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A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-β1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets. [ABSTRACT FROM AUTHOR]

Hyaline cartilage is a highly specialized tissue. When injured, its repair capacity is low, which results in the massive destruction of the articular surface. Using tissue engineering and genetic engineering techniques, it is possible to provide a suitable microenvironment providing chondrocyte growth factors involved in the development of hyaline cartilage proteins, as well as cell proliferation and differentiation. Our aim was to stimulate the synthesis of an extracellular matrix via the chondrocytes included in a fibrin matrix through the addition or overexpression of IGF1 and/or FGF2, while maintaining a constant agitation of the culture medium. Collagen type II and glycosaminoglycans increased during the entire incubation time. In contrast, collagen type I decreased its expression under the same culture conditions, transfecting or supplementing growth factors to chondrocytes. However, chondrocytes that were not transfected or supplemented showed a general increase in the proteins analyzed in this study. The presence of IGF1 and FGF2 increased the protein synthesis of the hyaline cartilage, regardless of which one was the source of growth factors. Continuous agitation using the spinner flask allows for the adequate nutrition of chondrocytes included in the fibrin matrix. However, they require growth factors to up-regulate or down-regulate collagenous proteins. [ABSTRACT FROM AUTHOR]

The current limitations of commercial microcarriers for mesenchymal stromal cell (MSC) expansion are driving the demand for novel tuneable microcarriers. Novel microcarriers are effective in improving MSC expansion under static and dynamic culture conditions without compromising their stemness and differentiation potential. Integrating microcarrier-based cultures within dynamic bioreactors provides an effective scalable approach for MSC expansion and harvest for downstream clinical translation. The development of good manufacturing practice (GMP)-grade systems, non-xenogenic culture media, and optimised bioreactors, which are compatible with novel microcarriers, is essential to improve cell yield and reproducibility. Microcarrier expansion systems show exciting potential to revolutionise mesenchymal stromal cell (MSC)-based clinical therapies by providing an opportunity for economical large-scale expansion of donor- and patient-derived cells. The poor reproducibility and efficiency of cell expansion on commercial polystyrene microcarriers have driven the development of novel microcarriers with tuneable physical, mechanical, and cell-instructive properties. These new microcarriers show innovation toward improving cell expansion outcomes, although their limited biological characterisation and compatibility with dynamic culture systems suggest the need to realign the microcarrier design pathway. Clear headway has been made toward developing infrastructure necessary for scaling up these technologies; however, key challenges remain in characterising the wholistic effects of microcarrier properties on the biological fate and function of expanded MSCs. [ABSTRACT FROM AUTHOR]

Despite recent advances in the field of tissue engineering, the development of complex tissue‐like structures in vitro is compromised by the lack of integration of a functioning vasculature. In this study, we propose a mesoscale three‐dimensional (3D) in vitro vascularized connective tissue model and demonstrate its feasibility to prompt the self‐assembly of endothelial cells into vessel‐like structures. Moreover, we investigate the effect of perfusion on the organization of the cells. For this purpose, primary endothelial cells (HUVECs) and a cell line of human foreskin fibroblasts are cultivated in ECM‐like matrices made up of freeze‐dried collagen scaffolds permeated with collagen type I hydrogel. A tailored bioreactor is designed to investigate the effect of perfusion on self‐organization of HUVECs. Immunofluorescent staining, two‐photon microscopy, second‐harmonic generation imaging, and scanning electron microscopy are applied to visualize the spatial arrangement of the cells. The analyses reveal the formation of hollow, vessel‐like structures of HUVECs in hydrogel‐permeated collagen scaffolds under both static and dynamic conditions. In conclusion, we demonstrate the feasibility of a 3D porous collagen scaffolding system that enables and maintains the self‐organization of HUVECs into vessel‐like structures independent of a dynamic flow. [ABSTRACT FROM AUTHOR]

Preimplantation embryo culture, pivotal in assisted reproductive technology (ART), has lagged in innovation compared to embryo selection advancements. This review examines the persisting gap between in vivo and in vitro embryo development, emphasizing the need for improved culture conditions. While in humans this gap is hardly estimated, animal models, particularly bovines, reveal clear disparities in developmental competence, cryotolerance, pregnancy and live birth rates between in vitro-produced (IVP) and in vivo-derived (IVD) embryos. Molecular analyses unveil distinct differences in morphology, metabolism, and genomic stability, underscoring the need for refining culture conditions for better ART outcomes. To this end, a deeper comprehension of oviduct physiology and embryo transport is crucial for grasping embryo–maternal interactions' mechanisms. Research on autocrine and paracrine factors, and extracellular vesicles in embryo–maternal tract interactions, elucidates vital communication networks for successful implantation and pregnancy. In vitro, confinement, and embryo density are key factors to boost embryo development. Advanced dynamic culture systems mimicking fluid mechanical stimulation in the oviduct, through vibration, tilting, and microfluidic methods, and the use of innovative softer substrates, hold promise for optimizing in vitro embryo development. [ABSTRACT FROM AUTHOR]

Two-dimensional in vitro cultures have represented a milestone in biomedical and pharmacological research. However, they cannot replicate the architecture and interactions of in vivo tissues. Moreover, ethical issues regarding the use of animals have triggered strategies alternative to animal models. The development of three-dimensional (3D) models offers a relevant tool to investigate disease pathogenesis and treatment, modeling in vitro the in vivo environment. We aimed to develop a dynamic 3D in vitro model for culturing human endothelial cells (ECs) and skin fibroblasts, simulating the structure of the tissues mainly affected in systemic sclerosis (SSc), a prototypical autoimmune fibrotic vasculopathy. Dermal fibroblasts and umbilical vein ECs grown in scaffold or hydrogel, respectively, were housed in bioreactors under flow. Fibroblasts formed a tissue-like texture with the deposition of a new extracellular matrix (ECM) and ECs assembled tube-shaped structures with cell polarization. The fine-tuned dynamic modular system allowing 3D fibroblast/EC culture connection represents a valuable model of the in vivo interplay between the main players in fibrotic vasculopathy as SSc. This model can lead to a more accurate study of the disease's pathogenesis, avoiding the use of animals, and to the development of novel therapies, possibly resulting in improved patient management. [ABSTRACT FROM AUTHOR]

Employing 3D printing bone scaffolds with various polymers is growing due to their biocompatibility, biodegradability, and good mechanical properties. However, their biological properties need modification to have fewer difficulties in clinical experiments. Herein, the fused‐deposition modeling technique is used to design triply‐periodic‐minimal‐surfaces polylactic‐acid scaffolds and evaluate their biological response under static and dynamic cell culture conditions. To enhance the biological response of 3D‐printed bone scaffolds, graphene‐oxide (GO) is coated on the surface of the scaffolds. Fourier‐transform infrared spectroscopy, X‐ray diffraction, and energy‐dispersion X‐ray analysis are conducted to check the GO presence and its effects. Also, computational fluid dynamics analysis is implemented to investigate the shear stress on the scaffold, which is a critical parameter for cell proliferation under dynamic cell culture conditions. Compression tests and contact‐angle measurements are performed to assess the GO effect on mechanical properties and wettability, respectively. Also, it was shown that surface‐treated scaffolds have lower mechanical properties and higher wettability than uncoated scaffolds. A perfusion bioreactor is used to study cell culture. Also, field‐emission‐scanning‐electron‐microscope and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5 diphenyl‐tetrazolium‐bromide (MTT) assay analyses are conducted to observe cell viability and cell attachment. An increase of up to 220% in viability was achieved with GO and dynamic cell culture. [ABSTRACT FROM AUTHOR]

