Tissue engineering aims to develop tissue constructs as models or substitutes for native tissues. For organ-level biological studies and regenerative medicine applications, it is essential to fabricate tissue constructs with physiologically relevant cell densities (on the order of 10 million to 1 billion cells·ml-1), large size (centimeter scale and larger), and a controllable geometry to guide tissue maturation. State-of-the-art biofabrication methods, however, struggle to simultaneously meet all of these demands. The recently proposed acoustic holographic assembly (AHA) method shows promise, as it is compatible with culture media and enables the contactless, label-free, and volumetric assembly of biological cells in a predefined geometry within few minutes. Here we present an AHA biofabrication scheme designated for fabricating cell-dense, centimeter-scale, and arbitrarily-shaped tissue constructs using a compact benchtop instrument compatible with a biolab environment. We demonstrate the assembly of C2C12 myoblasts in gelatin methacryloyl (GelMA) into large and asymmetric branch-shaped constructs, which are rapidly formed with an average cell density of 40 million cells·ml-1and a local density of up to 260 million cells·ml-1. Featuring a high viability of 90.5 ± 4.3%, the assembled cell constructs are observed to grow within the GelMA hydrogel under perfusion over five days. Further, we show how AHA can-in a single step-assemble cells into layered and three-dimensional geometries inside standard cell culture labware. It can therefore help obtain engineered tissue constructs with structural and functional characteristics seen in more complex native tissues.

CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.

The increasing interest in utilizing three-dimensional (3D) in vitro models with innovative biomaterials to engineer functional tissues arises from the limitations of conventional cell culture methods in accurately reproducing the complex physiological conditions of living organisms. This study presents a strategy for replicating the intricate microenvironment of the intestine by cultivating intestinal cells within bioinspired 3D interfaces that recapitulate the villus-crypt architecture and 3D tissue arrangement of the intestine. Intestinal cells cultured on these biomimetic substrates exhibited phenotypes and differentiation characteristics resembling intestinal-specific cell types, effectively replicating intestinal tissue. Notably, tissue proliferation and differentiation were achieved within 72-120 h-significantly faster than the several weeks required by conventional bioengineered materials, which often pose risks of tissue necrosis or cross-contamination. Additionally, the differentiated cells on these villi-crypts mimicking bio-interfaces exhibit higher production of natural antimicrobial peptides, resulting in reduced pathogenic infection compared to control samples. Furthermore, our method stands out for simplicity in fabrication, eliminating the need for cleanroom procedures and complex microfabrication techniques.

The enduring prevalence of cancer worldwide constitutes a significant public health challenge, thereby emphasizing the imperative for the development of therapeutic models capable of accounting for the heterogeneity inherent in tumors. In this context, cancer organoids have emerged as powerful tools for studying tumor biology, providing valuable insights into the complex interactions within the tumor microenvironment. Concurrently, research is increasingly focused on non-apoptotic forms of regulated cell death (RCD)-including ferroptosis, pyroptosis, and necroptosis-which exert pivotal influences on cancer development and progression. Cancer organoids not only recapitulate the genetic and phenotypic heterogeneity of the original tumors but also enable more precise investigations into the roles of non-apoptotic RCDs within oncology. This review explores the utility of cancer organoids in delineating the molecular mechanisms underlying RCDs and their implications for cancer biology and treatment responses. By synthesizing recent research findings, it highlights the essential role of organoid models in uncovering the intricate details of non-apoptotic RCDs. Furthermore, it emphasizes promising directions for future research that aim to deepen our understanding of these pathways and their therapeutic potential. The integration of organoid models into investigations of ferroptosis, pyroptosis, and necroptosis provides novel insights into oncogenic mechanisms and facilitates the development of targeted therapeutic strategies. By bridging cancer organoids with human pathophysiology, this approach not only provides a transformative framework for dissecting oncogenic pathways but also enables the design of precision therapeutics that selectively target the molecular machinery underlying non-apoptotic RCDs.

Current developments in bioequivalent technology have led to the creation of excellent models that mimic the structure and function of human organs. These models are based on the original tissues and organs of the human body, but they lack the complex interaction with the extensive network of vasculature, and this is a major challenge for these models. A functional vasculature is essential for oxygen, nutrient, and waste exchange. It is also responsible for inductive biochemical exchange, and provides a structural pattern for organ growth. In vitro systems, containing no perfusable vessels, suffer from the quick formation of a necrotic core of organoids, and further development does not occur due to increased metabolic demands. Another key limitation of 3D-based techniques is the absence of accurate architectural structures and large-scale tissue sizes. Recently, new 3D bioprinting methods have been developed for organoids and spheroids as living building blocks. These methods aim to address some of the challenges associated with 3D technologies. In this review, we discuss recent strategies for vascularization via organoids and spheroids, which are used as structural units in bioprinting to recreate natural organs and tissues with ever-increasing accuracy in structure and function.

