3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D cultures systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures.

A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future.

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The heterogeneity of native tissues requires complex materials to provide suitable substitutes for model systems and replacement tissues. Functionally graded materials have the potential to address this challenge by mimicking the gradients in heterogeneous tissues such as porosity, mineralization, and fiber alignment to influence strength, ductility, and cell signaling. Advancements in microfluidics, electrospinning, and 3D printing enable the creation of increasingly complex gradient materials that further the understanding of physiological gradients. The combination of these methods enables rapid prototyping of constructs with high spatial resolution. However, successful translation of these gradients requires both spatial and temporal presentation of cues to model the complexity of native tissues that few materials have demonstrated. This Review highlights recent strategies to engineer functionally graded materials for the modeling and repair of heterogeneous tissues, together with a description of how cells interact with various gradients.

29Animal models of bone marrow have limited spatial and temporal resolution to observe biological 30 events (intravasation and cellular egress) and are inadequate to dissect dynamic events at the niche level 31 (100 microns). Utilizing microfluidic and stem cell technology, we present a 3D in vitro model of human 32 bone marrow that contains perivascular and endosteal niches complete with dynamic, perfusable vascular 33 networks. We demonstrate that our model can perform in vivo functions including maintenance and 34 differentiation of CD34 + hematopoietic stem/progenitor cells (HSPC) for up to fourteen days, egress of 35 myeloid progenitors, and expression of markers consistent with in vivo human bone marrow. The platform 36 design enables the addition of tissue niches at a later timepoint to probe mechanisms such as tumor cell 37 migration. Overall, we present a novel organ-on-a-chip platform that is capable of recapitulating the human 38 bone marrow microenvironment to observe hematopoietic phenomena at high spatial and temporal 39 resolution. 40 41 Manuscript Template Page 2 of 34 MAIN TEXT 42 43the abluminal surface of a blood vessel comprises the perivascular niche (4, 9). 51Both the perivascular and endosteal niches contain sinusoidal blood vessels as a key feature of the bone 52 marrow. These specialized vessels are surrounded by many different cell types including mesenchymal stem cells 53 (MSC), specialized CXCL12 abundant reticular (CAR) cells, HSC, leukocytes at different stages of differentiation, 54 and adipocytes (10). Bone marrow stromal cells are known to secrete chemotactic signals such as stem cell factor 55 (SCF) (4) and CXCL-12/SDF-1 (11). Endothelial cells express specialized ligands on the luminal surface such as E-56 selectin, ICAM, and VCAM (3, 12). These chemokines and adhesion molecules cooperatively work to facilitate 57 homing of circulating HSC to bone marrow (3, 13). In vivo, osteoblasts also express VCAM and CXCL-12 in 58 addition to osteopontin, which has been implicated in maintaining HSC quiescence (7, 14). Moreover, blood vessels 59 found within the endosteal niche express higher levels of E-selectin, which play an important role in regulating HSC 60 cell cycling (15), and has been implicated as a regulator of breast cancer metastasis to bone (12, 16). We endeavored 61to create a unified model of these two bone marrow niches (perivascular and endosteal) using an in vitro using a 62 microfluidic organ-on-a-chip system. 63Advances in tissue engineering and microfluidics have created "organ-on-a-chip" technologies to generate 64 3D microphysiological mimics that utilize a broad range of human cells, thus providing specific advantages over 65 traditional 2D culture and mouse models. Moreover, microfluidic technology lends itself well to capturing the spatial 66 scale of the adjacent endosteal and perivascular bone marrow microenvironments. Although several labs have utilized 67 similar tissue engineering and microfluidic approaches to recreate aspects of the perivascular and endost...