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Three-dimensional (3D) cell culture has revolutionized life sciences, particularly in organoid technologies. Traditional bioscaffold materials, however, complicate the detachment of tumor organoids and hamper the routine use of organoid-immune cell cocultures. Here, we show an acoustic virtual 3D scaffold (AV-Scaf) method to achieve 3D tumor organoid culture, enabling a direct-interacting tumor organoid-immune cell coculture system. The self-organization process of tumor cells is facilitated by a vortex acoustic field, which enables the cell bioassembly and ion channel activation. This approach can significantly enhance the influx of calcium ions, thereby accelerating intercellular interactions of cellular assemblies. We established scaffold-free melanoma and breast cancer organoids using AV-Scaf and cocultured melanoma organoids with T cells. We found that our coculture system resulted in a high activation state of T cells, characterized by notable up-regulation of granzyme B (2.82 to 17.5%) and interferon-γ (1.36 to 16%). AV-Scaf offers an efficient method for tumor organoid-immune cell studies, advancing cancer research and immunotherapy development.

Electrical and dynamic stimulation are commonly employed to enhance the maturation of engineered cardiac tissue (ECT) derived from human induced pluripotent stem cells (iPSCs), reflecting the physiological environment of the heart. While electrical stimulation mimics natural bioelectrical signals and dynamic stimulation replicates mechanical forces, the combined effects of these stimuli on ECT maturation have not been thoroughly explored. We hypothesized that simultaneous electro-dynamic stimulation would enhance ECT maturation and function more effectively than either stimulus alone. Human iPSC-derived cardiovascular cells were co-cultured with Collagen I and Matrigel for 2 weeks, followed by a comparative analysis of four groups: no stimulation, dynamic stimulation, electrical stimulation, and simultaneous electro-dynamic stimulation. The functionality of ECTs was assessed by measuring contractile capacity and calcium indicators, and histological assessments examined structural maturation. Our results demonstrated that simultaneous electro-dynamic stimulation significantly increased the CM component, elevated TNNT2 mRNA expression levels, and enhanced calcium transient capacity. Additionally, ECTs subjected to simultaneous stimulation exhibited a positive force-frequency relationship in contractility and an elevation in peak calcium flux, indicative of advanced tissue maturation. Moreover, simultaneous stimulation promoted vascular network formation within the ECTs, suggesting improved structural organization. These findings underscore the importance of simultaneous stimulation for developing effective cardiac tissue engineering strategies., Competing Interests: Declaration of competing interest This research was supported by RIKEN BDR Organoid Project (to H.M.), and Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to H.M.) (#23K24420). W.M., K.Murata and H.M. are inventors of engineered cardiac tissue-related patents. All authors declare that they have no financial conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Neuroblastoma (NB) is the most common pediatric extracranial solid tumor. It accounts for 50 % of cancers diagnosed in infants less than 1 year old, and 10 % of all pediatric cancer deaths in the United States. High-risk patients have a less than 50 % 5-year survival rate with current treatment strategies. The complex tumor microenvironment of NB makes the development of treatment strategies for high-risk patients challenging. There is increasing evidence that intratumoral immune suppression plays an important role in the progression and invasion of NB tumors. Few three-dimensional (3D) cancer models include components of the innate immune system. This work develops a preclinical 3D NB-immune co-culture model using SK-N-AS NB cells, NK-92 natural killer cells, and THP-1 derived macrophages, co-cultured on porous 3D silk scaffolds to provide tumor architecture. Conditioned media and indirect co-culturing showed changes in SK-N-AS gene expression associated with immunoregulatory signaling, and changes in NK-92 gene expression that are associated with reduced cytotoxicity. This motivated the development of a 3D direct co-culture system in which NB cells were seeded prior to immune cells to allow incorporation and deposition of extracellular matrix within the construct. Immune cells were then incorporated into the model to achieve direct co-culture with SK-N-AS cells. Changes in THP-1 macrophage polarization toward a more M2-like phenotype were observed in 3D direct co-culture, as well as altered NK-92 cell protein secretion and cytotoxic activity. Preliminary testing of immunotherapeutics within the model was conducted on both NB-macrophage and NB-NK co-cultures, but the model demonstrated limited response to immunotherapeutics. This work lays the foundation for building high-throughput therapeutic screening models for the improved treatment NB and other solid tumors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In recent years, reducing the concentration of carbon dioxide in the atmosphere has become an important issue. Microalgae have a higher photosynthetic efficiency and growth rate than higher plants; thus, biological carbon dioxide fixation using microalgae is attracting particular attention as an efficient carbon dioxide fixation method. However, under dilute atmospheric conditions, microalgae exhibit lower growth rates and reduced carbon dioxide fixation efficiency. In recent years, technology that can efficiently fix carbon dioxide, even in the atmosphere, using a microalgae co-culture system that co-cultivates microalgae and heterotrophic microorganisms has attracted attention. In such a co-culture system, it is believed that a mutualistic relationship is established between microorganisms through the exchange of various compounds. This review focuses on the application of a co-culture system of microalgae and heterotrophic microorganisms for bioproduction and wastewater treatment. In addition, research to elucidate the mutualistic relationships in microalgal co-culture systems using analytical methods that have been widely used in recent years, such as next-generation sequencing technology, is also discussed. In the future, it is expected that the use of microalgae co-culture systems will expand on an industrial scale through the development of key technologies, such as efficient genetic modification techniques for microalgae and their heterotrophic microorganism partners, large-scale cultivation facilities that can efficiently cultivate microalgae, and stable control techniques for co-culture systems using advanced technology., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Metabolite exchange mediates crucial interactions in microbial communities, significantly impacting global carbon and nitrogen cycling. Understanding these chemically-mediated interactions is essential for elucidating natural community functions and developing engineered synthetic communities. This study investigated membrane-separated bioreactors (mBRs) as a novel tool to identify transient metabolites and their producers/consumers in mixed microbial communities. We compared three co-culture methods (direct mixed, 2-chamber mBR, and 3-chamber mBR) to grow a synthetic binary community of the cyanobacterium Synechococcus elongatus PCC 7942 and the fungus Rhodotorula toruloides NBRC 0880, as well as axenic S. elongatus. Despite not being natural lichen constituents, these organisms exhibited interactions resembling those in cyanolichens. S. elongatus fixed CO 2 into sugars as the primary shared metabolite, while R. toruloides secreted various biochemicals, predominantly sugar alcohols, mirroring the metabolite exchange observed in natural lichens. The mBR systems successfully captured metabolite gradients and revealed rapidly consumed compounds, including TCA cycle intermediates and amino acids. Our approach demonstrated that the 2-chamber mBR optimally balanced metabolite exchange and growth dynamics. This study provides insights into cross-species metabolic interactions and presents a valuable tool for investigating and engineering synthetic microbial communities with potential applications in biotechnology and environmental science., (© 2024. Battelle Memorial Institute and The Authors 2024.)

