Imaging living central neurones using viral gene transfer

Studies of central neurones and other cellular components of the brain, such as glial and vascular cells, can be greatly advanced by the use of the modern optical techniques such as confocal live cell imaging. Fluorescent proteins have allowed imaging of particular cell types or intracellular elements to be visualised and distinguished from irrelevant background structures. To introduce the genetic information encoding for fluorescent proteins into relevant cellular targets, molecular tools are required. Viral vectors are one of the best ways of gene delivery into differentiated postnatal brain neurones and glia. Current progress in this field allows targeting of various cell types and therefore makes it possible to express a variety of fluorescent constructs in selected subpopulations of neurones, for example. In this review, we will discuss and compare the properties of the most popular viral gene delivery systems and the advantages of different brain cell preparations to illustrate how they can be used for high-resolution live cell confocal imaging in order to study new aspects of central nervous system (CNS) structure and function.     

Point-, line-, and plane-shaped cellular constructs for 3D tissue assembly

Microsized cellular constructs such as cellular aggregates and cell-laden hydrogel blocks are attractive cellular building blocks to reconstruct 3D macroscopic tissues with spatially ordered cells in bottom-up tissue engineering. In this regard, microfluidic techniques are remarkable methods to form microsized cellular constructs with high production rate and control of their shapes such as point, line, and plane. The fundamental shapes of the cellular constructs allow for the fabrication of larger arbitrary-shaped tissues by assembling them. This review introduces microfluidic formation methods of microsized cellular constructs and manipulation techniques to assemble them with control of their arrangements. Additionally, we show applications of the cellular constructs to biological studies and clinical treatments and discuss future trends as their potential applications.     

Nanoparticles: Emerging carriers for drug delivery

The core objective of nanoparticles is to control and manipulate biomacromolecular constructs and supramolecular assemblies that are critical to living cells in order to improve the quality of human health. By definition, these constructs and assemblies are nanoscale and include entities such as drugs, proteins, DNA/RNA, viruses, cellular lipid bilayers, cellular receptor sites and antibody variable regions critical for immunology and are involved in events of nanoscale proportions. The emergence of such nanotherapeutics/diagnostics will allow a deeper understanding of human longevity and human ills that include cancer, cardiovascular disease and genetic disorders. A technology platform that provides a wide range of synthetic nanostructures that may be controlled as a function of size, shape and surface chemistry and scale to these nanotechnical dimensions will be a critical first step in developing appropriate tools and a scientific basis for understanding nanoparticles.     

High-throughput screening of cell responses to biomaterials

Biomaterials have emerged as powerful regulators of the cellular microenvironment for drug discovery, tissue engineering research and chemical testing. Although biomaterial-based matrices control the cellular behavior, these matrices are still far from being optimal. In principle, efficacy of biomaterial development for the cell cultures can be improved by using high-throughput techniques that allow screening of a large number of materials and manipulate microenvironments in a controlled manner. Several cell responses such as toxicity, proliferation, and differentiation have been used to evaluate the biomaterials thus providing basis for further selection of the lead biomimetic materials or microenvironments. Although high-throughput techniques provide an initial screening of the desired properties, more detailed follow-up studies of the selected materials are required to understand the true value of a 'positive hit'. High-throughput methods may become important tools in the future development of biomaterials-based cell cultures that will enable more realistic pre-clinical prediction of pharmacokinetics, pharmacodynamics, and toxicity. This is highly important, because predictive pre-clinical methods are needed to improve the high attrition rate of drug candidates during clinical testing.     

Phenotypic screening for pharmaceuticals using tissue constructs

Compounds can be screened for pharmaceutical activity either by detecting interactions with specified target molecules such as receptors or enzymes (molecular screening) or observing effects on the structure or physiological activities of cells or tissues (phenotypic screening). Screening at the molecular level has been greatly enhanced by fluorescence methods. Especially the combination of confocal detection with measurements of the amplitudes and time courses of fluorescence fluctuations have reduced sample volumes to < microliters and have increased throughputs to >100000 compounds per day. Screening at the molecular level, however, does not provide information about the effects of test compounds on cellular functions. Phenotypic screening, although much slower than molecular screening, does provide information about effects on cell or tissue structure or function and therefore can be used to eliminate at an early stage compounds that are toxic or do not produce the desired cellular response. Tissue constructs reconstituted using cells of specified types and defined extracellular matrix components provide test systems for detecting the effects of test compounds on cellular mechanical functions such as the development of contractile force and on cell and matrix structure and stiffness. For example, constructs based on vascular smooth muscle cells provide information about effects on cellular contractile force that can be used to identify agents that control blood pressure. Tissue constructs that mimic skeletal, smooth and heart muscles and connective tissues have been produced and can be used to study mechanical and structural responses to active compounds.     

