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Spheroids and other 3D cellular models more accurately recapitulate physiological responses when compared to 2D models and represent potential alternatives to animal testing. The cryopreservation of spheroids remains challenging, limiting their wider use. Standard DMSO-only cryopreservation results in supercooling to low subzero temperatures, reducing viability, shedding surface cells, and perforating spheroid interiors. Here, cocultured spheroids with differentially labeled outer cell layers allow spatial evaluation of the protective effect of macromolecular ice nucleators by microscopy and histology. Extracellular nucleation is shown to reduce damage to both interior and exterior regions of the spheroids, which will support the development of "off-the-shelf" 3D models.

The two-species microbial cocultures are effective in terms of awakening the cryptic biosynthetic pathways. They may also lead to the improved production of previously discovered molecules. Importantly, only a few outcomes of the cocultures may prove desirable, namely those leading to the formation of useful secondary metabolites. To address this issue, a method allowing for the evaluation of the final outcome of the co-culture process and fine-tune the cocultivation strategy was proposed. The systematic approach was supported by the experimental data from the bioreactor runs with the participation of Aspergillus terreus and Penicillium rubens confronted with Streptomyces rimosus and Streptomyces noursei. Kinetic, morphological and metabolic aspects of dominance were analysed via the newly proposed formula describing the dominance pattern. The suggested method involved the determination of the numerical value representing the dominance level. When it was high (value 1) no useful metabolites were formed apart from those originating from the winning counterpart. But either for the partial dominances or when the winning organism changed within the run or when the competition ended in draw, the number of the secondary metabolites of interest in the broth was the highest. Next, the systematic approach illustrated how the delayed inoculation strategy influenced the level of dominance leading to the change of winning counterpart and the set of metabolites produced. The proposed systematic approach allows for the reliable determination of the level of dominance in the two-species cocultures to seek for the potentially useful substances for future applications., Competing Interests: Declarations Conflict of interest The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s).)

Aims: Tumorigenicity is a significant concern in stem cell-based therapies. However, traditional tumorigenicity tests using animal models often produce inaccurate results. Consequently, a more sensitive method for assessing tumorigenicity is required. This study aimed to enhance sensitivity by exposing functional progenitors derived from human pluripotent stem cells (hPSCs) to the tumor microenvironment (TME) in vitro before transplantation, potentially making them more prone to abnormal proliferation or tumorigenicity., Methods: Midbrain dopamine (mDA) cells derived from hPSCs were exposed to the TME by coculturing with medulloblastoma. The cellular characteristics of these cocultured mDA cells were evaluated both in vitro and in vivo, and the mechanisms underlying the observed alterations were investigated., Results: Our findings demonstrated increased proliferation of cocultured mDA cells both in vitro and in vivo. Moreover, these proliferating cells showed a higher expression of Ki67 and SOX1, suggesting abnormal proliferation. The observed abnormal proliferation in cocultured mDA cells was attributed to the hyperactivation of proliferation-related genes, the JAK/STAT3 pathway, and cytokine stimulation., Conclusion: This study indicates that exposing functional progenitors to the TME in vitro before transplantation can induce abnormal proliferation, thereby increasing the sensitivity of tumorigenicity tests., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

The influence of the initial pH on the morphology and secondary metabolite production in cocultures and axenic cultures of Aspergillus terreus and Streptomyces rimosus was investigated. The detected secondary metabolites (6 of bacterial and 4 of fungal origin) were not found in the cultures initiated at pH values less than or equal to 4.0. The highest mean levels of oxytetracycline were recorded in S. rimosus axenic culture at pH 5.0. Initiating the axenic culture at pH 5.9 led to visibly lower product levels, yet the presence of A. terreus reduced the negative effect of non-optimal pH and led to higher oxytetracycline titer than in the corresponding S. rimosus axenic culture. The cocultivation initiated at pH 5.0 or 5.9 triggered the formation of oxidized rimocidin. The products of A. terreus were absent in the cocultures. At pH 4.0, the striking morphological differences between the coculture and the axenic cultures were recorded., (© 2024. The Author(s).)

Many microbial biosynthetic gene clusters (BGCs) are inactive under standard laboratory conditions, making characterization of their products difficult. Silent BGCs are likely activated by specific cues in their natural environment, such as the presence of competitors. Growth conditions such as coculture with other microbes, which more closely mimic natural environments, are practical strategies for inducing silent BGCs. Here, we utilize coculture to activate BGCs in nine actinobacteria strains. We observed increased production of the ferrous siderophores siderochelin A and B during coculture of Amycolatopsis strain WAC04611 and Tsukamurella strain WAC06889b. Furthermore, we identified the siderochelin BGC in WAC04611 and discovered that the GntR-family transcription factor sidR3 represses siderochelin production. Deletion of the predicted aminotransferase sidA abolished production of the carboxamides siderochelin A/B and led to the accumulation of the carboxylate siderochelin D. Finally, we deleted the predicted hydroxylase sidB and established that it is essential for siderochelin production. Our findings show that microbial coculture can successfully activate silent BGCs and lead to the discovery and characterization of unknown BGCs for molecules like siderochelin.IMPORTANCESiderophores are vital iron-acquisition elements required by microbes for survival in a variety of environments. Furthermore, many siderophores are essential for the virulence of various human pathogens, making them a possible target for antibacterials. The significance of our work is in the identification and characterization of the previously unknown BGC for the siderophore siderochelin. Our work adds to the growing knowledge of siderophore biosynthesis, which may aid in the future development of siderophore-targeting pharmaceuticals and inform on the ecological roles of these compounds. Furthermore, our work demonstrates that combining microbial coculture with metabolomics is a valuable strategy for identifying upregulated compounds and their BGCs., Competing Interests: The authors declare no conflict of interest.

