3D Bioprinted Model Research Articles

Ultrasounds trigger reversible permeability of the cerebrovascular endothelium in a novel 3D-bioprinted model of the human blood-brain barrier

The blood-brain barrier (BBB) restricts the penetration of therapeutic agents to the brain. Focused ultrasound (FUS) insonation of microbubbles has enhanced drug delivery via a temporary opening of the tight junctions of the BBB. Rodents are often used for testing novel therapeutic strategies. However, biological discrepancies existing between rodents and humans impedes the translation of some results. We investigated the effect of FUS transient disruption of a human microphysiological model of the BBB consisting of human brain endothelial cells cultured within a 3D-bio printed scaffold designed to reconstitute the architecture of a perfused blood vessel. Cavitation activity was detected in the model when perfused with Lumason microbubbles and exposed to 500 kHz ultrasound at 0.4 MPa for 10 ms pulse duration and 2 Hz pulse repetition frequency. An increase in extravasation of a dye beyond the endothelial layer was detected within 2–3 h of ultrasound insonation. Our results support that this system could be used for targeted BBB opening with a human cell culture model. Moreover, this system holds a promise to further the translational study on US-mediated drug delivery and the development of personalized therapies for patients with brain cancer and other neurovascular disorders.

Comparative Analysis of Dasatinib Effect between 2D and 3D Tumor Cell Cultures

Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model.

Development of 3D-Bioprinted Colitis-Mimicking Model to Assess Epithelial Barrier Function Using Albumin Nano-Encapsulated Anti-Inflammatory Drugs

Physiological barrier function is very difficult to replicate in vitro. This situation leads to poor prediction of candidate drugs in the drug development process due to the lack of preclinical modelling for intestinal function. By using 3D bioprinting, we generated a colitis-like condition model that can evaluate the barrier function of albumin nanoencapsulated anti-inflammatory drugs. Histological characterization demonstrated the manifestation of the disease in 3D-bioprinted Caco-2 and HT-29 constructs. A comparison of proliferation rates in 2D monolayer and 3D-bioprinted models was also carried out. This model is compatible with currently available preclinical assays and can be implemented as an effective tool for efficacy and toxicity prediction in drug development.

Hybrid biofabrication of neurosecretory structures as a model for neurosecretion.

The present study aimed to combine extrusion-based three-dimensional (3D) bioprinting and polymer nanofiber electrospinning technology to fabricate tissue-like structures with neurosecretory function in vitro. Using neurosecretory cells as cell resources, sodium alginate/gelatin/fibrinogen as matrix, polylactic acid/gelatin electrospun nanofibers as diaphragm, and neurosecretory cells-loaded 3D hydrogel scaffolds were bioprinted and then covered with electrospun nanofibers layer-by-layer. The morphology was observed by scanning electron microscopy and transmission electron microscopy (TEM), and the mechanical characteristics and cytotoxicity of the hybrid biofabricated scaffold structure were evaluated. The 3D-bioprinted tissue activity, including cell death and proliferation, was verified. Western blotting and ELISA experiments were used to confirm the cell phenotype and secretory function, while animal in vivo transplantation experiments confirmed the histocompatibility, inflammatory reaction, and tissue remodeling ability of the heterozygous tissue structures. Neurosecretory structures with 3D structures were successfully prepared by hybrid biofabrication in vitro. The mechanical strength of the composite biofabricated structures was significantly higher than that of the hydrogel system (P < 0.05). The survival rate of PC12 cells in the 3D-bioprinted model was 92.849 ± 2.995%. Hematoxylin and eosin-stained pathological sections showed that the cells grew in clumps, and there was no significant difference in the expression of MAP2 and tubulin-β between 3D organoids and PC12 cells. The results of ELISA showed that the PC12 cells in 3D structures retained the ability to continuously secrete noradrenaline and met-enkephalin, and the secretory vesicles around and within the cells could be observed by TEM. In in vivo transplantation, PC12 cells gathered and grew in clusters, maintained high activity, neovascularization, and tissue remodeling in 3D structures. The neurosecretory structures were biofabricated by 3D bioprinting and nanofiber electrospinning in vitro, which had high activity and neurosecretory function. In vivo transplantation of neurosecretory structures showed active proliferation of cells and potential for tissue remodeling. Our research provides a new method for biological manufacture of neurosecretory structures in vitro, which maintains neurosecretory function and lays the foundation for the clinical application of neuroendocrine tissues.