Gene-expression analysis at the single-cell level

The manner in which a cell responds to and influences its environment is ultimately determined by the genes that it expresses. To fully understand and manipulate cellular function, identification of these expressed genes is essential. Techniques such as RT-PCR enable examination of gene expression at the tissue level. However, the study of complex heterogeneous tissue, such as the CNS or immune system, requires gene analysis to be performed at much higher resolution. In this article, the various methods that have been developed to enable RT-PCR to be performed at the level of the single cell are reviewed. In addition, how, when carried out in combination with techniques such as patch-clamp recording, single-cell gene-expression studies extend our understanding of biological systems is discussed.     

Dyes, indispensable tools for the 19th century's biologic and therapeutic revolution

Born from growing organic chemistry laboratories, dyes were extensively used par textile industry before to be applied in field of biology and therapeutics. Besides their interest for diagnostic techniques due to cell visualization (Virchow, Papanicolaou), dyes allowed scientists to propose scientific hypothesis founding, in conjunction with new microscopy tools, modern basis for biology : tissue constitution, cellular and sub cellular structure, s.o. One of the brightest illustrations of these progresses is the birth of neuronal theory which due to silver print of brain tissue allowed to see intimacy of cerebral structures et propose an operating scheme (Golgi, Cajal). Therapeutic progresses born from dyes chemistry are multiple. First concentrated on the research of antimalarial drugs (Ehrlich) following the use of methylene blue, then generally, anti-infectious drugs, they gave birth to various chimiotherapeutic families: antiseptics, antiparasitic drugs, antibacterial, among which one of the most spectacular illustrations remain sulphonamides preparation.     

Biomaterials for stem cell differentiation

The promise of cellular therapy lies in the repair of damaged organs and tissues in vivo as well as generating tissue constructs in vitro for subsequent transplantation. Unfortunately, the lack of available donor cell sources limits its ultimate clinical applicability. Stem cells are a natural choice for cell therapy due to their pluripotent nature and self-renewal capacity. Creating reserves of undifferentiated stem cells and subsequently driving their differentiation to a lineage of choice in an efficient and scalable manner is critical for the ultimate clinical success of cellular therapeutics. In recent years, a variety of biomaterials have been incorporated in stem cell cultures, primarily to provide a conducive microenvironment for their growth and differentiation and to ultimately mimic the stem cell niche. In this review, we examine applications of natural and synthetic materials, their modifications as well as various culture conditions for maintenance and lineage-specific differentiation of embryonic and adult stem cells.     

Compartmental Tissue Distribution of Antibody Therapeutics: Experimental Approaches and Interpretations

Monoclonal antibodies have provided many validated and potential new therapeutic candidates for various diseases encompassing the realms of neurology, ophthalmology, immunology, and especially oncology. The mechanism of action for these biological molecules typically involves specific binding to a soluble ligand or cell surface protein in order to block or alter a molecular pathway, induce a desired cellular response, or deplete a target cell. Many antigens reside within the interstitial space, the fluid-filled compartment that lies between the outer endothelial vessel wall and the plasma membranes of cells. This mini-review examines the concepts relevant to the kinetics and behavior of antibodies within the interstitium with a special emphasis on radiometric measurement of quantitative pharmacology. Molecular probes are discussed to outline chemical techniques, selection criteria, data interpretation, and relevance to the study of antibody pharmacokinetics. The importance of studying the tissue uptake of antibodies at a compartmental level is highlighted, including a brief overview of receptor occupancy and its interpretation in radiotracer studies. Experimental methods for measuring the spatial composition of tissues are examined in terms of relative vascular, interstitial, and cellular volumes using solid tumors as a representative example. Experimental methods and physiologically based pharmacokinetic modeling are introduced as distinct approaches to distinguish between free and bound fractions of interstitial antibody. Overall, the review outlines the available methods for pharmacokinetic measurements of antibodies and physiological measurements of the compartments that they occupy, while emphasizing that such approaches may not fully capture the complexities of dynamic, heterogeneous tumors and other tissues.     

Electrospun materials as potential platforms for bone tissue engineering

Nanofibrous materials produced by electrospinning processes have attracted considerable interest in tissue regeneration, including bone reconstruction. A range of novel materials and processing tools have been developed to mimic the native bone extracellular matrix for potential applications as tissue engineering scaffolds and ultimately to restore degenerated functions of the bone. Degradable polymers, bioactive inorganics and their nanocomposites/hybrids nanofibers with suitable mechanical properties and bone bioactivity for osteoblasts and progenitor/stem cells have been produced. The surface functionalization with apatite minerals and proteins/peptides as well as drug encapsulation within the nanofibers is a promising strategy for achieving therapeutic functions with nanofibrous materials. Recent attempts to endow a 3D scaffolding technique to the electrospinning regime have shown some promise for engineering 3D tissue constructs. With the improvement in knowledge and techniques of bone-targeted nanofibrous matrices, bone tissue engineering is expected to be realized in the near future.     