Organoids are 3D artificial miniature organs composed of a cluster of self-renewing and self-organizing cells in vitro, which mimic the functions of real organs. Nanotechnologies, including the preparation of nanomaterials and the fabrication of micro/nanostructures, have been proven to promote cell proliferation, guide cell differentiation, and regulate cell self-organization, showing great promise in engineering organoids. In this Perspective, different types of nanocomposite hydrogels for organoid culture are summarized, the effects of micro/nanostructures on organoid growth and development are discussed, and 3D bioprinting technologies for constructing organoid models are introduced.

Organoids are rapidly self-organizing 3D in vitro cultures derived from pluripotent stem cells (PSCs) or adult stem cells (ASCs) that possess disease-like characteristics with high success rates. Due to their ability to retain tissue structure, biological phenotypes, and genetic information, they have been utilized as a novel in vitro model for disease research. In recent years, scientists have established self-organizing 3D organoids for human endometrium, fallopian tubes, ovaries, and cervix by culturing stem cells with cytokines in 3D scaffolds. The integration of organoids with animal models, organ-on-a-chip systems, and 3D printing technologies offers a novel preclinical model for exploring disease mechanisms and developing treatments. This review elaborate on the recent research progress of stem cells-formed organoids in the field of gynecology from the aspects of constructing gynecological disease organoids, drug screening and new drug development, simulation modeling, allogeneic transplantation, regenerative medicine and personalized treatment."

In vitromodeling of vascular diseases provides a useful platform for drug screening and mechanistic studies, by recapitulating the essential structures and physiological characteristics of the native tissue. Bioprinting is an emerging technique that offers high-resolution 3D capabilities, which have recently been employed in the modeling of various tissues and associated diseases. Blood vessels are composed of multiple layers of distinct cell types, and experience different mechanical conditions depending on the vessel type. The intimal layer, in particular, is directly exposed to such hemodynamic conditions inducing shear stress, which in turn influence vascular physiology. 3D bioprinting techniques have addressed the structural limitations of the previous vascular models, by incorporating supporting cells such as smooth muscle cells, geometrical properties such as dilation, curvature, or branching, or mechanical stimulation such as shear stress and pulsatile pressure. This paper presents a review of the physiology of blood vessels along with the pathophysiology of the target diseases including atherosclerosis, thrombosis, aneurysms, and tumor angiogenesis. Additionally, it discusses recent advances in fabricatingin vitro3D vascular disease models utilizing bioprinting techniques, while addressing the current challenges and future perspectives for the potential clinical translation into therapeutic interventions.

Human stem cell-derived neural organoids were recently introduced as powerful in vitro 3D experimental model systems that innately undergo critical steps of organogenesis in culture and exhibit molecular, cellular, and structural features similar to the fetal human nervous system. These organoids have yielded new insights into human neurodevelopment and associated disorders. However, neural organoids have some crucial limitations that arise from the loosely controlled conditions for their development, an inability to maintain their spatial orientation in culture and a lack of technologies for taking long-term measurements on their morphology and electrical activity. Here, we review recent progress in using bioengineering methods to improve neural organoid formation and analysis by leveraging microfabrication, biomaterials, 3D printing, and flexible electrodes. We discuss how the applications of each technique can help to address critical limitations with standard neural organoid models. We conclude with a perspective on future applications of bioengineered next-generation neural organoids.

Biomimetic gut models show promise for enhancing our understanding of intestinal disorder pathogenesis and accelerating therapeutic strategy development. Currentin vitromodels predominantly comprise traditional static cell culture and animal models. Static cell culture lacks the precise control of the complex microenvironment governing human intestinal function. Animal models provide greater microenvironment complexity but fail to accurately replicate human physiological conditions due to interspecies differences. As the available models do not accurately reflect the microphysiological environment and functions of the human intestine, their applications are limited. An optimal approach to intestinal modeling is yet to be developed, but the field will probably benefit from advances in biofabrication techniques. This review highlights biofabrication strategies for constructing biomimetic intestinal models and research approaches for simulating key intestinal physiological features. We also discuss potential biomedical applications of these models and provide an outlook on multi-scale intestinal modeling.

Colorectal epithelium was the first long-term 3D organoid culture established in vitro. Identification of the key components essential for the long-term survival of the stem cell niche allowed an indefinite propagation of these cultures and the modulation of their differentiation into various lineages of mature intestinal epithelial cells. While these methods were eventually adapted to establish organoids from different organs, colorectal organoids remain a pioneering model for the development of new applications in health and disease. Several basic and applicative aspects of organoid culture, modeling, monitoring and testing are analyzed in this review. We also tackle the ethical problems of biobanking and distribution of these precious research tools, frequently confined in the laboratory of origin or condemned to destruction at the end of the project.

Extrusion-based embedded 3D bioprinting, where bioinks and biomaterials are extruded within a support bath, has greatly expanded the achievable tissue architectures and complexity of biologic constructs that can be fabricated. However, significant scientific, engineering, and process-related challenges remain to recreate the full anatomic structure and physiologic function required for many therapeutic applications. This perspective explores the future advances in extrusion-based embedded 3D bioprinting that could address these challenges, paving the way for clinical translation of bioprinted tissues.

