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Tissue engineering is an interdisciplinary field that applies the principles and methods of engineering and the cell culture toward the development of biomaterials that restore, maintain or improve tissue function. The amalgamation of engineering and medicine has interested many scientists for at last two hundred years. What was the goal of cell culture? First, for progress in life sciences achievement and subsequent for virology and toxicology development. In vitro studies are done because of many problems with carrying out animal experiments. In this work the authors present the attempts of physicians, anatomopathologists, embryologists and biologists which contributed to fast development of new area in medicine--tissue engineering.

Developing scaffolds that mimic the architecture of tissue at the nanoscale is one of the major challenges in the field of tissue engineering. The development of nanofibers has greatly enhanced the scope for fabricating scaffolds that can potentially meet this challenge. Currently, there are three techniques available for the synthesis of nanofibers: electrospinning, self-assembly, and phase separation. Of these techniques, electrospinning is the most widely studied technique and has also demonstrated the most promising results in terms of tissue engineering applications. The availability of a wide range of natural and synthetic biomaterials has broadened the scope for development of nanofibrous scaffolds, especially using the electrospinning technique. The three dimensional synthetic biodegradable scaffolds designed using nanofibers serve as an excellent framework for cell adhesion, proliferation, and differentiation. Therefore, nanofibers, irrespective of their method of synthesis, have been used as scaffolds for musculoskeletal tissue engineering (including bone, cartilage, ligament, and skeletal muscle), skin tissue engineering, vascular tissue engineering, neural tissue engineering, and as carriers for the controlled delivery of drugs, proteins, and DNA. This review summarizes the currently available techniques for nanofiber synthesis and discusses the use of nanofibers in tissue engineering and drug delivery applications.

Plant cell, tissue, and organ culture is a set of techniques designed for the growth and multiplication of cells and tissues using nutrient solutions in an aseptic and controlled environment. This technology explores conditions that promote cell division and genetic reprogramming in in vitro conditions. Mainly developed in the early 1960s, plant tissue culture has turned into a standard procedure for modem biotechnology and today one can recognize five major areas where in vitro cell cultures are currently applied: large-scale propagation of elite materials, generation of genetic modified fertile individuals, as a model system for fundamental plant cell physiology aspects, preservation of endangered species, and metabolic engineering of fine chemicals. This chapter reviews the recent advances in such areas.

The theme of this brief presentation is that there are tremendous advances not only in PCR-based detection but in other novel ways of detecting viruses and other agents. Much of the research is now being driven by the bio-defence agenda. Somehow those with manufacturing and regulatory responsibilities have to keep abreast of these developments and incorporate them as they become available into defining the safety of cell substrates. I am concerned that these techniques are and may remain academic research tools without validation or standardization, and that individual academic laboratories will not develop the capacity and special skills necessary to satisfy regulatory requirements, but that there is a tremendous research incentive in this area that needs to be linked with the development of safe prophylactic and therapeutic biologicals. With that, I will finish what I would consider as a report rather than an original talk.

This paper reviews reports on three-dimensional mammalian tissue growth in bioreactors and the corresponding mammalian tissue growth requirements. The needs for nutrient and waste removal of several mammalian tissues are reviewed and compared with the environment of many reactors currently in use such as the continuous stirred tank, the hollow fiber, the Couette-Taylor, the airlift, and the rotating-wall reactors developed by NASA. Many studies conclude that oxygen supply appears to be one of the most important factors limiting tissue growth. Various correlations to describe oxygen mass transfer are presented and discussed with the aim to provide some guidance to design, construct, and test reactors for tissue mass culture. To obtain tissue thickness clinically valuable, dimensionless and other types of analysis tend to point out that diffusive transport will have to be matched with an important convection to bring sufficient oxygen molecular flux to the growing cells located within a tissue mass. As learned from solid-state fermentation and hairy root culture, during the growth of large biomass, heterogeneity (i.e., channeling, temperature gradients, non-uniform cell growth, transfer gradients, etc.) can cause some important problems and these should be addressed in tissue engineering as well. Reactors (along with the scaffolds) should be designed to minimize these issues. The role of the uterus, the reactor built by Nature, is examined, and the environment provided to a growing embryo is reported, yielding possible paths for further reactor developments. Finally, the importance of cell seeding methods is also addressed.