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-β1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

This study explores the intersection of dynamic culture and participatory design in urban spaces, emphasizing the role of user experiences in shaping urban environments. By integrating residents' input in the design and planning processes, this research aims to foster sustainable, viable urban futures that reflect local cultural dynamics and community needs. Utilizing a case study in Tataouine, Tunisia, the research employs parametric mapping and user experience mapping to analyze interactions with heritage sites and public spaces. This approach captures qualitative data on user experiences and overlays it with spatial context, enabling the identification of patterns and relationships between cultural practices and territorial dynamics. The study aims to identify key factors for citizen participation, analyze user experiences in relation to cultural dynamics, and formulate recommendations for integrating participatory design principles. By aligning urban development with a dynamic culture and user-centric design, the study envisions urban spaces that are inclusive, sustainable, and reflective of local identities. The findings underscore the potential of participatory design to enhance the value of urban heritage and promote a more engaged and resilient urban community. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to determine whether a dynamic orbital shaking culture system could enhance the cartilage production and viability of bioengineered nasoseptal cartilage.Methods: Human nasal chondrocytes were seeded onto nanocellulose-alginate biomaterials and cultured in static or dynamic conditions for 14 days. Quantitative polymerase chain reaction for chondrogenic gene expression (type 2 collagen, aggrecan and SOX9) was performed, demonstrating a transient rise in SOX9 expression at 1 and 7 days of culture, followed by a rise at 7 and 14 days in Aggrecan (184.5-fold increase, p < 0.0001) and Type 2 Collagen (226.3-fold increase, p = 0.049) expression. Samples were analysed histologically for glycosaminoglycan content using Alcian blue staining and demonstrated increased matrix formation in dynamic culture.Results: Superior cell viability was identified in the dynamic conditions through live-dead and alamarBlue assays. Computational analysis was used to determine the shear stress experienced by cells in the biomaterial in the dynamic conditions and found that the mechanical stimulation exerted was minimal (fluid shear stress

Human organotypic bone models are an emerging technology that replicate bone physiology and mechanobiology for comprehensive in vitro experimentation over prolonged periods of time. Recently, we introduced a mineralized bone model based on 3D bioprinted cell-laden alginate-gelatin-graphene oxide hydrogels cultured under dynamic loading using commercially available human mesenchymal stem cells. In the present study, we created cell-laden scaffolds from primary human osteoblasts isolated from surgical waste material and investigated the effects of a previously reported optimal cell printing density (5 × 106 cells/mL bioink) vs. a higher physiological cell density (10 × 106 cells/mL bioink). We studied mineral formation, scaffold stiffness, and cell morphology over a 10-week period to determine culture conditions for primary human bone cells in this microenvironment. For analysis, the human bone-derived cell-laden scaffolds underwent multiscale assessment at specific timepoints. High cell viability was observed in both groups after bioprinting (>90%) and after 2 weeks of daily mechanical loading (>85%). Bioprinting at a higher cell density resulted in faster mineral formation rates, higher mineral densities and remarkably a 10-fold increase in stiffness compared to a modest 2-fold increase in the lower printing density group. In addition, physiological cell bioprinting densities positively impacted cell spreading and formation of dendritic interconnections. We conclude that our methodology of processing patient-specific human bone cells, subsequent biofabrication and dynamic culturing reliably affords mineralized cell-laden scaffolds. In the future, in vitro systems based on patient-derived cells could be applied to study the individual phenotype of bone disorders such as osteogenesis imperfecta and aid clinical decision making.

Introduction: We report the development and preliminary evaluation of a novel dynamic bioreactor to culture ovarian cortical tissue strips that leverages tissue response to enhanced oxygen transport and adequate mechanical stimulation. In vitro multistep ovarian tissue static culture followed by mature oocyte generation, fertilization, and embryo transfer promises to use the reserve of dormant follicles. Unfortunately, static in vitro culture of ovarian tissue does not promote development of primordial to secondary follicles or sustain follicle viability and thereby limits the number of obtainable mature oocytes. Enhancing oxygen transport to and exerting mechanical stimulation on ovarian tissue in a dynamic bioreactor may more closely mimic the physiological microenvironment and thus promote follicle activation, development, and viability.Materials and Methods: The most transport-effective dynamic bioreactor design was modified using 3D models of medium and oxygen transport to maximize strip perifusion and apply tissue fluid dynamic shear stresses and direct compressive strains to elicit tissue response. Prototypes of the final bioreactor design were manufactured with materials of varying cytocompatibility and assessed by testing the effect of leachables on sperm motility. Effectiveness of the bioreactor culture was characterized against static controls by culturing fresh bovine ovarian tissue strips for 7 days at 4.8 × 10−5 m/s medium filtration flux in air at −15% maximal total compressive strain and by assessing follicle development, health, and viability.Results and Conclusions: Culture in dynamic bioreactors promoted effective oxygen transport to tissues and stimulated tissues with strains and fluid dynamic shear stresses that, although non-uniform, significantly influenced tissue metabolism. Tissue strip culture in bioreactors made of cytocompatible polypropylene preserved follicle viability and promoted follicle development better than static culture, less so in bioreactors made of cytotoxic ABS-like resin.

The possible pathogenic impact of pro-inflammatory molecules produced by the gut microbiota is one of the hypotheses considered at the basis of the biomolecular dialogue governing the microbiota-gut-brain axis. Among these molecules, lipopolysaccharides (LPS) produced by Gram-negative gut microbiota strains may have a potential key role due to their toxic effects in both the gut and the brain.In this work, we engineered a new dynamic fluidic system, the MINERVA device (MI-device), with the potential to advance the current knowledge of the biological mechanisms regulating the microbiota-gut molecular crosstalk. The MI-device supported the growth of bacteria that are part of the intestinal microbiota under dynamic conditions within a 3D moving mucus model, with features comparable to the physiological conditions (storage modulus of 80 ± 19 Pa, network mesh size of 41 ± 3 nm), without affecting their viability (∼ 109 bacteria/mL). The integration of a fluidically optimized and user-friendly design with a bioinspired microenvironment enabled the sterile extraction and quantification of the LPS produced within the mucus by bacteria (from 423 ± 34 ng/mL to 1785 ± 91 ng/mL). Compatibility with commercially available Transwell-like inserts allows the user to precisely control the transport phenomena that occur between the two chambers by selecting the pore density of the insert membrane without changing the design of the system. The MI-device is able to provide the flow of sterile medium enriched with LPS directly produced by bacteria, opening up the possibility of studying the effects of bacteria-derived molecules on cells in depth, as well as the assessment and characterization of their effects in a physiological or pathological scenario.