3D-bioprinted, phototunable hydrogel models for studying adventitial fibroblast activation in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculature, characterized by elevated pulmonary blood pressure, remodeling of the pulmonary arteries, and ultimately right ventricular failure. Therapeutic interventions for PAH are limited in part by the lack of in vitro screening platforms that accurately reproduce dynamic arterial wall mechanical properties. Here we present a 3D-bioprinted model of the pulmonary arterial adventitia comprised of a phototunable poly(ethylene glycol) alpha methacrylate (PEG-αMA)-based hydrogel and primary human pulmonary artery adventitia fibroblasts (HPAAFs). This unique biomaterial emulates PAH pathogenesis in vitro through a two-step polymerization reaction. First, PEG-αMA macromer was crosslinked off-stoichiometry by 3D bioprinting an acidic bioink solution into a basic gelatin support bath initiating a base-catalyzed thiol-ene reaction with synthetic and biodegradable crosslinkers. Then, matrix stiffening was induced by photoinitiated homopolymerization of unreacted αMA end groups. A design of experiments approach produced a hydrogel platform that exhibited an initial elastic modulus (E) within the range of healthy pulmonary arterial tissue (E = 4.7 ± 0.09 kPa) that was stiffened to the pathologic range of hypertensive tissue (E = 12.8 ± 0.47 kPa) and supported cellular proliferation over time. A higher percentage of HPAAFs cultured in stiffened hydrogels expressed the fibrotic marker alpha-smooth muscle actin than cells in soft hydrogels (88 ± 2% versus 65 ± 4%). Likewise, a greater percentage of HPAAFs were positive for the proliferation marker 5-ethynyl-2ʹ-deoxyuridine (EdU) in stiffened models (66 ± 6%) compared to soft (39 ± 6%). These results demonstrate that 3D-bioprinted, phototunable models of pulmonary artery adventitia are a tool that enable investigation of fibrotic pathogenesis in vitro.

3D bioprinted colorectal cancer models based on hyaluronic acid and signalling glycans

In cancer microenvironment, aberrant glycosylation events of ECM proteins and cell surface receptors occur. We developed a protocol to generate 3D bioprinted models of colorectal cancer (CRC) crosslinking hyaluronic acid and gelatin functionalized with three signalling glycans characterized in CRC, 3′-Sialylgalactose, 6′-Sialylgalactose and 2′-Fucosylgalactose. The crosslinking, performed exploiting azide functionalized gelatin and hyaluronic acid and 4arm-PEG-dibenzocyclooctyne, resulted in biocompatible hydrogels that were 3D bioprinted with commercial CRC cells HT-29 and patient derived CRC tumoroids. The glycosylated hydrogels showed good 3D printability, biocompatibility and stability over the time. SEM and synchrotron radiation SAXS/WAXS analysis revealed the influence of glycosylation in the construct morphology, whereas MALDI-MS imaging showed that protein profiles of tumoroid cells vary with glycosylation, indicating that sialylation and fucosylation of ECM proteins induce diverse alterations to the proteome of the tumoroid and surrounding cells.

Mesenchymal-endothelial nexus in breast cancer spheroids induces vasculogenesis and local invasion in a CAM model

Interplay between non-cancerous cells (immune, fibroblasts, mesenchymal stromal cells (MSC), and endothelial cells (EC)) has been identified as vital in driving tumor progression. As studying such interactions in vivo is challenging, ex vivo systems that can recapitulate in vivo scenarios can aid in unraveling the factors impacting tumorigenesis and metastasis. Using the synthetic tumor microenvironment mimics (STEMs)—a spheroid system composed of breast cancer cells (BCC) with defined human MSC and EC fractions, here we show that EC organization into vascular structures is BC phenotype dependent, and independent of ERα expression in epithelial cancer cells, and involves MSC-mediated Notch1 signaling. In a 3D-bioprinted model system to mimic local invasion, MDA STEMs collectively respond to serum gradient and form invading cell clusters. STEMs grown on chick chorioallantoic membrane undergo local invasion to form CAM tumors that can anastomose with host vasculature and bear the typical hallmarks of human BC and this process requires both EC and MSC. This study provides a framework for developing well-defined in vitro systems, including patient-derived xenografts that recapitulate in vivo events, to investigate heterotypic cell interactions in tumors, to identify factors promoting tumor metastasis-related events, and possibly drug screening in the context of personalized medicine.

Can 3D bioprinting solve the mystery of senescence in cancer therapy?

