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Microorganisms are an important part of productivity, water quality, and biogeochemical cycles in an aquaculture ecosystems and play a key role in determining the growth and fitness of aquaculture animals. Coculture ecosystems are widely applied with great significance in agricultural production worldwide. The crayfish-rice coculture ecosystem (CRCE) and crayfish-waterweed coculture ecosystem (CWCE) are two high-profile artificial ecosystems for crayfish culture. However, the bacterial communities of the environmental water, sediment, and intestine in the CRCE and CWCE remain elusive. In this study, we investigated the diversity, composition, and function of bacterial communities in water, sediment, and intestine samples from the CRCE to CWCE. The physicochemical factors of water [such as ORP (oxidation-reduction potential), TC (total carbon), TOC (total oxygen carbon), and NO3--N] and sediment [such as TC, TOC, TN (total nitrogen), and TP (total phosphate)] were significantly different in the CRCE and CWCE. The abundances of Proteobacteria, Actinobacteria, Verrucomicrobia, Cyanobacteria, Chlorobi, Chloroflexi, and Firmicutes were significantly different in the water bacterial communities of the CRCE and CWCE. The abundance of Vibrio in the crayfish intestine was higher in the CRCE than in the CWCE. The most abundant phyla in the CRCE and CWCE sediment were Proteobacteria and Bacteroidetes. The abundances of genes involved in transporters and ABC transporters were different in water of CRCE and CWCE. The abundances of genes involved in oxidative phosphorylation were significantly higher in the crayfish intestine of the CRCE than in that of the CWCE. Furthermore, the functional genes associated with carbon metabolism were significantly more abundant in the sediment of the CRCE than in that of the CWCE. Spearman correlation analysis and redundancy analysis (RDA) showed that the bacterial communities of the water and sediment in the CRCE and CWCE were correlated with environmental factors (pH, total carbon (TC), total oxygen carbon (TOC), total nitrogen (TN), and total phosphorus (TP)). Our findings showed that the composition, diversity and function of the bacterial communities were distinct in the environmental water, sediment, and intestine of the CRCE and CWCE crayfish coculture ecosystems due to their different ecological patterns. These results can help guide healthy farming practices and deepen the understanding of bacterial communities in crayfish-plant coculture ecosystems from the perspective of bacterial ecology.Key points• The composition of bacterial communities in the environmental water, sediment, and intestine of the CRCE and CWCE were distinct.̉• The abundances of genes involved in transporters and ABC transporters were different in the water of the CRCE and CWCE.• The bacterial communities of the water and sediment in the CRCE and CWCE were correlated with some environmental factors.

Multicellular 3D culture and interaction with stromal components are considered essential elements in establishing a 'more clinically relevant' tumor model. Matrix-embedded 3D cultures using a microfluidic chip platform can recapitulate the microscale interaction within tumor microenvironments. As a major component of tumor microenvironment, cancer-associated fibroblasts (CAFs) play a role in cancer progression and drug resistance. Here, we present a microfluidic chip-based tumor tissue culture model that integrates 3D tumor spheroids (TSs) with CAF in proximity within a hydrogel scaffold. HT-29 human colorectal carcinoma cells grew into 3D TSs and the growth was stimulated when co-cultured with fibroblasts as shown by 1.5-folds increase of % changes in diameter over 5 days. TS cultured for 6 days showed a reduced expression of Ki-67 along with increased expression of fibronectin when co-cultured with fibroblasts compared to mono-cultured TSs. Fibroblasts were activated under co-culture conditions, as demonstrated by increases in α-SMA expression and migratory activity. When exposed to paclitaxel, a survival advantage was observed in TSs co-cultured with activated fibroblasts. Overall, we demonstrated the reciprocal interaction between TSs and fibroblasts in our 7-channel microfluidic chip. The co-culture of 3D TS-CAF in a collagen matrix-incorporated microfluidic chip may be useful to study the tumor microenvironment and for evaluation of drug screening and evaluation.

