A patient-specific lung cancer assembloid model with heterogeneous tumor microenvironments.

Cancer models play critical roles in basic cancer research and precision medicine. However, current in vitro cancer models are limited by their inability to mimic the three-dimensional architecture and heterogeneous tumor microenvironments (TME) of in vivo tumors. Here, we develop an innovative patient-specific lung cancer assembloid (LCA) model by using droplet microfluidic technology based on a microinjection strategy. This method enables precise manipulation of clinical microsamples and rapid generation of LCAs with good intra-batch consistency in size and cell composition by evenly encapsulating patient tumor-derived TME cells and lung cancer organoids inside microgels. LCAs recapitulate the inter- and intratumoral heterogeneity, TME cellular diversity, and genomic and transcriptomic landscape of their parental tumors. LCA model could reconstruct the functional heterogeneity of cancer-associated fibroblasts and reflect the influence of TME on drug responses compared to cancer organoids. Notably, LCAs accurately replicate the clinical outcomes of patients, suggesting the potential of the LCA model to predict personalized treatments. Collectively, our studies provide a valuable method for precisely fabricating cancer assembloids and a promising LCA model for cancer research and personalized medicine.

Satellite-Based On-Orbit Printing of 3D Tumor Models.

Space three dimension (3D） bioprinting provides a precise and bionic tumor model for evaluating the compound effect of the space environment on tumors, thereby providing insight into the progress of the disease and potential treatments. However, space 3D bioprinting faces several challenges, including prelaunch uncertainty, possible liquid leakage, long-term culture in space, automatic equipment control, data acquisition, and transmission. Here, a novel satellite-based 3D bioprinting device with high structural strength, small volume, and low weight (<6 kg) is developed. A microgel-based biphasic thermosensitive bioink and suspension medium that supports the on-orbit printing and in situ culture of complex tumor models is developed. An intelligent control algorithm that enables the automatic control of 3D printing, autofocusing, fluorescence imaging, and data transfer back to the ground is developed. To the authors' knowledge, this is the first time that on-orbit printing of tumor models is achieved in space with stable morphology and moderate viability via a satellite. It is found that 3D tumor models are more sensitive to antitumor drugs in space than on Earth. This study opens up a new avenue for 3D bioprinting in space and offers new possibilities for future research in space life science and medicine.

Engineering Highly Vascularized Bone Tissues by 3D Bioprinting of Granular Prevascularized Spheroids.

The convergence of 3D bioprinting with powerful manufacturing capability and cellular self-organization that can reproduce intricate tissue microarchitecture and function is a promising direction toward building functional tissues and has yet to be demonstrated. Here, we develop a granular aggregate-prevascularized (GAP) bioink for engineering highly vascularized bone tissues by capitalizing on the condensate-mimicking, self-organization, and angiogenic properties of prevascularized mesenchymal spheroids. The GAP bioink utilizes prevascularized aggregates as building blocks, which are embedded densely in extracellular matrices conducive to spontaneous self-organization. We printed various complex structures with high cell density (∼1.5 × 108 cells/cm3), viability (∼80%), and shape fidelity using GAP bioink. After printing, the prevascularized mesenchymal spheroids developed an interconnected vascular network through angiogenic sprouting. We printed highly vascularized bone tissues using GAP bioink and found that prevascularized spheroids were more conducive to osteogenesis and angiogenesis. We envision that the design of the GAP bioink could be further integrated with human-induced pluripotent stem cell-derived organoids, which opens new avenues to create patient-specific vascularized tissues for therapeutic applications..

3D printing of vascularized hepatic tissues with a high cell density and heterogeneous microenvironment.