Tissue engineering and regenerative medicine are interdisciplinary fields that apply principles of engineering and life sciences to develop biological substitutes, typically composed of biological and synthetic components, that restore, maintain or improve tissue function. Many tissue engineering technologies are still at a laboratory or pre-commercial scale. The short review paper describes the most significant manufacturing and bio-process challenges inherent in the commercialisation and exploitation of the exciting results emerging from the biological and clinical laboratories exploring tissue engineering and regenerative medicine. A three-generation road map of the industry has been used to structure a view of these challenges and to define where the manufacturing community can contribute to the commercial success of the products from these emerging fields. The first-generation industry is characterised by its demonstrated clinical applications and products in the marketplace, the second is characterised by emerging clinical applications, and the third generation is characterised by aspirational clinical applications. The paper focuses on the cost reduction requirement of the first generation of the industry to allow more market penetration and consequent patient impact. It indicates the technological requirements, for instance the creation of three-dimensional tissue structures, and value chain issues in the second generation of the industry. The third-generation industry challenges lie in fundamental biological and clinical science. The paper sets out a road map of these generations to identify areas for research.

Investigation of the role of extracellular nucleotides in nervous system has been one of the main topics of the P2Y receptor research throughout the years. In parallel to numerous studies on primary culture systems, various neuronal and non-neuronal cell lines have been used to model in vitro the processes mediated by extracellular nucleotides. In this review article, a survey of expression profiles of G protein-coupled P2Y receptor subtypes in nervous-system-derived cell lines is presented, by analysing the receptor expression at the mRNA, protein, and functional level. The variability of receptor expression profiles in established cell lines is further discussed, bringing forward some general properties for neuronal and glial malignant cell lines.

Embryonic stem cells (ESCs), which are isolated from the inner cell mass of the blastocyst stage embryo, have the potential to give rise to an entire organism and to generate every body cell type. Much improvement has been made in the field of induction and differentiation of ESCs during the last two years, such as the ESCs differentiation into germ cells (2003) and the cloning of human ESCs (2004), both of which were chosen respectively as one of the top ten achievements evaluated by academic journals. Great attention was also paid to the research of the new genes which could maintain ESCs in the undifferentiated state and the research of the induction and differentiation of ESCs.

This review analyzes activities in the field of marine invertebrate cell culture during the years 1999 to 2004 and compares the outcomes with those of the preceding decade (1988 to 1998). During the last 5 years, 90 reports of primary cell culture studies of marine organisms belonging to only 6 taxa (Porifera, Cnidaria, Crustacea, Mollusca, Echinodermata, and Urochordata) have been published. This figure represents a 2-fold increase in the annual number of publications over the decade 1988 to 1998. Three other trends distinguish the two reviewed periods. First, in recent years studies attempting to improve cell culture methodologies have decreased, while interest in applications of already existing methodologies has increased. This reflects the effects of short-term cultures in attracting new researchers and scientific disciplines to the field. Second, only 17.8% of the recent publications used long-term cultures, compared with 30.0% of the publications in the previous decade. Third, during recent years research in cell cultures has studied fewer model species more extensively (mainly, Botryllus schlosseri, Crassostrea, Mytilus, Penaeus, and Suberites domuncula), signifying a shift from previous investigations that had studied a more diverse range of organisms. From 1988 to 1998 the phylum Mollusca was the most studied taxon (34.4%), but recent years have seen more studies of Porifera and Crustacea (30.0% and 32.2% of publications) than of Mollusca (21.1%). Still, not even a single established cell line from any marine invertebrate has yet been made available. However, the use of new cellular, genomic, and proteomic tools may fundamentally change our strategy for the development of cell cultures from marine invertebrates.

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number of approved biotherapeutics increases.

The developmental stages involved in the conversion of stem cells to fully functional neurons of specific neurotransmitter phenotype are complex and not fully understood. Over the past decade many studies have been published that demonstrate that in vitro manipulation of the epigenetic environment of the stem cells allows experimental control of final neuronal phenotypic choice. This review presents the evidence for the involvement of a number of endogenous neurobiochemicals, which have been reported to potently influence DAergic (and other neurotransmitter) phenotype expression in vitro. They act at different stages on the pathway to neurotransmitter phenotype determination, and in different ways. Many are better known for their involvement in other aspects of development, and in other biochemical roles. Their proper place, and precise roles, in neurotransmitter phenotype determination in vivo will no doubt be determined in the future. Meanwhile, considerable medical benefits are offered from producing large, long-term, viable cryostores of self-regenerating multipotential neural precursor cells (i.e., brain stem cells), which can be used for cell replacement therapies in the treatment of degenerative brain diseases, such as Parkinson's disease.