Hyaline cartilage is a highly specialized tissue. When injured, its repair capacity is low, which results in the massive destruction of the articular surface. Using tissue engineering and genetic engineering techniques, it is possible to provide a suitable microenvironment providing chondrocyte growth factors involved in the development of hyaline cartilage proteins, as well as cell proliferation and differentiation. Our aim was to stimulate the synthesis of an extracellular matrix via the chondrocytes included in a fibrin matrix through the addition or overexpression of IGF1 and/or FGF2, while maintaining a constant agitation of the culture medium. Collagen type II and glycosaminoglycans increased during the entire incubation time. In contrast, collagen type I decreased its expression under the same culture conditions, transfecting or supplementing growth factors to chondrocytes. However, chondrocytes that were not transfected or supplemented showed a general increase in the proteins analyzed in this study. The presence of IGF1 and FGF2 increased the protein synthesis of the hyaline cartilage, regardless of which one was the source of growth factors. Continuous agitation using the spinner flask allows for the adequate nutrition of chondrocytes included in the fibrin matrix. However, they require growth factors to up-regulate or down-regulate collagenous proteins.

Preimplantation embryo culture, pivotal in assisted reproductive technology (ART), has lagged in innovation compared to embryo selection advancements. This review examines the persisting gap between in vivo and in vitro embryo development, emphasizing the need for improved culture conditions. While in humans this gap is hardly estimated, animal models, particularly bovines, reveal clear disparities in developmental competence, cryotolerance, pregnancy and live birth rates between in vitro-produced (IVP) and in vivo-derived (IVD) embryos. Molecular analyses unveil distinct differences in morphology, metabolism, and genomic stability, underscoring the need for refining culture conditions for better ART outcomes. To this end, a deeper comprehension of oviduct physiology and embryo transport is crucial for grasping embryo–maternal interactions’ mechanisms. Research on autocrine and paracrine factors, and extracellular vesicles in embryo–maternal tract interactions, elucidates vital communication networks for successful implantation and pregnancy. In vitro, confinement, and embryo density are key factors to boost embryo development. Advanced dynamic culture systems mimicking fluid mechanical stimulation in the oviduct, through vibration, tilting, and microfluidic methods, and the use of innovative softer substrates, hold promise for optimizing in vitro embryo development.

The main function of articular cartilage is to provide a low friction surface and protect the underlying subchondral bone. The extracellular matrix composition of articular cartilage mainly consists of glycosaminoglycans and collagen type II. Specifically, collagen type II fibers have an arch-like organization that can be mimicked with segments of a hypotrochoidal curve. In this study, a script was developed that allowed the fabrication of scaffolds with a hypotrochoidal design. This design was investigated and compared to a regular 0–90 woodpile design. The mechanical analyses revealed that the hypotrochoidal design had a lower component Young's modulus while the toughness and strain at yield were higher compared to the woodpile design. Fatigue tests showed that the hypotrochoidal design lost more energy per cycle due to the damping effect of the unique microarchitecture. In addition, data from cell culture under dynamic stimulation demonstrated that the collagen type II deposition was improved and collagen type X reduced in the hypotrochoidal design. Finally, Alcian blue staining revealed that the areas where the stress was higher during the stimulation produced more glycosaminoglycans. Our results highlight a new and simple scaffold design based on hypotrochoidal curves that could be used for cartilage tissue engineering.

Two-dimensional in vitro cultures have represented a milestone in biomedical and pharmacological research. However, they cannot replicate the architecture and interactions of in vivo tissues. Moreover, ethical issues regarding the use of animals have triggered strategies alternative to animal models. The development of three-dimensional (3D) models offers a relevant tool to investigate disease pathogenesis and treatment, modeling in vitro the in vivo environment. We aimed to develop a dynamic 3D in vitro model for culturing human endothelial cells (ECs) and skin fibroblasts, simulating the structure of the tissues mainly affected in systemic sclerosis (SSc), a prototypical autoimmune fibrotic vasculopathy. Dermal fibroblasts and umbilical vein ECs grown in scaffold or hydrogel, respectively, were housed in bioreactors under flow. Fibroblasts formed a tissue-like texture with the deposition of a new extracellular matrix (ECM) and ECs assembled tube-shaped structures with cell polarization. The fine-tuned dynamic modular system allowing 3D fibroblast/EC culture connection represents a valuable model of the in vivo interplay between the main players in fibrotic vasculopathy as SSc. This model can lead to a more accurate study of the disease’s pathogenesis, avoiding the use of animals, and to the development of novel therapies, possibly resulting in improved patient management.

Human neural stem cells (hNSCs) possess remarkable potential for regenerative medicine in the treatment of presently incurable diseases. However, a key challenge lies in producing sufficient quantities of hNSCs, which is necessary for effective treatment. Dynamic culture systems are recognized as a powerful approach to producing large quantities of hNSCs required, where microcarriers play a critical role in supporting cell expansion. Nevertheless, the currently available microcarriers have limitations, including a lack of appropriate surface chemistry to promote cell adhesion, inadequate mechanical properties to protect cells from dynamic forces, and poor suitability for mass production. Here, we present the development of three-dimensional (3D) chitosan scaffolds as microcarriers for hNSC expansion under defined conditions in bioreactors. We demonstrate that chitosan scaffolds with a concentration of 4 wt% (4CS scaffolds) exhibit desirable microstructural characteristics and mechanical properties suited for hNSC expansion. Furthermore, they could also withstand degradation in dynamic conditions. The 4CS scaffold condition yields optimal metabolic activity, cell adhesion, and protein expression, enabling sustained hNSC expansion for up to three weeks in a dynamic culture. Our study introduces an effective microcarrier approach for prolonged expansion of hNSCs, which has the potential for mass production in a three-dimensional setting. [ABSTRACT FROM AUTHOR]

An effective therapeutic strategy to treat tendon or ligament injury continues to be a clinical challenge due to the limited natural healing capacity of these tissues. Furthermore, the repaired tendons or ligaments usually possess inferior mechanical properties and impaired functions. Tissue engineering can restore the physiological functions of tissues using biomaterials, cells, and suitable biochemical signals. It has produced encouraging clinical outcomes, forming tendon or ligament-like tissues with similar compositional, structural, and functional attributes to the native tissues. This paper starts by reviewing tendon/ligament structure and healing mechanisms, followed by describing the bioactive nanostructured scaffolds used in tendon and ligament tissue engineering, with emphasis on electrospun fibrous scaffolds. The natural and synthetic polymers for scaffold preparation, as well as the biological and physical cues offered by incorporating growth factors in the scaffolds or by dynamic cyclic stretching of the scaffolds, are also covered. It is expected to present a comprehensive clinical, biological, and biomaterial insight into advanced tissue engineering-based therapeutics for tendon and ligament repair. [ABSTRACT FROM AUTHOR]