Many tissues have a laminar structure, but there are limited technologies for establishing laminar co-cultures for in vitro testing. Here, we demonstrate that collagen-alginate-fibrin (CAF) hydrogel scaffolds produced using the reactive jet impingement bioprinting technique can produce osteochondral laminar co-cultures with well-defined interfaces between cell types and high cell densities to support cell-cell interaction across the interfaces. The influence of cell density and the presence of the two cell types on the production of extracellular matrix (ECM) and the emergent mechanical properties of gels is investigated using IHC, ELISA, gel mass, and the compression modulus. The results indicate that high-cell-density cultures and co-cultures with these specific cell types produce greater levels of ECM and a more biomimetic in vitro culture than low-cell-density cultures. In laminar scaffolds produced using TC28a2 chondrocytes and SaoS-2 osteoblasts, both cell density and the presence of the two cell types enhance ECM production and the mechanical properties of the cultures, presenting a promising approach for the production of more biomimetic in vitro models.

Bacterial cellulose (BC) has a long-standing human consumption history in different geographies without any report of adverse effects. Despite its unique textural and functional properties, the use of BC in food products in Europe is still restricted due to concerns over its nanosize. Here, we evaluated the potential uptake of celluloses (from plant and microbial sources, processed using different blenders) by macrophages (differentiated THP-1 cells) and human intestinal epithelial cells (Caco-2 and HT29-MTX cells) without (coculture) or with (triculture) Raji-B cells. A carbohydrate-binding module coupled to a green fluorescent protein was employed to observe cellulose in the cell cultures by confocal laser scanning microscopy and stimulated emission depletion microscopy. The methodology demonstrated excellent sensitivity, allowing detection of single nanocrystals within cells. All celluloses were taken up by the macrophages, without significantly compromising the cell's metabolic viability. The viability of the cocultures was also not affected. Furthermore, no internalization was observed in the triculture cell model that was exposed 24 h to BC and Avicel LM310. When (rarely) detected, cellulose particles were found on the apical side of the membrane. Overall, the obtained results suggest that BC should not be absorbed into the human gut.

A healthy heart comprises various cell types, including cardiomyocytes, endothelial cells, fibroblasts, immune cells, and among others, which work together to maintain optimal cardiac function. These cells engage in complex communication networks, known as cell-cell interactions (CCIs), which are essential for homeostasis, cardiac structure, and efficient function. However, in the context of cardiac diseases, the heart undergoes damage, leading to alterations in the cellular composition. Such pathological conditions trigger significant changes in CCIs, causing cell rearrangement and the transition between cell types. Studying these interactions can provide valuable insights into cardiac biology and disease mechanisms, enabling the development of new therapeutic strategies. While the development of cardiac organoids and advanced 3D co-culture technologies has revolutionized in vitro studies of CCIs, recent advancements in single-cell and spatial multi-omics technologies provide researchers with powerful and convenient tools to investigate CCIs at unprecedented resolution. This article provides a concise overview of CCIs observed in both normal and injured heart, with an emphasis on the cutting-edge methods used to study these interactions. It highlights recent advancements such as 3D co-culture systems, single-cell and spatial omics technologies, that have enhanced the understanding of CCIs. Additionally, it summarizes the practical applications of CCI research in advancing cardiovascular therapies, offering potential solutions for treating heart disease by targeting intercellular communication., (© 2024. The Author(s).)

In vitro, spheroid models have become well established in cancer research because they can better mimic certain characteristics of in vivo tumours. However, interaction with the tumour microenvironment, such as cancer-associated fibroblasts, plays a key role in tumour progression. We initially focused on the interaction of tumour cells with fibroblasts. To model this interaction, we developed a spheroid model of ovarian cancer and fibroblasts. To this end, ovarian cancer cell lines and ex vivo primary cells were simultaneously and sequentially seeded with fibroblasts in a scaffold-free system at different ratios and subsequently characterized with respect to changes in morphology, proliferation, and viability. We demonstrated that co-cultures are able to form by far more compact spheroids, especially in cells that form aggregates in mono-culture. In addition, the co-cultures were able to increase proliferation and sensitivity to cisplatin. Simultaneous seeding led fibroblasts invade the core in both cell lines and primary cells. These results show differences in formation, firmness, and size between co-culture and mono-culture. Our model is designed to better represent and characterize the mutual influencing factors of fibroblasts and tumour cells. Fibroblast-supplemented multicellular spheroids are a valuable tool for tumour microenvironment interaction and new drug discovery., (© 2024. The Author(s).)

Mitochondrial transfer is a normal physiological phenomenon that occurs widely among various types of cells. In the study to date, the most important pathway for mitochondrial transport is through tunneling nanotubes (TNTs). There have been many studies reporting that mesenchymal stem cells (MSCs) can transfer mitochondria to other cells by TNTs. However, few studies have demonstrated the phenomenon of bidirectional mitochondrial transfer. Here, our protocol describes an experimental approach to study the phenomenon of mitochondrial transfer between MSCs and retinal pigment epithelial cells in vitro by two mitochondrial tracing methods. We co-cultured mito-GFP-transfected MSCs with mito-RFP-transfected ARPE19 cells (a retinal pigment epithelial cell line) for 24 h. Then, all cells were stained with phalloidin and imaged by confocal microscopy. We observed mitochondria with green fluorescence in ARPE19 cells and mitochondria with red fluorescence in MSCs, indicating that bidirectional mitochondrial transfer occurs between MSCs and ARPE19 cells. This phenomenon suggests that mitochondrial transport is a normal physiological phenomenon that also occurs between MSCs and ARPE19 cells, and mitochondrial transfer from MSCs to ARPE19 cells occurs much more frequently than vice versa. Our results indicate that MSCs can transfer mitochondria into retinal pigment epithelium, and similarly predict that MSCs can fulfill their therapeutic potential through mitochondrial transport in the retinal pigment epithelium in the future. Additionally, mitochondrial transfer from ARPE19 cells to MSCs remains to be further explored.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC 50 values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity Basic Protocol 3: Image acquisition, processing, and data analysis., (© 2024 Wiley Periodicals LLC.)

