Dynamic Culture Research Articles

EXPRESS: Impact of Dynamic Operations Complexity on Performance: the Role of National Culture

We build on the national culture, complex adaptive systems, and operations complexity literature to develop hypotheses about the moderating role of national culture on the negative relationship between dynamic operations culture and operational complexity. Monthly performance data were collected from 179 plants in a multinational corporation’s global plant network that spans all national culture clusters. We use random effects time series modeling to incorporate the transitory nature of this relationship and control unobserved heterogeneity over time. The results indicate that the relationship between dynamic operations complexity and operational performance is moderated by every dimension of national culture except gender egalitarianism and institutional collectivism. We contribute to the literature on complexity by introducing the dynamic operations complexity construct and showing the moderating effect of national culture. We contribute to the national culture literature by comprehensively examining the role of national culture supporting the national culture divergence thesis. Managerial implications relate to locating new plants in regions whose national culture is supportive of key goals and allocating tasks and products accordingly.

Culture system for longitudinal monitoring of bone dynamics ex vivo.

To quantify and visualize both bone formation and resorption within osteochondral explants cultured ex vivo is challenging with the current analysis techniques. An approach that enables monitoring of bone remodeling dynamics is longitudinal microcomputed tomography (µCT), a non-destructive technique that relies on repeated µCT scanning and subsequent registration of consecutive scans. In this study, a two-compartment culture system suitable for osteochondral explants that allowed for µCT scanning during ex vivo culture was established. Explants were scanned repeatedly in a fixed orientation, which allowed assessment of bone remodeling due to adequate image registration. Using this method, bone formation was found to be restricted to the outer surfaces when cultured statically. To demonstrate that the culture system could capture differences in bone remodeling, explants were cultured statically and under dynamic compression as loading promotes osteogenesis. No quantitative differences between static and dynamic culture were revealed. Still, only in dynamic conditions, bone formation was visualized on trabecular surfaces located within the inner cores, suggesting enhanced bone formation towards the center of the explants upon mechanical loading. Taken together, the ex vivo culture system in combination with longitudinal µCT scanning and subsequent registration of images demonstrated potential for evaluating bone remodeling within explants.

3D printed gelatin methacryloyl hydrogels for perfusion culture of human trabecular meshwork cells and glaucoma studies.

Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.

Hybrid modeling for in silico optimization of a dynamic perfusion cell culture process.

The bio-pharmaceutical industry heavily relies on mammalian cells for the production of bio-therapeutic proteins. The complexity of implementing and high cost-of-goods of these processes are currently limiting more widespread patient access. This is driving efforts to enhance cell culture productivity and cost reduction. Upstream process intensification (PI), using perfusion approaches in the seed train and/or the main bioreactor, has shown substantial promise to enhance productivity. However, developing optimal process conditions for perfusion-based processes remain challenging due to resource and time constraints. Model-based optimization offers a solution by systematically screening process parameters like temperature, pH, and culture media to find the optimum conditions in silico. To our knowledge, this is the first experimentally validated model to explain the perfusion dynamics under different operating conditions and scales for process optimization. The hybrid model accurately describes Chinese hamster ovary (CHO) cell culture growth dynamics and a neural network model explains the production of mAb, allowing for optimization of media exchange rates. Results from six perfusion runs in Ambr® 250 demonstrated high accuracy, confirming the model's utility. Further, the implementation of dynamic media exchange rate schedule determined through model-based optimization resulted in 50% increase in volumetric productivity. Additionally, two 5 L-scale experiments validated the model's reliable extrapolation capabilities to large bioreactors. This approach could reduce the number of wet lab experiments needed for culture process optimization, offering a promising avenue for improving productivity, cost-of-goods in bio-pharmaceutical manufacturing, in turn improving patient access to pivotal medicine.