The efficiency of in vitro embryo production in mammals is influenced by variables associated with culture conditions during maturation, fertilization, and embryonic development. The embryos obtained often exhibit low quality due to suboptimal in vitro culture conditions compared to the in vivo environment. Co-culturing gametes and embryos with somatic cells has been developed to enhance in vitro culture conditions. This study aimed to assess the impact of coculturing in vitro-produced porcine embryos with porcine oviductal epithelial cells (POEC) on embryo development and quality. Firstly, a pure culture of POEC suitable for coculture systems was established. The epithelial origin of the cells was confirmed by the expression of E-cadherin and cytokeratin. The expression pattern of hormone receptors aligned with the diestrous oviduct, and POEC also secreted oviductal glycoprotein type 1 (OVGP-1). Secondly, POEC from passage 1 (POEC-1) were used to coculture with in vitro-produced porcine embryos. A successful coculture system was established without the addition of fetal bovine serum as a supplement. Coculturing POEC-1 in monolayers with in vitro-produced porcine embryos during the initial two days of culture enhanced the percentage of blastocysts and their hatching. Although the coculture did not alter the number of cells in the blastocysts or apoptosis assessed by TUNEL, it significantly reduced reactive oxygen species (ROS) levels in cleaved porcine embryos. This study represents the first report evaluating the quality of porcine embryos produced by IVF in coculture systems and assessing ROS levels in cleaved porcine embryos obtained by IVF., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Astrocytes are essential for the formation and maintenance of neural networks. However, a major technical challenge for investigating astrocyte function and disease-related pathophysiology has been the limited ability to obtain functional human astrocytes. Despite recent advances in human pluripotent stem cell (hPSC) techniques, primary rodent astrocytes remain the gold standard in coculture with human neurons. We demonstrate that a combination of leukemia inhibitory factor (LIF) and bone morphogenetic protein-4 (BMP4) directs hPSC-derived neural precursor cells to a highly pure population of astroglia in 28 d. Using single-cell RNA sequencing, we confirm the astroglial identity of these cells and highlight profound transcriptional adaptations in cocultured hPSC-derived astrocytes and neurons, consistent with their further maturation. In coculture with human neurons, multielectrode array recordings revealed robust network activity of human neurons in a coculture with hPSC-derived or rat astrocytes [3.63 ± 0.44 min -1 (hPSC-derived), 2.86 ± 0.64 min -1 (rat); p  = 0.19]. In comparison, we found increased spike frequency within network bursts of human neurons cocultured with hPSC-derived astrocytes [56.31 ± 8.56 Hz (hPSC-derived), 24.77 ± 4.04 Hz (rat); p  < 0.01], and whole-cell patch-clamp recordings revealed an increase of postsynaptic currents [2.76 ± 0.39 Hz (hPSC-derived), 1.07 ± 0.14 Hz (rat); p  < 0.001], consistent with a corresponding increase in synapse density [14.90 ± 1.27/100 μm 2 (hPSC-derived), 8.39 ± 0.63/100 μm 2 (rat); p  < 0.001]. Taken together, we show that hPSC-derived astrocytes compare favorably with rat astrocytes in supporting human neural network activity and maturation, providing a fully human platform for investigating astrocyte function and neuronal-glial interactions., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Lendemeijer et al.)

Background: In vitro single-cell experiments may yield inconsistent results compared to clinical trials. To enhance the reliability of cosmetic active ingredient screening, a coculture model of B16F10-HaCaT cells was established in vitro based on the structural characteristics of human skin, thereby improving the credibility of experimental outcomes. Currently, most cosmetic whitening additives primarily target simple efficacy goals such as inhibiting tyrosinase activity or melanin transfer. Therefore, investigating novel and efficient whitening additives has become a prominent research focus., Objectives: The aim is to establish an in vitro cell coculture model for more reliable experimental results and investigate the mechanism by which Paeonia lactiflora Pall seeds oil inhibits melanin production and transfer., Methods: The impact of different concentrations of Paeonia lactiflora Pall seeds oil on cocultured cell proliferation rate was assessed using cck8 assay. Tyrosinase inhibition ability in cocultured cells was tested using levodopa as a substrate. Melanin production inhibition ability in coculture cells was evaluated by lysing cells with sodium hydroxide. The effect of Paeonia lactiflora Pall seeds oil on dendrite-related gene expression levels was examined through qPCR analysis. Additionally, Western blotting was employed to study the effect of Paeonia lactiflora Pall seeds oil on dendrite-related protein expression levels., Results: Different concentrations of Paeonia lactiflora Pall seeds oil did not affect the proliferation activity of cocultured cells. A specific concentration of α-MSH increased cell tyrosinase activity, cellular melanin content, as well as Rac1, Cdc42, and PAR-2 gene and protein expression related to dendritic formation. Treatment with a certain concentration of Paeonia lactiflora Pall seeds oil resulted in decreased tyrosinase activity and melanin content in cells along with downregulated expression levels of Rac1, Cdc42, and PAR-2 genes and proteins associated with dendritic formation., Conclusions: Paeonia lactiflora Pall seeds oil at specific concentrations exhibits the ability to inhibit tyrosinase activity, decrease melanin content, and possesses the potential to impede melanin transfer., (© 2024 The Author(s). Journal of Cosmetic Dermatology published by Wiley Periodicals LLC.)

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes., (© 2024 Wiley Periodicals LLC.)

Generation of human induced pluripotent stem cell (hiPSC)-derived motor neurons (MNs) offers an unprecedented approach to modeling movement disorders such as dystonia and amyotrophic lateral sclerosis. However, achieving survival poses a significant challenge when culturing induced MNs, especially when aiming to reach late maturation stages. Utilizing hiPSC-derived motor neurons and primary mouse astrocytes, we assembled two types of coculture systems: direct coculturing of neurons with astrocytes and indirect coculture using culture inserts that physically separate neurons and astrocytes. Both systems significantly enhance neuron survival. Compared with these two systems, no significant differences in neurodevelopment, maturation, and survival within 3 weeks, allowing to prepare neurons at maturation stages. Using the indirect coculture system, we obtained highly pure MNs at the late mature stage from hiPSCs. Transcriptomic studies of hiPSC-derived MNs showed a typical neurodevelopmental switch in gene expression from the early immature stage to late maturation stages. Mature genes associated with neurodevelopment and synaptogenesis are highly enriched in MNs at late stages, demonstrating that these neurons achieve maturation. This study introduces a novel tool for the preparation of highly pure hiPSC-derived neurons, enabling the determination of neurological disease pathogenesis in neurons at late disease onset stages through biochemical approaches, which typically necessitate highly pure neurons. This advancement is particularly significant in modeling age-related neurodegeneration., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 Akter et al.)

