Evolution of organoid technology: Lessons learnt in Co-Culture systems from developmental biology.

In recent years, the development of 3D organoids has opened new avenues of investigation into development, physiology, and regenerative medicine. Organoid formation and the process of organogenesis share common developmental pathways; thus, our knowledge of developmental biology can help model the complexity of different organs to refine organoids into a more sophisticated platform. The developmental process is strongly dependent on complex networks and communication of cell-cell and cell-matrix interactions among different cell populations and their microenvironment, during embryogenesis. These interactions affect cell behaviors such as proliferation, survival, migration, and differentiation. Co-culture systems within the organoid technology were recently developed and provided the highly physiologically relevant systems. Supportive cells including various types of endothelial and stromal cells provide the proper microenvironment, facilitate organoid assembly, and improve vascularization and maturation of organoids. This review discusses the role of the co-culture systems in organoid generation, with a focus on how knowledge of developmental biology has directed and continues to shape the development of more evolved 3D co-culture system-derived organoids.

Using Co-Culture to Functionalize Clostridium Fermentation.

Clostridium fermentations have been developed for producing butanol and other value-added chemicals, but their development is constrained by some limitations, such as relatively high substrate cost and the need to maintain an anaerobic condition. Recently, co-culture is emerging as a popular way to address these limitations by introducing a partner strain with Clostridium. Generally speaking, the co-culture strategy enables the use of a cheaper substrate, maintains the growth of Clostridium without any anaerobic treatment, improves product yields, and/or widens the product spectrum. Herein, we review recent developments of co-culture strategies involving Clostridium species according to their partner stains' functions with representative examples. We also discuss research challenges that need to be addressed for the future development of Clostridium co-cultures.

Progress of co-culture systems in cartilage regeneration.

INTRODUCTION: Cartilage tissue engineering has rapidly developed in recent decades, exhibiting promising potential to regenerate and repair cartilage. However, the origin of a large amount of a suitable seed cell source is the major bottleneck for the further clinical application of cartilage tissue engineering. The use of a monoculture of passaged chondrocytes or mesenchymal stem cells results in undesired outcomes, such as fibrocartilage formation and hypertrophy. In the last two decades, co-cultures of chondrocytes and a variety of mesenchymal stem cells have been intensively investigated in vitro and in vivo, shedding light on the perspective of co-culture in cartilage tissue engineering. AREAS COVERED: We summarize the recent literature on the application of heterologous cell co-culture systems in cartilage tissue engineering and compare the differences between direct and indirect co-culture systems as well as discuss the underlying mechanisms. EXPERT OPINION: Co-culture system is proven to address many issues encountered by monocultures in cartilage tissue engineering, including reducing the number of chondrocytes needed and alleviating the dedifferentiation of chondrocytes. With the further development and knowledge of biomaterials, cartilage tissue engineering that combines the co-culture system and advanced biomaterials is expected to solve the difficult problem regarding the regeneration of functional cartilage.

From Axenic to Mixed Cultures: Technological Advances Accelerating a Paradigm Shift in Microbiology.

Since the onset of microbiology in the late 19th century, scientists have been growing microorganisms almost exclusively as pure cultures, resulting in a limited and biased view of the microbial world. Only a paradigm shift in cultivation techniques - from axenic to mixed cultures - can allow a full comprehension of the (chemical) communication of microorganisms, with profound consequences for natural product discovery, microbial ecology, symbiosis, and pathogenesis, to name a few areas. Three main technical advances during the last decade are fueling the realization of this revolution in microbiology: microfluidics, next-generation 3D-bioprinting, and single-cell metabolomics. These technological advances can be implemented for large-scale, systematic cocultivation studies involving three or more microorganisms. In this review, we present recent trends in microbiology tools and discuss how these can be employed to decode the chemical language that microorganisms use to communicate.

Metabolic activation and drug-induced liver injury: in vitro approaches for the safety risk assessment of new drugs.

Drug-induced liver injury (DILI) is a significant leading cause of hepatic dysfunction, drug failure during clinical trials and post-market withdrawal of approved drugs. Many cases of DILI are unexpected reactions of an idiosyncratic nature that occur in a small group of susceptible individuals. Intensive research efforts have been made to understand better the idiosyncratic DILI and to identify potential risk factors. Metabolic bioactivation of drugs to form reactive metabolites is considered an initiation mechanism for idiosyncratic DILI. Reactive species may interact irreversibly with cell macromolecules (covalent binding, oxidative damage), and alter their structure and activity. This review focuses on proposed in vitro screening strategies to predict and reduce idiosyncratic hepatotoxicity associated with drug bioactivation. Compound incubation with metabolically competent biological systems (liver-derived cells, subcellular fractions), in combination with methods to reveal the formation of reactive intermediates (e.g., formation of adducts with liver proteins, metabolite trapping or enzyme inhibition assays), are approaches commonly used to screen the reactivity of new molecules in early drug development. Several cell-based assays have also been proposed for the safety risk assessment of bioactivable compounds. Copyright © 2015 John Wiley & Sons, Ltd.

