Additive manufacturing, also called 3D printing, is an effective method for preparing scaffolds with defined structure and porosity. The disadvantage of the technique is the excessive smoothness of the printed fibers, which does not support cell adhesion. In the present study, a 3D printed scaffold was combined with electrospun classic or structured nanofibers to promote cell adhesion. Structured nanofibers were used to improve the infiltration of cells into the scaffold. Electrospun layers were connected to 3D printed fibers by gluing, thus enabling the fabrication of scaffolds with unlimited thickness. The composite 3D printed/nanofibrous scaffolds were seeded with primary chondrocytes and tested in vitro for cell adhesion, proliferation and differentiation. The experiment showed excellent cell infiltration, viability, and good cell proliferation. On the other hand, partial chondrocyte dedifferentiation was shown. Other materials supporting chondrogenic differentiation will be investigated in future studies.

• Keywords
• 3D printing, cell infiltration, chondrocytes, electrospinning, nanofibres,
• MeSH
• Printing, Three-Dimensional * MeSH
• Cell Adhesion physiology   MeSH
• Cell Differentiation physiology   MeSH
• Chondrocytes cytology   MeSH
• Cells, Cultured physiology   MeSH
• Humans MeSH
• Nanofibers * chemistry   MeSH
• Cell Proliferation physiology   MeSH
• Tissue Engineering methods   MeSH
• Tissue Scaffolds * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

In the rapidly evolving landscape of cell biology and biomedical research, three-dimensional (3D) cell culture has contributed not only to the diversification of experimental tools available but also to their improvement toward greater physiological relevance. 3D cell culture has emerged as a revolutionary technique that bridges the long-standing gap between traditional two-dimensional (2D) cell culture and the complex microenvironments found in living organisms. By providing conditions for establishing critical features of in vivo environment, such as cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper tissue-like architecture and differentiated function of cells. Since the early days of 3D cell culture in the 1970s, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. One particular 3D cell culture technique has caught the attention of many scientists and has experienced an unprecedented boom and enthusiastic application in both basic and translational research over the past decade - the organoid technology. This book chapter provides an introduction to the fundamental concepts of 3D cell culture including organoids, an overview of 3D cell culture techniques, and an overview of methodological- and protocol-oriented chapters in the book 3D Cell Culture.

• Keywords
• 3D cell culture, Assembloid, Bioprinting, Extracellular matrix, Microenvironment, Microfluidics, Organoid, Spheroid,
• MeSH
• Biomedical Research * MeSH
• Cell Culture Techniques methods   MeSH
• Organoids * MeSH
• Cell Culture Techniques, Three Dimensional MeSH
• Translational Research, Biomedical MeSH
• Publication type
• Journal Article MeSH
• Review MeSH

Repairing and regenerating damaged tissues or organs, and restoring their functioning has been the ultimate aim of medical innovations. 'Reviving healthcare' blends tissue engineering with alternative techniques such as hydrogels, which have emerged as vital tools in modern medicine. Additive manufacturing (AM) is a practical manufacturing revolution that uses building strategies like molding as a viable solution for precise hydrogel manufacturing. Recent advances in this technology have led to the successful manufacturing of hydrogels with enhanced reproducibility, accuracy, precision, and ease of fabrication. Hydrogels continue to metamorphose as the vital compatible bio-ink matrix for AM. AM hydrogels have paved the way for complex 3D/4D hydrogels that can be loaded with drugs or cells. Bio-mimicking 3D cell cultures designed via hydrogel-based AM is a groundbreaking in-vivo assessment tool in biomedical trials. This brief review focuses on preparations and applications of additively manufactured hydrogels in the biomedical spectrum, such as targeted drug delivery, 3D-cell culture, numerous regenerative strategies, biosensing, bioprinting, and cancer therapies. Prevalent AM techniques like extrusion, inkjet, digital light processing, and stereo-lithography have been explored with their setup and methodology to yield functional hydrogels. The perspectives, limitations, and the possible prospects of AM hydrogels have been critically examined in this study.