The synergism of host Paris polyphylla medium, the monoculture, and the coculture led to seventeen new metabolites, including eight sesquiterpenes, 1-7 having uncommon structural motifs compared to similar caryophyllene derivatives, 8 with an unprecedented bicyclic framework, and three xyloketals (13-15) with unprecedented frameworks from Nigrospora lacticolonia; one polyketide, 17 with novel bicyclo [2.2.2] undecane skeleton, and five polyketide-terpenoid hybrids, 20 (one novel sulfated), 21-24 from Penicillium rubens. The structures were determined mainly by the NMR, HRESIMS, ECD calculation, and single-crystal X-ray diffraction. Nine cryptic compounds (2-4, 5, 12-15, 17) were produced by the inductions of host medium and the coculture. The compounds 13 from N. lacticolonia, 24-26, 28, 29, and 31 from P. rubens indicated significant antiphytopathogenic activities against N. lacticolonia with MICs at 2-4 μg/mL. Moreover, compounds 22-26, 28, 29, and 31 from P. rubens showed antifungal activities against P. rubens with MICs at 2-4 μg/mL. The synergistic effects of host medium and the coculture can induce the structural diversity of metabolites., 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.)

The emerging interest in skeletal muscle tissue originates from its unique properties that control body movements. In particular, recent research advances in engineered skeletal muscle tissue have broadened the possibilities of applications in nonclinical models. However, due to the lack of adipose tissue, current engineered skeletal muscle tissue has the limitation of satisfying in vivo-like position and proportion of intermuscular fat. Adipose tissue within the skeletal muscle affects their functional properties. Here, a fabrication method for cocultured tissue composed of skeletal muscle and adipose tissues is proposed to reproduce the functional and morphological characteristics of muscle. By implementing prematured adipose microfibers in a myoblast-laden hydrogel sheet, both the accumulation of large lipid droplets and control of the position of adipose tissue within the skeletal muscle tissue becomes feasible. The findings of this study provide helpful information regarding engineered skeletal muscle, which has strong potential in drug screening models., (© 2021 Wiley Periodicals LLC.)

Due to the lack of blood vessels, nerves and lymphatics, articular cartilage is difficult to repair once damaged. Tissue engineering is considered to be a potential strategy for cartilage regeneration. Successful tissue engineering strategies depend on the effective combination of biomaterials, seed cells and biological factors. In our previous study, a genetically modified coculture system with chondrocytes and ATDC5 cells in an alginate hydrogel has exhibited a superior ability to enhance chondrogenesis. In this study, we further evaluated the influence of chondrocytes at various passages on chondrogenesis in the coculture system. The results demonstrated that transfection efficiency was hardly influenced by the passage number of chondrocytes. The coculture system with passage 5 (P5) chondrocytes had a better effect on chondrogenesis of ATDC5 cells, while chondrocytes in this coculture system presented higher levels of dedifferentiation than other groups with P1 or P3 chondrocytes. Therefore, P5 chondrocytes were shown to be more suitable for the coculture system, as they accumulated in sufficient cell numbers with more passages and had a higher level of dedifferentiation, which was prone to form a favorable niche for chondrogenesis of ATDC5 cells. This study may provide fresh insights for future cartilage tissue engineering strategies with a combination of a coculture system and advanced biomaterials., (© 2022 IOP Publishing Ltd.)

The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, the candidate phyla radiation (CPR) group has been recognized as representing a large proportion of the microbial kingdom (>26%). CPR are characterized by their obligate symbiotic or exoparasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the coculture of CPR with a suitable bacterial host. Based on various sputum samples, we tried to enrich CPR ( Saccharibacteria members) and to cocultivate them with pure hosts (Schaalia odontolytica). This protocol was monitored by TaqMan real-time quantitative PCR (qPCR) using a system specific for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in coculturing and sequencing the complete genomes of two new Saccharibacteria species, " Candidatus Minimicrobia naudis" and " Candidatus Minimicrobia vallesae." In addition, we noticed a decrease in the C T values of Saccharibacteria and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members, and in the future, their currently unknown characteristics may be revealed. IMPORTANCE In this study, the first TaqMan real-time quantitative PCR (qPCR) system, targeting Saccharibacteria phylum, has been developed. This technique can specifically quantify Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific, and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol facilitates subsequent studies of the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.

Eosinophils affect nerve structure and function in organs such as lungs and skin, which contributes to disease pathogenesis. We have developed methods for culturing primary sensory and parasympathetic neurons in multiple species and have refined these techniques for coculture with eosinophils. Eosinophil-nerve coculture has been an essential tool for testing interactions between these cell types. Here we describe methods for coculturing primary parasympathetic ganglia, vagal sensory nerves, and dorsal root sensory nerves with eosinophils.

During angiogenesis, endothelial cells must undergo a coordinated set of morphological changes in order to form a new vessel. There is a need for endothelial cells to communicate with each other in order to take up different identities in the sprout and to migrate collectively as a connected chord. Endothelial cells must also interact with a wide range of other cells that contribute to vessel formation. In ischemic disease, hypoxic cells in tissue will generate proangiogenic signals that promote and guide angiogenesis. In solid tumors, this function is co-opted by tumor cells, which make a complex range of interactions with endothelial cells, even integrating into the walls of vessels. In vessel repair, cells from the immune system contribute to the promotion and remodeling of new vessels. The coculture angiogenesis assay is a long-term in vitro protocol that uses fibroblasts to secrete and condition an artificial stromal matrix for tubules to grow through. We show here how the assay can be easily adapted to include additional cell types, facilitating the study of cellular interactions during neovascularization.

Although IL-10-producing B cells have been shown to play key roles in regulating immune responses involved in autoimmunity, inflammation, and cancer, the mechanisms at the base of the generation and maintenance of the pool of regulatory B cells are still poorly characterized. Several evidences show that the cross talk between B cells and other immune cell types promotes IL-10 production by B lymphocytes. Soluble mediators released into the microenvironment, together with direct cell-cell contact, are key signals in the process of regulatory B-cell development and differentiation. Here we describe the methods required to follow IL-10-producing B cells in MC- and MDSC-B-cell cocultures as examples of in vitro systems that induce the expansion of the regulatory B-cell population. These protocols can be also adapted for the study of other immune cell systems.

Direct contact-based coculture of human dermal fibroblasts and epidermal keratinocytes has been a long-standing and challenging issue owing to different serum and growth factor requirements of the two cell types. Existing protocols employ high serum concentrations (up to 10% fetal bovine serum), complex feeder systems and a range of supplemental factors. These approaches are technically demanding and labor intensive, and pose scientific and ethical limitations associated with the high concentrations of animal serum. On the other hand, serum-free conditions often fail to support the proliferation of one or both cell types when they are cultured together. We have developed two reduced serum approaches (1-2% serum) that support the contact-based coculture of human dermal fibroblasts and immortalized keratinocytes and enable the study of cell migration and wound closure.

