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INTRODUCTION: Cardiac safety assessment, such as lethal arrhythmias and contractility dysfunction, is critical during drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been shown to be useful in predicting drug-induced proarrhythmic risk through international validation studies. Although cardiac contractility is another key function, fit-for-purpose hiPSC-CMs in evaluating drug-induced contractile dysfunction remain poorly understood. In this study, we investigated whether alignment of hiPSC-CMs on nanopatterned culture plates can assess drug-induced contractile changes more efficiently than non-aligned monolayer culture. METHODS: Aligned hiPSC-CMs were obtained by culturing on 96-well culture plates with a ridge-groove-ridge nanopattern on the bottom surface, while non-aligned hiPSC-CMs were cultured on regular 96-well plates. Next-generation sequencing and qPCR experiments were performed for gene expression analysis. Contractility of the hiPSC-CMs was assessed using an imaging-based motion analysis system. RESULTS: When cultured on nanopatterned plates, hiPSC-CMs exhibited an aligned morphology and enhanced expression of genes encoding proteins that regulate contractility, including myosin heavy chain, calcium channel, and ryanodine receptor. Compared to cultures on regular plates, the aligned hiPSC-CMs also showed both enhanced contraction and relaxation velocity. In addition, the aligned hiPSC-CMs showed a more physiological response to positive and negative inotropic agents, such as isoproterenol and verapamil. DISCUSSION: Taken together, the aligned hiPSC-CMs exhibited enhanced structural and functional properties, leading to an improved capacity for contractility assessment compared to the non-aligned cells. These findings suggest that the aligned hiPSC-CMs can be used to evaluate drug-induced cardiac contractile changes.
Assuntos
Células-Tronco Pluripotentes Induzidas , Contração Miocárdica , Miócitos Cardíacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Células Cultivadas , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Técnicas de Cultura de Células/métodos , Isoproterenol/farmacologiaRESUMO
Alveolar echinococcosis is considered to be one of the most potentially lethal parasitic zoonotic diseases. However, the molecular mechanisms by which Echinococcus multilocularis interacts with hosts are poorly understood, hindering the prevention and treatment of this disease. Due to the great advantages of cell culture systems for molecular research, numerous attempts have been made to establish primary cell cultures for E. multilocularis. In this study we developed a simple, rapid, and economical method that allows E. multilocularis metacestode tissue blocks to generate daughter vesicles without the continuous presence of host feeder cells in a regular medium. We performed anaerobic, hypoxic (1% O2), normoxic, and semi-anaerobic (in sealed tubes) cultures and found that E. multilocularis metacestode tissues can produce daughter vesicles only in the sealed tubes after 4 wk of incubation. The daughter vesicles cultivated in this system were remarkably enlarged under anaerobic conditions after 8 days of culture, whereas vesicles cultured under hypoxic (1% O2) and normoxic conditions showed only a mild increase in volume. Our in vitro cultivated vesicles showed strong viability and could be used to test antiparasitic drugs, isolate primary cells, and infect animals.
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Echinococcus multilocularis , Animais , Echinococcus multilocularis/crescimento & desenvolvimento , Equinococose/parasitologia , Camundongos , Anaerobiose , Técnicas de Cultura de CélulasRESUMO
The lack of a precise noninvasive, clinical evaluation method for cardiac fibrosis hinders the development of successful treatments that can effectively work in physiological settings, where tissues and organs are interconnected and moderating drug responses. To address this challenge and advance personalized medicine, researchers have turned to human-induced pluripotent stem (iPS) cells, which can be differentiated to resemble the human heart in terms of structure, function and cellular composition. In this chapter, we present an assay protocol that uses these iPS cells to generate heart organoids for the in vitro evaluation of cardiac fibrosis. By establishing this biological platform, we pave the way for conducting phenotype evaluation and treatment screening in a multiscale approach, aiming to discover effective interventions for the treatment of cardiac fibrosis.