Organoids have revolutionized the whole field of biology with their ability to model complex three-dimensional human organs in vitro. Intestinal organoids were especially consequential as the first successful long-term culture of intestinal stem cells, which raised hopes for translational medical applications. Despite significant contributions to basic research, challenges remain to develop intestinal organoids into clinical tools for diagnosis, prognosis, and therapy. In this review, we outline the current state of translational research involving adult stem cell and pluripotent stem cell derived intestinal organoids, highlighting the advances and limitations in disease modeling, drug-screening, personalized medicine, and stem cell therapy. Preclinical studies have demonstrated a remarkable functional recapitulation of infectious and genetic diseases, and there is mounting evidence for the reliability of intestinal organoids as a patient-specific avatar. Breakthroughs now allow the generation of structurally and cellularly complex intestinal models to better capture a wider range of intestinal pathophysiology. As the field develops and evolves, there is a need for standardized frameworks for generation, culture, storage, and analysis of intestinal organoids to ensure reproducibility, comparability, and interpretability of these preclinical and clinical studies to ultimately enable clinical translation.

Bone tissue engineering aims to address critical-sized defects by developing biomimetic scaffolds that promote repair and regeneration. This study introduces a material extrusion-based embedded bioprinting approach to fabricate dense cellular constructs within methacrylated hyaluronic acid (MeHA) hydrogels enhanced with bioactive microparticles. Composite matrices containing human bone allograft or tricalcium phosphate (TCP) particles were evaluated for their rheological, mechanical, and osteoinductive properties. High cell viability (>95%) and uniform strand dimensions were achieved across all bioprinting conditions, demonstrating the method's ability to preserve cellular integrity and structural fidelity. The inclusion of bone or TCP particles did not significantly alter the viscosity, crosslinking kinetics, or compressive modulus of the MeHA hydrogels, ensuring robust mechanical stability and shape retention. However, bone allograft particles significantly enhanced osteogenic differentiation of human mesenchymal stem cells (hMSCs), as evidenced by increased alkaline phosphatase (ALP) activity and calcium deposition. Notably, osteogenesis was observed even in basal media, with a dose-dependent response to bone particle concentration, highlighting the intrinsic bioactivity of allograft particles. This study demonstrates the potential of combining embedded bioprinting with bioactive matrices to create dense, osteoinductive cellular constructs. The ability to induce osteogenesis without external growth factors positions this platform as a scalable and clinically relevant solution for bone repair and regeneration.

Kidney transplantation remains a pivotal treatment modality for kidney disease, yet its progress is significantly hindered by the scarcity of donor kidneys and ethical dilemmas surrounding their procurement. As organoid technology evolves and matures, the creation of bionic human kidney organoids offers profound potential for advancing kidney disease research, drug nephrotoxicity screening, and regenerative medicine. Nevertheless, current kidney organoid models grapple with limitations such as constrained cellular differentiation, underdeveloped functional structures, and a crucial absence of vascularization. This deficiency in vascularization, in particular, stunts organoid development, restricts their size, diminishes filtration capabilities, and may trigger immune inflammatory reactions through the resulting ischemic microenvironment. Hence, the achievement of vascularization within kidney organoids and the successful establishment of functional microvascular networks constitutes a paramount goal for their future progression. In this review, we provide an overview of recent advancements in biotechnology domains, encompassing organ-on-a-chip technology, biomimetic matrices, and bioprinting, with the aim of catalyzing technological breakthroughs that can enhance the vascularization of kidney organoids and broaden their applicability. These technologies hold the key to unlocking the full potential of kidney organoids as a transformative therapeutic option for kidney disease.

Organs-on-chips (OoCs) have significantly advanced biomedical research by precisely reconstructing human microphysiological systems with biomimetic functions. However, achieving greater structural complexity of cell cultures on-chip for enhanced biological mimicry remains a challenge. To overcome these challenges, 3D bioprinting techniques can be used in directly building complex 3D cultures on chips, facilitating the in vitro engineering of organ-level models. Herein, we review the distinctive features of OoCs, along with the technical and biological challenges associated with replicating complex organ structures. We discuss recent bioprinting innovations that simplify the fabrication of OoCs while increasing their architectural complexity, leading to breakthroughs in the field and enabling the investigation of previously inaccessible biological problems. We highlight the challenges for the development of 3D bioprinted OoCs, concluding with a perspective on future directions aimed at facilitating their clinical translation.

In vitro development relies primarily on treating progenitor cells with media-borne morphogens and thus lacks native-like spatial information. Here, we engineer morphogen-secreting organizer cells programmed to self-assemble, via cell adhesion, around mouse embryonic stem (ES) cells in defined architectures. By inducing the morphogen WNT3A and its antagonist DKK1 from organizer cells, we generated diverse morphogen gradients, varying in range and steepness. These gradients were strongly correlated with morphogenetic outcomes: the range of minimum-maximum WNT activity determined the resulting range of anterior-to-posterior (A-P) axis cell lineages. Strikingly, shallow WNT activity gradients, despite showing truncated A-P lineages, yielded higher-resolution tissue morphologies, such as a beating, chambered cardiac-like structure associated with an endothelial network. Thus, synthetic organizer cells, which integrate spatial, temporal, and biochemical information, provide a powerful way to systematically and flexibly direct the development of ES or other progenitor cells in different directions within the morphogenetic landscape.