Recognizing the cell‐instructive capacity of the extracellular matrix (ECM), this study investigated the effects of expanding human adipose‐derived stromal cells (hASCs) on ECM‐derived microcarriers fabricated from decellularized adipose tissue (DAT) or decellularized cartilage tissue (DCT) within spinner flask bioreactors. Protocols were established for decellularizing porcine auricular cartilage and electrospraying methods were used to generate microcarriers comprised exclusively of DAT or DCT, which were compositionally distinct, but had matching Young's moduli. Both microcarrier types supported hASC attachment and growth over 14 days within a low‐shear spinner culture system, with a significantly higher cell density observed on the DCT microcarriers at 7 and 14 days. Irrespective of the ECM source, dynamic culture on the microcarriers altered the expression of genes and proteins associated with cell adhesion and ECM remodeling. Label‐free mass spectrometry analysis showed upregulation of proteins associated with cartilage development and ECM in the hASCs expanded on the DCT microcarriers. Based on Luminex analysis, the hASCs expanded on the DCT microcarriers secreted significantly higher levels of IL‐8 and PDGFAA, supporting that the ECM source can modulate hASC paracrine factor secretion. Finally, the hASCs expanded on the microcarriers were extracted for analysis of adipogenic and chondrogenic differentiation relative to baseline controls. The microcarrier‐cultured hASCs showed enhanced intracellular lipid accumulation at 7 days post‐induction of adipogenic differentiation. In the chondrogenic studies, a low level of differentiation was observed in all groups. Future studies are warranted using alternative cell sources with greater chondrogenic potential to further assess the chondro‐inductive properties of the DCT microcarriers. [ABSTRACT FROM AUTHOR]

In vitro tumor models that are 3-dimensional (3D) have emerged as a significant area in the field of cancer research over the past several years. In order for breast cancer cell lines to grow and develop, it is important to select a scaffold, determine a strategy for seeding cells into the scaffold, then cultivate the cells under a variety of conditions. Therefore, we cultivated MCF-7 cells and fibroblasts on a gelatin-alginate scaffold alone or in co-culture under static or dynamic culture conditions in order to produce a 3D tumor model. MCF-7 and fibroblast were seeded into Gellatin-Alginate by Centrifugation and Incubation. After that, the cell proliferation was examined by MTT assay and the cell number determination in the scaffold. The morphology of MCF-7 was observed by H&E staining and SEM. The results showed that the co-culture of MCF-7 cells and fibroblasts in the scaffold exhibited an increase in cell mass size. Their mass morphologies feature a significant number of MCF-7 cells with a round structure that persists for an extended period of time. Perfusion bioreactors also demonstrate an increase in the size of cell mass (3 times higher than static culture). As a result, the long-term stability of the structure offers the possibility of cancer biology research and drug testing, especially the sustained release or actions experiements. [ABSTRACT FROM AUTHOR]

BACKGROUND: The effect of composite culture of scaffold materials and biological factors on chondrocyte function in different conditions is a hot topic in the research of tissue-engineered materials. OBJECTIVE: To investigate the effect of hyaluronic acid combined with domestic porous tantalum on the function of rat chondrocytes under the dynamic environment. METHODS: Knee chondrocytes of SD rats were extracted and cultured. Five groups of experiments were established as static culture group (group A), tantalum static group (group B), tantalum hyaluronic acid static group (group C), tantalum dynamic group (in the dynamic bottle rotation, two-way alternating forward and reverse rotation; the rotation angle is 40°, 40 r/h; group D), and tantalum hyaluronic acid dynamic group (in the dynamic bottle rotation, two-way alternating forward and reverse rotation; the rotation angle is 40°, 40 r/h; group E). The samples were continuously cultured for 7 days in the five groups. Cell morphology on materials was observed under a scanning electron microscope. The secretion of proteoglycan, type II collagen and SOX9 was detected by ELISA. The expression levels of proteoglycan, type II collagen and SOX9 protein were detected by western blot assay. RESULTS AND CONCLUSION: (1) Scanning electron microscope observation showed that with the prolongation of static culture time, the number of chondrocytes in group C gradually increased, and gradually covered the surface of tantalum material. (2) On the 5th day of culture, the secretion of proteoglycan and type II collagen in group C was higher than that in groups A and B (P < 0.05). The secretion of SOX9 in group D was lower than that in group E (P < 0.05). The secretion of proteoglycan and type II collagen in group B was lower than that in group D (P < 0.05). The secretion of proteoglycan and type II collagen in group C was lower than that in group E (P < 0.05). (3) On the 5th day of culture, the expression of type II collagen in group C was higher than that in group B (P < 0.05), and the expression of proteoglycan and type II collagen in group D was higher than that in group B (P < 0.05). The expression levels of proteoglycan and type II collagen in group E were higher than those in group C (P < 0.05). (4) These findings suggest that the combination of domestic porous tantalum and hyaluronic acid can promote the secretion and protein expression of proteoglycan and type II collagen in chondrocytes. Under dynamic environment culture, the secretion and protein expression of proteoglycan and type II collagen are better than in static culture. SOX9 secretion and protein expression are not affected by the dynamic and static culture environments. [ABSTRACT FROM AUTHOR]

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here we engineered a 3D esophageal tumor tissue model using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, we developed a detergent-free, rapid decellularization method to decellularize porcine esophagus. We characterized the decellularized esophageal matrix (DEM) and utilized the DEM for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. We then analyzed the expression of cancer-related markers of KYSE30 cells and compared them with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Our results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens. These findings built a foundation with potential for further study of esophageal cancer behavior in a biomimetic microenvironment using this new esophageal cancer model. [ABSTRACT FROM AUTHOR]

Tissue engineering is an alternative method for preparing small‐caliber (<6 mm) vascular grafts. Dynamic mechanical conditioning is being researched as a method to improve mechanical properties of tissue engineered blood vessels. This method attempts to induce unique reaction in implanted cells that regenerate the matrix around them, thereby improving the overall mechanical stability of the grafts. In this study, we used a bioreactor to seed endothelial cells and smooth muscle cells into the inner and outer layers of the electrospun spider silk protein scaffold respectively to construct vascular grafts. The cell proliferation, mechanical properties, blood compatibility and other indicators of the vascular grafts were characterized in vitro. Furthermore, the vascular grafts were implanted in Sprague Dawley rats, and the vascular grafts' patency, extracellular matrix formation, and inflammatory response were evaluated in vivo. We aimed to construct spider silk protein vascular grafts with the potential for in vivo implantation by using a pulsating flow bioreactor. The results showed that, when compared with the static culture condition, the dynamic culture condition improved cell proliferation on vascular scaffolds and enhanced mechanical function of vascular scaffolds. In vivo experiments also showed that the dynamic culture of vascular grafts was more beneficial for the extracellular matrix deposition and anti‐thrombogenesis, as well as reducing the inflammatory response of vascular grafts. In conclusion, dynamic mechanical conditioning aid in the resolution of challenges impeding the application of electrospun scaffolds and have the potential to construct small‐caliber blood vessels with regenerative function for cardiovascular tissue repair. [ABSTRACT FROM AUTHOR]

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling. [ABSTRACT FROM AUTHOR]