Articular cartilage repair after injury is a great challenge worldwide due to its nerveless and avascular features. Tissue engineering is proposed as a promising alternative for cartilage regeneration. In this study, an adenoviral vector carrying the transforming growth factor-β3 (TGF-β3) gene was constructed and introduced into dedifferentiated chondrocytes, which were then cocultured with ATDC5 cells in an alginate hydrogel system. The results showed that the experimental groups exhibited better cell viability and higher levels of cartilage-related genes than the control groups. In this coculture system, the chondrogenic differentiation of ATDC5 cells was effectively induced by TGF-β3 and other latent cytokines that were produced by the transfected chondrocytes. Thus, this method can avoid the degradation of exogenous TGF-β3, and it can protect ATDC5 cells during virus transfection to maintain cell viability and chondrogenic differentiation capability. Taken together, this study provides fresh insights for applying this genetically manipulated coculture system to cartilage repair in the future., (© 2020 Wiley Periodicals LLC.)

Coculture played a major role in clinical microbiology by elucidating strict intracellular bacteria era. Since some of these bacteria are human pathogens, in-depth studies at the strain level are necessary to better understand their pathologies and treatment. Over the last decades, culture-independent tools have taken over the diagnostic procedure at the expense of coculture. These tools, although proven to be rapid and efficient, cannot overcome the need to culture the bacteria, as strain isolation remains a key factor to understanding its epidemiology, virulence and antibiotic susceptibility testing. Moreover, strain availability allows the development of molecular and serological tools, and remains crucial for taxonomy. This review revisits the current status of culture, its advantages, drawbacks and future needs.

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone-butanol-ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful "coculture" for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE., (© 2019 Wiley Periodicals, Inc.)

Background: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease., Aim: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development., Methods and Results: Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions., Conclusions: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner., (© 2019 The Authors Cancer Reports Published by Wiley Periodicals, Inc.)

Vascularization is a critical process during bone regeneration. The lack of vascular networks leads to insufficient oxygen and nutrients supply, which compromises the survival of regenerated bone. One strategy for improving the survival and osteogenesis of tissue-engineered bone grafts involves the coculture of endothelial cells (ECs) with mesenchymal stromal cells (MSCs). Moreover, bone regeneration is especially challenging due to its unique structural properties with aligned topographical cues, with which stem cells can interact. Inspired by the aligned fibrillar nanostructures in human cancellous bone, we fabricated polycaprolactone (PCL) electrospun fibers with aligned and random morphology, cocultured human MSCs with human umbilical vein ECs (HUVECs), and finally investigated how these two factors modulate osteogenic differentiation of human MSCs (hMSCs). After optimizing cell ratio, a hMSCs/HUVECs ratio (90:10) was considered to be the best combination for osteogenic differentiation. Coculture results showed that hMSCs and HUVECs adhered to and proliferated well on both scaffolds. The aligned structure of PCL fibers strongly influenced the morphology and orientation of hMSCs and HUVECs; however, fiber alignment was observed to not affect alkaline phosphate (ALP) activity or mineralization of hMSCs compared with random scaffolds. More importantly, cocultured cells on both random and aligned scaffolds had significantly higher ALP activities than monoculture groups, which indicated that coculture with HUVECs provided a larger relative contribution to the osteogenesis of hMSCs compared with fiber alignment. Taken together, we conclude that coculture of hMSCs with ECs is an effective strategy to promote osteogenesis on electrospun scaffolds, and aligned fibers could be introduced to regenerate bone tissues with oriented topography without significant deleterious effects on hMSCs differentiation. This study shows the ability to grow oriented tissue-engineered cocultures with significant increases in osteogenesis over monoculture conditions. Impact statement This work demonstrates an effective method of enhancing osteogenesis of mesenchymal stromal cells on electrospun scaffolds through coculturing with endothelial cells. Furthermore, we provide the optimized conditions for cocultures on electrospun fibrous scaffolds and engineered bone tissues with oriented topography on aligned fibers. This study demonstrates promising findings for growing oriented tissue-engineered cocultures with significant increase in osteogenesis over monoculture conditions.

Objectives: To investigate the possibility of inducing differentiation of human urine-derived stem cells (hUSCs) into hepatocyte-like cells by coculturing with human hepatocyte L02 cells in vitro., Methods: HUSCs were isolated from fresh urine samples collected from healthy adult volunteers by centrifugation. Cells were observed under an inverted phase contrast microscope, and proliferative activity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Stem cell surface markers were detected by flow cytometry. HUSCs were induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells, which were confirmed by cellular morphology, messenger RNA expression of albumin (ALB), α-fetoprotein (AFP) and hepatocyte cytochrome P450 (CYP450) analyzed with quantitative reverse transcription polymerase chain reaction and the expression of glycogen detected by glycogen staining kits at 5, 10, and 15 days after coculturing., Results: HUSCs from urine were successfully isolated and cultured in vitro. At passages 3, the growth curve of hUSCs was S-shaped with good proliferation activity. Mesenchymal stem cell surface markers CD44 and CD90 were detected positive by flow cytometry. CD31 for endothelial cells and CD34 for hematopoietic stem cell markers were not detected. HUSCs gained the cellular morphology and function of hepatocyte cells including higher expression of several hepatocyte-specific genes such as ALB and some CYP450, lower expression of AFP and positive glycogen expression (P <  .05) in coculturing with human hepatocyte L02 cells for 10-15d., Conclusions: HUSCs can be induced to differentiate into hepatocyte-like cells by coculturing with human hepatocyte L02 cells for a certain number of days., (© 2019 Wiley Periodicals, Inc.)

Group 2 innate lymphoid cells are important innate effectors and regulators of adaptive immunity in response to parasitic infections and allergic inflammation. Their low frequency in vivo during steady state condition may complicate research on the cells. During type 2 biased immune responses they are activated, increase in frequency and release cytokines as well as regulate T cell functions through direct interactions including MHC class II-T cell receptor interactions. Importantly, coreceptors significantly influence the ILC2-T cell cross talk and shape the adaptive immune response.Here, we provide an experimental framework to study the function of coreceptors expressed on tissue ILC2. In brief, we describe flow cytometric analysis of the coreceptor of interest, the isolation and culture of mouse pulmonary ILC2 and splenic T cells, as well as approaches to manipulate their coculture. Finally, downstream readout options are outlined.