Three-dimensional bioprinting has emerged as an appealing approach for creating functional tissues; however, a lack of suitable bioinks with high cell density and printability has greatly limited our ability to print functional tissues. We address this limitation by developing a granular cell aggregate-based biphasic (GCAB) bioink based on densely packed cell aggregates. The GCAB bioink exhibited the desired shear-thinning and shear-recovery properties for extrusion bioprinting and hyperelastic behaviors postprinting for modeling the mechanical characteristics of soft biological tissues. The GCAB bioink displayed a high cell density (∼1.7 × 108cells cm-3) without compromising viability (∼83%). We printed dense hepatic tissue constructs with enhanced vascularization and metabolic functions by preorganization of GCAB bioink with a defined heterogeneous microenvironment. By simultaneously printing the GCAB bioink and an endothelial cell-laden gelatin bioink, we successfully produced functional hepatic tissues with a high cell density and a perfusable vascular network. The design of the generalizable GCAB bioink opens new avenues to create functional tissues for therapeutic applications.

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.

3D Printed Conductive Multiscale Nerve Guidance Conduit with Hierarchical Fibers for Peripheral Nerve Regeneration.

Nerve guidance conduits (NGCs) have become a promising alternative for peripheral nerve regeneration; however, the outcome of nerve regeneration and functional recovery is greatly affected by the physical, chemical, and electrical properties of NGCs. In this study, a conductive multiscale filled NGC (MF-NGC) consisting of electrospun poly(lactide-co-caprolactone) (PCL)/collagen nanofibers as the sheath, reduced graphene oxide /PCL microfibers as the backbone, and PCL microfibers as the internal structure for peripheral nerve regeneration is developed. The printed MF-NGCs presented good permeability, mechanical stability, and electrical conductivity, which further promoted the elongation and growth of Schwann cells and neurite outgrowth of PC12 neuronal cells. Animal studies using a rat sciatic nerve injury model reveal that the MF-NGCs promote neovascularization and M2 transition through the rapid recruitment of vascular cells and macrophages. Histological and functional assessments of the regenerated nerves confirm that the conductive MF-NGCs significantly enhance peripheral nerve regeneration, as indicated by improved axon myelination, muscle weight increase, and sciatic nerve function index. This study demonstrates the feasibility of using 3D-printed conductive MF-NGCs with hierarchically oriented fibers as functional conduits that can significantly enhance peripheral nerve regeneration.

3D-printed Bioresorbable Stent Coated with Dipyridamole-Loaded Nanofiber for Restenosis Prevention and Endothelialization.

Intimal hyperplasia and restenosis caused by excessive proliferation of smooth muscle cells (SMC) are the main factors for the failure of stent implantation. Drug-eluting stents carried with antiproliferative drugs have emerged as a successful approach to alleviate early neointimal development. However, these agents have been reported to have an undesirable effect on re-endothelialization. In this study, we proposed an integrated bioresorbable stent coated with dipyridamole (DP)-loaded poly(D,L-lactide) (PDLLA) nanofibers. Three-dimensional (3D) bioresorbable stents were fabricated by printing on a rotation mandrel using polycaprolactone (PCL), and the stents were further coated with PDLLA/DP nanofibers. The in vitro degradation and drug release evaluation illustrated the potential for long-term release of DP. Stents coated with PDLLA/DP nanofibers showed excellent hemocompatibility. The cell viability, proliferation, and morphology analysis results revealed that stents coated with PDLLA/DP nanofibers could prevent the proliferation of SMC and have no adverse effects on endothelial cells. The in vivo implantation of stents coated with PDLLA/DP nanofibers showed initial patency and continuous endothelialization and alleviated neointimal formation. The attractive in vitro and in vivo performance indicated its potential for restenosis prevention and endothelialization.

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.

Recent advances on bioengineering approaches for fabrication of functional engineered cardiac pumps: A review.