Preclinical and clinical studies have demonstrated the therapeutic effects of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and secretome to cure various degenerative diseases. Thus, the mass-scale production of MSCs is necessary to ensure their availability and cost-effectiveness. In the current study, we evaluated the effect of dynamic 3D and static 2D culture systems on cell proliferation and conditioned media of UC-MSCs. The lysate of concentrated thrombocyte was used to substitute animal-derived serum in the culture media. From two experimental sets with different UC and lysates of concentrated thrombocyte donors, it was found that the shortest PDTs for experimental set 1 were 12.3 h (2D culture) and 14.8 h (3D culture), whereas in experimental set 2, they were 17.7 h (2D culture) and 16.9 h (3D culture). Microscopic observation confirmed the formation of cell aggregates in the 3D system, particularly during the exponential phase. SDS-PAGE analysis revealed similar protein profiles of conditioned media from both culture systems. An anti-inflammatory cytokine, namely tumor necrosis factor beta (TGF-?), was analyzed using ELISA to evaluate the effect of culturing methods on TGF-? release. Interestingly, the relative TGF-? contents in the 2D culture were stagnant throughout the incubation times, whereas a higher accumulation of TGF-? was detected in the 3D culture, which was most likely caused by shear stress. Our study confirmed that a dynamic culture system with a microcarrier-supported bioreactor is a promising approach to scaling up MSC and secretome productions.

Objective: This research aims to study the effects of continuous microvibration stimulation on the parthenogenetic development of human germinal vesicle oocytes. Methods: Ninety-five discarded germinal vesicle oocytes from intracytoplasmic sperm injection treatment (ICSI) cycles performed at Amcare Women's & Children's Hospital between January and December 2021 were used for conventional static culture as well as 10 Hz microvibration culture. We investigated the differences between the two groups in terms of oocyte maturation rate, parthenogenetic activation rate, and parthenogenetic blastocyst formation rate. Results: The static culture and 10 Hz microvibration culture of 95 oocytes showed that the parthenogenetic blastocyst formation rate in the microvibration culture group was significantly higher than those in the traditional static culture group. Conclusion: A continuous microvibration stimulation can significantly improve the parthenogenetic developmental potential of human immature oocytes. [ABSTRACT FROM AUTHOR]

A gelatin-based hydrogel scaffold with highly uniform pore size and biocompatibility was fabricated for cartilage tissue engineering using microfluidic 3D-foaming technology. Mainly, bubbles with different diameters, such as 100 μm and 160 μm, were produced by introducing an optimized nitrogen gas and gelatin solution at an optimized flow rate, and N2/gelatin bubbles were formed. Furthermore, a cross-linking agent (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, EDC) was employed for the cross-linking reaction of the gelatin-based hydrogel scaffold with uniform bubbles, and then the interface between the close cells were broken by degassing. The pore uniformity of the gelatin-based hydrogel scaffolds was confirmed by use of a bright field microscope, conjugate focus microscope and scanning electron microscope. The in vitro degradation rate, mechanical properties, and swelling rate of gelatin-based hydrogel scaffolds with highly uniform pore size were studied. Rabbit knee cartilage was cultured, and its extracellular matrix content was analyzed. Histological analysis and immunofluorescence staining were employed to confirm the activity of the rabbit knee chondrocytes. The chondrocytes were seeded into the resulting 3D porous gelatin-based hydrogel scaffolds. The growth conditions of the chondrocyte culture on the resulting 3D porous gelatin-based hydrogel scaffolds were evaluated by MTT analysis, live/dead cell activity analysis, and extracellular matrix content analysis. Additionally, a dynamic culture of cartilage tissue was performed, and the expression of cartilage-specific proteins within the culture time was studied by immunofluorescence staining analysis. The gelatin-based hydrogel scaffold encouraged chondrocyte proliferation, promoting the expression of collagen type II, aggrecan, and sox9 while retaining the structural stability and durability of the cartilage after dynamic compression and promoting cartilage repair. [ABSTRACT FROM AUTHOR]

Abstract Background Exposure to indoor air pollution from solid fuel combustion is associated with lung diseases and cancer. This study investigated the cytotoxicity and molecular mechanisms of biomass combustion-derived particles in human pulmonary alveolar epithelial cells (HPAEpiC) using a platform that combines air–liquid interface (ALI) and dynamic culture (DC) systems. Methods HPAEpiC were cultured on the surface of polycarbonate (PC) membranes on the ALI–DC platform. The cells were sprayed with an aerosolized solution of biomass combustion soluble constituents (BCSCs) and simultaneously nourished with culture medium flowing beneath the permeable PC membranes. The ALI–DC method was compared with the traditional submerged culture approach. BCSC particle morphology and dosages deposited on the chip were determined for particle characterization. Flow cytometry, scanning electron microscopy, and transmission electron microscopy were used to investigate the apoptosis rate of HPAEpiC and changes in the cell ultrastructure induced by BCSCs. Additionally, the underlying apoptotic pathway was examined by determining the protein expression levels by western blotting. Results Scanning electron microscope images demonstrated that the sample processing and delivering approach of the ALI–DC platform were suitable for pollutant exposure. Compared with the submerged culture method, a significant decline in cell viability and increase in apoptosis rate was observed after BCSC exposure on the ALI–DC platform, indicating that the ALI–DC platform is a more sensitive system for investigating cytotoxicity of indoor air pollutants in lung cells. The morphology and ultrastructure of the cells were damaged after exposure to BCSCs, and the p53 pathway was activated. The Bcl-2/Bax ratio was reduced, upregulating caspase-9 and caspase-3 expression and subsequently inducing apoptosis of HPAEpiC. The addition of N-acetyl cysteine antioxidant significantly alleviated the cytotoxicity induced by BCSCs. Conclusion A novel ALI–DC platform was developed to study the cytotoxicity of air pollutants on lung cells. Using the platform, we demonstrated that BCSCs could damage the mitochondria, produce reactive oxygen species, and activate p53 in HPAEpiC, ultimately inducing apoptosis.

In recent years, conditioned medium (CM) obtained from the culture of mesenchymal stromal/stem cells (MSCs) has been shown to effectively promote tissue repair and modulate the immune response in vitro and in different animal models, with potential for application in regenerative medicine. Using CM offers multiple advantages over the implantation of MSCs themselves: 1) simpler storage, transport, and preservation requirements, 2) avoidance of the inherent risks of cell transplantation, and 3) potential application as a ready-to-go biologic product. For these reasons, a large amount of MSCs research has focused on the characterization of the obtained CM, including soluble trophic factors and vesicles, preconditioning strategies for enhancing paracrine secretion, such as hypoxia, a three-dimensional (3D) environment, and biochemical stimuli, and potential clinical applications. In vitro preconditioning strategies can increase the viability, proliferation, and paracrine properties of MSCs and therefore improve the therapeutic potential of the cells and their derived products. Specifically, dynamic cultivation conditions, such as fluid flow and 3D aggregate culture, substantially impact cellular behaviour. Increased levels of growth factors and cytokines were observed in 3D cultures of MSC grown on orbital or rotatory shaking platforms, in stirred systems, such as spinner flasks or stirred tank reactors, and in microgravity bioreactors. However, only a few studies have established dynamic culture conditions and protocols for 3D aggregate cultivation of MSCs as a scalable and reproducible strategy for CM production. This review summarizes significant advances into the upstream processing, mainly the dynamic generation and cultivation of MSC aggregates, for de CM manufacture and focuses on the standardization of the soluble factor production.

