modeling.…”

3D Miniaturization of Human Organs for Drug Discovery

“…Organoids are 3D microtissues that overcome the major constraints of 2D tissue models and provide prolonged cell viability and functionality. [41] Organoid-based techniques are compatible with a variety of different pipetting methods and coculturing techniques allowing the development of multiorgan models. [53] In these multicellular aggregates, the need for supporting gels or matrices is eliminated, the adverse effects 3D culture approach for generating a laminated cerebral cortex like structure from pluripotent stem cells.…”

“…Bottom-up approaches rely on the emergent behavior of biological systems to generate complex tissue-and organ-like constructs. [39][40][41] Pluripotent stem cells (PSCs) are primarily used as the cell source due to their capacity for self-organization through processes of self-assembly, self-patterning, and self-driven morphogenesis. [42][43][44] Independent of the approach used to develop 3D printing models, accurately recapitulating key aspects of the in vivo environment remains a major challenge.…”

Microdevices and Microsystems for Cell Manipulation

“…Another common design using extruded electrodes generate strong DEP forces, which improves throughput and affords better trapping efficiency up to a flow rate of 12 μL/min. Targeted cells such as HL-60 cell [157], polystyrene particles, drosophila cells [158], and viable yeast cells [155,159] are trapped by the electrodes in the microchannel with positive DEP while non-target cells are flushed to the outlet via negative DEP. Arrays of extruded electrode posts have been fabricated from gold [157], carbon [158] and highly doped silicon [160].…”