Applying low-cost substrate is critical for sustainable bioproduction. Co-culture of phototrophic and heterotrophic microorganisms can be a promising solution as they can use CO 2 and light as feedstock. This study aimed to create a light-driven consortium using a marine cyanobacterium Synechococcus sp. PCC 7002 and an industrial yeast Yarrowia lipolytica. First, the cyanobacterium was engineered to accumulate and secrete sucrose by regulating the expression of genes involved in sucrose biosynthesis and transport, resulting in 4.0 g/L of sucrose secretion. Then, Yarrowia lipolytica was engineered to efficiently use sucrose and produce β-caryophyllene that has various industrial applications. Then, co- and sequential-culture were optimized with different induction conditions and media compositions. A maximum β-caryophyllene yield of 14.1 mg/L was obtained from the co-culture. This study successfully established an artificial light-driven consortium based on a marine cyanobacterium and Y. lipolytica, and provides a foundation for sustainable bioproduction from CO 2 and light through co-culture systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Neurodegenerative diseases (NDs) are characterized by the progressive loss of neurons. As to developing effective therapeutic interventions, it is crucial to understand the underlying mechanisms of NDs. Cellular models have become invaluable tools for studying the complex pathogenesis of NDs, offering insights into disease mechanisms, determining potential therapeutic targets, and aiding in drug discovery. This review provides a comprehensive overview of various cellular models used in ND research, focusing on Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Cell lines, such as SH-SY5Y and PC12 cells, have emerged as valuable tools due to their ease of use, reproducibility, and scalability. Additionally, co-culture models, involving the growth of distinct cell types like neurons and astrocytes together, are highlighted for simulating brain interactions and microenvironment. While cell lines cannot fully replicate the complexity of the human brain, they provide a scalable method for examining important aspects of neurodegenerative diseases. Advancements in cell line technologies, including the incorporation of patient-specific genetic variants and improved co-culture models, hold promise for enhancing our understanding and expediting the development of effective treatments. Integrating multiple cellular models and advanced technologies offers the potential for significant progress in unraveling the intricacies of these debilitating diseases and improving patient outcomes., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of <12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth. Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 °C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm 3  (451 µm x 462.84 µm)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Biorefineries require low-cost production processes, low waste generation and equipment that can be used not only for a single process, but for the manufacture of several products. In this context, in this research a continuous 3D printing microbioreactor coupled to an Arduino-controlled automatic feeding system was developed for the intensification of the ethanol production process from xylose/xylulose (3:1), using a new biocatalyst containing the co-culture of Scheffersomyces stipitis and Saccharomyces cerevisiae (50/50). Initially, batch fermentations of monocultures of S. cerevisiae and S. stipitis and co-culture were carried out. Subsequently, the immobilized co-culture was used as a biocatalyst in continuous fermentations using the developed microreactor. Fermentations carried out in the microbioreactor presented a 2-fold increase in the ethanol concentration and a 3-fold increase in productivity when compared to monocultures. The microbioreactor developed proved to be efficient and can be extended for other bioproducts production. This approach proved to be a promising alternative for the use of the hemicellulose fraction of biomasses without the need to use modified strains.

In vitro co-culture techniques that allow the growth of plants and pathogens under controlled environmental conditions are being used to re-create host plant infection. These approaches reduce infection times, promote reproducibility, and enable a rapid evaluation of plant-pathogen interactions. As a result, these systems have become essential in breeding programs aimed at developing plant resistance to diseases. In this study, we developed and validated an in vitro co-culture system to investigate the Armillaria root rot (ARR) affecting Prunus spp. This disease, caused by fungi Armillaria spp. and Desarmillaria caespitosa, poses a severe threat to the stone and nut fruit industry due to the susceptibility of most commercial rootstocks to infection and the lack of effective management options for its control. The system consists of a fiber-supported liquid approach in sterile plastic vessels that allows a fast and reproducible fungal infection under controlled environmental conditions. The floor of the vessels was covered with a polyester-fiber matte and a germination paper that served as an interface between the mycelia and the plant roots. The vessels were subjected to inoculation with Armillaria mellea and D. caespitosa, and three Prunus genotypes ('Guardian®', 'MP-29', and Prunus cerasifera '14-4') were co-cultured with both fungi. Disease progression and plant and fungal biomass were monitored during co-culture. The presented in vitro co-culture approach facilitates the concurrent growth of Armillaria/Desarmillaria spp. and Prunus spp., excluding most of the limitations associated with greenhouses and field experiments. This system provides consistent and reproducible conditions for investigating a prominent plant disease affecting Prunus spp., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Calle et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Leukemia niche impacts quiescence; however, culturing patient-derived samples ex vivo is technically challenging. Here, we present a protocol for in vitro co-culture of patient-derived xenograft acute lymphoblastic leukemia (PDX-ALL) cells with human mesenchymal stem cells (MSCs). We describe steps for labeling PDX-ALL cells with CellTrace Violet dye to demonstrate MSC-primed PDX-ALL cycling. We then detail procedures to identify MSC-primed G0/quiescent PDX-ALL cells via Hoechst-33342/Pyronin Y live cell cycle analysis. For complete details on the use and execution of this protocol, please refer to Pal et al. 1 , 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The intertwined interactions various immune cells have with epithelial cells in our body require sophisticated experimental approaches to be studied. Due to the limitations of immortalized cell lines and animal models, there is an increasing demand for human in vitro model systems to investigate the microenvironment of immune cells in normal and in pathological conditions. Organoids, which are self-renewing, 3D cellular structures that are derived from stem cells, have started to provide gap-filling tissue modelling solutions. In this review, we first demonstrate with some of the available examples how organoid-based immune cell co-culture experiments can advance disease modelling of cancer, inflammatory bowel disease, and tissue regeneration. Then, we argue that to achieve both complexity and scale, organ-on-chip models combined with cutting-edge microfluidics-based technologies can provide more precise manipulation and readouts. Finally, we discuss how genome editing techniques and the use of patient-derived organoids and immune cells can improve disease modelling and facilitate precision medicine. To achieve maximum impact and efficiency, these efforts should be supported by novel infrastructures such as organoid biobanks, organoid facilities, as well as drug screening and host-microbe interaction testing platforms. All these together or in combination can allow researchers to shed more detailed, and often patient-specific, light on the crosstalk between immune cells and epithelial cells in health and disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

Helicobacter pylori is one of the most common bacterial pathogens worldwide and the main etiological agent of numerous gastric diseases. The frequency of multidrug resistance of H. pylori is growing and the leading factor related to this phenomenon is its ability to form biofilm. Therefore, the establishment of a proper model to study this structure is of critical need. In response to this, the aim of this original article is to validate conditions of the optimal biofilm development of H. pylori in monoculture and co-culture with a gastric cell line in media simulating human fluids. Using a set of culture-based and microscopic techniques, we proved that simulated transcellular fluid and simulated gastric fluid, when applied in appropriate concentrations, stimulate autoaggregation and biofilm formation of H. pylori . Additionally, using a co-culture system on semi-permeable membranes in media imitating the stomach environment, we were able to obtain a monolayer of a gastric cell line with H. pylori biofilm on its surface. We believe that the current model for H. pylori biofilm formation in monoculture and co-culture with gastric cells in media containing host-mimicking fluids will constitute a platform for the intensification of research on H. pylori biofilms in in vitro conditions that simulate the human body.

This study presents the development and validation of an innovative microfluidic liver-on-a-chip device utilizing gravity-driven perfusion for the evaluation of drug hepatotoxicity. This research involved the construction of a hydrogel-based coculture chip that integrates liver parenchymal and stellate cells within a tri-channel configuration. The assembly and operation of the liver-on-a-chip and its accompanying custom rocker were straightforward. The cells in the chip maintained high viability and continuously synthesized liver albumin over extended culture durations. Acetaminophen (APAP), a hepatic injury-inducing drug, was utilized as a positive control in hepatic toxicity assays on the chip. The liver chip exhibited hepatotoxic responses comparable to those observed in 2D models. Furthermore, in this study we evaluated the effects of two plant-derived natural compounds, aristolochic acid I (AA) and its analog aristolactam AII (AL), in both 2D cell models and the liver-on-a-chip system. AA, known for its hepatorenal toxicity, was observed to cause hepatotoxicity in both the 2D models and on the chip. The flow cytometry and mRNA sequencing results confirmed the propensity of these compounds to induce liver cell apoptosis. Notably, AL, previously considered nontoxic, provoked a significant decrease in the hepatic functionality marker albumin exclusively in the liver chip but not in 2D models, indicating the liver chip's enhanced sensitivity to toxic substances. In summary, this pumpless liver-on-a-chip is a simple yet powerful tool for drug hepatotoxicity studies.

