Dual-Phase, Surface Tension-Based Fabrication Method for Generation of Tumor Millibeads

Numerous methods have been developed for the fabrication of poly­(ethylene glycol)-based hydrogel microstructures for drug-delivery and tissue-engineering applications. However, present methods focus on the fabrication of submicrometer scale hydrogel structures which have limited applications in creating larger tissue constructs, especially in recreating cancer tissue microenvironments. We aimed to establish a platform where cancer cells can be cultured in a three-dimensional (3D) environment, which closely replicates the native cancer microenvironment and facilitates efficient testing of anticancer drugs. This study demonstrated a novel surface tension-based fabrication technique for the generation of millimeter-scale hydrogel beads using a liquid–liquid dual phase system. The “hydrogel millibeads” obtained by this method were larger than previously reported, highly uniform in shape and size with better ease of size control and a high degree of consistency and reproducibility between batches. In addition, human breast cancer cells were encapsulated within these hydrogel constructs to generate “tumor millibeads”, which were subsequently maintained in long-term 3D culture. Microscopic visualization using fluorescence imaging and microstructure analysis showed the morphology and uniform distribution of the cells within the 3D matrix and arrangement of cells with the surrounding scaffold material. Cell viability analysis revealed the creation of a core region of dead cells surrounded by healthy, viable cell layers at the periphery following long-term culture. These observations closely matched with those of native and in vivo tumors. Based on these results, this study established a rapidly reproducible surface tension-based fabrication technique for making spherical hydrogel millibeads and demonstrated the potential of this method in creating engineered 3D tumor tissues. It is envisioned that the developed hydrogel millibead system will facilitate the formation of physiologically relevant in vitro tumor models which will closely simulate the native tumor microenvironmental conditions and could enable future high-throughput testing of different anticancer drugs in preclinical trials

Dual-Phase, Surface Tension-Based Fabrication Method for Generation of Tumor Millibeads

Numerous methods have been developed for the fabrication of poly­(ethylene glycol)-based hydrogel microstructures for drug-delivery and tissue-engineering applications. However, present methods focus on the fabrication of submicrometer scale hydrogel structures which have limited applications in creating larger tissue constructs, especially in recreating cancer tissue microenvironments. We aimed to establish a platform where cancer cells can be cultured in a three-dimensional (3D) environment, which closely replicates the native cancer microenvironment and facilitates efficient testing of anticancer drugs. This study demonstrated a novel surface tension-based fabrication technique for the generation of millimeter-scale hydrogel beads using a liquid–liquid dual phase system. The “hydrogel millibeads” obtained by this method were larger than previously reported, highly uniform in shape and size with better ease of size control and a high degree of consistency and reproducibility between batches. In addition, human breast cancer cells were encapsulated within these hydrogel constructs to generate “tumor millibeads”, which were subsequently maintained in long-term 3D culture. Microscopic visualization using fluorescence imaging and microstructure analysis showed the morphology and uniform distribution of the cells within the 3D matrix and arrangement of cells with the surrounding scaffold material. Cell viability analysis revealed the creation of a core region of dead cells surrounded by healthy, viable cell layers at the periphery following long-term culture. These observations closely matched with those of native and in vivo tumors. Based on these results, this study established a rapidly reproducible surface tension-based fabrication technique for making spherical hydrogel millibeads and demonstrated the potential of this method in creating engineered 3D tumor tissues. It is envisioned that the developed hydrogel millibead system will facilitate the formation of physiologically relevant in vitro tumor models which will closely simulate the native tumor microenvironmental conditions and could enable future high-throughput testing of different anticancer drugs in preclinical trials

Direct Production of Human Cardiac Tissues by Pluripotent Stem Cell Encapsulation in Gelatin Methacryloyl

Direct stem cell encapsulation and cardiac differentiation within supporting biomaterial scaffolds are critical for reproducible and scalable production of the functional human tissues needed in regenerative medicine and drug-testing applications. Producing cardiac tissues directly from pluripotent stem cells rather than assembling tissues using pre-differentiated cells can eliminate multiple cell-handling steps that otherwise limit the potential for process automation and production scale-up. Here we asked whether our process for forming 3D developing human engineered cardiac tissues using poly­(ethylene glycol)-fibrinogen hydrogels can be extended to widely used and printable gelatin methacryloyl (GelMA) hydrogels. We demonstrate that low-density GelMA hydrogels can be formed rapidly using visible light (<1 min) and successfully employed to encapsulate human induced pluripotent stem cells while maintaining high cell viability. Resulting constructs had an initial stiffness of approximately 220 Pa, supported tissue growth and dynamic remodeling, and facilitated high-efficiency cardiac differentiation (>70%) to produce spontaneously contracting GelMA human engineered cardiac tissues (GEhECTs). GEhECTs initiated spontaneous contractions on day 8 of differentiation, with synchronicity, frequency, and velocity of contraction increasing over time, and displayed developmentally appropriate temporal changes in cardiac gene expression. GEhECT-dissociated cardiomyocytes displayed well-defined and aligned sarcomeres spaced at 1.85 ± 0.1 μm and responded appropriately to drug treatments, including the β-adrenergic agonist isoproterenol and antagonist propranolol, as well as to outside pacing up to 3.0 Hz. Overall results demonstrate that GelMA is a suitable biomaterial for the production of developing cardiac tissues and has the potential to be employed in scale-up production and bioprinting of GEhECTs

Xenogenesis-Production of Channel Catfish × Blue Catfish Hybrid Progeny by Fertilization of Channel Catfish Eggs with Sperm from Triploid Channel Catfish Males with Transplanted Blue Catfish Germ Cells

<p>Putative spermatogonia A from a fresh-cell isolate or a density-gradient-centrifuged isolate from the testes of Blue Catfish <i>Ictalurus furcatus</i> were transplanted into the gonads of triploid Channel Catfish <i>I. punctatus</i>. The cells were introduced into gonads of the host via catheterization (2 × 10⁴–1.43 × 10⁶ cells) or by surgically inserting the cells directly into the gonad (7 × 10⁴–1.25 × 10⁵ cells). Ten months after implantation, DNA was analyzed from biopsies of the gonads and seven of eight males were found to be xenogenic, having Blue Catfish cells in their gonads. The xenogenic males successfully courted normal Channel Catfish that had been induced with hormones to ovulate, but none of the eggs hatched, indicating inadequate sperm production, an inability to ejaculate, and/or low sperm quality. Male xenogenic catfish treated with luteinizing hormone releasing hormone analog had well-developed testes, and sperm production was detected in three of seven xenogenic males examined 2 years after transplantation. Sperm were removed from a male that had been surgically transplanted with Blue Catfish cells and used to fertilize eggs from a hand-stripped Channel Catfish female. One percent of these eggs hatched. All seven surviving 6-month-old progeny of this male had the external morphology, swim bladder shape, nuclear DNA profile, and mitochondrial DNA profile of female Channel Catfish × male Blue Catfish F₁ hybrids, indicating that this triploid Channel Catfish male produced Blue Catfish sperm. This is the first report of successful production of xenogenic catfish and the first report of producing 100% hybrid progeny using xenogenesis in fish.</p> <p>Received March 23, 2016; accepted July 23, 2016 Published online November 18, 2016</p