Organoids are innovative three-dimensional and self-organizing cell cultures of various lineages that can be used to study diverse tissues and organs. Human organoids have dramatically increased our understanding of developmental and disease biology. They provide a patient-specific model to study known diseases, with advantages over animal models, and can also provide insights into emerging and future health threats related to climate change, zoonotic infections, environmental pollutants or even microgravity during space exploration. Furthermore, organoids show potential for regenerative cell therapies and organ transplantation. Still, several challenges for broad clinical application remain, including inefficiencies in initiation and expansion, increasing model complexity and difficulties with upscaling clinical-grade cultures and developing more organ-specific human tissue microenvironments. To achieve the full potential of organoid technology, interdisciplinary efforts are needed, integrating advances from biology, bioengineering, computational science, ethics and clinical research. In this Review, we showcase pivotal achievements in epithelial organoid research and technologies and provide an outlook for the future of organoids in advancing human health and medicine.

The creation of self-organizing liver organoids represents a significant, although modest, step toward addressing the ongoing organ shortage crisis in allogeneic liver transplantation. However, researchers have recognized that achieving a fully functional whole liver remains a distant goal, and the original ambition of organoid-based liver generation has been temporarily put on hold. Instead, liver organoids have revolutionized the field of hepatology, extending their influence into various domains of precision and molecular medicine. These 3D cultures, capable of replicating key features of human liver function and pathology, have opened new avenues for human-relevant disease modeling, CRISPR gene editing, and high-throughput drug screening that animal models cannot accomplish. Moreover, advancements in creating more complex systems have led to the development of multicellular assembloids, dynamic organoid-on-chip systems, and 3D bioprinting technologies. These innovations enable detailed modeling of liver microenvironments and complex tissue interactions. Progress in regenerative medicine and transplantation applications continues to evolve and strives to overcome the obstacles of biocompatibility and tumorigenecity. In this review, we examine the current state of liver organoid research by offering insights into where the field currently stands, and the pivotal developments that are shaping its future.

Organoids form through the sel f-organizing capabilities of stem cells to produce a variety of differentiated cell and tissue types. Most organoid models, however, are limited in terms of the structure and function of the tissues that form, in part because it is difficult to regulate the cell type, arrangement, and cell-cell/cell-matrix interactions within these systems. In this article, we will discuss the engineering approaches to generate more complex organoids with improved function and translational relevance, as well as their advantages and disadvantages. Additionally, we will explore how biofabrication strategies can manipulate the cell composition, 3D organization, and scale-up of organoids, thus improving their utility for disease modeling, drug screening, and regenerative medicine applications.

Organoids are three-dimensional (3D) cell cultures derived from human pluripotent stem cells or adult stem cells that recapitulate the cellular heterogeneity, structure, and function of human organs. These microstructures are invaluable for biomedical research due to their ability to closely mimic the complexity of native tissues while retaining human genetic material. This fidelity to native organ systems positions organoids as a powerful tool for advancing our understanding of human biology and for enhancing preclinical drug testing. Recent advancements have led to the successful development of a variety of organoid types, reflecting a broad range of human organs and tissues. This progress has expanded their application across several domains, including regenerative medicine, where organoids offer potential for tissue replacement and repair; disease modeling, which allows for the study of disease mechanisms and progression in a controlled environment; drug discovery and evaluation, where organoids provide a more accurate platform for testing drug efficacy and safety; and microecological research, where they contribute to understanding the interactions between microbes and host tissues. This review provides a comprehensive overview of the historical development of organoid technology, highlights the key achievements and ongoing challenges in the field, and discusses the current and emerging applications of organoids in both laboratory research and clinical practice.

Organoids, with their capacity to mimic the structures and functions of human organs, have gained significant attention for simulating human pathophysiology and have been extensively investigated in the recent past. Additionally, 3D bioprinting, as an emerging bio-additive manufacturing technology, offers the potential for constructing heterogeneous cellular microenvironments, thereby promoting advancements in organoid research. In this review, the latest developments in 3D bioprinting technologies aimed at enhancing organoid engineering are introduced. The commonly used bioprinting methods and materials for organoids, with a particular emphasis on the potential advantages of combining 3D bioprinting with organoids are summarized. These advantages include achieving high cell concentrations to form large cellular aggregates, precise deposition of building blocks to create organoids with complex structures and functions, and automation and high throughput to ensure reproducibility and standardization in organoid culture. Furthermore, this review provides an overview of relevant studies from recent years and discusses the current limitations and prospects for future development.