From Single Cells to Engineered and Explanted Tissues: New Perspectives in Bacterial Infection Biology.

Cell culture techniques are essential for studying host-pathogen interactions. In addition to the broad range of single cell type-based two-dimensional cell culture models, an enormous amount of coculture systems, combining two or more different cell types, has been developed. These systems enable microscopic visualization and molecular analyses of bacterial adherence and internalization mechanisms and also provide a suitable setup for various biochemical, immunological, and pharmacological applications. The implementation of natural or synthetical scaffolds elevated the model complexity to the level of three-dimensional cell culture. Additionally, several transwell-based cell culture techniques are applied to study bacterial interaction with physiological tissue barriers. For keeping highly differentiated phenotype of eukaryotic cells in ex vivo culture conditions, different kinds of microgravity-simulating rotary-wall vessel systems are employed. Furthermore, the implementation of microfluidic pumps enables constant nutrient and gas exchange during cell cultivation and allows the investigation of long-term infection processes. The highest level of cell culture complexity is reached by engineered and explanted tissues which currently pave the way for a more comprehensive view on microbial pathogenicity mechanisms.

[Progress in microbial co-culture--A review].

We reviewed the history and applications of microorganism co-cultivation in food, agriculture, industry and sewage purification, and summarized ecology relationships between co-culture microorganisms. Joint mixed culture, sequence mixed culture and immobilized cells mixed culture have been used widely and lots of achievements have been made, for example, obtaining metabolites that are difficult to achieve or too low production in pure culture, transforming traditional fermentation industry, producing energy substance, improving substrate utilization ratio, expanding the scope of substrates and degrading toxic substances. Research reports indicate there are many ecology relationships between microorganisms, such as collaborative metabolism, induction effect, quorum sensing and gene transfer. The ecological interplay mechanism of co-culture microorganisms should have a further research, which will lay the foundation for developing applications of microorganism co-culture.

Developing cellular systems in vitro to simulate regeneration.

In the past two decades, cellular systems in vitro have progressed from predominantly monocellular testing models to study the toxic effects of new biomaterials for replacement to relevant human coculture systems for regeneration, often a combination of progenitor cells with novel biomaterials. Considerable progress has been made in understanding cellular cross talk and its contribution to the vascularization of bone. Future challenges include using the established physiological, that is, nonactivated, stem cell niches as a platform to develop coculture models, which will enable the true in situ regenerative niche to be investigated. Hypoxia and a changing inflammatory status are factors that need to be incorporated. Major advances in polymer synthesis permitting the incorporation of specific biologically relevant signals in hydrogels will help make this a reality.

Consideration of the cellular microenvironment: physiologically relevant co-culture systems in drug discovery.

There is renewed interest in phenotypic approaches to drug discovery, using cell-based assays to select new drugs, with the goal of improving pharmaceutical success. Assays that are more predictive of human biology can help researchers achieve this goal. Primary cells are more physiologically relevant to human biology and advances are being made in methods to expand the available cell types and improve the potential clinical translation of these assays through the use of co-cultures or three-dimensional (3D) technologies. Of particular interest are assays that may be suitable for industrial scale drug discovery. Here we review the use of primary human cells and co-cultures in drug discovery and describe the characteristics of co-culture models for inflammation biology (BioMAP systems), neo-vascularization and tumor microenvironments. Finally we briefly describe technical trends that may enable and impact the development of physiologically relevant co-culture assays in the near future.

Cell sources for articular cartilage repair strategies: shifting from monocultures to cocultures.

The repair of articular cartilage is challenging due to the sparse native cell population combined with the avascular and aneural nature of the tissue. In recent years, cartilage tissue engineering has shown great promise. As with all tissue engineering strategies, the possible therapeutic outcome is intimately linked with the used combination of cells, growth factors, and biomaterials. However, the optimal combination has remained a controversial topic and no consensus has been reached. In consequence, much effort has been dedicated, to further design, investigate, and optimize cartilage repair strategies. Specifically, various research groups have performed intensive investigations attempting to identify the single most optimal cell source for articular cartilage repair strategies. However, recent findings indicate that not the heavily investigated monocell source, but the less studied combinations of cell sources in coculture might be more attractive for cartilage repair strategies. This review will give a comprehensive overview on the cell sources that have been investigated for articular cartilage repair strategies. In particular, the advantages and disadvantages of investigated cell sources are comprehensively discussed with emphasis on the potential of cocultures in which benefits are combined, while the disadvantages of single-cell sources for cartilage repair are mitigated.

Multicellular tumor spheroids: an underestimated tool is catching up again.

