Creating a semi-opened micro-cavity ovary through sacrificial microspheres as an in vitro model for discovering the potential effect of ovarian toxic agents.

The bio-engineered ovary is an essential technology for treating female infertility. Especially the development of relevant in vitro models could be a critical step in a drug study. Herein, we develop a semi-opened culturing system (SOCS) strategy that maintains a 3D structure of follicles during the culture. Based on the SOCS, we further developed micro-cavity ovary (MCO) with mouse follicles by the microsphere-templated technique, where sacrificial gelatin microspheres were mixed with photo-crosslinkable gelatin methacryloyl (GelMA) to engineer a micro-cavity niche for follicle growth. The semi-opened MCO could support the follicle growing to the antral stage, secreting hormones, and ovulating cumulus-oocyte complex out of the MCO without extra manipulation. The MCO-ovulated oocyte exhibits a highly similar transcriptome to the in vivo counterpart (correlation of 0.97) and can be fertilized. Moreover, we found that a high ROS level could affect the cumulus expansion, which may result in anovulation disorder. The damage could be rescued by melatonin, but the end of cumulus expansion was 3h earlier than anticipation, validating that MCO has the potential for investigating ovarian toxic agents in vitro. We provide a novel approach for building an in vitro ovarian model to recapitulate ovarian functions and test chemical toxicity, suggesting it has the potential for clinical research in the future.

Electrical Stimulation Promotes the Vascularization and Functionalization of an Engineered Biomimetic Human Cardiac Tissue.

The formation of multiscale vascular networks is essential for the in vitro construction of large-scale biomimetic cardiac tissues/organs. Although a variety of bioprinting processes have been developed to achieve the construction of mesoscale and large-scale blood vessels, the formation of microvascular networks still mainly depends on the self-assembly behavior of endothelial cells (ECs), which is inefficient and demanding without appropriate stimulus. To address this problem, the elongation and connection of endothelial cells in engineered cardiac tissue (ECT) are sought to promote by electrical stimulation (ES) to achieve vascularization. As proof of the concept, bio-inks are composed of GelMA/fibrin hydrogel, human pluripotent stem cells induced cardiomyocytes (iPSC-CM), and human umbilical vein endothelial cells (HUVEC) are used for the bioprinting of ECTs. It is demonstrated that electrical stimulation significantly promotes the elongation, migration, and interconnection of HUVECs in ECT and increases the expression of related genes. Moreover, ES also enhances the secretion of signal factors interacting between CMs and HUVECs. It seems that the HUVECs further strengthen the contractility of cardiac tissue. Taken together, electrical stimulation promotes vascularization and CMs functionalization in ECT, which has important application potential in the fabrication of vascularized ECT and its clinical transplantation.

Expanding Embedded 3D Bioprinting Capability for Engineering Complex Organs with Freeform Vascular Networks.

Creating functional tissues and organs in vitro on demand is a major goal in biofabrication, but the ability to replicate the external geometry of specific organs and their internal structures such as blood vessels simultaneously remains one of the greatest impediments. Here, this limitation is addressed by developing a generalizable bioprinting strategy of sequential printing in a reversible ink template (SPIRIT). It is demonstrated that this microgel-based biphasic (MB) bioink can be used as both an excellent bioink and a suspension medium that supports embedded 3D printing due to its shear-thinning and self-healing behavior. When encapsulating human-induced pluripotent stem cells, the MB bioink is 3D printed to generate cardiac tissues and organoids by extensive stem cell proliferation and cardiac differentiation. By incorporating MB bioink, the SPIRIT strategy enables the effective printing of a ventricle model with a perfusable vascular network, which is not possible to fabricate using extant 3D printing strategies. This SPIRIT technique offers an unparalleled bioprinting capability to replicate the complex organ geometry and internal structure in a faster manner, which will accelerate the biofabrication and therapeutic applications of tissue and organ constructs.

Study of paraquat-induced pulmonary fibrosis using biomimetic micro-lung chips.

Paraquat (PQ) poisoning induces pulmonary fibrosisin vivo. The pathogenesis of pulmonary fibrosis is complex, which has prevented the development of specific treatments. Pulmonary fibrosis shows several characteristics including epithelial-mesenchymal transition (EMT), fibroblast activation, and extracellular matrix (ECM) deposition. To investigate pulmonary fibrosis, we designed a biomimetic multichannel micro-lung chip to imitate thein vivointerface between the lung epithelium and the lung interstitium. In our model, A549 (lung epithelial cells) and MRC-5 (fetal lung fibroblasts) cells were used to test the efficacy of our chip-based model. Rat tail type I collagen and hyaluronic acid were used to simulate ECM and to provide a 3D microenvironment. The micro-lung chips were cultured with PQ (0, 75, 150, 300, and 400µM). The viability of A549 and MRC-5 cells significantly decreased with increasing PQ concentrations. There were significant changes in surfactant proteins C (SP-C), alpha smooth muscle actin protein (α-SMA), and vimentin protein levels during PQ-induced pulmonary fibrosis. SP-C levels were decreased in A549 cells, while those ofα-SMA and vimentin were increased in A549 cells and MRC-5 cells treated with PQ in the micro-lung chip. We also designed a reference model without interaction between the lung epithelial cells and fibroblasts. Compared to the non-contact model, co-culturing A549 and MRC-5 cells in chips induced more severe EMT in A549 cells after treatment with 75µM PQ and together defended against PQ-induced damage. Thus, our novel co-culture micro-lung chip that models the lung epithelium and interstitium may provide a new approach for studying lung fibrosis and will facilitate drug development.