This work uses response surface methodology (RSM) to study the co-cultivation of symbiotic indigenous wastewater microalgae and bacteria under different conditions (inoculum ratio of bacteria to microalgae, CO 2 , light intensity, and harvest time) for optimal bioenergy feedstock production. The findings of this study demonstrate that the symbiotic microalgae-bacteria culture not only increases total microalgal biomass and lipid productivity, but also enlarges microalgal cell size and stimulates lipid accumulation. Meanwhile, inoculum ratio of bacteria to microalgae, light intensity, CO 2, and harvest time significantly affect biomass and lipid productivity. CO 2 concentration and harvest time have significant interactive effect on lipid productivity. The response of microalgal biomass and lipid productivity varies significantly from 2.1 × 10 5 to 1.9 × 10 7  cells/mL and 2.8 × 10 2 to 3.7 × 10 12 Total Fluorescent Units/mL respectively. Conditions for optimum biomass and oil accumulation are 100% of inoculation ratio (bacteria/microalgae), 3.6% of CO 2 (v/v), 205.8 µmol/m 2 /s of light intensity, and 10.6 days of harvest time. This work provides a systematic methodology with RSM to explore the benefits of symbiotic microalgae-bacteria culture, and to optimize various cultivation parameters within complex wastewater environments for practical applications of integrated wastewater-microalgae systems for cost-efficient bioenergy production., (© 2024. The Author(s).)

Background: Chronic lung disease of prematurity, called bronchopulmonary dysplasia (BPD), lacks effective therapies, stressing the need for preclinical testing systems that reflect human pathology for identifying causal pathways and testing novel compounds. Alveolar organoids derived from human pluripotent stem cells (hPSC) are promising test platforms for studying distal airway diseases like BPD, but current protocols do not accurately replicate the distal niche environment of the native lung. Herein, we investigated the contributions of cellular constituents of the alveolus and fetal respiratory movements on hPSC-derived alveolar organoid formation., Methods: Human PSCs were differentiated in 2D culture into lung progenitor cells (LPC) which were then further differentiated into alveolar organoids before and after removal of co-developing mesodermal cells. LPCs were also differentiated in Transwell® co-cultures with and without human fetal lung fibroblast. Forming organoids were subjected to phasic mechanical strain using a Flexcell® system. Differentiation within organoids and Transwell® cultures was assessed by flow cytometry, immunofluorescence, and qPCR for lung epithelial and alveolar markers of differentiation including GATA binding protein 6 (GATA 6), E-cadherin (CDH1), NK2 Homeobox 1 (NKX2-1), HT2-280, surfactant proteins B (SFTPB) and C (SFTPC)., Results: We observed that co-developing mesenchymal progenitors promote alveolar epithelial type 2 cell (AEC2) differentiation within hPSC-derived lung organoids. This mesenchymal effect on AEC2 differentiation was corroborated by co-culturing hPSC-NKX2-1 + lung progenitors with human embryonic lung fibroblasts. The stimulatory effect did not require direct contact between fibroblasts and NKX2-1 + lung progenitors. Additionally, we demonstrate that episodic mechanical deformation of hPSC-derived lung organoids, mimicking in situ fetal respiratory movements, increased AEC2 differentiation without affecting proximal epithelial differentiation., Conclusion: Our data suggest that biophysical and mesenchymal components promote AEC2 differentiation within hPSC-derived distal organoids in vitro., (© 2024. The Author(s).)

The blood-brain barrier (BBB) poses a significant challenge for drug delivery and is linked to various neurovascular disorders. In vitro BBB models provide a tool to investigate drug permeation across the BBB and the barrier's response to external injury events. Yet, existing models lack fidelity in replicating the BBB's complexity, hindering a comprehensive understanding of its functions. This study introduces a three-dimensional (3D) model using polyethylene glycol (PEG) hydrogels modified with biomimetic peptides that represent recognition sequences of key proteins in the brain. Hydrogels were functionalized with recognition sequences for laminin (IKVAV) and fibronectin peptides (RGD) and chemically cross-linked with matrix metalloprotease-sensitive peptides (MMPs) to mimic the extracellular matrix of the BBB. Astrocytes and endothelial cells were seeded within and on the surface of the hydrogels, respectively. The barrier integrity was assessed through different tests including transendothelial electrical resistance (TEER), the permeability of sodium fluorescence (Na-F), the permeability of Evan's blue bound to albumin (EBA), and the expression of zonula occluden-1 (ZO-1) in seeded endothelial cells. Hydrogels with a combination of RGD and IKVAV peptides displayed superior performance, exhibiting significantly higher TEER values (55.33 ± 1.47 Ω·cm 2 ) at day 5 compared to other 2D controls including HAECs-monoculture and HAECs-cocultured with NHAs seeded on well inserts and 3D controls including RGD hydrogel and RGD-IKVAV monoculture with HAECs and RGD hydrogel cocultured with HAECs and NHAs. The designed 3D system resulted in the lowest Evan's blue permeability at 120 min (0.215 ± 0.055 μg/mL) compared to controls. ZO-1 expression was significantly higher and formed a relatively larger network in the functionalized hydrogel cocultured with astrocytes and endothelial cells compared to the controls. Thus, the designed 3D model effectively recapitulates the main BBB structure and function in vitro and is expected to contribute to a deeper understanding of pathological CNS angiogenesis and the development of effective CNS medications.

The study of human intestinal physiology and host-microbe interactions is crucial for understanding gastrointestinal health and disease. Traditional two-dimensional cell culture models lack the complexity of the native intestinal environment, limiting their utility in studying intestinal biology. Here, we present a detailed protocol for the set up and utilization of a three-dimensional (3D) in vitro bioreactor system for human intestinal studies and bacterial co-culture. This article outlines the design and assembly of the bioreactor system, scaffold fabrication, bacterial culture techniques, analysis methods, and troubleshooting tips. By providing step-by-step instructions, the goal is to enable other laboratories to utilize physiologically relevant tissue models of the human intestine, incorporating key features, such as nutrient flow, multiple human cell types, 3D architecture, and microbial communities. The incorporation of commensal bacteria into the bioreactor system allows for the investigation of complex host-microbe interactions, providing insight into gastrointestinal health and pathology. This article serves as a comprehensive resource for scientists seeking to advance their understanding of intestinal biology toward the development of novel therapeutic strategies for gastrointestinal disorders. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Scaffold design Basic Protocol 2: Intestinal cell culture: Caco2 cells Basic Protocol 3: Intestinal cell culture: organoids Basic Protocol 4: Bioreactor design and set up Basic Protocol 5: Bacteria in 3D bioreactor set up Basic Protocol 6: Bacteria and drug dosing., (© 2024 Wiley Periodicals LLC.)