Complex signaling between Schwann cells and axons are vital for peripheral neuron development, myelination, and repair. The interaction between these two cell types can be modeled in vitro by coculturing rodent Schwann cells and neurons together. These have in the past been used with great success to help unravel the bidirectional signaling mechanisms that lead to Schwann cell proliferation and myelination. To provide more translatable potential, we have developed myelinating cocultures using human, induced pluripotent stem cell (iPSC)-derived neurons. Under the right conditions, the human neurons are efficiently myelinated by rat Schwann cells, demonstrating successful cross-species signaling. This chapter describes all the necessary steps to generate these myelinating cocultures and methods to investigate and quantify various aspects of myelination. The myelinating cocultures can be maintained in excellent health for over 1 year, facilitating their use to study developmental or chronic disease processes. With this in mind, we have used the cocultures to model a sensory neuropathy which displays clinically with both axonal and demyelinating features. In the cocultures, we found evidence of extensive axonal degeneration and demyelination demonstrated by axonal swelling and fragmentation, and myelin disintegration. The myelinating cocultures can therefore be used to study complex, human disease processes that result in both axonal and myelin-associated degenerative processes.

Autonomous mechanisms of axon degeneration are frequently studied in vitro by mechanical axon injury of isolated sensory neurons. This has led to major advances in understanding the molecular pathways governing axon degeneration. However, this approach does not pay attention to potential glial mechanisms for the regulation of axon death. Here, I describe a straightforward protocol to seed purified rat Schwann cells on neuronal cultures in order to study the interaction between axons and these glia during axon degeneration.

Impact Statement: In this study, we proposed for the first time the use of inorganic ions dissolved from BaG in a cell coculture system to induce vascularized bone formation in vitro. For that, we used dental pulp pluripotent-like stem cells from a single individual source obtained in a minimally invasive extraction manner. Moreover, we carried out all the experiments under xeno-free conditions, allowing the extrapolation of the results to the development of clinically orientated applications. Overall, these results would provide a new promising system to promote the success and survival of bone tissue engineering constructs after implantation.

Aim: Umbilical cord blood hematopoietic stem cells (UCB HSCs) have been considered for the treatment of hematological malignancies due to their noninvasive collection, greater capacity of expansion, and remarkable tolerance for HLA mismatch in transplantation. On the other hand, the most considerable limitation of these cells is their inadequate amount of CD34 + HSCs which leads to delayed engraftment. The aim of this study was the expansion of CD34 + HSCs by coculturing with mesenchymal stem cells (MSCs) overexpressing stromal cell-derived factor-1, soluble and membrane isoforms of stem cell factor (sSCF/mSCF). Keeping structure and function of overexpressed cytokines by MSCs was expected which could beneficially affect the CD34 + HSC expansion., Methods: MSCs and CD34 + HSCs were isolated from UCB mononuclear cells. UCB MSCs were nucleofected with one or more of sSCF, mSCF, and SDF-1 expression vectors. Isolated CD34 + HSCs were then cocultured with nucleofected MSCs in 10 groups in culture medium containing TPO and Flt3L with or without SCF (3F or 2F groups). Then the CD34 + HSCs numeration, clonogenic capacity, and transcriptional levels of well-known HSCs regulatory and stemness genes (CXCR4, HOXB4, BMI1, and SALL4) were assessed following coculture with modified MSCs., Results: CD34 + HSCs which expanded on MSCs overexpressing mSCF/sSCF/SDF-1 in the 3F group showed the most significant increase in the expansion (4.73 ± 0.26 fold), clonogenic capacity (5.3 ± 0.25 fold) and also transcriptional levels of CXCR4, HOXB4, and BMI1 (3.49 ± 0.13, 9.49 ± 0.78, and 11.6 ± 0.9 fold), respectively ( P < 0.05)., Conclusion: Overexpression of SCF and SDF-1 by UCB MSCs in coculture system has efficient effect on UCB HSCs expansion. Furthermore nucleofected MSC overexpressing either sSCF/mSCF or mSCF/ sSCF /SDF-1 could substitute the rhu SCF in HSC expansion culture medium., (© 2019 Wiley Periodicals, Inc.)

Vascular tissue engineering seeks to develop functional blood vessels that comprise of both endothelial cells and pericytes for translational medicine and is often faced with numerous challenges such as nutrients and wastes diffusion problem in the centre of the scaffolds. Various strategies have been adopted to solve the diffusion problem in thick engineered scaffolds. Typically, microchannels or dissolvable microspheres are introduced into three-dimensional (3D) scaffolds as an alternative way to improve the infiltration of scaffolds and endothelial cells are usually incorporated into the biomaterials. While some research groups now focus on finding supporting cells to build further vascularized structures in the scaffolds. In this study, a bioinspired 3D gelatin-methacrylate (Gel-MA) hydrogel with dissolvable microspheres was created to encapsulate human bone marrow stromal cells (HMSCs) and human umbilical vein endothelial cells (HUVECs) which was used to investigate whether HMSCs could play a pericytes-like role and enhance vascularization within the engineered scaffolds. The results showed co-culture of HMSCs and HUVECs demonstrated significantly improved vascularization when compared to either HUVECs or HMSCs monoculture. Angiogenic genes were expressed significantly higher in co-culture group. Moreover, when implanting the pre-vascularized scaffolds in vivo, co-culture system integrated more successfully with host tissue and showed higher host tissue invasion than any other groups. More importantly, both the qPCR and immunofluorescence results indicated MSCs differentiated towards pericytes to enhance vascularization in this study. This paper highlights the enhanced capability of 3D micro-cavitary Gel-MA hydrogel for co-culturing HUVECs and HMSCs to promote vascularization which presents a potential strategy for future tissue repair and regeneration., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Biosensors can be used to screen or select for small molecule production in engineered microbes. However, mutations to the biosensor that interfere with accurate signal transduction are common, producing an excess of false positives. Strategies have been developed to avoid this limitation by physically separating the production pathway and biosensor, but these approaches have only been applied to screens, not selections. We have developed a novel biosensor-mediated selection strategy using competition between cocultured bacteria. When applied to the biosynthesis of cis , cis -muconate, we show that this strategy yields a selective advantage to producer strains that outweighs the costs of production. By encapsulating the competitive cocultures into microfluidic droplets, we successfully enriched the muconate-producing strains from a large population of control nonproducers. Facile selections for small molecule production will increase testing throughput for engineered microbes and allow for the rapid optimization of novel metabolic pathways.