• Keywords
• 3D/4D printing, Biosensors, Digital light processing (DLP), Stereolithography (SLD), Targeted drug delivery, Tissue engineering, cancer therapy,
• MeSH
• Printing, Three-Dimensional MeSH
• Bioprinting methods   MeSH
• Cell Culture Techniques MeSH
• Hydrogels * chemistry   MeSH
• Drug Delivery Systems MeSH
• Humans MeSH
• Cell Culture Techniques, Three Dimensional methods   MeSH
• Tissue Engineering * methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Hydrogels * MeSH

Ectodermal organ development, including lacrimal gland, is characterized by an interaction between an epithelium and a mesenchyme. Murine lacrimal gland is a good model to study non-stereotypical branching morphogenesis. In vitro cultures allow the study of morphogenesis events with easy access to high-resolution imaging. Particularly, embryonic lacrimal gland organotypic 3D cell cultures enable the follow-up of branching morphogenesis thanks to the analysis of territories organization by immunohistochemistry. In this chapter, we describe a method to culture primary epithelial fragments together with primary mesenchymal cells, isolated from embryonic day 17 lacrimal glands.

• Keywords
• 3D culture, Branching, Development, Epithelium, Lacrimal gland, Morphogenesis,
• MeSH
• Epithelium MeSH
• Morphogenesis MeSH
• Mice MeSH
• Organ Culture Techniques MeSH
• Lacrimal Apparatus * MeSH
• Cell Culture Techniques, Three Dimensional MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

In vitro cell cultures are a very useful tool for the validation of biomaterial cytocompatibility, especially for bone tissue engineering scaffolds and bone implants. In this chapter, a protocol for a static three-dimensional osteoblast cell culture on titanium scaffolds and subsequent analysis of osteogenic capacity is presented. The protocol is explained for additively manufactured titanium scaffolds, but it can be extrapolated to other scaffolds with similar size and structure, while differing in composition or manufactured technology. Additionally, the protocol can be used for culture of other adherent cell types beyond osteoblast cells such as mesenchymal stem cells.

• Keywords
• Cell culture, In vitro bone model, Osteoblast, Scaffold, Titanium,
• MeSH
• Printing, Three-Dimensional * MeSH
• Cell Culture Techniques MeSH
• Osteoblasts MeSH
• Osteogenesis MeSH
• Cell Proliferation MeSH
• Titanium * chemistry   MeSH
• Tissue Engineering methods   MeSH
• Tissue Scaffolds chemistry   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Titanium * MeSH

The development of bioink-based 3D-printed scaffolds has revolutionized bone tissue engineering (BTE) by enabling patient-specific and biomimetic constructs for bone regeneration. This review focuses on the biocompatibility and mechanical properties essential for scaffold performance, highlighting advancements in bioink formulations, material combinations, and printing techniques. The key biomaterials, including natural polymers (gelatin, collagen, alginate), synthetic polymers (polycaprolactone, polyethylene glycol), and bioactive ceramics (hydroxyapatite, calcium phosphate), are discussed concerning their osteoconductivity, printability, and structural integrity. Despite significant progress, challenges remain in achieving optimal mechanical strength, degradation rates, and cellular interactions. The review explores emerging strategies such as gene-activated bioinks, nanocomposite reinforcements, and crosslinking techniques to enhance scaffold durability and bioactivity. By synthesizing recent developments, this work provides insights into future directions for bioink-based scaffolds, paving the way for more effective and personalized bone regenerative therapies.

• MeSH
• Printing, Three-Dimensional * MeSH
• Biocompatible Materials * chemistry   MeSH
• Bioprinting MeSH
• Ink MeSH
• Humans MeSH
• Bone Regeneration * MeSH
• Tissue Engineering methods   MeSH
• Tissue Scaffolds * chemistry   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Biocompatible Materials * MeSH

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrPC on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics.

• Keywords
• PrP, cell differentiation, neuronal cells, prion infection, prion protein, spheroid culture,
• MeSH
• Cell Differentiation * physiology   MeSH
• Cell Culture Techniques methods   MeSH
• Cell Line MeSH
• Spheroids, Cellular * metabolism   MeSH
• Mice MeSH
• Neurons metabolism   MeSH
• Prion Diseases * metabolism   pathology   MeSH
• Prions metabolism   MeSH
• Cell Culture Techniques, Three Dimensional methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Prions MeSH

The consideration of human and environmental exposure to dendrimers, including cytotoxicity, acute toxicity, and cell and tissue accumulation, is essential due to their significant potential for various biomedical applications. This study aimed to evaluate the biodistribution and toxicity of a novel methoxyphenyl phosphonium carbosilane dendrimer, a potential mitochondria-targeting vector for cancer therapeutics, in 2D and 3D cancer cell cultures and zebrafish embryos. We assessed its cytotoxicity (via MTT, ATP, and Spheroid growth inhibition assays) and cellular biodistribution. The dendrimer cytotoxicity was higher in cancer cells, likely due to its specific targeting to the mitochondrial compartment. In vivo studies using zebrafish demonstrated dendrimer distribution within the vascular and gastrointestinal systems, indicating a biodistribution profile that may be beneficial for systemic therapeutic delivery strategies. The methoxyphenyl phosphonium carbosilane dendrimer shows promise for applications in cancer cell delivery, but additional studies are required to confirm these findings using alternative labelling methods and more physiologically relevant models. Our results contribute to the growing body of evidence supporting the potential of carbosilane dendrimers as vectors for cancer therapeutics.