Sentiment Mining in E-Commerce

Sentiment analysis is crucial for comprehending customer feedback and enhancing workplace culture, as well as improving products and services. By employing natural language processing (NLP) techniques to meticulously analyze this feedback, organizations can identify specific areas that require improvement, address employee issues, and cultivate a positive work environment. These deep learning models powered by NLP offer invaluable tools for HR and sales departments in the e-commerce sector, enabling them to track sentiment trends among employees and users over time and implement targeted interventions. Focusing on the e-commerce industry, this study employs NLP-driven deep learning methodologies to analyze both employee and user feedback, with the objective of identifying underlying sentiments. The proposed framework leverages these advanced techniques to categorize user feedback into positive, negative, or neutral sentiments. This approach aims to develop a robust and effective system for sentiment analysis, providing significant insights that can help drive organizational improvements and enhance customer satisfaction. The key steps of this framework include data collection, NLP-enhanced feature extraction, sentiment detection, and final classification using finite-state automata. The effectiveness of this NLP-centric approach was tested on diverse datasets of customer feedback collected from an e-commerce industry. Evaluation metrics such as accuracy, precision, and recall were utilized to assess the performance of the system. The results demonstrate the effectiveness of the proposed framework, achieving a 93.75% accuracy rate and surpassing existing benchmark methods. The outcomes of this study are particularly consequential for the e-commerce sector, offering them a strategic advantage in refining their product portfolios and cultivating a more dynamic workplace culture

Sustainability of shear stress conditioning in endothelial colony-forming cells compared to human aortic endothelial cells to underline suitability for tissue-engineered vascular grafts

The endothelialization of cardiovascular implants is supposed to improve the long-term patency of these implants. In addition, in previous studies, it has been shown, that the conditioning of endothelial cells by dynamic cultivation leads to the expression of an anti-thrombogenic phenotype. For the creation of a tissue-engineered vascular graft (TEVG), these two strategies were combined to achieve optimal hemocompatibility. In a clinical setup, this would require the transfer of the already endothelialized construct from the conditioning bioreactor to the patient. Therefore, the reversibility of the dynamic conditioning of the endothelial cells with arterial-like high shear stress (20 dyn/cm2) was investigated to define the timeframe (tested in a range of up to 24 h) for the perseverance of dynamically induced phenotypical changes. Two types of endothelial cells were compared: endothelial colony-forming cells (ECFCs) and human aortic endothelial cells (HAECs). The results showed that ECFCs respond far more sensitively and rapidly to flow than HAECs. The resulting cell alignment and increased protein expression of KLF-2, Notch-4, Thrombomodulin, Tie2 and eNOS monomer was paralleled by increased eNOS and unaltered KLF-2 mRNA levels even under stopped-flow conditions. VCAM-1 mRNA and protein expression was downregulated under flow and did not recover under stopped flow. From these time kinetic results, we concluded, that the maximum time gap between the TEVG cultivated with autologous ECFCs in future reactor cultivations and the transfer to the potential TEVG recipient should be limited to ∼6 h.

Critical Suitability Evaluation of Caco-2 Cells for Gut-on-a-Chip.

Currently, culturing Caco-2 cells in a Gut-on-a-chip (GOC) is well-accepted for developing intestinal disease models and drug screening. However, Caco-2 cells were found to overexpress surface proteins (e.g., P-gp) compared with the normal intestinal epithelial cells in vivo. To critically evaluate the challenge and suitability of Caco-2 cells, a GOC integrated with a carcinoembryonic antigen (CEA) biosensor was developed. This three-electrode system electrochemical sensor detects CEA by antigen-antibody specific binding, and it exhibits high selectivity, excellent stability, and good reproducibility. Under dynamic culturing in the GOC, Caco-2 cells exhibited an intestinal villus-like structure and maintained tissue barrier integrity. Meanwhile, CEA was discovered to be secreted from 0 to 0.22 ng/mL during the 10-day culturing of Caco-2 cells. Especially, CEA secretion increased significantly with the differentiation of Caco-2 cells after 6 days of culturing. The sustained high-level CEA secretion may induce cells to avoid apoptotic stimuli, which faithfully reflects the efficacy of a new drug and the mechanism of intestinal disease. Different kinds of cell types (e.g., intestinal primary cells, stem cell-induced differentiation) in the GOC should be attempted for drug screening in the future.

Fibrin Scaffolds Perfused with Transforming Growth Factor-β1 as an In Vitro Model to Study Healthy and Tendinopathic Human Tendon Stem/Progenitor Cells.

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.

Evaluation of Dose-Response Relationship in Novel Extended Release of Targeted Nucleic Acid Nanocarriers to Treat Secondary Cataracts.