Human neural stem cells (hNSCs) possess remarkable potential for regenerative medicine in the treatment of presently incurable diseases. However, a key challenge lies in producing sufficient quantities of hNSCs, which is necessary for effective treatment. Dynamic culture systems are recognized as a powerful approach to producing large quantities of hNSCs required, where microcarriers play a critical role in supporting cell expansion. Nevertheless, the currently available microcarriers have limitations, including a lack of appropriate surface chemistry to promote cell adhesion, inadequate mechanical properties to protect cells from dynamic forces, and poor suitability for mass production. Here, we present the development of three-dimensional (3D) chitosan scaffolds as microcarriers for hNSC expansion under defined conditions in bioreactors. We demonstrate that chitosan scaffolds with a concentration of 4 wt% (4CS scaffolds) exhibit desirable microstructural characteristics and mechanical properties suited for hNSC expansion. Furthermore, they could also withstand degradation in dynamic conditions. The 4CS scaffold condition yields optimal metabolic activity, cell adhesion, and protein expression, enabling sustained hNSC expansion for up to three weeks in a dynamic culture. Our study introduces an effective microcarrier approach for prolonged expansion of hNSCs, which has the potential for mass production in a three-dimensional setting.

Human umbilical cord mesenchymal stem/stromal cells (hUC-MSCs) have attracted significant research interests in regenerative medicine and cell-based therapies due to their minimally invasive isolation procedure, abundant availability, allogenic nature, improved proliferation capacity and tri-lineage differentiation potential. However, the challenge in harvesting a sufficient number of hUC-MSCs through conventional static culture for downstream application hinders the downstream clinical translation of hUC-MSCs. Hence, an alternative culture method that can facilitate large-scale expansion is highly desirable. Herein, we developed a microcarrier-based dynamic culture system to culture hUC-MSCs combined fed-batch mode with medium refreshment to decrease concentrations of metabolic wastes, improve nutrient supplement and reduce the amount of medium used for cell culture. Instead of refreshing medium based on the pre-determined frequency, the replacement and feeding of medium using the novel feeding regime were carried out based on consumption of nutrients (glucose and glutamine) and production of metabolic waste (lactate and ammonia) to maintain a balanced and benign culture microenvironment. The optimal process allowed over 20 folds increase of cell with a maximum cell density at (24.13 ± 0.59) × 105 cells/mL. In addition, no significant alteration of cell surface markers of hUC-MSCs derived from dynamic conditions was observed compared to static conditions. Impressively, over 99.8% of the cellular population showed the desired positive expression of CD73, CD90 and CD105, while less than 0.2% of the population showed undesired negative expression of CD34, CD45 and HLA-DR. More importantly, hUC-MSCs derived from our dynamic culture condition still maintained their trilineage differentiation capacity. [ABSTRACT FROM AUTHOR]

Constructing an engineered hepatic lobule-mimetic model is challenging owing to complicated lobular architecture and crucial hepatic functionality. Our previous study has demonstrated the feasibility of using silk fibroin (SF) scaffolds as functional templates for engineering hepatic lobule-like constructs. But the unsatisfactory chemical and physical performances of the SF-only scaffold and the inherent defect in the functional activity of the carcinoma-derived seeding cells remain to be addressed to satisfy the downstream application demand. In this study, SF-collagen I (SFC) composite scaffolds with improved physical and chemical properties were fabricated, and their utilization for bioengineering a more hepatic lobule-like construct was explored using the immortalized human hepatocyte-derived liver progenitor-like cells (iHepLPCs) and endothelial cells incorporated in the dynamic culture system. The SFC scaffolds prepared through the directional lyophilization process showed radially aligned porous structures with increased swelling ratio and porosity, ameliorative mechanical stiffness that resembled the normal liver matrix more closely, and improved biocompatibility. The iHepLPCs displayed a hepatic plate-like distribution and differentiated into matured hepatocytes with improved hepatic function in vitro and in vivo. Moreover, hepatocyte–endothelial cell interphase arrangement was generated in the co-culture compartment with improved polarity, bile capillary formation, and enhanced liver functions compared with the monocultures. Thus, a more biomimetic hepatic lobule-like model was established and could provide a valuable and robust platform for various applications, including bioartificial liver and drug screening.

Abstract Effective nutrient transport and appropriate mechanical stimulation play important roles in production of tissue-engineered bone grafts. In this study, an experimental set-up for magnetic-driven dynamic culture of cells was designed to mimic the microenvironment of the bone tissue. Here, its ability to contribute to osteogenic differentiation was investigated by inoculating human umbilical cord mesenchymal stem cells (HUMSCs) on magnetic scaffolds. The cytocompatibility of the developed magnetic scaffolds was verified for HUMSCs. Magnetic scaffolds seeded with HUMSCs were exposed to magnetic fields. The results showed that magnetic fields did not affect cell activity and promoted HUMSCs osteogenic differentiation. The magnetic scaffolds were magnetically driven for dynamic culture in the experimental set-up to evaluate the influence of HUMSCs osteoblast differentiation. The results indicated that magnetic-driven dynamic culture increased cell alkaline phosphatase (ALP) activity (p

• A 3D-printed gastric model developed in this research combines biomimetic properties, including fold structures, flexibility, and fluid dynamics. • The rugae structure of the 3D model was effective in shielding bacteria and cancer cells from fluid stress and drugs. • This gastric model, crafted through our study, is a promising platform for drug development due to its biomimetic properties. The in vitro biomimetic digestive system simulating the digestive tract environment of animals and humans is necessary for research on drug development, food ingestion, and digestion processes. However, most current digestive models do not present the surface structures of a human stomach, the mechanical properties and fluid dynamics at the same time. In this study, a gastric model was fabricated by photo-curing 3D printing, where the mechanical strength, topographical cues, surface chemistry, and fluid dynamics were biocompatible. A gastric simulation system was assembled with soft and flexible photoresin having biomimetic mechanical strength. The gastric rugae were then replicated by 3D printing on the surface of gastric models, followed by designing the fluid dynamic based on the flow of in vivo gastric juice. Due to the combination of gastric flexibility, rugae morphology, fluid dynamics, and gastric juice emptying behavior, a highly biomimetic gastric model was created. The model was examined with antibiotic and anticancer drugs, where H. pylori and gastric cancer cells, MKN-45, were cultured on model surfaces. The survival rates of H. pylori and MKN-45 on 2D/3D models were systematically evaluated. The cultured bacteria and cells presented more biomimetic responses to drugs, compared to conventional in vitro tests for gastric diseases. The overestimated therapeutic activity in convectional 2D models was modified using the gastric model developed in this research. [Display omitted] [ABSTRACT FROM AUTHOR]