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Diferenciação Celular , Fibrose , Células-Tronco Pluripotentes Induzidas , Organoides , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Organoides/patologia , Organoides/citologia , Miocárdio/patologia , Miocárdio/citologia , Técnicas de Cultura de Células/métodos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/patologia , Células CultivadasRESUMO
Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the µm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.
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Técnicas de Cultura de Células , Microtecnologia , Microfluídica/métodos , Impressão Tridimensional , Dispositivos Lab-On-A-ChipRESUMO
Bone differentiation is crucial for skeletal development and maintenance. Its dysfunction can cause various pathological conditions such as rickets, osteoporosis, osteogenesis imperfecta, or Paget's disease. Although traditional two-dimensional cell culture systems have contributed significantly to our understanding of bone biology, they fail to replicate the intricate biotic environment of bone tissue. Three-dimensional (3D) spheroid cell cultures have gained widespread popularity for addressing bone defects. This review highlights the advantages of employing 3D culture systems to investigate bone differentiation. It highlights their capacity to mimic the complex in vivo environment and crucial cellular interactions pivotal to bone homeostasis. The exploration of 3D culture models in bone research offers enhanced physiological relevance, improved predictive capabilities, and reduced reliance on animal models, which have contributed to the advancement of safer and more effective strategies for drug development. Studies have highlighted the transformative potential of 3D culture systems for expanding our understanding of bone biology and developing targeted therapeutic interventions for bone-related disorders. This review explores how 3D culture systems have demonstrated promise in unraveling the intricate mechanisms governing bone homeostasis and responses to pharmacological agents.
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Técnicas de Cultura de Células , Osteogênese , Animais , Células Cultivadas , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Osso e OssosRESUMO
BACKGROUND AIMS: Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. METHODS: Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. RESULTS: The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. CONCLUSIONS: These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products.
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Técnicas de Cultura de Células , Células-Tronco Mesenquimais , Humanos , Técnicas de Cultura de Células/métodos , Reatores Biológicos , Osteogênese , Regeneração Óssea , Proliferação de Células , Diferenciação Celular , Células CultivadasRESUMO
Designing modified therapeutic antibodies with enhanced FcRn-binding affinity holds promise in the extension of circulation half-lives and potential refinement of pharmacokinetics. During the development of these new-generation therapeutic antibodies, FcRn binding affinity of IgGs is emphasized and monitored as a critical quality attribute (CQA), alongside other critical assessments including titer and aggregation level. However, the traditional workflow for assessing the overall quality of expressed IgGs in harvested cell culture fluid (HCCF) is blamed to be cumbersome and time-consuming. This study presents an integrated methodology for the rapid quality assessment of IgGs in HCCF by selectively extracting IgGs with favorable high FcRn affinity for subsequent analysis using size exclusion chromatography (SEC). The approach utilizes innovative adsorbents known as FcRn immobilized hydrophilic magnetic graphene (MG@PDA@PAMAM-FcRn) in a magnetic solid-phase extraction (MSPE) process. To simulate the in vivo binding dynamics, MSPE binding and dissociation was performed at pH 6.0 and 7.4, respectively. The composite have demonstrated enhanced extraction efficiency and impurity removal ability in comparison to commercially available magnetic beads. The SEC monomer peak area value provides the output of this method, the ranking of which enabled the facile identification of superior HCCF samples with high overall quality of IgG. Optimization of MSPE parameters was performed, and the method was validated for specificity, precision, sensitivity, and accuracy. The proposed method exhibited an analytical time of 0.6 h, which is 7-22 times shortened in comparison to the conventional workflow.