The germinal center (GC) is the stage of B cell differentiation that gives rise to a majority of B cell lymphomas. Here, we present an experimental coculture system for the ex vivo expansion and genetic manipulation of human GC B cells purified from discarded tonsil tissue. This system can be used to investigate the impact of defined genetic alterations, either individually or in combination, upon the growth and survival of human GC B cells in vitro. We provide examples of genetic combinations that lead to the immortalized growth of GC B cells in vitro, and others that result in malignant transformation in immunodeficient mice, allowing the creation of genetically bespoke, synthetic, human lymphoma models., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Acute myeloid leukemia (AML) is a deadly form of leukemia with ineffective traditional treatment and frequent chemoresistance-associated relapse. Personalized drug screening holds promise in identifying optimal regimen, nevertheless, primary AML cells undergo spontaneous apoptosis during cultures, invalidating the drug screening results. Here, we reconstitute a 3D osteogenic niche (3DON) mimicking that in bone marrow to support primary AML cell survival and phenotype maintenance in cultures. Specifically, 3DON derived from osteogenically differentiated mesenchymal stem cells (MSC) from healthy and AML donors are co-cultured with primary AML cells. The AML cells under the AML&#95;3DON niche showed enhanced viability, reduced apoptosis and maintained CD33 + CD34 - phenotype, associating with elevated secretion of anti-apoptotic cytokines in the AML&#95;3DON niche. Moreover, AML cells under the AML&#95;3DON niche exhibited low sensitivity to two FDA-approved chemotherapeutic drugs, further suggesting the physiological resemblance of the AML&#95;3DON niche. Most interestingly, AML cells co-cultured with the healthy&#95;3DON niche are highly sensitive to the same sample drugs. This study demonstrates the differential responses of AML cells towards leukemic and healthy bone marrow niches, suggesting the impact of native cancer cell niche in drug screening, and the potential of re-engineering healthy bone marrow niche in AML patients as chemotherapeutic adjuvants overcoming chemoresistance, respectively., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:The authors (Barbara Pui Chan, Hoi Lam Cheung, Anskar Yu Hung Leung and Jessica Evangeline Tan Kabigting) hold a patent associated with the current work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Tendinopathy is a chronic tendon disease. Mesenchymal stem cells (MSCs), known for their anti-inflammatory properties, may lose effectiveness with extensive culturing. Previous research introduced "small umbilical cord-derived fast proliferating cells" (smumf cells), isolated using a novel minimal cube explant method. These cells maintained their MSC characteristics through long-term culture. Thus, the purpose of the present study was to assess the anti-inflammatory effects of late-passage smumf cells at P10 on tenocytes derived from degenerative rotator cuff tears in a tendinopathic environment., Methods: The mRNA expression with respect to aging of MSCs and secretion of growth factors (GFs) by smumf cells at P10 were measured. mRNA and protein synthesis in tenocytes with respect to the tenocyte phenotype, inflammatory cytokines, and matrix- degradation enzymes were measured. The inflammatory signal pathways involving nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) in tenocytes were also investigated. The proliferative response of degenerative tenocytes to co-culture with smumf cells over 7 days in varying IL-1β induced tendinopathic environments was investigated., Results: smumf cells at P10 showed no signs of aging compared to those at P3. smumf cells at P10, secreting 2,043 pg/ml of hepatocyte growth factor (HGF), showed a 1.88-fold (p = .002) increase in HGF secretion in a tendinopathic environment. Degenerative tenocytes co-cultured with smumf cells showed significantly increased protein expression levels of collagen type I (Col I) and the Col I/III ratio by 1.46-fold (p < .001) and 1.66-fold (p < .001), respectively. The smumf cells at P10 reduced both mRNA and protein expression levels of matrix metalloproteinases-1, -2, -3, -8, -9, and -13 in tenocytes and attenuated NF-κB (phosphorylated IκBα/IκBα and phosphorylated p65/p65) and MAPK (phosphorylated p38/p38 and phosphorylated JNK/JNK) pathways activated by IL-1β. Removal of IL-1β from the co-culture accelerated the growth of tenocytes by 1.42-fold (p < .001). Removal of IL-1β accelerated tenocyte growth in co-cultures., Conculsion: Late-passage smumf cells exert anti-inflammatory effects on tenocytes derived from degenerative rotator cuff tears under a tendinopathic environment, primarily through the secretion of growth factors (GFs)., Competing Interests: Declarations. Conflict of interest: CHJ owns shares of AcesoStem Biostrategies Inc. The authors declare that they have no competing interests. Ethical approval: The study was approved by the Seoul Metropolitan Government Seoul National University Boramae Medical Center Institutional Review Board (IRB No. 06–2012-78). Informed Consent was obtained from all patients before performing the study., (© 2024. The Author(s).)

Acetobacterium woodii and Megasphaera hexanoica were co-cultured for caproic acid (CA) production from lactic acid (LA) and CO 2 . Also, various concentrations (1 g/L, 3 g/L, 5 g/L, and 10 g/L) of Zero Valent Iron (ZVI) were supplied to study its impact on the co-culture system. In flask experiments, 10 g/L LA and 1.0 bar CO 2 produced 0.6 g/L CA with some biomass growth. ZVI increased LA consumption and CA production. Indeed, 3 g/L ZVI boosted CA production by 186 % and biomass accumulation by 103 %, suggesting that ZVI controls the carbon flux. Subsequent automated bioreactor studies showed that 3 g/L ZVI produced 1.842 g/L CA at stable pH, compared to 0.969 g/L without ZVI (control). Further, metabolic activity showed that both bacteria could directly use H 2 , generated by ZVI (3 g/L), as electron donor. Higher ZVI concentrations (10 g/L) resulted in Fe 2+ causing excessive oxidation pressure on M. hexanoica, with its carbon flux flowing preferentially towards biomass. Enzyme assays confirmed that A. woodii preferred 10 g/L ZVI while M. hexanoica preferred 3 g/L for optimal bioconversion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In fetal development, tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis. Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC). We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells, both derived from hiPSCs. After 3 weeks, liver progenitors within hiPSC-BV-incorporated liver organoids (BVLO) differentiate to cholangiocytes and acquire epithelial characteristics, including intercellular junctions, microvilli on the apical membrane, and secretory functions. Furthermore, liver surface transplanted-BVLO temporarily attenuates cholestatic injury symptoms. Single cell RNA sequence analysis suggests that BD interact with the BV in BVLO through TGFβ and Notch pathways. Knocking out JAG1 in hiPSC-BV significantly attenuates bile duct formation, highlighting BVLO potential as a model for Alagille syndrome, a congenital biliary disease. Overall, we develop a novel 3D co-culture method that successfully establishes functional human BDs by emulating liver epithelial-BV interaction., (© 2024. The Author(s).)