Purpose: The present study aimed to determine the dose-response relationship between targeted nanocarriers released from a novel, sustained release formulation and their ability to specifically deplete cells responsible for the development of posterior capsular opacification (PCO) in month-long, dynamic cell cultures. Methods: Injectable, thermosensitive poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid) triblock copolymer hydrogels were loaded with either a low or a high dose of doxorubicin-loaded antibody-targeted nanocarriers (G8:3DNA:Dox). Human rhabdomyosarcoma cells, selected for their expression of PCO marker brain-specific angiogenesis inhibitor 1 (BAI1), were kept under dynamic media flow and received either a low or high dose of nanocarriers. Cells were fixed and stained at predetermined time points to evaluate targeted depletion of BAI1+ cells. Results: A lower dose of nanocarriers in hydrogel depleted BAI1+ cells at a slower rate than the higher dose, whereas both reached over 90% BAI1+ cellular nonviability at 28 days. Both treatment groups also significantly lowered the relative abundance of BAI1+ cells in the population compared with the control group. Conclusions: Controlled release of a lower dose of nanocarriers can still achieve therapeutically relevant effects in the prevention of PCO, while avoiding potential secondary effects associated with the administration of a higher dose.

The activity of the quorum sensing regulator HapR is modulated by the bacterial extracellular vesicle (BEV)-associated protein ObfA of Vibrio cholerae.

Vibrio cholerae, a facultative human pathogen and causative agent of the severe diarrheal disease cholera, transits between the human intestinal tract and aquatic reservoirs. Like other bacterial species, V. cholerae continuously releases bacterial extracellular vesicles (BEVs) from its surface, which have been recently characterised for their role during in vivo colonisation. However, between epidemic outbreaks, V. cholerae persists in the biofilm mode for extended periods in aquatic reservoirs, which enhances environmental fitness and host transition. In this study, we investigated the effect of V. cholerae BEVs on biofilm formation, a critical feature for ex vivo survival. In contrast to BEVs from planktonic cultures, our results show that physiological concentrations of BEVs from dynamic biofilm cultures facilitate V. cholerae biofilm formation, which could be linked to a proteinaceous factor. Comparative proteomic analyses of planktonic- and biofilm-derived BEVs identified a previously uncharacterised outer membrane protein as an abundant component of dynamic biofilm-derived BEVs, which was found to be responsible for the BEV-dependent enhancement of biofilm production. Consequently, this protein was named outer membrane-associated biofilm facilitating protein A (ObfA). Comprehensive molecular studies unravelled ObfA as a negative modulator of HapR activity. HapR is a key transcriptional regulator of the V. cholerae quorum sensing (QS) cascade acting as a potent repressor of biofilm formation and virulence. Consistently, obfA mutants not only exhibited reduced biofilm production but also reduced colonisation fitness. Surprisingly, our results demonstrate that ObfA does not affect HapR through the canonical QS system but via the Csr-cascade altering the expression of the small regulatory RNAs CsrC and CsrD. In summary, this study elucidates a novel intraspecies BEV-based communication in V. cholerae that influences biofilm formation and colonisation fitness via a new regulatory pathway involving HapR, Csr-cascade and the BEV-associated protein ObfA.

Oxygenator assisted dynamic microphysiological culture elucidates the impact of hypoxia on valvular interstitial cell calcification

IntroductionMicrophysiological systems (MPS) offer simulation of (patho)physiological parameters. Investigation includes items which lead to fibrosis and calcification in development and progress of calcific aortic valve disease, based e.g. on culturing of isolated valvular interstitial cells (VICs). Hypoxia regulated by hypoxia inducible factors impacts pathological differentiation in aortic valve (AV) disease. This is mimicked via an MPS implemented oxygenator in combination with calcification inducing medium supplementation.MethodsHuman valvular interstitial cells were isolated and dynamically cultured in MPS at hypoxic, normoxic, arterial blood oxygen concentration and cell incubator condition. Expression profile of fibrosis and calcification markers was monitored and calcification was quantified in induction and control media with and without hypoxia and in comparison to statically cultured counterparts.ResultsHypoxic 24-hour culture of human VICs leads to HIF1α nuclear localization and induction of EGLN1, EGLN3 and LDHA mRNA expression but does not directly impact expression of fibrosis and calcification markers. Dependent on medium formulation, induction medium induces monolayer calcification and elevates RUNX2, ACTA2 and FN1 but reduces SOX9 mRNA expression in dynamic and static MPS culture. But combining hypoxic oxygen concentration leads to higher calcification potential of human VICs in calcification and standard medium formulation dynamically cultured for 96 h.ConclusionIn hypoxic oxygen concentration an increased human VIC calcification in 2D VIC culture in an oxygenator assisted MPS was detected. Oxygen regulation therefore can be combined with calcification induction media to monitor additional effects of pathological marker expression. Validation of oxygenator dependent VIC behavior envisions future advancement and transfer to long term aortic valve tissue culture MPS.