Tumor dormancy leading to cancer relapse is still a poorly understood mechanism. Several cell states such as quiescence and diapause can explain the persistence of tumor cells in a dormant state, but the potential role of tumor cell senescence has been met with hesitance given the historical understanding of the senescent growth arrest as irreversible. However, recent evidence has suggested that senescence might contribute to dormancy and relapse, although its exact role is not fully developed. This limited understanding is largely due to the paucity of reliable study models. The current 2D cell modeling is overly simplistic and lacks the appropriate representation of the interactions between tumor cells (senescent or non-senescent) and the other cell types within the tumor microenvironment (TME), as well as with the extracellular matrix (ECM). 3D cell culture models, including 3D bioprinting techniques, offer a promising approach to better recapitulate the native cancer microenvironment and would significantly improve our understanding of cancer biology and cellular response to treatment, particularly Therapy-Induced Senescence (TIS), and its contribution to tumor dormancy and cancer recurrence. Fabricating a novel 3D bioprinted model offers excellent opportunities to investigate both the role of TIS in tumor dormancy and the utility of senolytics (drugs that selectively eliminate senescent cells) in targeting dormant cancer cells and mitigating the risk for resurgence. In this review, we discuss literature on the possible contribution of TIS in tumor dormancy, provide examples on the current 3D models of senescence, and propose a novel 3D model to investigate the ultimate role of TIS in mediating overall response to therapy.

Toward 3D-Bioprinted Models of the Liver to Boost Drug Development.

The main problems in drug development are connected to enormous costs related to the paltry success rate. The current situation empowered the development of high-throughput and reliable instruments, in addition to the current golden standards, able to predict the failures in the early preclinical phase. Being hepatotoxicity responsible for the failure of 30% of clinical trials, and the 21% of withdrawal of marketed drugs, the development of complex in vitro models (CIVMs) of liver is currently one of the hottest topics in the field. Among the different fabrication techniques, 3D-bioprinting is emerging as a powerful ally for their production, allowing the manufacture of three-dimensional constructs characterized by computer-controlled and customized geometry, and inter-batches reproducibility. Thanks to these, it is possible to rapidly produce tailored cell-laden constructs, to be cultured within static and dynamic systems, thus reaching a further degree of personalization when designing in vitro models. This review highlights and prioritizes the most recent advances related to the development of CIVMs of the hepatic environment to be specifically applied to pharmaceutical research, with a special focus on 3D-bioprinting, since the liver is primarily involved in the metabolism of drugs.

3D bioprinting as an emerging standard for cancer modeling and drug testing.

Neoplastic diseases are a leading cause of death worldwide accounting for 10 million mortalities in 2020. Despite constantly revised and improved therapeutic regimens, the number of fatal cases increases annually. Therefore, better preclinical models are needed to study tumorigenesis and assess new drugs. Although 2D cell cultures significantly contributed to the understanding of tumor biology, they present high clinical trial failure rates. This is because 2D cannot reproduce the intricate tumor architecture and multiple cell interactions.Nevertheless, novel 3D biofabrication technologies and 3D bioprinted tumor models successfully mirror the complexity of human tumors and are currently revolutionizing preclinical cancer research by using live cells encapsulated in a variety of biomaterials. Since bioinks possess excellent chemical and biophysical ECM-like characteristics, this allows for recreation of the intricate tumor-specific architecture with an unmatched level of control, accuracy, and reproducibility. The resulting cellular constructs approximate actual pathological microenvironment of the tumor and some key in vivo processes such as proliferation, differentiation, and metastasis. 3D bioprinted models of glioblastoma, cervical, ovarian, and breast cancer are already being successfully used to study tumorigenesis and cellular response to antitumor drugs. This success showcases the potential of these novel experimental platforms.

Human platelet lysate-derived extracellular vesicles enhance angiogenesis through miR-126.

Extracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL. We tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL-derived EVs. A subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly-like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR-126, the most abundant angiogenic miRNA in platelets. The transfer of miR-126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system. PL is a biological source of available EVs with angiogenic effects involving a miRNAs-based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

Exploring the function of stromal cells in cholangiocarcinoma by three-dimensional bioprinting immune microenvironment model.