The present article highlights the rationale, potential and flexibility of tumor spheroid mono- and cocultures for implementation into state of the art anti-cancer therapy test platforms. Unlike classical monolayer-based models, spheroids strikingly mirror the 3D cellular context and therapeutically relevant pathophysiological gradients of in vivo tumors. Some concepts for standardization and automation of spheroid culturing, monitoring and analysis are discussed, and the challenges to define the most convenient analytical endpoints for therapy testing are outlined. The potential of spheroids to contribute to either the elimination of poor drug candidates at the pre-animal and pre-clinical state or the identification of promising drugs that would fail in classical 2D cell assays is emphasised. Microtechnologies, in the form of micropatterning and microfluidics, are also discussed and offer the exciting prospect of standardized spheroid mass production to tackle high-throughput screening applications within the context of traditional laboratory settings. The extension towards more sophisticated spheroid coculture models which more closely reflect heterologous tumor tissues composed of tumor and various stromal cell types is also covered. Examples are given with particular emphasis on tumor-immune cell cocultures and their usefulness for testing novel immunotherapeutic treatment strategies. Finally, tumor cell heterogeneity and the extraordinary possibilities of putative cancer stem/tumor-initiating cell populations that can be maintained and expanded in sphere-forming assays are introduced. The relevance of the cancer stem cell hypothesis for cancer cure is highlighted, with the respective sphere cultures being envisioned as an integral tool for next generation drug development offensives.

Tendon tissue engineering using scaffold enhancing strategies.

Tendon traumas or diseases are prevalent and debilitating lesions that affect the quality of life among populations worldwide. As a novel solution, tendon tissue engineering aims to address these lesions by integrating engineered, living substitutes with their native counterparts in vivo, thereby restoring the defective functions in situ. For such a purpose, competent scaffolding materials are essential. To date, three major categories of scaffolding materials have been employed: polyesters, polysaccharides, and collagen derivatives. Furthermore, with these materials as a base, a variety of specialized methodologies have been developed or adopted to enhance neo-tendogenesis. These strategies include cellular hybridization, interfacing improvement, and physical stimulation.

The development of 'feeder' cells for the preparation of clinical grade hES cell lines: challenges and solutions.

The development of human embryonic stem cell (hESC) lines for research and therapy is hampered by the need to improve the basic methodologies for cell culture expansion. In most current methods hESC lines are cultured on a mouse or human feeder cell layer which appears to be the most reliable way to maintain cells stably in the undifferentiated state. However, co-culture introduces complications for studying stem cell biology and the delivery of safe therapies for the future. This article reviews the specific risks associated with any proposed clinical use of feeder cells of mouse origin and compares these with the benefits and risks of using human feeder cells. The further work required to establish clinical grade feeder cell lines for hESC line culture is significant and costly. Much work is being done to find feeder-free culture systems but these are at an early stage of development and there may be consequences that affect the value of the hESCs for research and development. These challenges should be viewed in the context of the huge amount of work that will be required over many years to develop robust differentiation protocols and establish fully defined procedures and adequate safety data for embryonic stem cell products.

Perspectives in the modeling and optimization of PHB production by pure and mixed cultures.

Poly(beta-hydroxybutyrate) or PHB is an important member of the family of polyhydroxyalkanoates with properties that make it potentially competitive with synthetic polymers. In addition, PHB is biodegradable. While the biochemistry of PHB synthesis by microorganisms is well known, improvement of large-scale productivity requires good fermentation modeling and optimization. The latter aspect is reviewed here. Current models are of two types: (i) mechanistic and (ii) cybernetic. The models may be unstructured or structured, and they have been applied to single cultures and co-cultures. However, neither class of models expresses adequately all the important features of large-scale non-ideal fermentations. Model-independent neural networks provide faithful representations of observations, but they can be difficult to design. So hybrid models, combining mechanistic, cybernetic and neural models, offer a useful compromise. All three kinds of basic models are discussed with applications and directions toward hybrid model development.

Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance.

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms. PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. The use of mixed cultures and cheap substrates can reduce the production cost of PHA. Accumulation of PHA by mixed cultures occurs under transient conditions mainly caused by intermittent feeding and variation in the electron donor/acceptor presence. The maximum capacity for PHA storage and the PHA production rate are dependent on the substrate and the operating conditions used. This work reviews the development of PHA research. Aspects discussed include metabolism and various mechanisms for PHA production by mixed cultures; kinetics of PHA accumulation and conversion; effects of carbon source and temperature on PHA production using mixed cultures; PHA production process design; and characteristics of PHA produced by mixed cultures.

Differentiating adult hippocampal stem cells into neural crest derivatives.

To investigate the degree of plasticity of hippocampal neural stem cells from adult mice (mHNSC), we have analyzed their differentiation in co-culture with quail neural crest cells. In mixed culture, mHNSC give rise to several non-neuronal neural crest derivatives, including melanocytes, chondrocytes and smooth muscle cells. The data suggest that neural crest cell-derived short-range cues that are recognized across species can instruct adult mHNSC to differentiate into neural crest phenotypes.