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Engineered cardiac tissues that can be directly produced from human induced pluripotent stem cells (hiPSCs) in scalable, suspension culture systems are needed to meet the demands of cardiac regenerative medicine. Here, we demonstrate successful production of functional cardiac tissue microspheres through direct differentiation of hydrogel encapsulated hiPSCs. To form the microspheres, hiPSCs were suspended within the photocrosslinkable biomaterial, PEG-fibrinogen (25 million cells/mL), and encapsulated at a rate of 420,000 cells/minute using a custom microfluidic system. Even at this high cell density and rapid production rate, high intra-batch and batch-to-batch reproducibility was achieved. Following microsphere formation, hiPSCs maintained high cell viability and continued to grow within and beyond the original PEG-fibrinogen matrix. These initially soft microspheres (<250 Pa) supported efficient cardiac differentiation; spontaneous contractions initiated by differentiation day 8, and the microspheres contained >75% cardiomyocytes (CMs). CMs responded appropriately to pharmacological stimuli and exhibited 1:1 capture up to 6.0 Hz when electrically paced. Over time, cells formed cell-cell junctions and aligned myofibril fibers; engineered cardiac microspheres were maintained in culture over 3 years. The capability to rapidly generate uniform cardiac microsphere tissues is critical for advancing downstream applications including biomanufacturing, multi-well plate drug screening, and injection-based regenerative therapies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Three-dimensional (3D) in vitro cancer models offer an attractive approach towards the investigation of tumorigenic phenomena and other cancer studies by providing dimensional context and higher degree of physiological relevance than that offered by conventional two-dimensional (2D) models. The multicellular tumor spheroid model, formed by cell aggregation, is considered to be the "gold standard" for 3D cancer models, due to its ease and simplicity of use. Although better than 2D models, tumor spheroids are unable to replicate key features of the native tumor microenvironment, particularly due to a lack of surrounding extracellular matrix components and heterogeneity in shape, size and aggregate forming tendencies. In order to address this issue, we have developed a 3D "tumor microsphere" model, formed by a dual-photoinitiator, aqueous-oil emulsion technique, for the encapsulation of cancer cells within PEG-fibrinogen hydrogel microspheres and for subsequent long-term 3D culture. In comparison to self-aggregated tumor spheroids, the tumor microspheres displayed a higher degree of size and shape homogeneity throughout long-term culture. In sharp contrast to cells in tumor spheroids, cells within tumor microspheres demonstrated significant loss in apico-basal polarity and cellular architecture, cellular and nuclear atypia, increased disorganization, elevated nuclear cytoplasmic ratio and nuclear volume density and reduction in cell-cell junction length, all of which are hallmarks of malignant transformation and tumorigenic progression. Additionally, the tumor microsphere model was extended for the 3D encapsulation and maintenance of a wide range of other cancer cell (metastatic and non-metastatic) types. Taken together, our results reinforce the importance of incorporating a biomimetic matrix in the cellular microenvironment of 3D tumor models and the influential effects of the matrix on the tumorigenic morphology of 3D cultured cells. The tumor microsphere system established in this study has the potential to be used in future investigations of 3D cancer cell-cell and cell-ECM interactions and in drug-testing applications., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Human engineered heart tissues have potential to revolutionize cardiac development research, drug-testing, and treatment of heart disease; however, implementation is limited by the need to use pre-differentiated cardiomyocytes (CMs). Here we show that by providing a 3D poly(ethylene glycol)-fibrinogen hydrogel microenvironment, we can directly differentiate human pluripotent stem cells (hPSCs) into contracting heart tissues. Our straight-forward, ontomimetic approach, imitating the process of development, requires only a single cell-handling step, provides reproducible results for a range of tested geometries and size scales, and overcomes inherent limitations in cell maintenance and maturation, while achieving high yields of CMs with developmentally appropriate temporal changes in gene expression. We demonstrate that hPSCs encapsulated within this biomimetic 3D hydrogel microenvironment develop into functional cardiac tissues composed of self-aligned CMs with evidence of ultrastructural maturation, mimicking heart development, and enabling investigation of disease mechanisms and screening of compounds on developing human heart tissue., (Copyright © 2015 Elsevier Ltd. All rights reserved.)