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.

Low-density cultures of embryonic rat hippocampal neurons have been widely used to investigate localization and function of neuronal proteins using immunocytochemistry and electrophysiology. These cultures provide a relatively homogeneous population of hippocampal pyramidal neurons and interneurons compared to post-natal mixed neuron/glial cultures from hippocampus, cerebral cortex, and cerebellum. However, the limited quantity of neurons and the difficulty in harvesting adequate amounts makes biochemical analyses of endogenous neuronal proteins in these low-density cultured neurons difficult. Here, we provide detailed methods to prepare cultures of embryonic rat hippocampal neurons suitable for biochemical analyses of both endogenously and exogenously expressed proteins. The procedures described here are also suitable for comprehensive studies of expression, localization, post-translational modification, and function of neuronal proteins in the same neuronal culture system.

The concept of encapsulated-cell therapy is very appealing, but in practice a great deal of technology and know-how is needed for the production of long-term functional transplants. Alginate is one of the most promising biomaterials for immunoisolation of allogeneic and xenogeneic cells and tissues (such as Langerhans islets). Although great advances in alginate-based cell encapsulation have been reported, several improvements need to be made before routine clinical applications can be considered. Among these is the production of purified alginates with consistently high transplantation-grade quality. This depends to a great extent on the purity of the input algal source as well as on the development of alginate extraction and purification processes that can be validated. A key engineering challenge in designing immunoisolating alginate-based microcapsules is that of maintaining unimpeded exchange of nutrients, oxygen and therapeutic factors (released by the encapsulated cells), while simultaneously avoiding swelling and subsequent rupture of the microcapsules. This requires the development of efficient, validated and well-documented technology for cross-linking alginates with divalent cations. Clinical applications also require validated technology for long-term cryopreservation of encapsulated cells to maintaining a product inventory in order to meet end-user demands. As shown here these demands could be met by the development of novel, validated technologies for production of transplantation-grade alginate and microcapsule engineering and storage. The advances in alginate-based therapy are demonstrated by transplantation of encapsulated rat and human islet grafts that functioned properly for about 1 year in diabetic mice.

Synthetic bioactive and bioresorbable composite materials are becoming increasingly important as scaffolds for tissue engineering. Next-generation biomaterials should combine bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration, stimulating the body to heal itself and leading to replacement of the scaffold by the regenerating tissue. Certain bioactive ceramics such as tricalcium phosphate and hydroxyapatite as well as bioactive glasses, such as 45S5 Bioglass, react with physiologic fluids to form tenacious bonds with hard (and in some cases soft) tissue. However, these bioactive materials are relatively stiff, brittle and difficult to form into complex shapes. Conversely, synthetic bioresorbable polymers are easily fabricated into complex structures, yet they are too weak to meet the demands of surgery and the in vivo physiologic environment. Composites of tailored physical, biologic and mechanical properties as well as predictable degradation behavior can be produced combining bioresorbable polymers and bioactive inorganic phases. This review covers recent international research presenting the state-of-the-art development of these composite systems in terms of material constituents, fabrication technologies, structural and bioactive properties, as well as in vitro and in vivo characteristics for applications in tissue engineering and tissue regeneration. These materials may represent the effective optimal solution for tailored tissue engineering scaffolds, making tissue engineering a realistic clinical alternative in the near future.

This review looks at potential developments in medical devices which may be based upon nanofeaturing implant and tissue engineering scaffolds, and describes the basic science upon which such expectations are based.

Because the liver is a multifunctional and a vital organ for survival, the management of acute liver failure requires the support of a huge number of metabolic functions performed by the organ. Many early detoxification-based artificial liver techniques failed to treat the patients owing to the inadequate support of the many essential hepatic functions. For this reason, a bioartificial liver (BAL) comprising of viable hepatocytes on a mechanical support is believed to more likely provide these essential functions than a purely mechanical device. From 1990, nine clinical studies of various BAL systems have been reported, most of which utilize a hollow fiber technology, and a much larger number of various BAL systems have been suggested to show an enhanced performance. Safety issues such as immunological reactions, zoonosis and tumorgenicity have been successfully addressed for regulatory approval, but a recent report from a large-scale, randomized, and controlled phase III trial of a leading BAL system (HepatAssist) failed to meet our expectation of efficacy in terms of the overall survival rate. In this paper, we review the current BAL systems actively studied and discuss critical issues such as the hepatocyte bioreactor configuration and the hepatocyte source. On the basis of the insights gained from previously developed BAL systems and the rapid progress in stem cell technology, the short-term and long-term future perspectives of BAL systems are suggested.