Three-dimensional tissue fabrication

In recent years, advances in fabrication technologies have brought a new dimension to the field of tissue engineering. Using manufacturing-based methods and hydrogel chemistries, researchers have been able to fabricate tissue engineering scaffolds with complex 3-D architectures and customized chemistries that mimic the in vivo tissue environment. These techniques may be useful in developing therapies for replacing lost tissue function, as in vitro models of living tissue, and also for further enabling fundamental studies of structure/function relationships in three dimensional contexts. Here, we present an overview of 3-D tissue fabrication techniques based on methods for: scaffold fabrication, cellular assembly, and hybrid hydrogel/cell methods and review their potential utility for tissue engineering.     

Two-photon excitation imaging of exocytosis and endocytosis and determination of their spatial organization

Two-photon excitation imaging is the least invasive optical approach to study living tissues. We have established two-photon extracellular polar-tracer (TEP) imaging with which it is possible to visualize and quantify all exocytic events in the plane of focus within secretory tissues. This technology also enables estimate of the precise diameters of vesicles independently of the spatial resolution of the optical microscope, and determination of the fusion pore dynamics at nanometer resolution using TEP-imaging based quantification (TEPIQ). TEP imaging has been applied to representative secretory glands, e.g., exocrine pancreas, endocrine pancreas, adrenal medulla and a pheochromocytoma cell line (PC12), and has revealed unexpected diversity in the spatial organization of exocytosis and endocytosis crucial for the physiology and pathology of secretory tissues and neurons. TEP imaging and TEPIQ analysis are powerful tools for elucidating the molecular and cellular mechanisms of exocytosis and certain related diseases, such as diabetes mellitus, and the development of new therapeutic agents and diagnostic tools.     

Spatial regulation of controlled bioactive factor delivery for bone tissue engineering

Limitations of current treatment options for critical size bone defects create a significant clinical need for tissue engineered bone strategies. This review describes how control over the spatiotemporal delivery of growth factors, nucleic acids, and drugs and small molecules may aid in recapitulating signals present in bone development and healing, regenerating interfaces of bone with other connective tissues, and enhancing vascularization of tissue engineered bone. State-of-the-art technologies used to create spatially controlled patterns of bioactive factors on the surfaces of materials, to build up 3D materials with patterns of signal presentation within their bulk, and to pattern bioactive factor delivery after scaffold fabrication are presented, highlighting their applications in bone tissue engineering. As these techniques improve in areas such as spatial resolution and speed of patterning, they will continue to grow in value as model systems for understanding cell responses to spatially regulated bioactive factor signal presentation in vitro, and as strategies to investigate the capacity of the defined spatial arrangement of these signals to drive bone regeneration in vivo.     

Delivering bioactive molecules as instructive cues to engineered tissues

INTRODUCTION: Growth factors and other bioactive molecules play a crucial role in the creation of functional engineered tissues from dissociated cells. AREAS COVERED: This review discusses the delivery of bioactive molecules - particularly growth factors - to affect cellular function in the context of tissue engineering. We discuss the primary biological themes that are addressed by delivering bioactives, the types of molecules that are to be delivered, the major materials used in producing scaffolds and/or drug delivery systems, and the principal drug delivery strategies. EXPERT OPINION: Drug delivery systems have allowed the sustained release of bioactive molecules to engineered tissues, with marked effects on tissue function. Sophisticated drug delivery techniques will allow precise recapitulation of developmental milestones by providing temporally distinct patterns of release of multiple bioactives. High-resolution patterning techniques will allow tissue constructs to be designed with precisely defined areas where bioactives can act. New biological discoveries, just as the development of small molecules with potent effects on cell differentiation, will likely have a marked impact on the field.     

Micro- and nanoscale technologies for tissue engineering and drug discovery applications

Micro- and nanoscale technologies are emerging as powerful enabling tools for tissue engineering and drug discovery. In tissue engineering, micro- and nanotechnologies can be used to fabricate biomimetic scaffolds with increased complexity and vascularization. Furthermore, these technologies can be used to control the cellular microenvironment (i.e., cell–cell, cell–matrix and cell–soluble factor interactions) in a reproducible manner and with high temporal and spatial resolution. In drug discovery, miniaturized platforms based on micro- and nanotechnology can be used to precisely control the fluid flow, enable high-throughput screening, and minimize sample or reagent volumes. In addition, these systems enhance reproducibility and significantly reduce reaction times. This paper reviews the recent developments in the field of micro- and nanoscale technology and gives examples of their tissue engineering and drug discovery applications.     