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC-derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC-derived ECs with hPSC-derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC-derived CMs., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Isogenic bacterial populations are known to exhibit phenotypic heterogeneity at the single-cell level. Because of difficulties in assessing the phenotypic heterogeneity of a single taxon in a mixed community, the importance of this deeper level of organization remains relatively unknown for natural communities. In this study, we have used membrane-based microcosms that allow the probing of the phenotypic heterogeneity of a single taxon while interacting with a synthetic or natural community. Individual taxa were studied under axenic conditions, as members of a coculture with physical separation, and as a mixed culture. Phenotypic heterogeneity was assessed through both flow cytometry and Raman spectroscopy. Using this setup, we investigated the effect of microbial interactions on the individual phenotypic heterogeneities of two interacting drinking water isolates. Through flow cytometry we have demonstrated that interactions between these bacteria lead to a reduction of their individual phenotypic diversities and that this adjustment is conditional on the bacterial taxon. Single-cell Raman spectroscopy confirmed a taxon-dependent phenotypic shift due to the interaction. In conclusion, our data suggest that bacterial interactions may be a general driver of phenotypic heterogeneity in mixed microbial populations. IMPORTANCE Laboratory studies have shown the impact of phenotypic heterogeneity on the survival and functionality of isogenic populations. Because phenotypic heterogeneity plays an important role in pathogenicity and virulence, antibiotic resistance, biotechnological applications, and ecosystem properties, it is crucial to understand its influencing factors. An unanswered question is whether bacteria in mixed communities influence the phenotypic heterogeneity of their community partners. We found that coculturing bacteria leads to a reduction in their individual phenotypic heterogeneities, which led us to the hypothesis that the individual phenotypic diversity of a taxon is dependent on the community composition., (Copyright © 2019 American Society for Microbiology.)

Skeletal muscle tissue engineering aims at creating functional skeletal muscle in vitro. Human muscle organoids can be used for potential applications in regenerative medicine, but also as an in vitro model for myogenesis or myopathology. However, the thickness of constructs is limited due to passive diffusion of nutrients and oxygen. Introduction of a vascular network in vitro may solve this limitation. Here, we describe tissue engineering of in vitro skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. To create bio-artificial muscle (BAM), human muscle progenitor cells are cocultured with human umbilical vein endothelial cells (HUVECs) in a fibrin hydrogel. The cell-gel mix is cast into silicone molds with end attachment sites and cultured in endothelial growth medium (EGM-2) for 1 week. The passive forces generated in the contracted hydrogel align the myogenic cells parallel to the long axis of the contracted gel such that they fuse into aligned multinucleated myofibers. This results in the formation of a 2 cm long and ~1.5 mm tick human BAM construct with endothelial networks.

Background and Aims: Although HCV is one of the major health problems worldwide with the highest prevalence of genotype 4a in Egypt, it is poorly understood because of the limitations of having a robust in vitro model that allows the investigation and understanding of viral pathogenesis and life cycle. Genomic replicons for HCV are widely used and proved to have strong replication efficiency in cell culture, however, they are not able to produce infectious particles to enable the investigation of the whole viral life cycle and they mostly represent few sub-genomic classes for HCV. Hence, Genotype specific replication system is necessary to address specific sub-genomic phenotypes related to Hepatitis C pathogenicity., Methods: In this study we attempt to develop a sustainable co-culture model, which potentially provides essential route of infection for HCV by using HCV-positive sera from infected patients. In this novel in vitro model, we tested the viral replication in co-cultured Huh 7.5 and HepG2 cells in order to sustain full viral replication cycle. We used high viral load serum of HCV-infected patients (10 × 106 to 20 × 106 IU/ml) as a source for HCV particles to infect co-cultured cells for 7 days., Results and Conclusions: Viral replication capacity was increased 3-5 folds in the coculture condition compared to the individual cell lines, which indicates an improvement to viral infectivity in vitro., Significance Statement: This novel coculture system represents a new in vitro model that will help study the underlying mechanisms of HCV pathogenicity.

The preparation of oligodendrocytes and neurons independently in vitro has provided substantial insight into the biology of the process of myelin sheath formation. This chapter describes a myelination system of dorsal root ganglion neurons by independent isolation of oligodendrocyte progenitor cells from either rat or mouse cortex. This in vitro assay can be used to examine the molecular determinants of myelin sheath formation.

During vertebrate neural development, oligodendrocytes insulate nerve axons with myelin sheaths. Zebrafish (Danio rerio) has emerged as a useful model organism for studying oligodendrocyte development. However, the absence of an in vitro culture system necessitates in vivo manipulations and analyses, which, in some instances, limits the questions that can be addressed. To fill this gap we developed a mixed coculture system for embryonic zebrafish neurons and oligodendrocyte-lineage cells. Cultures harvested from embryos ≥30 hours postfertilization (hpf) yielded oligodendrocyte progenitor cells (OPCs) positive for olig2 and sox10 transgenic reporters. Cultured OPCs exhibited dynamic, exploratory membrane processes, and cell morphologies resembled those established in vivo. Cells harvested from advanced stage embryos possessed more arborized processes than those from early stage embryos. Advanced stage (>60 hpf) embryo culture produced differentiated, mbp + oligodendrocytes. Genetically tractable neuron subtypes extended neurites when harvested from embryos ≥19 hpf. Coculture produced juxtaposed oligodendrocytes and neurons, demonstrating the practical usefulness of this technique for future studies examining axon-oligodendrocyte interactions under defined conditions. We expect that zebrafish oligodendrocyte culture will complement existing in vivo strengths and may facilitate future studies elucidating the mechanisms of oligodendrocyte specification, proliferation, differentiation, motility, and axon-oligodendrocyte interactions that shape adult myelination patterns.