The tumor immune microenvironment significantly affects tumor progression, metastasis, and clinical therapy. Its basic cell components include tumor-associated endothelial cells, fibroblasts, and macrophages, all of which constitute the tumor stroma and microvascular network. However, the functions of tumor stromal cells have not yet been fully elucidated. The three-dimensional (3D) model created by 3D bioprinting is an efficient way to illustrate cellular interactions in vitro. However, 3D bioprinted model has not been used to explore the effects of stromal cells on cholangiocarcinoma cells. In this study, we fabricated 3D bioprinted models with tumor cells and stromal cells. Compared with cells cultured in two-dimensional (2D) environment, cells in 3D bioprinted models exhibited better proliferation, higher expression of tumor-related genes, and drug resistance. The existence of stromal cells promoted tumor cell activity in 3D models. Our study shows that 3D bioprinting of an immune microenvironment is an effective way to study the effects of stromal cells on cholangiocarcinoma cells.

Fusion between Glioma Stem Cells and Mesenchymal Stem Cells Promotes Malignant Progression in 3D-Bioprinted Models.

The interaction between glioma stem cells (GSCs) and mesenchymal stem cells (MSCs) in the glioma microenvironment is considered to be an important factor in promoting tumor progression, but the mechanism is still not fully elucidated. To further elucidate the interaction between GSCs and MSCs, two 3D-bioprinted tumor models (low-temperature molding and coaxial bioprinting) were used to simulate the tumor growth microenvironment. Cell fusion between GSCs and MSCs was found by the method of Cre-LoxP switch gene and RFP/GFP dual-color fluorescence tracing. The fused cells coexpressed biomarkers of GSCs and MSCs, showing stronger proliferation, cloning, and invasion abilities than GSCs and MSCs. In addition, the fused cells have stronger tumorigenic properties in nude mice, showing the pathological features of malignant tumors. In conclusion, GSCs and MSCs undergo cell fusion in 3D-bioprinted models, and the fused cells have a higher degree of malignancy than parental cells, which promotes the progression of glioma.

A Parkinson’s disease model composed of 3D bioprinted dopaminergic neurons within a biomimetic peptide scaffold

Parkinson’s disease (PD) is a progressive neurological disorder that affects movement. It is associated with lost dopaminergic (DA) neurons in the substantia nigra, a process that is not yet fully understood. To understand this deleterious disorder, there is an immense need to develop efficient in vitro three-dimensional (3D) models that can recapitulate complex organs such as the brain. However, due to the complexity of neurons, selecting suitable biomaterials to accommodate them is challenging. Here, we report on the fabrication of functional DA neuronal 3D models using ultrashort self-assembling tetrapeptide scaffolds. Our peptide-based models demonstrate biocompatibility both for primary mouse embryonic DA neurons and for human DA neurons derived from human embryonic stem cells. DA neurons encapsulated in these scaffolds responded to 6-hydroxydopamine, a neurotoxin that selectively induces loss of DA neurons. Using multi-electrode arrays, we recorded spontaneous activity in DA neurons encapsulated within these 3D peptide scaffolds for more than 1 month without decrease of signal intensity. Additionally, vascularization of our 3D models in a co-culture with endothelial cells greatly promoted neurite outgrowth, leading to denser network formation. This increase of neuronal networks through vascularization was observed for both primary mouse DA and cortical neurons. Furthermore, we present a 3D bioprinted model of DA neurons inspired by the mouse brain and created with an extrusion-based 3D robotic bioprinting system that was developed during previous studies and is optimized with time-dependent pulsing by microfluidic pumps. We employed a hybrid fabrication strategy that relies on an external mold of the mouse brain construct that complements the shape and size of the desired bioprinted model to offer better support during printing. We hope that our 3D model provides a platform for studies of the pathogenesis of PD and other neurodegenerative disorders that may lead to better understanding and more efficient treatment strategies.

Functional Drug Screening in the Era of Precision Medicine.

The focus of precision medicine is providing the right treatment to each unique patient. This scientific movement has incited monumental advances in oncology including the approval of effective, targeted agnostic therapies. Yet, precision oncology has focused largely on genomics in the treatment decision making process, and several recent clinical trials demonstrate that genomics is not the only variable to be considered. Drug screening in three dimensional (3D) models, including patient derived organoids, organs on a chip, xenografts, and 3D-bioprinted models provide a functional medicine perspective and necessary complement to genomic testing. In this review, we discuss the practicality of various 3D drug screening models and each model’s ability to capture the patient’s tumor microenvironment. We highlight the potential for enhancing precision medicine that personalized functional drug testing holds in combination with genomic testing and emerging mathematical models.