Spheroids provide a 3D environment with intensive cell–cell contacts. As a result of their excellent regenerative properties and rapid progress in their high-throughput production, spheroids are increasingly suggested as building blocks for tissue engineering. In this review, we focus on innovative biotechnological approaches that increase the quality of spheroids for this specific type of application. These include in particular the fabrication of coculture spheroids, mimicking the complex morphology and physiological tasks of natural tissues. In vitro preconditioning under different culture conditions and incorporation of biomaterials improve the function of spheroids and their directed fusion into macrotissues of desired shapes. The continuous development of these sophisticated approaches may markedly contribute to a broad implementation of spheroid-based tissue engineering in future regenerative medicine. Spheroids are increasingly used as building blocks in tissue engineering, because they ideally mimic the physiological 3D environment of tissues. Automatized large-scale production of spheroids is technically feasible. Compared to 2D cell systems, spheroids exhibit an enhanced regenerative capacity, which can be improved during the production process by adjusting the culture conditions and incorporation of biomaterials. The complexity of tissues can be mimicked by incorporation of multiple cell types in coculture spheroids. Macrotissues can be generated by seeding spheroids on scaffolds or by scaffold-free fusion of spheroids.

Angiogenesis plays a critical role in many diseases, including macular degeneration. At present, the pathological mechanisms remain unclear while appropriate models dissecting regulation of angiogenic processes are lacking. We propose an in vitro angiogenesis process and test it by examining the co-culture of human retinal pigmental epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVEC) inside a microfluidic device. From characterisation of the APRE-19 monoculture, the tight junction protein (ZO-1) was found on the cells cultured in the microfluidic device but changes in the medium conditions did not affect the integrity of monolayers found in the permeability tests. Vascular endothelial growth factor (VEGF) secretion was elevated under low glucose and hypoxia conditions compared to the control. After confirming the angiogenic ability of HUVEC, the cell-cell interactions were analyzed under lowered glucose medium and chemical hypoxia by exposing ARPE-19 cells to cobalt (II) chloride (CoCl 2 ). Heterotypic interactions between ARPE-19 and HUVEC were observed, but proliferation of HUVEC was hindered once the monolayer of ARPE-19 started breaking down. The above characterisations showed that alterations in glucose concentration and/or oxygen level as induced by chemical hypoxia causes elevations in VEGF produced in ARPE-19 which in turn affected directional growth of HUVEC.

Eight out of 10 breast cancer patients die within 5 years after the primary tumor has spread to the bones. Tumor cells disseminated from the breast roam the vasculature, colonizing perivascular niches around blood capillaries. Slow flows support the niche maintenance by driving the oxygen, nutrients, and signaling factors from the blood into the interstitial tissue, while extracellular matrix, endothelial cells, and mesenchymal stem cells regulate metastatic homing. Here, we show the feasibility of developing a perfused bone perivascular niche-on-a-chip to investigate the progression and drug resistance of breast cancer cells colonizing the bone. The model is a functional human triculture with stable vascular networks within a 3D native bone matrix cultured on a microfluidic chip. Providing the niche-on-a-chip with controlled flow velocities, shear stresses, and oxygen gradients, we established a long-lasting, self-assembled vascular network without supplementation of angiogenic factors. We further show that human bone marrow-derived mesenchymal stem cells, which have undergone phenotypical transition toward perivascular cell lineages, support the formation of capillary-like structures lining the vascular lumen. Finally, breast cancer cells exposed to interstitial flow within the bone perivascular niche-on-a-chip persist in a slow-proliferative state associated with increased drug resistance. We propose that the bone perivascular niche-on-a-chip with interstitial flow promotes the formation of stable vasculature and mediates cancer cell colonization.

Since the reconstruction of large bone defects remains a challenge, knowledge about the biology of bone healing is desirable to develop novel strategies for improving the treatment of bone defects. In osteoimmunology, macrophages are the central component in the early stage of physiological response after bone injury and bone remodeling in the late stage. During this process, a switch of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) is observed. An appealing option for bone regeneration would be to exploit this regulatory role for the benefit of osteogenic differentiation of osteoprogenitor cells (e.g., mesenchymal stem cells; MSCs) and to eventually utilize this knowledge to improve the therapeutic outcome of bone regenerative treatment. In view of this, we focused on the in vitro interaction of different macrophage subtypes with adipose tissue MSCs to monitor the behavior (i.e. proliferation, differentiation and mineralization) of the latter in dedicated co-culture models. Our data show that co-culture of MSCs with M2 macrophages, but not with M1 macrophages or M0 macrophages, results in significantly increased MSC mineralization caused by soluble factors. Specifically, M2 macrophages promoted the proliferation and osteogenic differentiation of MSCs, while M0 and M1 macrophages solely stimulated the osteogenic differentiation of MSCs in the early and middle stages during co-culture. Secretion of the soluble factors oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) by macrophages showed correlation with MSC gene expression levels for OSM-receptor and BMP-2, suggesting the involvement of both signaling pathways in the osteogenic differentiation of MSCs.