Leveraging human cells as materials precursors is a promising approach for fabricating living materials with tissue-like functionalities and cellular programmability. Here we describe a set of cellular units with metabolically engineered glycoproteins that allow cells to tether together to function as macrotissue building blocks and bioeffectors. The generated human living materials, termed as Cellgels, can be rapidly assembled in a wide variety of programmable three-dimensional configurations with physiologically relevant cell densities (up to 108 cells per cm3), tunable mechanical properties and handleability. Cellgels inherit the ability of living cells to sense and respond to their environment, showing autonomous tissue-integrative behaviour, mechanical maturation, biological self-healing, biospecific adhesion and capacity to promote wound healing. These living features also enable the modular bottom-up assembly of multiscale constructs, which are reminiscent of human tissue interfaces with heterogeneous composition. This technology can potentially be extended to any human cell type, unlocking the possibility for fabricating living materials that harness the intrinsic biofunctionalities of biological systems.

The epithelium is one of the important tissues in the body as it plays a crucial barrier role serving as a gateway into and out of the body. Most organs in the body contain an epithelial tissue component, where the tightly connected, organ-specific epithelial cells organize into cysts, invaginations, or tubules, thereby performing distinct to endocrine or exocrine secretory functions. Despite the significance of epithelium, engineering functional epithelium in vitro has remained a challenge due to it is special architecture, heterotypic composition of epithelial tissues, and most importantly, difficulty in attaining the apico-basal and planar polarity of epithelial cells. Bioprinting has brought a paradigm shift in fabricating such apico-basal polarized tissues. In this review, we provide an overview of epithelial tissues and provide insights on recapitulating their cellular arrangement and polarization to achieve epithelial function. We describe the different bioprinting techniques that have been successful in engineering polarized epithelium, which can serve as in vitro models for understanding homeostasis and studying diseased conditions. We also discuss the different attempts that have been investigated to study these 3D bioprinted engineered epithelium for preclinical use. Finally, we highlight the challenges and the opportunities that need to be addressed for translation of 3D bioprinted epithelial tissues towards paving way for personalized healthcare in the future.

Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) are promising resources for intestinal regenerative therapy as they recapitulate both endodermal and mesodermal components of the intestine. However, due to their hPSC-line-dependent mesenchymal development and spherical morphology, HIOs have limited applicability beyond basic research and development. Here, we demonstrate the incorporation of separately differentiated mesodermal and mid/hindgut cells into assembled spheroids to stabilize mesenchymal growth in HIOs. In parallel, we generate tubular intestinal constructs (assembled human intestinal tubules [a-HITs]) by leveraging the high aggregative property of assembled spheroids. Through rotational culture in a bioreactor, a-HITs self-organize to develop epithelium and supportive mesenchyme. Upon mesenteric transplantation, a-HITs mature into centimeter-scale tubular intestinal tissue with complex architectures. Our aggregation- and suspension-based approach offers basic technology for engineering tubular intestinal tissue from hPSCs, which could be ultimately applied to the generation of the human intestine for clinical application.

This comprehensive overview of the historical milestones in cell culture underscores key breakthroughs that have shaped the field over time. It begins with Wilhelm Roux's seminal experiments in the 1880s, followed by the pioneering efforts of Ross Granville Harrison, who initiated groundbreaking experiments that fundamentally shaped the landscape of cell culture in the early 20th century. Carrel's influential contributions, notably the immortalization of chicken heart cells, have marked a significant advancement in cell culture techniques. Subsequently, Johannes Holtfreter, Aron Moscona, and Joseph Leighton introduced methodological innovations in three-dimensional (3D) cell culture, initiated by Alexis Carrel, laying the groundwork for future consolidation and expansion of the use of 3D cell culture in different areas of biomedical sciences. The advent of induced pluripotent stem cells by Takahashi and Yamanaka in 2006 was revolutionary, enabling the reprogramming of differentiated cells into a pluripotent state. Since then, recent innovations have included spheroids, organoids, and organ-on-a-chip technologies, aiming to mimic the structure and function of tissues and organs in vitro, pushing the boundaries of biological modeling and disease understanding. In this review, we overview the history of cell culture shedding light on the main discoveries, pitfalls and hurdles that were overcome during the transition from 2D to 3D cell culture techniques. Finally, we discussed the future directions for cell culture research that may accelerate the development of more effective and personalized treatments.

Providing oxygen and preventing infection at wound sites are effective ways to heal diabetic chronic wounds. Inspired by natural lichens, a bioprinted biogenic hydrogel (BBH) containing microalgae and probiotics is developed for diabetic chronic wound therapeutics, which offers prolonged biogenetic oxygen supply by microalgae and infection inhibition by probiotics. The rational design of symbiotic BBH with customizable structure and microorganism composition enhances wound resilience against elevated glucose levels and hypoxia, leading to the increased migration ability of fibroblasts and the angiogenic potential of human umbilical vein endothelial cells. Notably, BBH-treated diabetic wounds exhibit dense vascular distribution, reduced hypoxia levels and inflammatory responses, and enhanced epithelial differentiation and keratinization abilities. Consequently, the BBH achieves rapid tissue repairing within 3 d and restores approximately 90% of the whole skin structure within 12 d. This work presents an engineered platform for regulating biological microenvironment of diabetic wounds and provides insights for developing bioprinted hybrid microorganism systems.