• MeSH
• Zebrafish MeSH
• Dendrimers * toxicity   MeSH
• Humans MeSH
• Neoplasms * drug therapy   MeSH
• Cell Culture Techniques, Three Dimensional MeSH
• Tissue Distribution MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• carbosilane MeSH Browser
• Dendrimers * MeSH

This study deals with utilization of the hyaluronic acid (HA) and carbonyl iron (CI) microparticles to fabricate the magneto-responsive hydrogel scaffolds that can provide triggered functionality upon application of an external magnetic field. The various combinations of the HA and CI were investigated from the rheological and viscoelastic point of view to clearly show promising behavior in connection to 3D printing. Furthermore, the swelling capabilities with water diffusion kinetics were also elucidated. Magneto-responsive performance of bulk hydrogels and their noncytotoxic nature were investigated,, and all hydrogels showed cell viability in the range 75-85%. The 3D printing of such developed systems was successful, and fundamental characterization of the scaffolds morphology (SEM and CT) has been presented. The magnetic activity of the final scaffolds was confirmed at a very low magnetic field strength of 140 kA/m, and such a scaffold also provides very good biocompatibility with NIH/3T3 fibroblasts.

• Keywords
• 3D printing, hyaluronic acid, magnetic particles, magneto-responsive, scaffold,
• MeSH
• Printing, Three-Dimensional * MeSH
• Biocompatible Materials * chemistry   pharmacology   MeSH
• Biopolymers chemistry   MeSH
• NIH 3T3 Cells MeSH
• Hydrogels chemistry   pharmacology   MeSH
• Iron Carbonyl Compounds chemistry   MeSH
• Hyaluronic Acid * chemistry   pharmacology   MeSH
• Mice MeSH
• Materials Testing * MeSH
• Tissue Scaffolds * chemistry   MeSH
• Particle Size * MeSH
• Cell Survival * drug effects   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Biocompatible Materials * MeSH
• Biopolymers MeSH
• Hydrogels MeSH
• Iron Carbonyl Compounds MeSH
• Hyaluronic Acid * MeSH

3D bioprinting is revolutionizing tissue engineering and regenerative medicine by enabling the precise fabrication of biologically functional constructs. At its core, the success of 3D bioprinting hinges on the development of bioinks, hydrogel-based materials that support cellular viability, proliferation, and differentiation. However, conventional bioinks face limitations in mechanical strength, biological activity, and customization. Recent advancements in genetic engineering have addressed these challenges by enhancing the properties of bioinks through genetic modifications. These innovations allow the integration of stimuli-responsive elements, bioactive molecules, and extracellular matrix (ECM) components, significantly improving the mechanical integrity, biocompatibility, and functional adaptability of bioinks. This review explores the state-of-the-art genetic approaches to bioink development, emphasizing microbial engineering, genetic functionalization, and the encapsulation of growth factors. It highlights the transformative potential of genetically modified bioinks in various applications, including bone and cartilage regeneration, cardiac and liver tissue engineering, neural tissue reconstruction, and vascularization. While these advances hold promise for personalized and adaptive therapeutic solutions, challenges in scalability, reproducibility, and integration with multi-material systems persist. By bridging genetics and bioprinting, this interdisciplinary field paves the way for sophisticated constructs and innovative therapies in tissue engineering and regenerative medicine.

• Keywords
• 3D bioprinting, Bioink, Gel, Genetics, Tissue engineering,
• MeSH
• Printing, Three-Dimensional * MeSH
• Biocompatible Materials * chemistry   MeSH
• Bioprinting * methods   MeSH
• Extracellular Matrix chemistry   MeSH
• Hydrogels chemistry   MeSH
• Ink * MeSH
• Humans MeSH
• Regenerative Medicine MeSH
• Tissue Engineering * methods   MeSH
• Tissue Scaffolds MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Biocompatible Materials * MeSH
• Hydrogels MeSH