The electrical activity of living cells can be monitored in various ways, but for the study of ion channels and the drugs that affect them, the patch-clamp techniques are the most sensitive. Recent developments in microfabricated patch-clamp electrodes are reviewed, and technical challenges for the future are discussed.

Tissue culture has been and continues to be widely used in medical research. Since the beginning of central nervous system (CNS) tissue culture nearly 100 years ago, the scientific community has contributed innumerable protocols and materials leading to the current wide variety of culture systems. While nonhuman cultures have traditionally been more widely used, interest in human CNS tissue culture techniques has accelerated since the middle of the last century. This has been fueled largely by the desire to model human physiology and disease in vitro with human cells. We review the history of human CNS tissue culture summarizing advances that have led to the current breadth of options available. The review addresses tissue sources, culture initiation, formats, culture ware, media, supplements and substrates, and maintenance. All of these variables have been influential in the development of culturing options and the optimization of culture survival and propagation.

Increases in reconstructive orthopaedic surgery, such as total hip replacement and spinal fusion, resulting from advances in surgical practice and the ageing population, have lead to a demand for bone graft that far exceeds supply. Consequently, a number of synthetic bone-graft substitutes (BGSs) have been developed with mixed success and surgical acceptance. Skeletal tissue regeneration requires the interaction of three basic elements: cells, growth factors (GFs) and a permissive scaffold. This can be achieved by pre-loading a synthetic scaffold with GFs or pre-expanded cells; however, a 'simpler' approach is to design intrinsic 'osteoinductivity' into your BGS, i.e. the capability to recruit and stimulate the patient's own GFs and stem cells. Through investigation of the mechanisms controlling bone repair in BGSs, linking interactions between the local chemical and physical environment, scientists are currently developing osteoinductive materials that can stimulate bone regeneration through control of the scaffold chemistry and structure. Moreover, this body of research is providing the foundations for future generations of BGSs and bone-repair therapies and may ultimately contribute towards improving the quality of life through maintenance of the skeleton and reversal of disease states, as opposed to the mending of broken bones that we currently practice. Will we be able to grow our own bones in a bioreactor for use as autologous graft materials in the future? Could surgery be limited to accidental trauma cases, with greater restoration of function through biochemical or gene therapies? The technology and research probes necessary to this task are currently being developed with the advent of nanotechnology, genomics and proteomics: are we about to embark on a chemical revolution in medicine? This paper aims to discuss some of the current thinking on the mechanisms behind bioactivity and biocompatibility in bone and how a fuller understanding of the interactions between cells and the materials used today could bring about completely new approaches for the treatment of bone fracture and disease tomorrow.

Hybrid models aim to describe different components of a process in different ways. This makes sense when the corresponding knowledge to be represented is different as well. In this way, the most efficient representations can be chosen and, thus, the model performance can be increased significantly. From the various possible variants of hybrid model, three are selected which were applied for important biotechnical processes, two of them from existing production processes. The examples show that hybrid models are powerful tools for process optimisation, monitoring and control.

Current trends in the development of methods for monitoring, modeling and controlling biological production systems are reviewed from a bioengineering perspective. The ability to measure intracellular conditions in bioprocesses using genomics and other bioinformatics tools is addressed. Devices provided via micromachining techniques and new real-time optical technology are other novel methods that may facilitate biosystem engineering. Mathematical modeling of data obtained from bioinformatics or real-time monitoring methods are necessary in order to handle the dense flows of data that are generated. Furthermore, control methods must be able to cope with these data flows in efficient ways that can be implemented in plant-wide computer communication systems.