3D bioprinted cancer cells are more tolerant to serum starvation than 2D cells due to autophagy

Cancer is a global issue and a serious threat to human health, one approach to treatment is starvation therapy. Recently, three-dimensional (3D) bioprinted tumor tissue models have been developed; however, whether 3D bioprinted models are good for in vitro study of starvation therapy is unclear. In this study, we studied the state of cells with serum-free medium in both 3D bioprinted scaffold and 2D cell cultures and found that 3D bioprinted cancer cells (3D cells) were more tolerant to serum starvation than 2D cells in terms of cell viability, cell proliferation, and M2 macrophage polarization. Moreover, the ratio of LC3II/I, an index of autophagy, increased much more in 3D cells, and 3D cells showed more autophagosomes than 2D cells after serum starvation, which indicated that the autophagy levels were higher in 3D cells. These results suggested that 3D cells are more tolerant to serum starvation than 2D cells, and autophagy may play an important role in this process.

Multi-omics analysis based on 3D-bioprinted models innovates therapeutic target discovery of osteosarcoma.

Current in vitro models for osteosarcoma investigation and drug screening, including two-dimensional (2D) cell culture and tumour spheroids (i.e. cancer stem-like cells), lack extracellular matrix (ECM). Therefore, results from traditional models may not reflect real pathological processes in genuine osteosarcoma histological structures. Here, we report a three-dimensional (3D) bioprinted osteosarcoma model (3DBPO) that contains osteosarcoma cells and shrouding ECM analogue in a 3D frame. Photo-crosslinkable bioinks composed of gelatine methacrylamide and hyaluronic acid methacrylate mimicked tumour ECM. We performed multi-omics analysis, including transcriptomics and DNA methylomics, to determine differences between the 3DBPO model and traditional models. Compared with 2D models and tumour spheroids, our 3DBPO model showed significant changes in cell cycle, metabolism, adherens junctions, and other pathways associated with epigenetic regulation. The 3DBPO model was more sensitive to therapies targeted to the autophagy pathway. We showed that simulating ECM yielded different osteosarcoma cell metabolic characteristics and drug sensitivity in the 3DBPO model compared with classical models. We suggest 3D printed osteosarcoma models can be used in osteosarcoma fundamental and translational research, which may contribute to novel therapeutic strategy discovery.

Regulation of vascular branch formation in 3D bioprinted tissues using confining force

The quality of the vascular network is key to the success of skin transplants. For skin grafts prepared using a tissue engineering approach, vascularization is a critical step determining tissue survival. Prevascularization of bioengineered tissues is the basis for the effective establishment of high-quality vascular networks. Previously, we established a 3D bioprinted model to simulate the mechanical stimulation of vascular tissue development. Based on this, here, we create a prevascularized tissue using 3D bioprinting and show that the vascular branches of the tissue can be controlled by the confining forces created by changing the size of the polycaprolactone (PCL) framework. In addition, it was found that the Yes-associated protein (YAP) participates in the regulation of vascular branch formation in the tissue. A close relationship was found between the width of the cell-fibrin strip, the magnitude of the force and the number of vascular branches in the bioprinted tissue exists. Together, our findings indicate that vascularization of engineered skin tissue is a complex yet controllable process, and precise mechanical control can lead to effective vascular network generation.

Current state and future of 3D bioprinted models for cardiovascular research and drug development.

In the last decade, 3D bioprinting technology has emerged as an innovative tissue engineering approach for regenerative medicine and drug development. This article aims at providing an overview about the most commonly used bioengineered tissues, focusing on 3D bioprinted cardiac cells and how they have been utilized for drug discovery and development. The review describes that, while this field is still developing, cardiovascular research may benefit from laboratory-engineered heart tissues built of specific cell types with precise 3D architecture mimicking the native cardiac microenvironment. It also describes the role played by regulatory agencies and potential commercialization pathways for direct translation from the bench to the bedside of studies using 3D bioprinted cardiac tissues.

Tissue Engineering Through 3D Bioprinting to Recreate and Study Bone Disease.

The applications of 3D bioprinting are becoming more commonplace. Since the advent of tissue engineering, bone has received much attention for the ability to engineer normal bone for tissue engraftment or replacement. While there are still debates on what materials comprise the most durable and natural replacement of normal tissue, little attention is given to recreating diseased states within the bone. With a better understanding of the cellular pathophysiology associated with the more common bone diseases, these diseases can be scaled down to a more throughput way to test therapies that can reverse the cellular pathophysiology. In this review, we will discuss the potential of 3D bioprinting of bone tissue in the following disease states: osteoporosis, Paget’s disease, heterotopic ossification, osteosarcoma, osteogenesis imperfecta, and rickets disease. The development of these 3D bioprinted models will allow for the advancement of novel therapy testing resulting in possible relief to these chronic diseases.