Background: Identification of lung cancer subtypes is critical for successful treatment in patients, especially those in advanced stages. Many advanced and personal treatments require knowledge of specific mutations, as well as up- and down-regulations of genes, for effective targeting of the cancer cells. While many studies focus on individual cell structures and delve deeper into gene sequencing, the present study proposes a machine learning method for lung cancer classification based on low-magnification cancer outgrowth patterns in a 2D co-culture environment., Methods: Using a magnetic well plate holder, circular pattern lung cancer cell clusters were generated among fibroblasts, and daily images were captured to monitor cancer outgrowth over a 9-day period. These outgrowth images were then augmented and used to train a convolutional neural network (CNN) model based on the lightweight TinyVGG architecture. The model was trained with pairs of classes representing three subtypes of NSCLC: A549 (adenocarcinoma), H520 (squamous cell carcinoma), and H460 (large cell carcinoma). The objective was to assess whether this lightweight machine learning model could accurately classify the three lung cancer cell lines at different stages of cancer outgrowth. Additionally, cancer outgrowth images of two patient-derived lung cancer cells, one with the KRAS oncogene and the other with the EGFR oncogene, were captured and classified using the CNN model. This demonstration aimed to investigate the translational potential of machine learning-enabled lung cancer classification., Results: The lightweight CNN model achieved over 93% classification accuracy at 1 day of outgrowth among A549, H460, and H520, and reached 100% classification accuracy at 7 days of outgrowth. Additionally, the model achieved 100% classification accuracy at 4 days for patient-derived lung cancer cells. Although these cells are classified as Adenocarcinoma, their outgrowth patterns vary depending on their oncogene expressions (KRAS or EGFR)., Conclusions: These results demonstrate that the lightweight CNN architecture, operating locally on a laptop without network or cloud connectivity, can effectively create a machine learning-enabled model capable of accurately classifying lung cancer cell subtypes, including those derived from patients, based upon their outgrowth patterns in the presence of surrounding fibroblasts. This advancement underscores the potential of machine learning to enhance early lung cancer subtyping, offering promising avenues for improving treatment outcomes in advanced stage-patients., (© 2024. The Author(s).)

Large-scale production of cultured meat requires bulk culture medium containing growth-promoting proteins from animal serum. However, animal serum for mammalian cell culture is associated with high costs, ethical concerns, and contamination risks. Owing to its growth factor content, conditioned medium from rat liver epithelial RL34 cells can replace animal serum for myoblast proliferation. More seeded cells and longer culture periods are thought to yield higher growth factor levels, resulting in more effective muscle cell proliferation. However, RL34 cells can deplete nutrients and release harmful metabolites into the culture medium over time, potentially causing growth inhibition and apoptosis. This issue highlights the need for waste clearance during condition medium production. To address this issue, we introduced a lactate permease gene (lldP) and an L-lactate-to-pyruvate conversion enzyme gene (lldD) to generate a recombinant L-lactate-assimilating cyanobacterium Synechococcus sp. KC0110 strain. Transwell co-culture of this strain with RL34 cells exhibited a marked reduction in the levels of harmful metabolites, lactate and ammonium, while maintaining higher concentrations of glucose, pyruvate, and pyruvate-derived amino acids than those seen with RL34 cell monocultures. The co-culture medium supported myoblast proliferation without medium dilution or additional nutrients, which was attributed to the waste clearance and nutrient replenishment effects of the KC0110 strain. This culture system holds potential for the production of low-cost, and animal-free cultured meat., (© 2024. The Author(s).)

DPANN is a widespread and diverse group of archaea characterized by their small size, reduced genome, limited metabolic pathways, and symbiotic existence. Known DPANN species are predominantly obligate ectosymbionts that depend on their host for proliferation. The structural and molecular details of host recognition, host-DPANN intercellular communication, and host adaptation in response to DPANN attachment remain unknown. Here, we use electron cryotomography (cryo-ET) to show that the Microcaldus variisymbioticus ARM-1 may interact with its host, Metallosphaera javensis AS-7 through intercellular proteinaceous nanotubes. Combining cryo-ET and sub-tomogram averaging, we show the in situ architectures of host and DPANN S-layers and the structures of the nanotubes in their primed and extended states. In addition, comparative proteomics and genomic analyses identified host proteomic changes in response to DPANN attachment. These results provide insights into the structural basis of host-DPANN communication and deepen our understanding of the host ectosymbiotic relationships., (© 2024. Crown.)

In recent years, research on organ-on-a-chip technology has been flourishing, particularly for drug screening and disease model development. Fibroblasts and vascular endothelial cells engage in crosstalk through paracrine signaling and direct cell-cell contact, which is essential for the normal development and function of the heart. Therefore, to faithfully recapitulate cardiac function, it is imperative to incorporate fibroblasts and vascular endothelial cells into a heart-on-a-chip model. Here, we report the development of a human heart-on-a-chip composed of induced pluripotent stem cell (iPSC)-derived cardiomyocytes, fibroblasts, and vascular endothelial cells. Vascular endothelial cells cultured on microfluidic channels responded to the flow of culture medium mimicking blood flow by orienting themselves parallel to the flow direction, akin to in vivo vascular alignment in response to blood flow. Furthermore, the flow of culture medium promoted integrity among vascular endothelial cells, as evidenced by CD31 staining and lower apparent permeability. The tri-culture condition of iPSC-derived cardiomyocytes, fibroblasts, and vascular endothelial cells resulted in higher expression of the ventricular cardiomyocyte marker IRX4 and increased contractility compared to the bi-culture condition with iPSC-derived cardiomyocytes and fibroblasts alone. Such tri-culture-derived cardiac tissues exhibited cardiac responses similar to in vivo hearts, including an increase in heart rate upon noradrenaline administration. In summary, we have achieved the development of a heart-on-a-chip composed of cardiomyocytes, fibroblasts, and vascular endothelial cells that mimics in vivo cardiac behavior., (© 2024. The Author(s).)

Organoids have emerged as valuable tools for the study of development and disease. Assembloids are formed by integrating multiple organoid types to create more complex models. However, the process by which organoids integrate to form assembloids remains unclear and may play an important role in the resulting organoid structure. Here, a microfluidic platform is developed that allows separate culture of distinct organoid types and provides the capacity to partially control the geometry of the resulting organoid surfaces. Removal of a microfabricated barrier then allows the shaped and positioned organoids to interact and form an assembloid. When midbrain and unguided brain organoids were allowed to assemble with a defined spacing between them, axonal projections from midbrain organoids and cell migration out of unguided organoids were observed and quantitatively measured as the two types of organoids fused together. Axonal projection directions were statistically biased toward other midbrain organoids, and unguided organoid surface geometry was found to affect cell invasion. This platform provides a tool to observe cellular interactions between organoid surfaces that are spaced apart in a controlled manner, and may ultimately have value in exploring neuronal migration, axon targeting, and assembloid formation mechanisms., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)

Increasing evidence shows that microbial synthesis plays an important role in producing high value-added products. However, microbial monoculture generally hampers metabolites production and limits scalability due to the increased metabolic burden on the host strain. In contrast, co-culture is a more flexible approach to improve the environmental adaptability and reduce the overall metabolic burden. The well-defined co-culturing microbial consortia can tap their metabolic potential to obtain yet-to-be discovered and pre-existing metabolites. This review focuses on the use of a co-culture strategy and its underlying mechanisms to enhance the production of products. Notably, the significance of comprehending the microbial interactions, diverse communication modes, genetic information, and modular co-culture involved in co-culture systems were highlighted. Furthermore, it addresses the current challenges and outlines potential future directions for microbial co-culture. This review provides better understanding the diversity and complexity of the interesting interaction and communication to advance the development of co-culture techniques., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Mesenchymal stromal cells (MSCs) continue to be proposed for clinical investigation to treat myriad diseases given their purported potential to stimulate endogenous regenerative processes, such as angiogenesis. However, MSC functional heterogeneity has hindered clinical success and still poses a substantial manufacturing challenge from a product quality control perspective. Here, a quantitative bioassay based on an enhanced-throughput is described, microphysiological system (MPS) to measure the specific bioactivity of MSCs to stimulate angiogenesis as a potential measure of MSC potency. Using this novel bioassay, MSCs derived from multiple donors at different passages are co-cultured with human umbilical vein endothelial cells and exhibit significant heterogeneity in angiogenic potency between donors and cell passage. Depending on donor source and cellular passage number, MSCs varied in their ability to stimulate tip cell dominant or stalk cell dominant phenotypes in angiogenic sprout morphology which correlated with expression levels of hepatocyte growth factor (HGF). These findings suggest that MSC angiogenic bioactivity may be considered as a possible potency attribute in MSC quality control strategies. Development of a reliable and functionally relevant potency assay for measuring clinically relevant potency attributes of MSCs will help to improve consistency in quality and thereby, accelerate clinical development of these cell-based products., (© 2023 Wiley‐VCH GmbH.)