3D Bioprinted GelMA-Nanoclay Hydrogels Induce Colorectal Cancer Stem Cells Through Activating Wnt/ß-Catenin Signaling.

Cancer stem cells (CSCs) are a rare cell population in tumors that are responsible for tumor recurrence and metastasis. They are a priority as therapeutic targets, however, assays targeting CSCs have been limited by expanding and maintaining CSCs in vitro. Here, the authors find that gelatin methacryloyl (GelMA)-nanoclay hybrid hydrogels can induce and enrich colorectal CSCs assisted by three-dimensional (3D) bioprinting. The presence of the nanoclay increases the printability, Young's modulus, pore size, and cytocompatibility of the hydrogels. Bioprinted GelMA-nanoclay hydrogels promote the formation of spheroids expressing elevated levels of the stemness markers LGR5, CD133, CD26, and SOX2. Cancer cells grown in GelMA-nanoclay hydrogel possess higher self-renewal and differentiation capacity in vitro and higher tumorigenic capacity in vivo. GelMA-nanoclay hydrogels induce CSCs by stimulating the activation of the Wnt/ß-catenin signaling pathway. Further studies demonstrate that spheroids from GelMA-nanoclay hydrogels possess increased stemness, higher consistency, yield, and sensitivity to the anti-CSC compounds compared to the classic CSC-enrichment model. Collectively, this study may provide a valuable biomaterial and method for inducing and enriching CSCs, to facilitate the effective CSC-targeting drug screening.

Dual cure (thermal/photo) composite hydrogel derived from chitosan/collagen for in situ 3D bioprinting.

In situ 3D printing technologies is a new frontier for highly personalized medicine, which requires suitable bioink with rheology, biocompatibility, and gelation kinetics to support the right shape and mechanical properties of the printed construct. To this end, a facile design of thermo/photo dual cure composite hydrogel was proposed using MHBC and soluble collagen in this study. M/C composite hydrogel exhibited rapid thermo-induced sol-gel transition and contraction, tunable mechanical properties, proper microstructure and biodegradability for 3D cell culture, as well as improve cyto-compatibility, all of which were dependent upon the methacrylation degree of MHBC and M/C ratios. The printability of the optimal formulation (3% MHBC/1% collagen) was validated by its mild printing condition, rapid gelation of bioink at 37 °C and simple postprocessing manipulation. Both desirable printability and cyto-compatibility enable M/C composite hydrogel a potential candidate as bioink to be applied for in situ 3D bioprinting.

Optimizing Bifurcated Channels within an Anisotropic Scaffold for Engineering Vascularized Oriented Tissues.

Despite progress in engineering both vascularized tissues and oriented tissues, the fabrication of 3D vascularized oriented tissues remains a challenge due to an inability to successfully integrate vascular and anisotropic structures that can support mass transfer and guide cell alignment, respectively. More importantly, there is a lack of an effective approach to guiding the scaffold design bearing both structural features. Here, an approach is presented to optimize the bifurcated channels within an anisotropic scaffold based on oxygen transport simulation and biological experiments. The oxygen transport simulation is performed using the experimentally measured effective oxygen diffusion coefficient and hydraulic permeability of the anisotropic scaffolds, which are also seeded with muscle precursor cells and cultured in a custom-made perfusion bioreactor. Symmetric bifurcation model is used as fractal unit to design the channel network based on biomimetic principles. The bifurcation level of channel network is further optimized based on the oxygen transport simulation, which is then validated by DNA quantification assay and pimonidazole immunostaining. This study provides a practical guide to optimizing bifurcated channels in anisotropic scaffolds for oriented tissue engineering.

Rapid Fabrication of Cell-Laden Microfibers for Construction of Aligned Biomimetic Tissue.

Bottom-up engineering of tissue constructs is being rapidly developed and broadly applied in biomanufacturing. As one type of building block, cell-laden microfibers are promising for reconstruction of oriented structures and functions of linear tissues, such as skeletal muscles, myocardia, and spinal cord tissues. Herein, we propose wet-spinning method with agitating collection, wherein alginate-based material is extruded into an agitated CaCl2 bath with a magnetic rotor acting as the microfiber collector. By applying this method, we achieve rapid fabrication and oriented collection of hydrogel microfibers with diameters ranging from 100 to 400 µm. In addition, we encapsulate myoblasts in the hydrogel to form cell-laden microfibers, which show a high viability (more than 94%) during in vitro culture. Moreover, the method allows to fabricate of cell-laden core-sheath microfibers and hollow microfibers. We also fabricate 3D constructs using various methods of microfiber assembly like weaving and braiding. The assembling results suggest that the proposed method is a promising technology for bottom-up engineering of aligned biomimetic tissue constructs.