Compared to cell therapy, where cells are injected into a defect region, the treatment of heart infarction with cells seeded in a vascularized scaffold bears advantages, such as an immediate nutrient supply or a controllable and persistent localization of cells. For this purpose, decellularized native tissues are a preferable choice as they provide an in vivo-like microenvironment. However, the quality of such scaffolds strongly depends on the decellularization process. Therefore, two protocols based on sodium dodecyl sulfate or sodium deoxycholate were tailored and optimized for the decellularization of a porcine heart. The obtained scaffolds were tested for their applicability to generate vascularized cardiac patches. Decellularization with sodium dodecyl sulfate was found to be more suitable and resulted in scaffolds with a low amount of DNA, a highly preserved extracellular matrix composition, and structure shown by GAG quantification and immunohistochemistry. After seeding human endothelial cells into the vasculature, a coagulation assay demonstrated the functionality of the endothelial cells to minimize the clotting of blood. Human-induced pluripotent-stem-cell-derived cardiomyocytes in co-culture with fibroblasts and mesenchymal stem cells transferred the scaffold into a vascularized cardiac patch spontaneously contracting with a frequency of 25.61 ± 5.99 beats/min for over 16 weeks. The customized decellularization protocol based on sodium dodecyl sulfate renders a step towards a preclinical evaluation of the scaffolds. [ABSTRACT FROM AUTHOR]

The increased demand for physiologically relevant in vitro human skin models for testing pharmaceutical drugs has led to significant advancements in skin engineering. One of the most promising approaches is the use of in vitro microfluidic systems to generate advanced skin models, commonly known as skin-on-a-chip (SoC) devices. These devices allow the simulation of key mechanical, functional and structural features of the human skin, better mimicking the native microenvironment. Importantly, contrary to conventional cell culture techniques, SoC devices can perfuse the skin tissue, either by the inclusion of perfusable lumens or by the use of microfluidic channels acting as engineered vasculature. Moreover, integrating sensors on the SoC device allows real-time, non-destructive monitoring of skin function and the effect of topically and systemically applied drugs. In this Review, the major challenges and key prerequisites for the creation of physiologically relevant SoC devices for drug testing are considered. Technical (e.g., SoC fabrication and sensor integration) and biological (e.g., cell sourcing and scaffold materials) aspects are discussed. Recent advancements in SoC devices are here presented, and their main achievements and drawbacks are compared and discussed. Finally, this review highlights the current challenges that need to be overcome for the clinical translation of SoC devices. [ABSTRACT FROM AUTHOR]

Preclinical and clinical studies have demonstrated the therapeutic effects of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and secretome to cure various degenerative diseases. Thus, the mass-scale production of MSCs is necessary to ensure their availability and costeffectiveness. In the current study, we evaluated the effect of dynamic 3D and static 2D culture systems on cell proliferation and conditioned media of UC-MSCs. The lysate of concentrated thrombocyte was used to substitute animal-derived serum in the culture media. From two experimental sets with different UC and lysates of concentrated thrombocyte donors, it was found that the shortest PDTs for experimental set 1 were 12.3 h (2D culture) and 14.8 h (3D culture), whereas in experimental set 2, they were 17.7 h (2D culture) and 16.9 h (3D culture). Microscopic observation confirmed the formation of cell aggregates in the 3D system, particularly during the exponential phase. SDS-PAGE analysis revealed similar protein profiles of conditioned media from both culture systems. An anti-inflammatory cytokine, namely tumor necrosis factor beta (TGF-β), was analyzed using ELISA to evaluate the effect of culturing methods on TGF-β release. Interestingly, the relative TGF-β contents in the 2D culture were stagnant throughout the incubation times, whereas a higher accumulation of TGF-β was detected in the 3D culture, which was most likely caused by shear stress. Our study confirmed that a dynamic culture system with a microcarriersupported bioreactor is a promising approach to scaling up MSC and secretome productions. [ABSTRACT FROM AUTHOR]

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here we engineered a 3D esophageal tumor tissue model using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, we developed a detergent-free, rapid decellularization method to decellularize porcine esophagus. We characterized the decellularized esophageal matrix (DEM) and utilized the DEM for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. We then analyzed the expression of cancer-related markers of KYSE30 cells and compared them with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Our results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens. These findings built a foundation with potential for further study of esophageal cancer behavior in a biomimetic microenvironment using this new esophageal cancer model.

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.

Recreating the natural heart's mechanical and electrical environment is crucial for engineering functional cardiac tissue and repairing infarcted myocardium in vivo. In this study, multimaterial-printed serpentine microarchitectures are presented with synergistic mechanical/piezoelectric stimulation, incorporating polycaprolactone (PCL) microfibers for mechanical support, polyvinylidene fluoride (PVDF) microfibers for piezoelectric stimulation, and magnetic PCL/Fe 3 O 4 for controlled deformation via an external magnet. Rat cardiomyocytes in piezoelectric constructs, subjected to dynamic mechanical stimulation, exhibit advanced maturation, featuring superior sarcomeric structures, improved calcium transients, and upregulated maturation genes compared to non-piezoelectric constructs. Furthermore, these engineered piezoelectric cardiac constructs demonstrate significant structural and functional repair of infarcted myocardium, as evidenced by enhanced ejection and shortening fraction, reduced fibrosis and inflammation, and increased angiogenesis. The findings underscore the therapeutic potential of piezoelectric cardiac constructs for myocardial infarction therapy., (© 2024 Wiley‐VCH GmbH.)

Background: Exposure to indoor air pollution from solid fuel combustion is associated with lung diseases and cancer. This study investigated the cytotoxicity and molecular mechanisms of biomass combustion-derived particles in human pulmonary alveolar epithelial cells (HPAEpiC) using a platform that combines air–liquid interface (ALI) and dynamic culture (DC) systems. Methods: HPAEpiC were cultured on the surface of polycarbonate (PC) membranes on the ALI–DC platform. The cells were sprayed with an aerosolized solution of biomass combustion soluble constituents (BCSCs) and simultaneously nourished with culture medium flowing beneath the permeable PC membranes. The ALI–DC method was compared with the traditional submerged culture approach. BCSC particle morphology and dosages deposited on the chip were determined for particle characterization. Flow cytometry, scanning electron microscopy, and transmission electron microscopy were used to investigate the apoptosis rate of HPAEpiC and changes in the cell ultrastructure induced by BCSCs. Additionally, the underlying apoptotic pathway was examined by determining the protein expression levels by western blotting. Results: Scanning electron microscope images demonstrated that the sample processing and delivering approach of the ALI–DC platform were suitable for pollutant exposure. Compared with the submerged culture method, a significant decline in cell viability and increase in apoptosis rate was observed after BCSC exposure on the ALI–DC platform, indicating that the ALI–DC platform is a more sensitive system for investigating cytotoxicity of indoor air pollutants in lung cells. The morphology and ultrastructure of the cells were damaged after exposure to BCSCs, and the p53 pathway was activated. The Bcl-2/Bax ratio was reduced, upregulating caspase-9 and caspase-3 expression and subsequently inducing apoptosis of HPAEpiC. The addition of N-acetyl cysteine antioxidant significantly alleviated the cytotoxicity induced by BCSCs. Conclusion: A novel ALI–DC platform was developed to study the cytotoxicity of air pollutants on lung cells. Using the platform, we demonstrated that BCSCs could damage the mitochondria, produce reactive oxygen species, and activate p53 in HPAEpiC, ultimately inducing apoptosis. [ABSTRACT FROM AUTHOR]