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Grafite , Receptores Fc , Receptores Fc/química , Receptores Fc/metabolismo , Imunoglobulina G/química , Meia-Vida , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/metabolismo , Técnicas de Cultura de Células , Fenômenos MagnéticosRESUMO
Organ-on-a-chip technology has shown great potential in disease modeling and drug evaluation. However, traditional organ-on-a-chip devices are mostly pump-dependent with low throughput, which makes it difficult to leverage their advantages. In this study, we have developed a generic, pump-free organ-on-a-chip platform consisting of a 32-unit chip and an adjustable rocker, facilitating high-throughput dynamic cell culture with straightforward operation. By utilizing the rocker to induce periodic fluid forces, we can achieve fluidic conditions similar to those obtained with traditional pump-based systems. Through constructing a gut-on-a-chip model, we observed remarkable enhancements in the expression of barrier-associated proteins and the spatial distribution of differentiated intestinal cells compared to static culture. Furthermore, RNA sequencing analysis unveiled enriched pathways associated with cell proliferation, lipid transport, and drug metabolism, indicating the ability of the platform to mimic critical physiological processes. Additionally, we tested seven drugs that represent a range of high, medium, and low in vivo permeability using this model and found a strong correlation between their Papp values and human Fa, demonstrating the capability of this model for drug absorption evaluation. Our findings highlight the potential of this pump-free organ-on-a-chip platform as a valuable tool for advancing drug development and enabling personalized medicine.
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Dispositivos Lab-On-A-Chip , Sistemas Microfisiológicos , Humanos , Técnicas de Cultura de Células , Intestinos , Transporte BiológicoRESUMO
Over the past 50 years, researchers from the mammary gland field have launched a collection of distinctive 3D cell culture systems to study multiple aspects of mammary gland physiology and disease. As our knowledge about the mammary gland evolves, more sophisticated 3D cell culture systems are required to answer more and more complex questions. Nowadays, morphologically complex mammary organoids can be generated in distinct 3D settings, along with reproduction of multiple aspects of the gland microenvironment. Yet, each 3D culture protocol comes with its advantages and limitations, where some culture systems are best suited to study stemness potential, whereas others are tailored towards the study of mammary gland morphogenesis. Therefore, prior to starting a 3D mammary culture experiment, it is important to consider and select the ideal culture model to address the biological question of interest. The number and technical requirements of novel 3D cell culture methods vastly increased over the past decades, making it currently challenging and time consuming to identify the best experimental testing. In this chapter, we provide a summary of the most promising murine and human 3D organoid models that are currently used in mammary gland biology research. For each model, we will provide a brief description of the protocol and an overview of the expected morphological outcome, the advantages of the model, and the potential pitfalls, to guide the reader to the best model of choice for specific applications.
Assuntos
Glândulas Mamárias Animais , Glândulas Mamárias Humanas , Humanos , Camundongos , Animais , Mama , Organoides , Técnicas de Cultura de Células/métodos , Árvores de DecisõesRESUMO
For the past decade, three-dimensional (3D) culture models have been emerging as powerful tools in translational research to overcome the limitations of two-dimensional cell culture models. Thanks to their ability to recapitulate the phenotypic and molecular heterogeneity found in numerous organs, organoids have been used to model a broad range of tumors, such as colorectal cancer. Several approaches to generate organoids exist, with protocols using either pluripotent stem cells, embryonic stem cells, or organ-restricted adult stem cells found in primary tissues, such as surgical resections as starting material. The latter, so-called patient-derived organoids (PDOs), have shown their robustness in predicting patient drug responses compared to other models. Because of their origin, PDOs are natural offspring of the patient tumor or healthy surrounding tissue, and therefore, have been increasingly used to develop targeted drugs and personalized therapies. Here, we present a new protocol to generate patient-derived colon organoids (PDCOs) from tumor and healthy tissue biopsies. We emphasize budget-friendly and reproducible techniques, which are often limiting factors in this line of research that restrict the development of this 3D-culture model to a small number of laboratories worldwide. Accordingly, we describe efficient and cost-effective techniques to achieve immunoblot and high-resolution microscopy on PDCOs. Finally, a novel strategy of lentiviral transduction of PDCOs, which could be applied to all organoid models, is detailed in this article. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of PDCOs from biopsies Basic Protocol 2: Long-term maintenance and expansion of PDCOs in BME domes Basic Protocol 3: Cryopreservation and thawing of PDCOs Basic Protocol 4: Lentiviral transduction of PDCOs Basic Protocol 5: Immunoblot and evaluation of variability between donors Basic Protocol 6: Immunofluorescence labeling and high-resolution microscopy of PDCOs Basic Protocol 7: Transcriptomic analyses of PDCOs by RT-qPCR.