Centrifugal Microfluidic Cell Culture Platform for Physiologically Relevant Virus Infection Studies: A Case Study with HSV-1 Infection of Periodontal Cells.

Static well plates remain the gold standard to study viral infections in vitro, but they cannot accurately mimic dynamic viral infections as they occur in the human body. Therefore, we established a dynamic cell culture platform, based on centrifugal microfluidics, to study viral infections in perfusion. To do so, we used human primary periodontal dental ligament (PDL) cells and herpes simplex virus-1 (HSV-1) as a case study. By microscopy, we confirmed that the PDL cells efficiently attached and grew in the chip. Successful dynamic viral infection of perfused PDL cells was monitored using fluorescent imaging and RT-qPCR-based experiments. Remarkably, viral infection in flow resulted in a gradient of HSV-1-infected cells gradually decreasing from the cell culture chamber entrance towards its end. The perfusion of acyclovir in the chip prevented HSV-1 spreading, demonstrating the usefulness of such a platform for monitoring the effects of antiviral drugs. In addition, the innate antiviral response of PDL cells, measured by interferon gene expression, increased significantly over time in conventional static conditions compared to the perfusion model. These results provide evidence suggesting that dynamic viral infections differ from conventional static infections, which highlights the need for more physiologically relevant in vitro models to study viral infections.

Dynamic cell culture modulates colon cancer cell migration in a novel 3D cell culture system

The progression of cancer cell migration, invasion and subsequent metastasis is the main cause of mortality in cancer patients. Through creating more accurate cancer models, we can achieve more precise results, which will lead to a better understanding of the invasion process. This holds promise for more effective prevention and treatment strategies. Although numerous 2D and 3D cell culture systems have been developed, they poorly reflect the in vivo situation and many questions have remained unanswered. This work describes a novel dynamic 3D cell culture system aimed at advancing our comprehension of cancer cell migration. With the newly designed cultivation chamber, 3D tumor spheroids were cultivated within a collagen I matrix in the presence of fluid flow to study the migration of cancer cells from spheroids in the matrix. Using light sheet microscopy and histology, we demonstrated that the morphology of spheroids is influenced by dynamic culture and that, in contrast to static culture, spheroids in dynamic culture are characterized by the absence of a large necrotic core. Additionally, this influence extends to an increase in the size of migration area, coupled with an increase in expression of some genes related to epithelial-mesenchymal transition (EMT). The results here highlight the importance of dynamic culture in cancer research. Although the dynamic 3D cell culture system in this study was used to investigate migration of one cell type into a matrix, it has the potential to be further developed and used for more complex models consisting of different cell types or to analyze other steps of metastasis development such as transendothelial migration or extravasation.

Embedded three-dimensional printing of thick pea-protein-enriched constructs for large, customized structured cell-based meat production

Recent 3D-printing research showed the potential of using plant-protein-enriched inks to fabricate cultivated meat (CM) via agar-based support baths. However, for fabricating large, customized, structured, thick cellular constructs and further cultivation, improved 3D-printing capabilities and diffusion limit circumvention are warranted. The presented study harnesses advanced printing and thick tissue engineering concepts for such purpose. By improving bath composition and altering printing design and execution, large-scale, marbled, 0.5-cm-thick rib-eye shaped constructs were obtained. The constructs featured stable fibrous architectures comparable to those of structured-meat products. Customized multi-cellular constructs with distinct regions were produced as well. Furthermore, sustainable 1-cm-thick cellular constructs were carefully designed and produced, which successfully maintained cell viability and activity for 3 weeks, through the combined effects of void-incorporation and dynamic culturing. As large, geometrically complex construct fabrication suitable for long-term cellular cultivation was demonstrated, these findings hold great promise for advancing structured CM research.

Organs on chips: fundamentals, bioengineering and applications.

Human body constitutes unique biological system containing specific fluid mechanics and biomechanics. Traditional cell culture techniques of 2D and 3D do not recapitulate these specific natures of the human system. In addition, they lack the spatiotemporal conditions of representing the cells. Moreover, they do not enable the study of cell-cell interactions in multiple cell culture platforms. Therefore, establishing biological system of dynamic cell culture was of great interest. Organs on chips systems were fabricated proving their concept to mimic specific organs functions. Therefore, it paves the way for validating new drugs and establishes mechanisms of emerging diseases. It has played a key role in validating suitable vaccines for Coronavirus disease (COVID-19). Herein, the concept of organs on chips, fabrication methodology and their applications are discussed.