Candida albicans frequently causes recurrent intimal infectious disease (ID). This demands the treatment of multiple phases of the infection. The objective of this study was to uncover the host-pathogen interaction using two-dimensional (2D) epithelium cell-barrier and three-dimensional (3D) subepithelium tissue cells of human mucosa. The 2D cell cultures assessed C. albicans adhesion. Addition of the antifungal drug Fluconazol did not inhibit the adhesion, despite its pathogen growth inhibition (minimal inhibitory concentration value 0.08 μg/mL). A 3D tissue was engineered in multitranswells by placing human fibroblast cultures on a thick porous scaffold. This contained the yeast placed in the top compartment and prevented passive penetration. After 28 h, the pathogen transmigrated the barrier and was collected in the bottom compartment. A change in pathogen morphology was observed where hypha formed and grew to be 231 μm long after 28 h. The hypha was thus long enough to cross the 200 μm thick 3D tissue. The 3D infection was inhibited by addition of Fluconazol (0.08 μg/mL), confirming that penetration is dependent on pathogen growth. In conclusion, ID was reconstituted step-by-step on 2D epithelium surface and in 3D connective tissue of human mucosa. Fluconazol growth-inhibition of the pathogen C. albicans was confirmed in the 3D tissue. We thus propose that this ID in vitro test is suitable for the identification and characterization of new treatments against C. albicans.

In bio-based fermentation, the overall bioprocess efficiency is significantly affected by the metabolic burden associated with the expression of complete biosynthetic pathway as well as precursor and cofactor generating enzymes into a single microbial cell. To attenuate such burden by compartmentalizing the enzyme expression, recently synthetic biologists have used coculture or poly-culture techniques for biomolecules synthesis. In this paper, coculture system of two metabolically engineered Escherichia coli populations were employed which comprises upstream module expressing two enzymes converting para-coumaric acid into resveratrol and the downstream module expressing glucosyltransferase to convert the resveratrol into its glucosidated forms; polydatin and resveratroloside. Upon optimization of the initial inoculum ratio of two E. coli populations, 92 mg resveratrol glucosides/L (236 µM) was produced i.e. achieving 84% bioconversion from 280 µM of p-coumaric acid in 60 h by 3 L fed batch fermentor. This is the report of applying coculture system to produce resveratrol glucosides by expressing the aglycone formation pathway and sugar dependent pathway into two different cells.

Candida is a member of the normal human microbiota and often resides on mucosal surfaces such as the oral cavity or the gastrointestinal tract. In addition to their commensality, Candida species can opportunistically become pathogenic if the host microbiota is disrupted or if the host immune system becomes compromised. An important factor for Candida pathogenesis is its ability to form biofilm communities. The two most medically important species- Candida albicans and Candida glabrata -are often coisolated from infection sites, suggesting the importance of Candida coculture biofilms. In this work, we report that biofilm formation of the coculture population depends on the relative ratio of starting cell concentrations of C. albicans and C. glabrata When using a starting ratio of C. albicans to C. glabrata of 1:3, ∼6.5- and ∼2.5-fold increases in biofilm biomass were observed relative to those of a C. albicans monoculture and a C. albicans / C. glabrata ratio of 1:1, respectively. Confocal microscopy analysis revealed the heterogeneity and complex structures composed of long C. albicans hyphae and C. glabrata cell clusters in the coculture biofilms, and reverse transcription-quantitative PCR (qRT-PCR) studies showed increases in the relative expression of the HWP1 and ALS3 adhesion genes in the C. albicans / C. glabrata 1:3 biofilm compared to that in the C. albicans monoculture biofilm. Additionally, only the 1:3 C. albicans / C. glabrata biofilm demonstrated an increased resistance to the antifungal drug caspofungin. Overall, the results suggest that interspecific interactions between these two fungal pathogens increase biofilm formation and virulence-related gene expression in a coculture composition-dependent manner. IMPORTANCE Candida albicans and Candida glabrata are often coisolated during infection, and the occurrence of coisolation increases with increasing inflammation, suggesting possible synergistic interactions between the two Candida species in pathogenesis. During the course of an infection, the prevalence of each Candida species may change over time due to differences in metabolism and in the resistance of each species to antifungal therapies. Therefore, it is necessary to understand the dynamics between C. albicans in coculture to develop better therapeutic strategies against C. glabrata in coculture to develop better therapeutic strategies against Candida infections. Existing in vitro work has focused on understanding how an equal-part culture of C. albicans and C. glabrata species in coculture impacts overall biofilm formation, virulence gene expression, and the therapeutic treatment of biofilms.Candida species in coculture impacts overall biofilm formation, virulence gene expression, and the therapeutic treatment of biofilms., (Copyright © 2018 American Society for Microbiology.)

Smooth muscle cells (SMCs) and macrophages are important cellular components involved in the development of complications following the implantation of cardiovascular devices. This leads to various disorders such as restenosis, chronic inflammation, and may ultimately result in device failure. In this study, we developed a postimplant stent coculture model using different ratios of SMCs and macrophages seeded on to cobalt-chromium alloy. The macrophages had an increased affinity to the coculture surfaces, which resulted in decreased SMC attachment to the alloy surfaces at the initial time point. Once adhered, the macrophages spread freely and displayed advanced stages of inflammation at 48 h when cocultured with SMCs. This resulted in an increased secretion of proinflammatory cytokines (tumor necrosis factor alpha, monocyte chemotactic protein 1, interleukin [IL]-6, and IL-8) by 48 h in the coculture samples with the greatest increase observed with the high number of macrophages. Therefore, the increased levels of proinflammatory cytokines promoted the growth of SMCs in coculture to a greater extent than when the SMCs were culture alone. Thus, this study demonstrated the constant cross-talk between SMCs and macrophages occurring on the postimplant stent surface. Similar coculture models can be used to test the biocompatibility of drugs and biomaterials at possible postimplantation scenarios. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 673-685, 2018., (© 2017 Wiley Periodicals, Inc.)

With insufficient options to meet the clinical demand for cornea transplants, one emerging area of emphasis is on cornea tissue engineering. In the present study, the goal was to combine the corneal stroma and epithelium into one coculture system, to monitor both human corneal stromal stem cell (hCSSC) and human corneal epithelial cell (hCE) growth and differentiation into keratocytes and differentiated epithelium in these three-dimensional tissue systems in vitro. Coculture conditions were first optimized, including the medium, air-liquid interface culture, and surface topography and chemistry of biomaterial scaffold films based on silk protein. The silk was used as scaffolding for both stromal and epithelial tissue layers because it is cell compatible, can be surface patterned, and is optically clear. Next, the effects of proliferating and differentiating hCEs and hCSSCs were studied in this in vitro system, including the effects on cell proliferation, matrix formation by immunochemistry, and gene expression by quantitative reverse transcription-polymerase chain reaction. The incorporation of both cell types into the coculture system demonstrated more complete differentiation and growth for both cell types compared to the corneal stromal cells and corneal epithelial cells alone. Silk films for corneal epithelial culture were optimized to combine a 4.0-μm-scale surface pattern with bulk-loaded collagen type IV. Differentiation of each cell type was in evidence based on increased expression of corneal stroma and epithelial proteins and transcript levels after 6 weeks in coculture on the optimized silk scaffolds., (Copyright © 2017 John Wiley & Sons, Ltd.)