3D hydrogel-based cell cultures provide models for studying cell behavior and can efficiently replicate the physiologic environment. Hydrogels can be tailored to mimic mechanical and biochemical properties of specific tissues and allow to produce gel-in-gel models. In this system, microspheres encapsulating cells are embedded in an outer hydrogel matrix, where cells are able to migrate. To enhance the efficiency of such studies, a lab-on-a-chip named 3D cell migration-chip (3DCM-chip) is designed, which offers substantial advantages over traditional methods. 3DCM-chip facilitates the analysis of biochemical and physical stimuli effects on cell migration/invasion in different cell types, including stem, normal, and tumor cells. 3DCM-chip provides a smart platform for developing more complex cell co-cultures systems. Herein the impact of human fibroblasts on MDA-MB 231 breast cancer cells' invasiveness is investigated. Moreover, how the presence of different cellular lines, including mesenchymal stem cells, normal human dermal fibroblasts, and human umbilical vein endothelial cells, affects the invasive behavior of cancer cells is investigated using 3DCM-chip. Therefore, predictive tumoroid models with a more complex network of interactions between cells and microenvironment are here produced. 3DCM-chip moves closer to the creation of in vitro systems that can potentially replicate key aspects of the physiological tumor microenvironment., (© 2024 Wiley‐VCH GmbH.)

Ex vivo 3D culture of human tissue explants addresses many limitations of traditional monolayer cell culture techniques, namely the lack of cellular heterogeneity and absence of 3D intercellular spatial relationships, but presents challenges with regard to repeatability owing to the difficulty of acquiring multiple tissue samples from the same donor. In this study, we used a cryopreserved bank of human lung microexplants, ∼1 mm 3 fragments of peripheral lung from donors undergoing lung resection surgery, and a liquid-like solid 3D culture matrix to describe a method for the analysis of non-small-cell lung cancer adhesion to human lung tissue. H226 (squamous cell carcinoma), H441 (lung adenocarcinoma), and H460 (large cell carcinoma) cell lines were cocultured with lung microexplants. Confocal fluorescence microscopy was used to visualize the adherence of each cell line to lung microexplants. Adherent cancer cells were quantified following filtration of nonadherent cells, digestion of cultured microexplants, and flow cytometry. This method was used to evaluate the role of integrins in cancer cell adherence. A statistically significant decrease in the adherence of H460 cells to lung microexplants was observed when anti-integrins were administered to H460 cells before coculture with lung microexplants.

Despite preventive measures and available treatments, cervical cancer still ranks as the fourth most prevalent cancer among women worldwide and remains the leading cause of cancer death in women in many developing countries. To gain further insights into pathogenesis and to develop novel (immuno)therapies, more sophisticated human models recreating patient heterogeneities and including aspects of the tumor microenvironment are urgently required. A novel polydimethylsiloxane-free microfluidic platform, designed specifically for the generation and ccultivation of cervical cancerous tissue, is introduced. The microscale open-top tissue chambers of the cervical cancer-on-chip (CCoC) enable facile generation and long-term cultivation of SiHa spheroids in co-culture with donor-derived cervical fibroblasts. The resulting 3D tissue emulates physiological architecture and allows dissection of distinct effects of the stromal tissue on cancer viability and growth. Treatment with cisplatin at clinically-relevant routes of administration and dosing highlights the platform's applicability for drug testing. Moreover, the model is amenable for integration and recruitment of donor-derived neutrophils from the microvasculature-like channel into the tissue, all while retaining their ability to produce neutrophil extracellular traps. In the future, the immunocompetent CCoC featuring donor-specific primary cells and tumor spheroids has the potential to contribute to the development of new (immuno)therapeutic options., (© 2023 The Authors. Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing "synaptopathy" and hearing loss. Cocultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that, when P3-5 denervated organs of Corti are cocultured with SGNs, newly formed IHC/SGN synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When using older organs of Corti at P10-11, synaptic activity probed by deconvolution showed more mature release properties, closer to the specialized mode of IHC synaptic transmission crucial for coding the sound signal. This functional assessment of newly formed IHC synapses developed here, provides a powerful tool for testing approaches to improve synapse regeneration., Competing Interests: Competing interests statement:The authors declare no competing interest.

Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.

Due to the limited accessibility of the human retina, retinal organoids (ROs) are the best model for studying human retinal disease, which could reveal the mechanism of retinal development and the occurrence of retinal disease. Microglia (MG) are unique resident macrophages in the retina and central nervous system (CNS), serving crucial immunity functions. However, retinal organoids lack microglia since their differentiation origin is the yolk sac. The specific pathogenesis of microglia in these retinal diseases remains unclear; therefore, the establishment of a microglia-incorporated retinal organoid model turns out to be necessary. Here, we successfully constructed a co-cultured model of retinal organoids with microglia derived from human stem cells. In this article, we differentiated microglia and then co-cultured to retinal organoids in the early stage. As the incorporation of immune cells, this model provides an optimized platform for retinal disease modeling and drug screening to facilitate in-depth research on the pathogenesis and treatment of retinal and CNS-related diseases.

Mechanistic investigation of host-microbe interactions in the human gut are hindered by difficulty of co-culturing microbes with intestinal epithelial cells. On one hand the gut bacteria are a mix of facultative, aerotolerant or obligate anaerobes, while the intestinal epithelium requires oxygen for growth and function. Thus, a coculture system that can recreate these contrasting oxygen requirements is critical step towards our understanding microbial-host interactions in the human gut. Here, we demonstrate Intestinal Organoid Physoxic Coculture (IOPC) system, a simple and cost-effective method for coculturing anaerobic intestinal bacteria with human intestinal organoids (HIOs). Using commensal anaerobes with varying degrees of oxygen tolerance, such as nano-aerobe Bacteroides thetaiotaomicron and strict anaerobe Blautia sp., we demonstrate that IOPC can successfully support 24-48 hours HIO-microbe coculture. The IOPC recapitulates the contrasting oxygen conditions across the intestinal epithelium seen in vivo. The IOPC cultured HIOs showed increased barrier integrity, and induced expression of immunomodulatory genes. A transcriptomic analysis suggests that HIOs from different donors show differences in the magnitude of their response to coculture with anaerobic bacteria. Thus, the IOPC system provides a robust coculture setup for investigating host-microbe interactions in complex, patient-derived intestinal tissues, that can facilitate the study of mechanisms underlying the role of the microbiome in health and disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fofanova et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Impairment of the intestinal epithelial barrier is frequently seen as collateral damage in various local and systemic inflammatory conditions. The inflammatory process is characterized by reciprocal interactions between the host intestinal epithelium and mucosal innate immune cells, e.g., macrophages. This article provides step-by-step instructions on how to set up a murine enteroid-macrophage co-culture by culturing cellular elements in proximity separated by a porous membrane. Unlike previously published co-culture systems, we have combined enteroids grown from C57BL6j mice with syngeneic bone marrow-derived macrophages to preclude potential allo-reactions between immune cells and epithelium. Transformation of intestinal crypts into proliferative enteroids was achieved by cultivation in Wnt3a-Noggin-R-Spondin-conditioned medium supplemented with ROCK inhibitor Y-27632. The differentiated phenotype was promoted by the use of the Wnt3-deprived EGF-Noggin-R-Spondin medium. The resulting co-culture of primary cells can be employed as a basic model to better understand the reciprocal relationship between intestinal epithelium and macrophages. It can be used for in vitro modelling of mucosal inflammation, mimicked by stimulation of macrophages either while being in co-culture or before being introduced into co-culture, to simulate enterogenic sepsis or systemic conditions affecting the intestinal tract.