Fibronectin Functionalization: A Way to Enhance Dynamic Cell Culture on Alginate/Hydroxyapatite Scaffolds.

Bone defects are a global health concern; bone tissue engineering (BTE) is the most promising alternative to reduce patient morbidity and overcome the inherent drawbacks of autograft and allograft bone. Three-dimensional scaffolds are pivotal in this field due to their potential to provide structural support and mimic the natural bone microenvironment. Following an already published protocol, a 3D porous structure consisting of alginate and hydroxyapatite was prepared after a gelation step and a freezing-drying step. Despite the frequent use of alginate in tissue regeneration, the biological inertness of this polysaccharide hampers proper cell colonization and proliferation. Therefore, the purpose of this work was to enhance the biological properties by promoting the interaction and adhesion between cells and biomaterial with the use of Fibronectin. This extracellular matrix protein was physically adsorbed on the scaffold, and its presence was evaluated with environmental scanning electron microscopy (eSEM) and the Micro-Bicinchoninic Acid (μBCA) protein assay. The MG-63 cell line was used for both static and dynamic (i.e., in bioreactor) 3D cell culturing on the scaffolds. The use of the bioreactor allowed for a better exchange of nutrients and oxygen and a better removal of cell catabolites from the inner portion of the construct, mimicking the physiological environment. The functionalized scaffolds showed an improvement in cell proliferation and colonization compared to non-functionalized ones; the effect of the addition of Fibronectin was more evident in the dynamic culturing conditions, where the cells clearly adhered on the surface of functionalized scaffolds.

Three-dimensional culture in a bioengineered matrix and somatic cell complementation to improve growth and survival of bovine preantral follicles.

Here we explored poly(ethylene glycol) (PEG) bioengineered hydrogels for bovine preantral follicle culture with or without ovarian cell co-culture and examined the potential for differentiation of bovine embryonic stem cells (bESCs) towards gonadal somatic cells to develop a system more similar to the ovarian microenvironment. Bovine preantral follicles were first cultured in two-dimensional (2D) control or within PEG hydrogels (3D) and then co-cultured within PEG hydrogels with bovine ovarian cells (BOCs) to determine growth and viability. Finally, we tested conditions to drive differentiation of bESCs towards the intermediate mesoderm and bipotential gonad fate. Primary follicles grew over the 10-day culture period in PEG hydrogels compared to 2D control. Early secondary follicles maintained a similar diameter within the PEG while control follicles decreased in size. Follicles lost viability after co-encapsulation with BOCs; BOCs lost stromal cell signature over the culture period within hydrogels. Induction of bESCs towards gonadal somatic fate under WNT signaling was sufficient to upregulate intermediate mesoderm ( LHX1 ) and early coelomic epithelium/bipotential gonad markers ( OSR1 , GATA4 , WT1 ). Higher BMP4 concentrations upregulated the lateral plate mesoderm marker FOXF1 . PAX3 expression was not induced, indicating absence of the paraxial mesoderm lineage. Culture of primary stage preantral follicles in PEG hydrogels promoted growth compared to controls; BOCs did not maintain identity in the PEG hydrogels. Collectively, we demonstrate that PEG hydrogels can be a potential culture system for early preantral follicles pending refinements, which could include addition of ESC-derived ovarian somatic cells using the protocol described here. We demonstrate that three-dimensional bioengineered hydrogels could aid in the survival and growth of small bovine preantral follicles. Moreover, bovine embryonic stem cells have the potential to differentiate towards precursors of somatic gonadal cell types, presenting an alternative cell source for preantral follicle co-culture.