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Lentivirus , Neoplasias , Adulto , Humanos , Lentivirus/genética , Colo , Técnicas de Cultura de Células/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Organoides/metabolismoRESUMO
Proper regulation of the in vitro cell culture environment is essential for disease modelling and drug toxicity screening. The main limitation of well plates used for cell culture is that they cannot accurately maintain energy sources and compounds needed during cell growth. Herein, to understand the importance of perfusion in cardiomyocyte culture, changes in contractile force and heart rate during cardiomyocyte growth are systematically investigated, and the results are compared with those of a perfusion-free system. The proposed perfusion system consists of a Peltier refrigerator, a peristaltic pump, and a functional well plate. A functional well plate with 12 wells is made through injection moulding, with two tubes integrated in the cover for each well to continuously circulate the culture medium. The contractile force of cardiomyocytes growing on the cantilever surface is analysed through changes in cantilever displacement. The maturation of cardiomyocytes is evaluated through fluorescence staining and western blot; cardiomyocytes cultured in the perfusion system show greater maturity than those cultured in a manually replaced culture medium. The pH of the culture medium manually replaced at intervals of 3 days decreases to 6.8, resulting in an abnormal heartbeat, while cardiomyocytes cultured in the perfusion system maintained at pH 7.4 show improved contractility and a uniform heart rate. Two well-known ion channel blockers, verapamil and quinidine, are used to measure changes in the contractile force of cardiomyocytes from the two systems. Cardiomyocytes in the perfusion system show greater stability during drug toxicity screening, proving that the perfusion system provides a better environment for cell growth.
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Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Miócitos Cardíacos , Humanos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Técnicas de Cultura de Células , Verapamil/farmacologia , Avaliação Pré-Clínica de Medicamentos , Células CultivadasRESUMO
Despite the fact that biotransformation in the liver plays an important role in the augmented toxicity and detoxification of chemicals, relatively little efforts have been made to incorporate biotransformation into in vitro neurotoxicity testing. Conventional in vitro systems for neurotoxicity tests lack the capability of investigating the qualitative and quantitative differences between parent chemicals and their metabolites in the human body. Therefore, there is a need for an in vitro toxicity screening system that can incorporate hepatic biotransformation of chemicals and predict the susceptibility of their metabolites to induce neurotoxicity. To address this need, we adopted 3D cultures of metabolically competent HepaRG cell line with ReNcell VM and established a high-throughput, metabolism-mediated neurotoxicity testing system. Briefly, spheroids of HepaRG cells were generated in an ultralow attachment (ULA) 384-well plate while 3D-cultured ReNcell VM was established on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds were added in the ULA 384-well plate with HepaRG spheroids and coupled with 3D-cultured ReNcell VM on the 384PillarPlate, which allowed us to generate metabolites in situ by HepaRG cells and test them against neural stem cells. We envision that this approach could be potentially adopted in pharmaceutical and chemical industries when high-throughput screening (HTS) is necessary to assess neurotoxicity of compounds and their metabolites.
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Técnicas de Cultura de Células , Células-Tronco Neurais , Humanos , Hepatócitos/metabolismo , Células Cultivadas , Fígado/metabolismo , Esferoides CelularesRESUMO
The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari (Capra hircus) bucks were established and the effect of different media viz. DMEM/F-12 [with low-glucose (5.5 mM; DL) and high-glucose (30 mM; DH)], α-MEM [with low-glucose (5.5 mM; ML) and with high-glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated. Cells were then compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population-doubling time, double-immunocytochemistry, colony-forming units, wound healing, transwell migration, and differential expression of fibroblast-specific markers (FSP-1 and vimentin). The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p < 0.05) lower in FGM compared with other media tested. Overall, our results illustrate the media-dependent deviation in in-vitro dynamics and culture characteristics of cadFibroblasts that may be useful to develop strategies to cultivate these cells efficiently for research and downstream applications.