Considering the importance of alternative methodologies to animal experimentation, we propose an organoid-based biological model for in vitro blood vessel generation, achieved through co-culturing endothelial and vascular smooth muscle cells (VSMCs). Initially, the organoids underwent comprehensive characterization, revealing VSMCs (α-SMA + cells) at the periphery and endothelial cells (CD31 + cells) at the core. Additionally, ephrin B2 and ephrin B4, genes implicated in arterial and venous formation respectively, were used to validate the obtained organoid. Moreover, the data indicates exclusive HIF-1α expression in VSMCs, identified through various methodologies. Subsequently, we tested the hypothesis that the generated blood vessels have the capacity to modulate the osteogenic phenotype, demonstrating the ability of HIF-1α to promote osteogenic signals, primarily by influencing Runx2 expression. Overall, this study underscores that the methodology employed to create blood vessel organoids establishes an experimental framework capable of producing a 3D culture model of both venous and arterial endothelial tissues. This model effectively guides morphogenesis from mesenchymal stem cells through paracrine signaling, ultimately leading to an osteogenic acquisition phenotype, with the dynamic involvement of HIF-1α., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A facile method was proposed for preparing controllable multicompartment gel microcarriers using an aqueous two-phase emulsion system. By leveraging the density difference between the upper polyethylene glycol solution and the lower dextran-calcium chloride (CaCl 2 ) solution in the collection solution and the high viscosity of the lower solution, controllable fusion of core-shell droplets made by coextrusion devices was achieved at the water/water (w/w) interface to fabricate microcarriers with separated core compartments. By adjusting the sodium alginate concentration, collected solution composition, and number of fused liquid droplets, the pore size, shape, and number of compartments could be controlled. Caco-2 and HepG2 cells were encapsulated in different compartments to establish gut-liver coculture models, exhibiting higher viability and proliferation compared to monoculture models. Notably, significant differences in cytokine expression and functional proteins were observed between the coculture and monoculture models. This method provides new possibilities for preparing complex and functional three-dimensional coculture materials.

The complexification of in vitro models requires the compatibility of cells with the same medium. Since immune cells are the most sensitive to growth conditions, growing intestinal epithelial cells in their usual medium seems to be necessary. This work was aimed at comparing the sensitivity of these epithelial cells to pro-inflammatory stimuli but also to dietary polyphenols in both DMEM and RPMI-1640 media. Co-cultures of Caco-2 and HT29-MTX cells were grown for 21 days in the two media before their stimulation with a cocktail of TNF-α (20 ng/mL), IL-1β (1 ng/mL), and IFN-γ (10 ng/mL) or with LPS (10 ng/mL) from E. coli (O111:B4). The role of catechins (15 µM), a dietary polyphenol, was evaluated after its incubation with the cells before their stimulation for 6 h. The RPMI-1640 medium did not alter the intensity of the inflammatory response observed with the cytokines. By contrast, LPS failed to stimulate the co-culture in inserts regardless of the medium used. Lastly, catechins were unable to prevent the pro-inflammatory response observed with the cytokines in the two media. The preservation of the response of this model of intestinal epithelium in RPMI-1640 medium is promising when considering its complexification to evaluate the complex cellular crosstalk leading to intestinal homeostasis.

Hepatocyte transplantation and bioartificial liver (BAL) systems hold significant promise as less invasive alternatives to traditional transplantation, providing crucial temporary support for patients with acute and chronic liver failure. Although human hepatocytes are ideal, their use is limited by ethical concerns and donor availability, leading to the use of porcine hepatocytes in BAL systems due to their functional similarities. Recent advancements in gene-editing technology have improved porcine organ xenotransplantation clinical trials by addressing immune rejection issues. Gene-edited pigs, such as alpha-1,3-galactosyltransferase (GGTA1) knockout pigs, offer a secure source of primary cells for BAL systems. Our research focuses on optimizing the safety and functionality of porcine primary hepatocytes during large-scale cultivation. We achieved this by creating GGTA1 knockout pigs through one-step delivery of CRISPR/Cas9 to pig zygotes via oviduct injection of rAAV, and enhancing hepatocyte viability and function by co-culturing hepatocytes with Roof plate-specific spondin 1 overexpressing HUVECs (R-HUVECs). Using a Rocker culture system, approximately 10 10 primary porcine hepatocytes and R-HUVECs rapidly formed organoids with a diameter of 92.1 ± 28.1 µm within 24 h. These organoids not only maintained excellent functionality but also supported partial hepatocyte self-renewal during long-term culture over 28 days. Gene-edited primary porcine hepatocyte organoids will significantly advance the applications of hepatocyte transplantation and BAL systems., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Modeling immuno-oncology by using patient-derived material and immune cell co-cultures can advance our understanding of immune cell tumor targeting in a patient-specific manner, offering leads to improve cellular immunotherapy. However, fully exploiting these living cultures requires analysis of the dynamic cellular features modeled, for which protocols are currently limited. Here, we describe the application of BEHAV3D, a platform that implements multi-color live 3D imaging and computational tools for: (i) analyzing tumor death dynamics at both single-organoid or cell and population levels, (ii) classifying T cell behavior and (iii) producing data-informed 3D images and videos for visual inspection and further insight into obtained results. Together, this enables a refined assessment of how solid and liquid tumors respond to cellular immunotherapy, critically capturing both inter- and intratumoral heterogeneity in treatment response. In addition, BEHAV3D uncovers T cell behavior involved in tumor targeting, offering insight into their mode of action. Our pipeline thereby has strong implications for comparing, prioritizing and improving immunotherapy products by highlighting the behavioral differences between individual tumor donors, distinct T cell therapy concepts or subpopulations. The protocol describes critical wet lab steps, including co-culture preparations and fast 3D imaging with live cell dyes, a segmentation-based image processing tool to track individual organoids, tumor and immune cells and an analytical pipeline for behavioral profiling. This 1-week protocol, accessible to users with basic cell culture, imaging and programming expertise, can easily be adapted to any type of co-culture to visualize and exploit cell behavior, having far-reaching implications for the immuno-oncology field and beyond., (© 2024. Springer Nature Limited.)