Heart valve disease is a significant medical problem worldwide. Current treatment for heart valve disease is heart valve replacement. State of the art replacement heart valves are less than ideal and are associated with significant complications. Using the basic principles of tissue engineering, promising alternatives to current replacement heart valves are being developed. Significant progress has been made in the development of a tissue-engineered semilunar heart valve substitute. Advancements include the development of different potential cell sources and cell-seeding techniques; advancements in matrix and scaffold development and in polymer chemistry fabrication; and the development of a variety of bioreactors, which are biomimetic devices used to modulate the development of tissue-engineered neotissue in vitro through the application of biochemical and biomechanical stimuli. This review addresses the need for a tissue-engineered alternative to the current heart valve replacement options. The basics of heart valve structure and function, heart valve disease, and currently available heart valve replacements are discussed. The last 10 years of investigation into a tissue-engineered heart valve as well as current developments are reviewed. Finally, the early clinical applications of cardiovascular tissue engineering are presented.

For patients with severe coronary artery and distal peripheral vascular disease not amenable to angioplasty and lacking sufficient autologous vessels there is a pressing need for improvements to current surgical bypass options. It has been decades since any real progress in bypass material has reached mainstream surgical practice. This review looks at possible remedies to this situation. Options considered are methods to reduce prosthetic graft thrombogenicity, including endothelial cell seeding and developments of new prosthetic materials. The promise of tissue-engineered blood vessels is examined with a specific look at how peptides can improve cell adhesion to scaffolds.

Tissue engineering offers new opportunities for meniscus repair and replacement. In this field different approaches are being studied to genererate a meniscus subsitute by a combination of a matrix scaffold, cells and specific stimuli. MENISCUS REPLACEMENT BY ACELLUAR MATRICES: For meniscus replacement the matrix material has to meet high biomechanical demands. Besides implant geometry, the material properties and a secure intraarticular attachment are important preconditions for implant function. A collagen scaffold has already been applied clinically for partial meniscus replacement. Scaffolds made of synthetic, bioabsorbable polymers and small intestine submucosa have been employed in animal studies. Following implantation, matrices are invaded by cells and undergo a process of remodeling. Formation of fibrocartilage repair tissue has been observerd. The biomechanical quality of implants and their effect on cartilage preservation have to be studied further. CELLULAR SEEDING OF MATRICES FOR MENSICUS REPLACEMENT: Advanced biological and biomechanical implant quality might be achieved by seeding matrices with cells in vitro. However, the ideal type of cell for this purpose has not yet been identified. Autologous mensicus cells, articular chondrocytes and mesenchymal stem cells represent possible cellular sources. Additional stimuli, such as cytokines and mechanical forces, and techniques of genetic engineering might further contribute to enhance the quality of engineered tissue.

The better repair of human tissue is an urgent medical goal and in order to achieve a safe outcome there is a parallel need for sensitive, non-invasive methods of assessing the quality of the engineered tissues and organs prior to surgical implantation. Optical coherence tomography (OCT) can potentially fulfil this role. The current status of OCT as an advanced imaging tool in clinical medicine, developmental biology and material science is reviewed and the parallels to the engineering of living tissue and organs are discussed. Preliminary data are also presented for a tissue engineering bioreactor with in situ OCT imaging. The data suggest that OCT can be utilized as a real time, non-destructive, non-invasive tool to critically monitor the morphology of tissue-engineered constructs during their fabrication and growth.

Animal models have been used extensively to investigate the biology of fracture healing and spinal fusion. The goal of each spinal fusion model is to try and reproduce the correct sequence of events during osseous healing in humans. Animal models allow us the capability of dialing in fusion rates and fusion parameters depending upon the study conditions. These models have become invaluable in assessing the clinical potential of emerging technologies such as recombinant growth factors and gene therapy.

In this paper, we discuss the current knowledge and achievements on bone tissue engineering with regard to spinal fusion and highlight the technique that employs hybrid constructs of porous scaffolds with bone marrow stromal cells. These hybrid constructs potentially function in a way comparable to the present golden standard, the autologous bone graft, which comprises besides many other factors, a construct of an optimal biological scaffold with osteoprogenitor cells. However, little is known about the role of the cells in autologous grafts, and especially survival of these cells is questionable. Therefore, more research will be needed to establish a level of functioning of hybrid constructs to equal the autologous bone graft. Spinal fusion models are relevant because of the increasing demand for graft material related to this procedure. Furthermore, they offer a very challenging environment to further investigate the technique. Anterior and posterolateral animal models of spinal fusion are discussed together with recommendations on design and assessment of outcome parameters.