A gelatin-based hydrogel scaffold with highly uniform pore size and biocompatibility was fabricated for cartilage tissue engineering using microfluidic 3D-foaming technology. Mainly, bubbles with different diameters, such as 100 μm and 160 μm, were produced by introducing an optimized nitrogen gas and gelatin solution at an optimized flow rate, and N2/gelatin bubbles were formed. Furthermore, a cross-linking agent (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, EDC) was employed for the cross-linking reaction of the gelatin-based hydrogel scaffold with uniform bubbles, and then the interface between the close cells were broken by degassing. The pore uniformity of the gelatin-based hydrogel scaffolds was confirmed by use of a bright field microscope, conjugate focus microscope and scanning electron microscope. The in vitro degradation rate, mechanical properties, and swelling rate of gelatin-based hydrogel scaffolds with highly uniform pore size were studied. Rabbit knee cartilage was cultured, and its extracellular matrix content was analyzed. Histological analysis and immunofluorescence staining were employed to confirm the activity of the rabbit knee chondrocytes. The chondrocytes were seeded into the resulting 3D porous gelatin-based hydrogel scaffolds. The growth conditions of the chondrocyte culture on the resulting 3D porous gelatin-based hydrogel scaffolds were evaluated by MTT analysis, live/dead cell activity analysis, and extracellular matrix content analysis. Additionally, a dynamic culture of cartilage tissue was performed, and the expression of cartilage-specific proteins within the culture time was studied by immunofluorescence staining analysis. The gelatin-based hydrogel scaffold encouraged chondrocyte proliferation, promoting the expression of collagen type II, aggrecan, and sox9 while retaining the structural stability and durability of the cartilage after dynamic compression and promoting cartilage repair.

Effective nutrient transport and appropriate mechanical stimulation play important roles in production of tissue-engineered bone grafts. In this study, an experimental set-up for magnetic-driven dynamic culture of cells was designed to mimic the microenvironment of the bone tissue. Here, its ability to contribute to osteogenic differentiation was investigated by inoculating human umbilical cord mesenchymal stem cells (HUMSCs) on magnetic scaffolds. The cytocompatibility of the developed magnetic scaffolds was verified for HUMSCs. Magnetic scaffolds seeded with HUMSCs were exposed to magnetic fields. The results showed that magnetic fields did not affect cell activity and promoted HUMSCs osteogenic differentiation. The magnetic scaffolds were magnetically driven for dynamic culture in the experimental set-up to evaluate the influence of HUMSCs osteoblast differentiation. The results indicated that magnetic-driven dynamic culture increased cell alkaline phosphatase (ALP) activity (p < 0.05) and calcium release (p < 0.05) compared with static culture. The effect was demonstrated in the expression of bone-associated genes. Overall, this study showed that magnetic-driven dynamic culture is a promising tool for regenerative bone engineering. [ABSTRACT FROM AUTHOR]

Compared to cell therapy, where cells are injected into a defect region, the treatment of heart infarction with cells seeded in a vascularized scaffold bears advantages, such as an immediate nutrient supply or a controllable and persistent localization of cells. For this purpose, decellularized native tissues are a preferable choice as they provide an in vivo-like microenvironment. However, the quality of such scaffolds strongly depends on the decellularization process. Therefore, two protocols based on sodium dodecyl sulfate or sodium deoxycholate were tailored and optimized for the decellularization of a porcine heart. The obtained scaffolds were tested for their applicability to generate vascularized cardiac patches. Decellularization with sodium dodecyl sulfate was found to be more suitable and resulted in scaffolds with a low amount of DNA, a highly preserved extracellular matrix composition, and structure shown by GAG quantification and immunohistochemistry. After seeding human endothelial cells into the vasculature, a coagulation assay demonstrated the functionality of the endothelial cells to minimize the clotting of blood. Human-induced pluripotent-stem-cell-derived cardiomyocytes in co-culture with fibroblasts and mesenchymal stem cells transferred the scaffold into a vascularized cardiac patch spontaneously contracting with a frequency of 25.61 ± 5.99 beats/min for over 16 weeks. The customized decellularization protocol based on sodium dodecyl sulfate renders a step towards a preclinical evaluation of the scaffolds.

The increased demand for physiologically relevant in vitro human skin models for testing pharmaceutical drugs has led to significant advancements in skin engineering. One of the most promising approaches is the use of in vitro microfluidic systems to generate advanced skin models, commonly known as skin-on-a-chip (SoC) devices. These devices allow the simulation of key mechanical, functional and structural features of the human skin, better mimicking the native microenvironment. Importantly, contrary to conventional cell culture techniques, SoC devices can perfuse the skin tissue, either by the inclusion of perfusable lumens or by the use of microfluidic channels acting as engineered vasculature. Moreover, integrating sensors on the SoC device allows real-time, non-destructive monitoring of skin function and the effect of topically and systemically applied drugs. In this Review, the major challenges and key prerequisites for the creation of physiologically relevant SoC devices for drug testing are considered. Technical (e.g., SoC fabrication and sensor integration) and biological (e.g., cell sourcing and scaffold materials) aspects are discussed. Recent advancements in SoC devices are here presented, and their main achievements and drawbacks are compared and discussed. Finally, this review highlights the current challenges that need to be overcome for the clinical translation of SoC devices.

Introduction: This study aimed to determine whether a dynamic orbital shaking culture system could enhance the cartilage production and viability of bioengineered nasoseptal cartilage. Methods: Human nasal chondrocytes were seeded onto nanocellulose-alginate biomaterials and cultured in static or dynamic conditions for 14 days. Quantitative polymerase chain reaction for chondrogenic gene expression (type 2 collagen, aggrecan and SOX9 ) was performed, demonstrating a transient rise in SOX9 expression at 1 and 7 days of culture, followed by a rise at 7 and 14 days in Aggrecan (184.5-fold increase, p < 0.0001) and Type 2 Collagen (226.3-fold increase, p = 0.049) expression. Samples were analysed histologically for glycosaminoglycan content using Alcian blue staining and demonstrated increased matrix formation in dynamic culture. Results: Superior cell viability was identified in the dynamic conditions through live-dead and alamarBlue assays. Computational analysis was used to determine the shear stress experienced by cells in the biomaterial in the dynamic conditions and found that the mechanical stimulation exerted was minimal (fluid shear stress <0.02 mPa, fluid pressure <48 Pa). Conclusion: We conclude that the use of an orbital shaking system exerts biologically relevant effects on bioengineered nasoseptal cartilage independently of the expected thresholds of mechanical stimulation, with implications for optimising future cartilage tissue engineering efforts., Competing Interests: The senior author IW would like to disclose a previously submitted patent application for the development of nanocellulose bioinks (Patent Pending: US16/976,803). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jovic, Zhao, Jia, Doak and Whitaker.)