Host and Viral Factors Influencing Interplay between the Macrophage and HIV-1

HIV-1 persists in cellular reservoirs that cannot be eliminated by antiretroviral therapy (ART). The major reservoir in infected individuals on effective ART is composed of resting memory CD4+ T cells that harbor proviral cDNA, and undergo a state of latency in which viral gene expression is minimal to absent. The CD4+ T cell reservoir has been extensively characterized. However, other HIV-1-permissive cells may contribute to HIV-1 persistence. Lentiviruses have a long recognized association with macrophages. However, the role, if any, played by macrophages in HIV-1 persistence is not well understood. Macrophages are resistant to cell death upon HIV-1 infection, and can survive for long periods of time, making them ideal host cells in which the virus might persist. Studying macrophages is challenging, as these cells reside in nearly all tissues. Moreover, detecting viral DNA or RNA in macrophages does not necessarily indicate that these cells will produce replication-competent viral particles. Currently, the gold standard assay to detect cellular reservoirs is the ex vivo quantitative viral outgrowth assay (QVOA), which requires a patient blood draw. However, macrophages reside deep within tissues that are inaccessible in living subjects, such as the central nervous system (CNS). Therefore, tools other than QVOA must be developed to identify cellular reservoirs that reside in the tissues. In this review, we will focus on the main aspects involved in HIV-1 persistence, including the molecular mechanisms of viral evasion, the main cell types responsible for harboring persistent HIV-1 and the tissue compartments that are likely to be reservoirs for HIV-1.

     

Three-dimensional cell culture technique and pathophysiology

Three-dimensional (3D) tissue constructs consisting of human cells have opened a new avenue for tissue engineering, pharmaceutical and pathophysiological applications, and have great potential to estimate the dynamic pharmacological effects of drug candidates, metastasis processes of cancer cells, and toxicity expression of nano-materials, as a 3D-human tissue model instead of in vivo animal experiments. However, most 3D-cellular constructs are a cell spheroid, which is a heterogeneous aggregation, and thus the reconstruction of the delicate and precise 3D-location of multiple types of cells is almost impossible.In recent years, various novel technologies to develop complex 3D-human tissues including blood and lymph capillary networks have demonstrated that physiological human tissue responses can be replicated in the nano/micro-meter ranges. Here, we provide a brief overview on current 3D-tissue fabrication technologies and their biomedical applications. 3D-human tissue models will be a powerful technique for pathophysiological applications.     

3D cell culture systems modeling tumor growth determinants in cancer target discovery

Phenotypic heterogeneity of cancer cells, cell biological context, heterotypic crosstalk and the microenvironment are key determinants of the multistep process of tumor development. They sign responsible, to a significant extent, for the limited response and resistance of cancer cells to molecular-targeted therapies. Better functional knowledge of the complex intra- and intercellular signaling circuits underlying communication between the different cell types populating a tumor tissue and of the systemic and local factors that shape the tumor microenvironment is therefore imperative. Sophisticated 3D multicellular tumor spheroid (MCTS) systems provide an emerging tool to model the phenotypic and cellular heterogeneity as well as microenvironmental aspects of in vivo tumor growth. In this review we discuss the cellular, chemical and physical factors contributing to zonation and cellular crosstalk within tumor masses. On this basis, we further describe 3D cell culture technologies for growth of MCTS as advanced tools for exploring molecular tumor growth determinants and facilitating drug discovery efforts. We conclude with a synopsis on technological aspects for on-line analysis and post-processing of 3D MCTS models.     

Host response to tissue engineered devices

The two main components of a tissue engineered device are the transplanted cells and the biomaterial, creating a device for the restoration or modification of tissue or organ function. The implantation of polymer/cell constructs combines concepts of biomaterials and cell transplantation. The interconnections between the host responses to the biomaterial and transplanted cells determines the biocompatibility of the device. This review describes the inflammatory response to the biomaterial component and immune response towards transplanted cells. Emphasis is on how the presence of the transplanted cell construct affects the host response. The inflammatory response towards a biomaterial can impact the immune response towards transplanted cells and vice versa. Immune rejection is the most important host response towards the cellular component of tissue engineered devices containing allogeneic, xenogeneic or immunogenic ex vivo manipulated autologous cells. The immune mechanisms towards allografts and xenografts are outlined to provide a basis for the mechanistic hypotheses of the immune response towards encapsulated cells, with antigen shedding and the indirect pathway of antigen presentation predominating. A review of experimental evidence illustrates examples of the inflammatory response towards biodegradable polymer scaffold materials, examples of devices appropriately integrated as assessed morphologically with the host for various applications including bone, nerve, and skin regeneration, and of the immune response towards encapsulated allogeneic and xenogeneic cells.