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Meios de Cultura , Derme , Fibroblastos , Cabras , Técnicas de Cultura de Células , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/microbiologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Técnicas In Vitro , Derme/citologia , Animais , Linhagem Celular , Masculino , Glucose/metabolismo , Perfilação da Expressão Gênica , Cicatrização , Ensaios de Migração Celular , BiomarcadoresRESUMO
Over the past 15 years, the field of oncology research has witnessed significant progress in the development of new cell culture models, such as tumor-on-chip (ToC) systems. In this comprehensive overview, we present a multidisciplinary perspective by bringing together physicists, biologists, clinicians, and experts from pharmaceutical companies to highlight the current state of ToC research, its unique features, and the challenges it faces. To offer readers a clear and quantitative understanding of the ToC field, we conducted an extensive systematic analysis of more than 300 publications related to ToC from 2005 to 2022. ToC offer key advantages over other in vitro models by enabling precise control over various parameters. These parameters include the properties of the extracellular matrix, mechanical forces exerted on cells, the physico-chemical environment, cell composition, and the architecture of the tumor microenvironment. Such fine control allows ToC to closely replicate the complex microenvironment and interactions within tumors, facilitating the study of cancer progression and therapeutic responses in a highly representative manner. Importantly, by incorporating patient-derived cells or tumor xenografts, ToC models have demonstrated promising results in terms of clinical validation. We also examined the potential of ToC for pharmaceutical industries in which ToC adoption is expected to occur gradually. Looking ahead, given the high failure rate of clinical trials and the increasing emphasis on the 3Rs principles (replacement, reduction, refinement of animal experimentation), ToC models hold immense potential for cancer research. In the next decade, data generated from ToC models could potentially be employed for discovering new therapeutic targets, contributing to regulatory purposes, refining preclinical drug testing and reducing reliance on animal models.
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Técnicas de Cultura de Células , Neoplasias , Humanos , Animais , Indústria Farmacêutica , Matriz Extracelular , Microambiente Tumoral , Neoplasias/tratamento farmacológicoRESUMO
Bioreactors are advanced biomanufacturing tools that have been widely used to develop various applications in the fields of health care and cellular agriculture. In recent years, there has been a growing interest in the use of bioreactors to enhance the efficiency and scalability of these technologies. In cell therapy, bioreactors have been used to expand and differentiate cells into specialized cell types that can be used for transplantation or tissue regeneration. In cultured meat production, bioreactors offer a controlled and efficient means of producing meat without the need for animal farming. Bioreactors can support the growth of muscle cells by providing the necessary conditions for cell proliferation, differentiation, and maturation, including the provision of oxygen and nutrients. This review article aims to provide an overview of the current state of bioreactor technology in both cell therapy and cultured meat production. It will examine the various bioreactor types and their applications in these fields, highlighting their advantages and limitations. In addition, it will explore the future prospects and challenges of bioreactor technology in these emerging fields. Overall, this review will provide valuable insights for researchers and practitioners interested in using bioreactor technology to develop innovative solutions in the biomanufacturing of therapeutic cells and cultured meat.