The field of cardiac tissue engineering has advanced over the past decades; however, most research progress has been limited to engineered cardiac tissues (ECTs) at the microscale with minimal geometrical complexities such as 3D strips and patches. Although microscale ECTs are advantageous for drug screening applications because of their high-throughput and standardization characteristics, they have limited translational applications in heart repair and the in vitro modeling of cardiac function and diseases. Recently, researchers have made various attempts to construct engineered cardiac pumps (ECPs) such as chambered ventricles, recapitulating the geometrical complexity of the native heart. The transition from microscale ECTs to ECPs at a translatable scale would greatly accelerate their translational applications; however, researchers are confronted with several major hurdles, including geometrical reconstruction, vascularization, and functional maturation. Therefore, the objective of this paper is to review the recent advances on bioengineering approaches for fabrication of functional engineered cardiac pumps. We first review the bioengineering approaches to fabricate ECPs, and then emphasize the unmatched potential of 3D bioprinting techniques. We highlight key advances in bioprinting strategies with high cell density as researchers have begun to realize the critical role that the cell density of non-proliferative cardiomyocytes plays in the cell-cell interaction and functional contracting performance. We summarize the current approaches to engineering vasculatures both at micro- and meso-scales, crucial for the survival of thick cardiac tissues and ECPs. We showcase a variety of strategies developed to enable the functional maturation of cardiac tissues, mimicking the in vivo environment during cardiac development. By highlighting state-of-the-art research, this review offers personal perspectives on future opportunities and trends that may bring us closer to the promise of functional ECPs.

Multi-scale hierarchical scaffolds with aligned micro-fibers for promoting cell alignment.

Cell alignment plays an essential role in cytoskeleton reorganization, extracellular matrix remodeling, and biomechanical properties regulation of tissues such as vascular tissues, cardiac muscles, and tendons. Based on the natural-oriented features of cells in native tissues, various biomimetic scaffolds have been reported with the introduction of well-arranged ultrafine fibers to induce cell alignment. However, it is still a challenge to fabricate scaffolds with suitable mechanical properties, biomimetic microenvironment, and ability to promote cell alignment. In this paper, we propose an integrated 3D printing system to fabricate multi-scale hierarchical scaffolds combined with meso-, micro-, and nano-fibrous filaments, in which the meso-, micro-, and nano-fibers fabricated via fused deposition modeling, melt electrospining writing, and solution electrospining can provide structural support, promote cell alignment, and create a biomimetic microenvironment to facilitate cell function, respectively. The plasma surface modification was performed improve the surface wettability of the scaffolds by measuring the contact angle. The obtainedin vitrobiological results validate the ability of multi-scale hierarchical scaffolds to enhance cell adhesion and proliferation, and promote cell alignment with the guidance of the aligned microfibers produced via melt electrospining writing in hierarchical scaffolds.

A Heart-Breast Cancer-on-a-Chip Platform for Disease Modeling and Monitoring of Cardiotoxicity Induced by Cancer Chemotherapy.

Cardiotoxicity is one of the most serious side effects of cancer chemotherapy. Current approaches to monitoring of chemotherapy-induced cardiotoxicity (CIC) as well as model systems that develop in vivo or in vitro CIC platforms fail to notice early signs of CIC. Moreover, breast cancer (BC) patients with preexisting cardiac dysfunctions may lead to different incident levels of CIC. Here, a model is presented for investigating CIC where not only induced pluripotent stem cell (iPSC)-derived cardiac tissues are interacted with BC tissues on a dual-organ platform, but electrochemical immuno-aptasensors can also monitor cell-secreted multiple biomarkers. Fibrotic stages of iPSC-derived cardiac tissues are promoted with a supplement of transforming growth factor-ß 1 to assess the differential functionality in healthy and fibrotic cardiac tissues after treatment with doxorubicin (DOX). The production trend of biomarkers evaluated by using the immuno-aptasensors well-matches the outcomes from conventional enzyme-linked immunosorbent assay, demonstrating the accuracy of the authors' sensing platform with much higher sensitivity and lower detection limits for early monitoring of CIC and BC progression. Furthermore, the versatility of this platform is demonstrated by applying a nanoparticle-based DOX-delivery system. The proposed platform would potentially help allow early detection and prediction of CIC in individual patients in the future.