Organoids are microscopic self-organizing, three-dimensional structures that are grown from stem cells in vitro. They recapitulate many structural and functional aspects of their in vivo counterpart organs. This versatile technology has led to the development of many novel human cancer models. It is now possible to create indefinitely expanding organoids starting from tumor tissue of individuals suffering from a range of carcinomas. Alternatively, CRISPR-based gene modification allows the engineering of organoid models of cancer through the introduction of any combination of cancer gene alterations to normal organoids.When combined with immune cells and fibroblasts, tumor organoids become models for the cancer microenvironment enabling immune-oncology applications. Emerging evidence indicates that organoids can be used to accurately predict drug responses in a personalized treatment setting. Here, we review the current state and future prospects of the rapidly evolving tumor organoid field.

Engineering microbial consortia to express complex biosynthetic pathways efficiently for the production of valuable compounds is a promising approach for metabolic engineering and synthetic biology. Here, we report the design, optimization, and scale-up of an Escherichia coli - E. coli coculture that successfully overcomes fundamental microbial production limitations, such as high-level intermediate secretion and low-efficiency sugar mixture utilization. For the production of the important chemical cis , cis -muconic acid, we show that the coculture approach achieves a production yield of 0.35 g/g from a glucose/xylose mixture, which is significantly higher than reported in previous reports. By efficiently producing another compound, 4-hydroxybenzoic acid, we also demonstrate that the approach is generally applicable for biosynthesis of other important industrial products.

Three-dimensional (3D) bioprinting, a flexible automated on-demand platform for the free-form fabrication of complex living architectures, is a novel approach for the design and engineering of human organs and tissues. Here, we demonstrate the potential of 3D bioprinting for tissue engineering using human skin as a prototypical example. Keratinocytes and fibroblasts were used as constituent cells to represent the epidermis and dermis, and collagen was used to represent the dermal matrix of the skin. Preliminary studies were conducted to optimize printing parameters for maximum cell viability as well as for the optimization of cell densities in the epidermis and dermis to mimic physiologically relevant attributes of human skin. Printed 3D constructs were cultured in submerged media conditions followed by exposure of the epidermal layer to the air–liquid interface to promote maturation and stratification. Histology and immunofluorescence characterization demonstrated that 3D printed skin tissue was morphologically and biologically representative of in vivo human skin tissue. In comparison with traditional methods for skin engineering, 3D bioprinting offers several advantages in terms of shape- and form retention, flexibility, reproducibility, and high culture throughput. It has a broad range of applications in transdermal and topical formulation discovery, dermal toxicity studies, and in designing autologous grafts for wound healing. The proof-of-concept studies presented here can be further extended for enhancing the complexity of the skin model via the incorporation of secondary and adnexal structures or the inclusion of diseased cells to serve as a model for studying the pathophysiology of skin diseases.

In this paper, we describe the effects of the combination of topographical, mechanical, chemical and intracellular electrical stimuli on a co-culture of fibroblasts and skeletal muscle cells. The co-culture was anisotropically grown onto an engineered micro-grooved (10 µm-wide grooves) polyacrylamide substrate, showing a precisely tuned Young's modulus (∼ 14 kPa) and a small thickness (∼ 12 µm). We enhanced the co-culture properties through intracellular stimulation produced by piezoelectric nanostructures (i.e., boron nitride nanotubes) activated by ultrasounds, thus exploiting the ability of boron nitride nanotubes to convert outer mechanical waves (such as ultrasounds) in intracellular electrical stimuli, by exploiting the direct piezoelectric effect. We demonstrated that nanotubes were internalized by muscle cells and localized in both early and late endosomes, while they were not internalized by the underneath fibroblast layer. Muscle cell differentiation benefited from the synergic combination of topographical, mechanical, chemical and nanoparticle-based stimuli, showing good myotube development and alignment towards a preferential direction, as well as high expression of genes encoding key proteins for muscle contraction (i.e., actin and myosin). We also clarified the possible role of fibroblasts in this process, highlighting their response to the above mentioned physical stimuli in terms of gene expression and cytokine production. Finally, calcium imaging-based experiments demonstrated a higher functionality of the stimulated co-cultures.