In the intricately defined spatial microenvironment, a single fertilized egg remarkably develops into a conserved and well-organized multicellular organism. This observation leads us to hypothesize that stem cells or other seed cell types have the potential to construct fully structured and functional tissues or organs, provided the spatial cues are appropriately configured. Current organoid technology, however, largely depends on spontaneous growth and self-organization, lacking systematic guided intervention. As a result, the structures replicated in vitro often emerge in a disordered and sparse manner during growth phases. Although existing organoids have made significant contributions in many aspects, such as advancing our understanding of development and pathogenesis, aiding personalized drug selection, as well as expediting drug development, their potential in creating large-scale implantable tissue or organ constructs, and constructing multicomponent microphysiological systems, together with functioning at metabolic levels remains underutilized. Recent discoveries have demonstrated that the spatial definition of growth factors not only induces directional growth and migration of organoids but also leads to the formation of assembloids with multiple regional identities. This opens new avenues for the innovative engineering of higher-order organoids. Concurrently, the spatial organization of other microenvironmental cues, such as physical stresses, mechanical loads, and material composition, has been minimally explored. This review delves into the burgeoning field of organoid engineering with a focus on potential spatial microenvironmental control. It offers insight into the molecular principles, expected outcomes, and potential applications, envisioning a future perspective in this domain.

Advancements in bioprinting technology are driving the creation of complex, functional tissue constructs for use in tissue engineering and regenerative medicine. Various methods, including extrusion, jetting, and light-based bioprinting, have their unique advantages and drawbacks. Over the years, researchers and industry leaders have made significant progress in enhancing bioprinting techniques and materials, resulting in the production of increasingly sophisticated tissue constructs. Despite this progress, challenges still need to be addressed in achieving clinically relevant, human-scale tissue constructs, presenting a hurdle to widespread clinical translation. However, with ongoing interdisciplinary research and collaboration, the field is rapidly evolving and holds promise for personalized medical interventions. Continued development and refinement of bioprinting technologies have the potential to address complex medical needs, enabling the development of functional, transplantable tissues and organs, as well as advanced in vitro tissue models.

Regenerative medicine is moving from the nascent to the transitional stage as researchers are actively engaged in creating mini-organs from pluripotent stem cells to construct artificial models of physiological and pathological conditions. Currently, mini-organs can express higher-order functions, but their size is limited to the order of a few millimeters. Therefore, one of the ultimate goals of regenerative medicine, "organ replication and transplantation with organoid," remains a major obstacle. Three-dimensional (3D) bioprinting technology is expected to be an innovative breakthrough in this field, but various issues have been raised, such as cell damage, versatility of bioink, and printing time. In this study, we established a method for fabricating, connecting, and assembling organoid units of various shapes independent of cell type, extracellular matrix, and adhesive composition (unit construction method). We also fabricated kidney tissue-like structures using three types of parenchymal and interstitial cells that compose the human kidney and obtained findings suggesting the possibility of crosstalk between the units. This study mainly focuses on methods for reproducing the structure of organs, and there are still issues to be addressed in terms of the expression of their higher-order functions. We anticipate that engineering innovation based on this technique will bring us closer to the realization of highly efficient and rapid fabrication of full-scale organoids that can withstand organ transplantation.

All-aqueous embedded 3D printing, which involves extruding inks in an aqueous bath, has emerged as a transformative platform for the freeform fabrication of 3D constructs with precise control. The use of a supporting bath not only enables the printing of arbitrarily designed 3D constructs but also broadens ink selection for various soft matters, advancing the wide application of this technology. This review focuses on recent progress in the freeform preparation of 3D constructs using all-aqueous embedded 3D printing. It begins by discussing the significance of ultralow interfacial tension in all-liquid embedded printing and highlights the fundamental concepts and properties of all-aqueous system. The review then introduces recent advances in all-aqueous embedded 3D printing and clarifies the key factors affecting printing stability and shape fidelity, aiming to guide expansion and assessment of emerging printing systems used for various representative applications. Furthermore, it proposes the potential scope and applications of this technology, including in vitro models, cytomimetic microreactors, and soft ionic electronics. Finally, the review discusses the challenges facing current all-aqueous embedded 3D printing and offers future perspectives on possible improvements and developments.