This work is motivated by both (1) the desire to make interesting products without the reliance on fixed carbon or fixed nitrogen using microbial cocultures, and (2) the desire to develop new methods for growing microbial communities. The bioplastic polyhydroxybutyrate (PHB), for example, is considered prohibitively expensive to make from sugar (compared to polypropylene). Utilizing and building on engineered strains of Synechococcus elongatus and Azotobacter vinelandii, we have combined a nitrogen-fixing organism with a carbon-fixing organism to make PHB from air, water, sunlight, and trace minerals. Our observations of coculture growth in batch culture led us to develop an improved system based on manipulating the osmotic pressure within a hydrogel. The methods we used to develop this coculture are described in detail in the following chapter, including notes detailing some of our additional observations or thoughts on this system.

Glial cell activation plays an important role in the pathogenesis of various neurodegenerative disorders as well as in chronic and neuropathic pain. This chapter describes a model which allows one to assess the individual and combined contributions of astrocytes and microglia in response to a pro-inflammatory stimulus, with emphasis on ionotropic purinergic receptors.

Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools to recapitulate this complex interaction. To address this barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived astrocytes (hAstros) combined with neurons generated from hPSC-derived neural stem cells (hNeurons) or directly induced via Neurogenin 2 overexpression (iNeurons). Our systematic methods rapidly produce structurally complex hAstros and synapses in high-density coculture with iNeurons in precise numbers, allowing for improved studies of neural circuit function, disease modeling, and drug screening. We conclude that these bioengineered neural circuit model systems are reliable and scalable tools to accurately study aspects of human astrocyte-neuron functional properties while being easily accessible for cell-type-specific manipulations and observations., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Low oxygen tolerance and substrate restriction continues to hamper the process of biobutanol industrialization. In this work, butanol fermentation with cocultures of Bacillus cereus China General Microbiological Culture Collection Center (CGMCC) 1.895 and Clostridium beijerinckii NCIMB 8052 under nonanaerobic conditions was investigated, and the interactions between these two strains were examined. The addition of B. cereus CGMCC 1.895 resulted in higher oxygen tolerance and a wider range of substrate utilization, compared with the pure culture of C. beijerinckii NCIMB 8052. Butanol concentration reached 10.49 g/L with an optimized inoculation size of 90% under nonanaerobic conditions, and this concentration was close to that of pure C. beijerinckii NCIMB 8052 culture under anaerobic conditions. Dynamic relative abundance analysis demonstrated that the ratio of C. beijerinckii NCIMB 8052 accounted for nearly 99% of the cocultured cells. Furthermore, the substrate utilization range was expanded, allowing the use of corn mash for butanol production. The final concentration of butanol and total solvents was 6.78 and 10.52 g/L, respectively. Coculture also was performed successfully in a 5-L fermenter and 8.75 g/L butanol was obtained. Dynamic dissolved oxygen analysis demonstrated that B. cereus consumed the dissolved oxygen in the broth and resulted in the anaerobic condition for C. beijerinckii., (© 2016 International Union of Biochemistry and Molecular Biology, Inc.)

Recent studies suggested that notochordal cells (NCs) and NC-conditioned medium (NCCM) can stimulate cell viability and matrix production of nucleus pulposus cells (NPCs). However, the potential of notochordal cell-rich nucleus pulposus (NRNP) incorporating the native environment of the intervertebral disc (IVD) has not been evaluated. The objective of this study was to develop an optimal NRNP model and test whether it can allow a significant level of NPC activation in vitro. Rabbit NRNP explants were divided into three groups according to different digestion time: digestion NRNP of 8 h, partial digestion NRNP of 2 h, and natural NRNP. Cell viability and NC phenotype were compared between these groups after 14 days of incubation. The products of the selected partial digestion NRNP group were then cocultured with human degenerated NPCs for 14 days. NPC viability, cell proliferation and senescence, the production of glycosaminoglycan (GAG) found in extracellular matrix, and NP matrix production by NPCs were assessed. The results showed that coculturing with partial digestion NRNP significantly improved the cell proliferation, cell senescence, and disc matrix gene expression of NPCs compared with those in the monoculture group. In addition, GAG/DNA ratio in the coculture group increased significantly, while the level of collagen II protein remained unchanged. In this study, we demonstrated that partial digestion NRNP may show a promising potential for NPC regeneration in IVD tissue engineering.

Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry., (© 2017 International Society for Advancement of Cytometry.)

Microbial co-cultures promise the development of more efficient bioproductions. However, the design of obligate mutualisms is complicated when using organisms that possess differing growth rates or incompatible media requirements. In this work, we investigate sucrose production by cscB Synechococcus elongatus PCC 7942 within a polyacrylate hydrogel matrix. This system secretes sucrose only when the hydrogel is spatially constrained, demonstrating a new utilization of hydrogel swelling pressure to control the osmotic strength of a microbial microenvironment. The sucrose produced via the constrained microbial hydrogel is used to grow the diazotrophic organism, Azotobacter vinelandii, in a mutually dependent fashion. The growth of this hydrogel-based coculture has several advantages over batch cultures, including better growth over a longer period of time and decreased salt stress on A. vinelandii. Biotechnol. Bioeng. 2017;114: 1195-1200. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Due to many advantages associated with mixed cultures, their application in biotechnology has expanded rapidly in recent years. At the same time, many challenges remain for effective mixed culture applications. One obstacle is how to efficiently and accurately monitor the individual cell populations. Current approaches on individual cell mass quantification are suitable for off-line, infrequent characterization. In this study, we propose a fast and accurate "soft sensor" approach for estimating individual cell concentrations in mixed cultures. The proposed approach utilizes optical density scanning spectrum of a mixed culture sample measured by a spectrophotometer over a range of wavelengths. A multivariate linear regression method, partial least squares or PLS, is applied to correlate individual cell concentrations to the spectrum. Three experimental case studies are used to examine the performance of the proposed soft sensor approach. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:347-354, 2017., (© 2017 American Institute of Chemical Engineers.)