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.

Engineering of brain-like tissue constructs via 3D Cell-printing technology.

The development of 3D Cell-printing technology contributes to the application of tissue constructs in vitro in neuroscience. Collecting neural cells from patients is an efficient way of monitoring health of an individual target, which, in turn, benefits the enhancement of medicines. The fabricated sample of neural cells is exposed to potential drugs for the analysis of neuron responses. 3D cell-printing as an emerging biofabrication technology has been widely used to mimic natural 3D models in in vitro tissue research, especially in vitro brain-like tissue constructs in neuroscience. Fabricated brain-like tissue constructs provide a 3D microenvironment for primary neural cells to grow within. After more than several weeks of in vitro culturing, the formation of neural circuits in structures equips them with the capability of sensitively responding to a stimulus. In this study, an in vitro layered brain-like tissue construct is first proposed and later developed by 3D cell-printing technology. The layered structure is systematically analyzed, starting from printing parameter optimization to biological functionality performance. The optimized diameter of printing nozzle and printing speed are 0.51 mm and 5 µl s-1, respectively, and the elastic modulus is approximately 6 kPa. Live/dead and immunostaining imaging is used to verify the growth of neural cells in the printed structure. The survival rate of neural cells in 2D and 3D samples is compared, and the results demonstrate that the 3D-printed structures exhibit a better artificial culturing environment and a higher survival rate. Both 2D and 3D samples are directly cultured in a 4 × 4 multiple electrode array. Local field potentials are collected and validated by the Med64 recording system. Tetrodotoxin is used to test the drug sensitivity of the printed structure, and the excitatory postsynaptic potential signals of the physiological performance indicate that the 3D-printed structure has great potential as a drug testing model in the pharmaypeceutical study.

Design, modeling and 3D printing of a personalized cervix tissue implant with protein release function.

Cervical cancer induced by human papillomavirus (HPV) causes severe morbidity worldwide. Although cervical conization has been widely accepted as the most conventional surgery against cervical cancer, tissue defects and high recurrence rates have a significant negative impact on women's mental and physical health. Herein we developed an implantable, personalized cervical implant with drug release function using 3D printing technology. The cervical implant was designed in cone-shape with hieratical porous structures according to the clinical data, 3D-printed using polyurethane by low-temperature deposition manufacturing (LDM), and finished by lyophilization. Anti-HPV protein was loaded into the porous structure under negative pressure afterwards. Elastic biomedical polyurethane and the porous structure ensured that these cervical implants were equipped with tailored mechanical properties comparable to physiological cervix tissue. Cytotoxicity and cytocompatibility tests indicated that these 3D-printed cervical implants supported cell adhesion and growth. More importantly, the cervical implants with regulated pores could help to quantitatively control the loading and release of anti-HPV protein to inhibit dissociative viruses near the cervix validly. As a result, the 3D-printed cervical implants in the present study showed considerable potential for use as functional tissue implants against HPV infection after cervical conization.

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.

Assessment of various crosslinking agents on collagen/chitosan scaffolds for myocardial tissue engineering.