Engineering 3D tissue-like constructs for applications such as regenerative medicine remains a major challenge in biomedical research. Recently, self-healing, viscoplastic fluids have been introduced as suspension media to allow lower viscosity, water-rich bioinks to be printed within them for the fabrication of more biomimetic structures. Here, we present gellan gum granular gels produced through the application of shear during gelation, as a candidate suspension medium. We demonstrate that these granular gels exhibit viscoplasticity over a wide range of temperatures, permitting their use for 3D bioprinting of filaments and droplets at low (4°C) as well as physiological temperatures. These granular gels exhibit very low yield stresses (down to 0.4 Pa) which facilitated printing at print speeds up to 60 mm.s-1. Furthermore, we demonstrate the printing of cell-laden droplets maintained over 7 days to show the potential for multiple days of cell culture, as well as the fabrication of hydrogel features within a crosslinkable version of the suspension medium containing granular gellan gum and gelatine-methacryloyl. The combination of ease of preparation, high printing speed, wide temperature tolerance, and crosslinkability makes this gellan gum sheared through cooling-induced gelation an attractive candidate for suspended biofabrication.

The cornea acts as the eye foremost protective layer and is essential for its focusing power. Corneal blindness may arise from physical trauma or conditions like dystrophies, keratitis, keratoconus, or ulceration. While conventional treatments involve medical therapies and donor allografts-sometimes supplemented with keratoprostheses-these options are not suitable for all corneal defects. Consequently, the development of bioartificial corneal tissue has emerged as a critical research area, aiming to address the global shortage of human cornea donors. Bioengineered corneas hold considerable promise as substitutes, with the potential to replace either specific layers or the entire thickness of damaged corneas. This review first delves into the structural anatomy of the human cornea, identifying key attributes necessary for successful corneal tissue bioengineering. It then examines various corneal pathologies, current treatments, and their limitations. Finally, the review outlines the primary approaches in corneal tissue engineering, exploring cell-free, cell-based, and scaffold-based options as three emerging strategies to address corneal failure.

3D in vitro model has emerged as a valuable tool for studying tissue development, drug screening, and disease modeling. 3D systems can accurately replicate tissue microstructures and physiological features, mirroring the in vivo microenvironment departing from conventional 2D cell cultures. Various 3D in vitro models utilizing biomacromolecules like collagen and synthetic polymers have been developed to meet diverse research needs and address the complex challenges of contemporary research. Silk proteins, bearing structural and functional similarities to collagen, have been increasingly employed to construct advanced 3D in vitro systems, surpassing the limitations of 2D cultures. This review examines silk proteins' composition, structure, properties, and functions, elucidating their role in 3D in vitro models. Furthermore, recent advances in biomedical applications involving silk-based organoid models are discussed. In particular, the unique physiological attributes of silk matrix constituents in in vitro tissue constructs are highlighted, providing a meticulous evaluation of their importance. Additionally, it outlines the current research hurdles and complexities while contemplating future avenues, thereby paving the way for developing complex and biomimetic silk protein-based microtissues.

As we navigate the transition from the Fourth to the Fifth Industrial Revolution, the emerging fields of biomanufacturing and biofabrication are transforming life sciences and healthcare. These sectors are benefiting from a synergy of synthetic and engineering biology, sustainable manufacturing, and integrated design principles. Advanced techniques such as 3D bioprinting, tissue engineering, directed assembly, and self-assembly are instrumental in creating biomimetic scaffolds, tissues, organoids, medical devices, and biohybrid systems. The field of biofabrication in the United Kingdom and Ireland is emerging as a pivotal force in bioscience and healthcare, propelled by cutting-edge research and development. Concentrating on the production of biologically functional products for use in drug delivery, in vitro models, and tissue engineering, research institutions across these regions are dedicated to innovating healthcare solutions that adhere to ethical standards while prioritising sustainability, affordability, and healthcare system benefits.

Constructing tissue/organ analogs with natural structures and cell types in vitro offers a valuable strategy for the in situ repair of damaged tissues/organs. Three-dimensional (3D) bioprinting is a flexible method for fabricating these analogs. However, extrusion-based 3D bioprinting faces the challenge of balancing the use of soft bioinks with the need for high-fidelity geometric shapes. To address these challenges, recent advancements have introduced various suspension mediums based on gelatin, agarose, and gellan gum microgels. The emergence of these gel-based suspension mediums has significantly advanced the fabrication of tissue/organ constructs using 3D bioprinting. They effectively stabilize and support soft bioinks, enabling the formation of complex spatial geometries. Moreover, they provide a stable, cell-friendly environment that maximizes cell viability during the printing process. This minireview will summarize the properties, preparation methods, and potential applications of gel-based suspension mediums in constructing tissue/organ analogs, while also addressing current challenges and providing an outlook on the future of 3D bioprinting.