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Reatores Biológicos , Biotecnologia , Terapia Baseada em Transplante de Células e Tecidos , Produtos da Carne , Terapia Baseada em Transplante de Células e Tecidos/economia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Produtos da Carne/economia , Biotecnologia/economia , Biotecnologia/métodos , Biotecnologia/tendências , Técnicas de Cultura de CélulasRESUMO
Current investigations in the field of toxicology mostly rely on 2D cell cultures and animal models. Although well-accepted, the traditional 2D cell-culture approach has evident drawbacks and is distant from the in vivo microenvironment. To overcome these limitations, increasing efforts have been made in the development of alternative models that can better recapitulate the in vivo architecture of tissues and organs. Even though the use of 3D cultures is gaining popularity, there are still open questions on their robustness and standardization. In this review, we discuss the current spheroid culture and organ-on-a-chip techniques as well as the main conceptual and technical considerations for the correct establishment of such models. For each system, the toxicological functional assays are then discussed, highlighting their major advantages, disadvantages, and limitations. Finally, a focus on the applications of 3D cell culture for mycotoxin toxicity assessments is provided. Given the known difficulties in defining the safety ranges of exposure for regulatory agency policies, we are confident that the application of alternative methods may greatly improve the overall risk assessment.
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Técnicas de Cultura de Células , Sistemas Microfisiológicos , Animais , Técnicas de Cultura de Células/métodosRESUMO
Alzheimer's disease (AD) is a major neurological disease affecting elderly individuals worldwide. Existing drugs only reduce the symptoms of the disease without addressing the underlying causes. Commonly, Aß25-35 peptide aggregation is the main reason for AD development. Recently, the discovery of multiple protein-targeting molecules has provided a new strategy for treating AD. This study demonstrates the neuroprotective potential of oxymatrine against multiple mechanisms, such as acetylcholinesterase, mitochondrial damage, and ß-amyloid-induced cell toxicity. The in vitro cell culture studies showed that oxymatrine possesses significant potential to inhibit acetylcholine esterase and promotes antioxidant, antiapoptotic effects while preventing Aß25-35 peptide aggregation in PC12 cells. Furthermore, oxymatrine protects PC12 cells against Aß25-35-induced cytotoxicity and down-regulates the reactive oxygen species generation. The in vivo acute toxicological studies confirm the safety of oxymatrine without causing organ damage or death in animals. Overall, this study provided evidence that oxymatrine is an efficient neuroprotective agent, with a potential to be a multifunctional drug for Alzheimer's disease treatment. These findings present a reliable and synergistic approach for treating AD.
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Doença de Alzheimer , Fármacos Neuroprotetores , Humanos , Ratos , Animais , Idoso , Peptídeos beta-Amiloides/metabolismo , Células PC12 , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Acetilcolinesterase/farmacologia , Apoptose , Fragmentos de Peptídeos/toxicidade , Técnicas de Cultura de Células , Cognição , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêuticoRESUMO
The function of vascular endothelial cells (ECs) within the complex vascular microenvironment is typically modulated by biochemical cues, cell-cell interactions, and fluid shear stress. These regulatory factors play a crucial role in determining cell mechanical properties, such as elastic and shear moduli, which are important parameters for assessing cell status. However, most studies on the measurement of cell mechanical properties have been conducted in vitro, which is labor-intensive and time-consuming. Notably, many physiological factors are lacking in Petri dish culture compared with in vivo conditions, leading to inaccurate results and poor clinical relevance. Herein, we developed a multi-layer microfluidic chip that integrates dynamic cell culture, manipulation and dielectrophoretic in situ measurement of mechanical properties. Furthermore, we numerically and experimentally simulated the vascular microenvironment to investigate the effects of flow rate and tumor necrosis factor-alpha (TNF-α) on the Young's modulus of human umbilical vein endothelial cells (HUVECs). Results showed that greater fluid shear stress results in increased Young's modulus of HUVECs, suggesting the importance of hemodynamics in modulating the biomechanics of ECs. In contrast, TNF-α, an inflammation inducer, dramatically decreased HUVEC stiffness, demonstrating an adverse impact on the vascular endothelium. Blebbistatin, a cytoskeleton disruptor, significantly reduced the Young's modulus of HUVECs. In summary, the proposed vascular-mimetic dynamic culture and monitoring approach enables the physiological development of ECs in organ-on-a-chip microsystems for accurately and efficiently studying hemodynamics and pharmacological mechanisms underlying cardiovascular diseases.