Suitable material for scaffolds that support cell attachment, proliferation, vascularization and contraction has always been a challenge in myocardial tissue engineering. Much research effort has been focused on natural polymers including collagen, gelatin, chitosan, fibrin, alginate, etc. Among them, a collagen/chitosan composite scaffold was widely used for myocardial tissue engineering. Due to the non-proliferative and contractile characteristics of cardiomyocytes, the biocompatibility and mechanical properties of the scaffolds are extremely important for supporting intercellular connection and tissue function for myocardial tissue engineering. To the best of our knowledge, the three crosslinking agents (glutaraldehyde (GTA), genipin (GP), tripolyphosphate (TPP)) have not yet been comparatively studied in myocardial tissue engineering. Thus, the aim of this study is to compare and identify the crosslinking effect of GTA, GP and TPP for myocardial tissue engineering. The collagen/chitosan scaffolds prepared with various crosslinking agents were fabricated and evaluated for physical characteristics, biocompatibility and contractile performance. All the groups of scaffolds exhibited high porosity (>65%) and good swelling ratio suitable for myocardial tissue engineering. TPP crosslinked scaffolds showed excellent mechanical properties, with their elastic modulus (81.0 ± 8.1 kPa) in the physiological range of native myocardium (20∼100 kPa). Moreover, GP and TPP crosslinked scaffolds exhibited better biocompatibility than GTA crosslinked scaffolds, as demonstrated by the live/dead staining and proliferation assay. In addition, cardiomyocytes within TPP crosslinked scaffolds showed the highest expression of cardiac-specific marker protein and the best contractile performance. To conclude, of the three crosslinking agents, TPP was recommended as the most suitable crosslinking agent for collagen/chitosan scaffold in myocardial tissue engineering.

A heart-on-a-chip platform for online monitoring of contractile behavior via digital image processing and piezoelectric sensing technique.

Heart-on-a-chip devices have recently emerged as a viable and promising model for drug screening applications, owing to its capability of capturing important biological and physiological parameters of cardiac tissue. However, most heart-on-a-chips are not developed for online and continuous monitoring of contractile behavior, which are the main functional characteristics of cardiac tissue. In this study, we designed and investigated on a heart-on-a-chip platform that provides online monitoring of contractile behavior of a 3D cardiac tissue construct. The contractile behavior include contraction force, frequency, and synchronization. They can be evaluated by an image processing system and a piezoelectric sensing system simultaneously. Based on the deformation of a micro-pillar array embedded within the 3D cardiac tissue upon subjected to cardiac contraction, the image processing system provides in situ multi-site detection of the contractile behavior. At the same time, the piezoelectric sensing system measures the contractile behavior of the entire cardiac tissue construct. A 3D cardiac tissue construct was successfully fabricated. Then the heart-on-a-chip platform was validated by applying various motion patterns on the micro-pillars, which mimicked the contraction patterns of the 3D cardiac tissue. The drug reactivity of the 3D cardiac tissue construct after a treatment of isoproterenol and doxorubicin was evaluated by measuring the contractile behavior via the image processing and the piezoelectric sensing systems. The results from the drug reactivity provided by both these measurement systems were consistent with previous reports, demonstrating the reliability of the heart-on-a-chip platform and its potential for use in cardio-related drug screening applications.

Biomimetic design and fabrication of scaffolds integrating oriented micro-pores with branched channel networks for myocardial tissue engineering.

The ability to fabricate three-dimensional (3D) thick vascularized myocardial tissue could enable scientific and technological advances in tissue engineering and drug screening, and may accelerate its application in myocardium repair. In this study, we developed a novel biomimetic scaffold integrating oriented micro-pores with branched channel networks to mimic the anisotropy and vasculature of native myocardium. The oriented micro-pores were fabricated using an 'Oriented Thermally Induced Phase Separation (OTIPS)' technique, and the channel network was produced by embedding and subsequently dissolving a 3D-printed carbohydrate template after crosslinking. Micro-holes were incorporated on the wall of channels, which greatly enhanced the permeability of channels. The effect of the sacrificial template on the formation of oriented micro- pores was assessed. The mechanical properties of the scaffold were tuned by varying the temperature gradient and chitosan/collagen ratio to match the specific stiffness of native heart tissue. The engineered cardiac tissue achieved synchronized beating with electrical stimulation. Calcium transient results suggested the formation of connection between cardiomyocytes within scaffold. All the results demonstrated that the reported scaffold has the potential to induce formation of a perfusable vascular network and to create thick vascularized cardiac tissue that may advance further clinical applications.