Organoids are powerful models of tissue physiology, yet their applications remain limited due to their relatively simple morphology and high organoid-to-organoid structural variability. To address these limitations we developed a soft, composite yield-stress extracellular matrix that supports optimal organoid morphogenesis following freeform 3D bioprinting of cell slurries at tissue-like densities. The material is designed with two temperature regimes: at 4 °C it exhibits reversible yield-stress behavior to support long printing times without compromising cell viability. When transferred to cell culture at 37 °C, the material cross-links and exhibits similar viscoelasticity and plasticity to basement membrane extracts such as Matrigel. We first characterize the rheological properties of MAGIC matrices that optimize organoid morphogenesis, including low stiffness and high stress relaxation. Next, we combine this material with a custom piezoelectric printhead that allows more reproducible and robust self-organization from uniform and spatially organized tissue "seeds." We apply MAGIC matrix bioprinting for high-throughput generation of intestinal, mammary, vascular, salivary gland, and brain organoid arrays that are structurally similar to those grown in pure Matrigel, but exhibit dramatically improved homogeneity in organoid size, shape, maturation time, and efficiency of morphogenesis. The flexibility of this method and material enabled fabrication of fully 3D microphysiological systems, including perfusable organoid tubes that experience cyclic 3D strain in response to pressurization. Furthermore, the reproducibility of organoid structure increased the statistical power of a drug response assay by up to 8 orders-of-magnitude for a given number of comparisons. Combined, these advances lay the foundation for the efficient fabrication of complex tissue morphologies by canalizing their self-organization in both space and time.

Advances in tissue engineering for both system modeling and organ regeneration depend on embracing and recapitulating the target tissue's functional and structural complexity. Microenvironmental features such as anisotropy, heterogeneity, and other biochemical and mechanical spatiotemporal cues are essential in regulating tissue development and function. Novel biofabrication strategies and innovative biomaterial design have emerged as promising tools to better reproduce such features. These facilitate a transition towards high-fidelity biomimetic structures, offering opportunities for a deeper understanding of tissue function and the development of superior therapies. In this review, we explore some of the key structural and compositional aspects of tissues, lay out how to achieve similar outcomes with current fabrication strategies, and identify the main challenges and promising avenues for future research.

Synthetic extracellular matrix (ECM) mimics that can recapitulate the complex biochemical and mechanical nature of native tissues are needed for advanced models of development and disease. Biomedical research has heavily relied on the use of animal-derived biomaterials, which is now impeding their translational potential and convoluting the biological insights gleaned from in vitro tissue models. Natural hydrogels have long served as a convenient and effective cell culture tool, but advances in materials chemistry and fabrication techniques now present promising new avenues for creating xenogenic-free ECM substitutes appropriate for organotypic models and microphysiological systems. However, significant challenges remain in creating synthetic matrices that can approximate the structural sophistication, biochemical complexity, and dynamic functionality of native tissues. This review summarizes key properties of the native ECM, and discusses recent approaches used to systematically decouple and tune these properties in synthetic matrices. The importance of dynamic ECM mechanics, such as viscoelasticity and matrix plasticity, is also discussed, particularly within the context of organoid and engineered tissue matrices. Emerging design strategies to mimic these dynamic mechanical properties are reviewed, such as multi-network hydrogels, supramolecular chemistry, and hydrogels assembled from biological monomers.

Bioprinting is a highly promising application area of additive manufacturing technology that has been widely used in various fields, including tissue engineering, drug screening, organ regeneration, and biosensing. Its primary goal is to produce biomedical products such as artificial implant scaffolds, tissues and organs, and medical assistive devices through software-layered discrete and numerical control molding. Despite its immense potential, bioprinting technology still faces several challenges. It requires concerted efforts from researchers, engineers, regulatory bodies, and industry stakeholders are principal to overcome these challenges and unlock the full potential of bioprinting. This review systematically discusses bioprinting principles, applications, and future perspectives while also providing a topical overview of research progress in bioprinting over the past two decades. The most recent advancements in bioprinting are comprehensively reviewed here. First, printing techniques and methods are summarized along with advancements related to bioinks and supporting structures. Second, interesting and representative cases regarding the applications of bioprinting in tissue engineering, drug screening, organ regeneration, and biosensing are introduced in detail. Finally, the remaining challenges and suggestions for future directions of bioprinting technology are proposed and discussed. Bioprinting is one of the most promising application areas of additive manufacturing technology that has been widely used in various fields. It aims to produce biomedical products such as artificial implant scaffolds, tissues and organs, and medical assistive devices. This review systematically discusses bioprinting principles, applications, and future perspectives, which provides a topical description of the research progress of bioprinting.

Cell spheroids (esp. organoids) as 3D culture platforms are popular models for representing cell-cell and cell-extracellular matrix (ECM) interactions, bridging the gap between 2D cell cultures and natural tissues. 3D cell models with spatially organized multiple cell types are preferred for gaining comprehensive insights into tissue pathophysiology and constructing in vitro tissues and disease models because of the complexities of natural tissues. In recent years, an assembly strategy using cell spheroids (or organoids) as living building blocks has been developed to construct complex 3D tissue models with spatial organization. Here, a comprehensive overview of recent advances in multispheroid assembly studies is provided. The different mechanisms of the multispheroid assembly techniques, i.e., automated directed assembly, noncontact remote assembly, and programmed self-assembly, are introduced. The processing steps, advantages, and technical limitations of the existing methodologies are summarized. Applications of the multispheroid assembly strategies in disease modeling, drug screening, tissue engineering, and organogenesis are reviewed. Finally, this review concludes by emphasizing persistent issues and future perspectives, encouraging researchers to adopt multispheroid assembly techniques for generating advanced 3D cell models that better resemble real tissues.