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Microfluídica , Fator de Necrose Tumoral alfa , Humanos , Técnicas de Cultura de Células , Células Endoteliais da Veia Umbilical Humana , Citoesqueleto , Estresse MecânicoRESUMO
The cultivated meat industry, also known as cell-based meat, cultured meat, lab-grown meat, or meat alternatives, is a growing field that aims to generate animal tissues ex-vivo in a cost-effective manner that achieves price parity with traditional agricultural products. However, cell culture media costs account for 55%-90% of production costs. To address this issue, efforts are aimed at optimizing media composition. Systems biology-driven approaches have been successfully used to improve the biomass and productivity of multiple bioproduction platforms, like Chinese hamster ovary cells, by accelerating the development of cell line-specific media and reducing research and development and production costs related to cell media and its optimization. In this review, we summarize systems biology modeling approaches, methods for cell culture media and bioprocess optimization, and metabolic studies done in animals of interest to the cultivated meat industry. More importantly, we identify current gaps in knowledge that prevent the identification of metabolic bottlenecks. These include the lack of genome-scale metabolic models for some species (pigs and ducks), a lack of accurate biomass composition studies for different growth conditions, and 13 C-metabolic flux analysis (MFA) studies for many of the species of interest for the cultivated meat industry (only shrimp and duck cells have been subjected to 13 C-MFA). We also highlight the importance of characterizing the metabolic requirements of cells at the organism, breed, and cell line-specific levels, and we outline future steps that this nascent field needs to take to achieve price parity and production efficiency similar to those of other bioproduction platforms. Practical Application: Our article summarizes systems biology techniques for cell culture media design and bioprocess optimization, which may be used to significantly reduce cell-based meat production costs. We also present the results of experimental studies done on some of the species of interest to the cultivated meat industry and highlight why modeling approaches are required for multiple species, cell-types, and cell lines.
Assuntos
Carne , Biologia de Sistemas , Cricetinae , Animais , Suínos , Células CHO , Biologia de Sistemas/métodos , Cricetulus , Técnicas de Cultura de Células/métodosRESUMO
In recent years, three-dimensional (3D) cell culture has been attracting attention as a cell culture model that mimics an environment closer to that of a living organism. It is known that there is a close relationship between cell nuclear shape and cellular function, which highlights the importance of cell nucleus shape analysis in the 3D culture. On the other hand, it is difficult to observe the cell nuclei inside the 3D culture models because the penetration depth of the laser light under a microscope is limited. In this study, we adopted an aqueous iodixanol solution to the 3D osteocytic spheroids derived from mouse osteoblast precursor cells to make the spheroids transparent for 3D quantitative analysis. With a custom-made image analysis pipeline in Python, we found that the aspect ratio of the cell nuclei near the surface of the spheroid was significantly greater than that at the center, suggesting that the nuclei on the surface were deformed more than those at the center. The results also quantitatively showed that the orientation of nuclei in the center of the spheroid was randomly distributed, whereas those on the surface of the spheroid were oriented parallel to the surface of the spheroid. Our 3D quantitative method with an optical clearing technique will contribute to the 3D culture models including various organoid models to elucidate the nuclear deformation during the development of the organs. Insight box Although 3D cell culture has been a powerful tool in the fields of fundamental biology and tissue engineering, it raises the demand for quantification techniques for cell nuclear morphology in the 3D culture model. In this study, we attempted to optically clear a 3D osteocytic spheroid model using iodixanol solution for the nuclear observation inside the spheroid. Moreover, using a custom-made image analysis pipeline in Python, we successfully quantified the nuclear morphology regarding aspect ratio and orientation. Our quantitative method with the optical clearing technique will contribute to the 3D culture models such as various organoid models to elucidate the nuclear deformation during the development of the organs.