Dynamic cultures, dynamic responses: The impact on corporate cash holdings amid uncertainty

This study investigates the changes in national culture, economic uncertainty, and their joint effects on corporate cash holdings across 58 countries over the period 2004 to 2020. The study also delves into the importance of national culture in amplifying the impact of economic uncertainty in influencing corporate cash holdings. Drawing on three national cultural dimensions, trust-distrust, collectivism-individualism, and duty-joy orientation-based on the European Value Survey (EVS) and World Value Survey (WVS), we employ a panel data model with a fixed effect approach and incorporate the lag of the independent variable to mitigate potential endogeneity issues. Our findings show that transitions in national culture from collectivism to individualism do not significantly decrease corporate cash holdings. Moreover, a cultural shift from distrust to trust does not result in a decrease in corporate cash holdings. Conversely, a move towards a short-term orientation motivates firms to reduce their cash holdings. The interaction between economic uncertainty and collectivism and individualism tends to increase corporate cash holdings. However, there is no difference in the impacts of the three cultural dimensions for financially constrained and unconstrained firms on corporate cash holdings. This study offers significant practical implications, particularly for policymakers in countries where they have strong collectivistic national culture, which may amplify the effects of economic uncertainty.

Abstract We047: Enhanced Vascularization in hPSC-derived Cardiac Organoids by Dynamic Culture

Introduction: Human pluripotent stem cells (hPSC)-derived cardiac organoids (COs) have gained increasing interest in biotechnology due to their characteristics like diverse cardiovascular cell types, 3D complex structure, and function that recapitulates the human heart in early development. However, current methods for vascularizing COs are not systematic and robust. Hypothesis: We hypothesize that culturing hPSC-derived COs dynamically will further enhance vascularization in COs. Materials and Methods: Two conditions were created to compare vascularization in COs: 1) dynamic culturing (Dyn) and 2) static culturing (St). Firstly, hPSCs were cultured for two days in ultra-low attachment plates to develop spheroids. The spheroids were then transferred to pillar plates followed by direct cardiac differentiation the next day. Live-cell imaging was used to monitor the process of vascularization for up to 16 days and the corresponding fluorescence intensity of each cell type from its reporter expression was analyzed. The cardiac function was measured by video-based contractility analysis. Data were presented as mean ± standard error of the mean (SEM). The student’s t-test was used for the statistical analysis. Results: Cardiomyocytes (CMs) and endothelial cells (ECs) were imaged on pillars under the two conditions as shown in Fig. 1A . Successful cardiac differentiation was present, displaying hPSC-CM contraction on day 12 and onwards. Higher intensity values of hPSC-CMs under Dyn were observed in all four days compared to St as shown in Figs. 1B&C . The hPSC-ECs intensity displayed greater intensity values compared to St, demonstrating enhanced vascularization in VCOs due to the active Dyn as shown in Figs. 1D&E . The functionality was improved in Dyn as indicated with higher contraction velocity as shown in Fig. 1F. Conclusion: This study demonstrates the effects of incorporating dynamic culture for enhancing cardiogenesis and vasculogenesis in hPSC-derived COs. The enhanced hPSC-VCOs hold promise for studying early cardiovascular development, congenital heart disease modeling, and drug screening and discovery.

Design, development, and benchmarking of a bioreactor integrated with 3D bioprinting: Application to skeletal muscle regeneration

In recent years, great efforts have been spent to create engineered muscle constructs recapitulating the 3D architecture and applying external stimulations. In this regard, tissue engineering approaches could be very promising in regenerating skeletal muscle, in which bioprinting techniques have produced encouraging results especially regarding 3D architecture. Tensile stimuli showed a fundamental role in regulating the behavior of muscle cells both in terms of 3D organizations and protein expression. Despite this promising premise, the combination of 3D bioprinting and mechanical stimulation has been poorly investigated, calling for novel approaches dealing with the mechanical stimulation of the 3D bioprinted construct and the integration of the bioprinting phase into the stimulation device. To this aim, the present work proposes the design, manufacturing, and benchmarking of a bioprinting-integrated mechanical platform conceived for mechanically stimulating a 3D muscle model directly printed into the bioreactor to foster the integration of printing and stimulation. The study consists of three main steps: 1) the design, fabrication, and mechanical characterization of stretchable supports suitable for bioprinting and long-term cell culture; 2) the design, assisted by computational tools, and the fabrication of the smart Petri dish containing the stimulation mechanism and of the final cyclic mechanical platform; 3) the in-vitro validation of the proposed platform in terms of transmission of the mechanical stimulation to the 3D construct and the biological effect of dynamic culture on 3D bioprinted muscle cells. The results highlighted excellent viability and demonstrated that the external stimulus influences the murine myoblasts behavior already after 7 days of culture. In conclusion, prototypes are now available of a mechanical platform that integrates the 3D bioprinting and is capable of stimulating 3D biological constructs for applications in the field of muscle tissue engineering.