Bone is a tissue with a strong regenerative potential. New strategies for tissue engineering of bone should therefore only focus on defects with a certain size that will not heal spontaneously. In the development of tissue-engineered constructs many variables may play a role, e.g. the source of the cells used, the design and mechanical properties of the scaffold and the concentration and mode of application of growth factor(s). Models for studying new strategies for tissue engineering of bone should be based on the target tissue to be restored. However, in light of the many potential variables, which may also interact if used in combination(s), there is also a large need for relatively simple models in which variables can be tested in a limited number of animals. Moreover, in compromised bone there may be a problem with the load-bearing capacity of the remaining healthy bone. In this light, an important prerequisite for tissue-engineering constructs is that they can be tested in loaded conditions. Particularly, this latter prerequisite is very difficult to achieve. Therefore, in vitro tests for mechanical stability are very useful for evaluating the mechanical consequences of a particular reconstruction procedure prior to the animal experiment. Before a tissue-engineered construct can be introduced into a clinical trial, a final test should be available in a large animal model that is as close and relevant to a particular problematic clinical situation as possible.In the past, a series of models were developed in our laboratory that are very useful for testing tissue-engineered constructs. In this paper, we focus on the use of relatively new simple in vitro and in vivo models for hip revision surgery, segmental bone defect restoration and tumour surgery.

The purpose of this article is to inform newcomers on the most important aspects of experimentation with living cells and tissues in space laboratories and platforms. There are strong arguments that justify the efforts and investments in such activity. Experimentation in space is subject to safety and technological constraints that require considerable attention to the development of the flight protocols and of the flight instrumentation. Nevertheless to fly an experiment in space is a unique opportunity to study living systems under conditions not reproducible on Earth and it is also a contribution to human exploration of space. Thereby important progress in basic and applied science can be expected. Parallel investigations on ground with devices averaging the exposure to the gravity vector but not reproducing microgravity shall always be part of a space flight project.

Many technologies have been developed to help explain the phenotypic consequences of genetic and/or environmental modifications in areas like functional genomics, pharmaceutical research and metabolic engineering. The missing link in contemporary functional analyses that focus on the analysis of cellular components is the capacity to directly observe functional units. By linking genes and proteins to higher level biological functions, the molecular fluxes through metabolic networks (the fluxome) determine the cellular phenotype. Quantitative monitoring of such whole network operations by methods of metabolic flux analysis, thus bridges the gap by providing a global perspective of the integrated regulation at the transcriptional, translational and metabolic level. This review highlights recent developments towards high-throughput flux analysis.

Many efforts are currently underway to try and mimic the properties of single cells with the aim of designing chips that are as efficient as cells. However, cells are nature's nanotechnology engineering at the scale of atoms and molecules, and it might be better to envision a microchip that utilizes a single cell as an experimentation platform. A novel, so-called laboratory-in-a-cell concept has been described, where advantage is taken of micro- and nanotechnological tools to enable precise control of the biochemical cellular environment; these tools also offer the possibility to analyse the composition of single cells. Methods for single-cell handling and analysis are being developed and will be required for this concept to progress further.

Based on current average yields of wheat per hectare and the saccharide content of wheat grain, it is feasible to produce wheat-based alternatives to many petrochemicals. However, the requirements in terms of wheat utilization would be equivalent to 82% of current production if intermediates and primary building blocks such as ethylene, propylene, and butadiene were to be produced in addition to conventional bioproducts. If only intermediates and bioproducts were produced this requirement would fall to just 11%, while bioproducts alone would require only 7%. These requirements would be easily met if the global wheat yield per hectare of cultivated land was increased from the current average of 2.7 to 5.5 tonnes ha(-1) (well below the current maximum). Preliminary economic evaluation taking into account only raw material costs demonstrated that the use of wheat as a generic feedstock could be advantageous in the case of bioproducts and specific intermediate petrochemicals. Gluten plays a significant role considering the revenue occurring when it is sold as a by-product. A process leading to the production of a generic fermentation feedstock from wheat has been devised and evaluated in terms of efficiency and economics. This feedstock aims at providing a replacement for conventional fermentation media and petrochemical feedstocks. The process can be divided into four major stages--wheat milling; fermentation of whole wheat flour by A. awamori leading to the production of enzymes and fungal cells; glucose enhancement via enzymatic hydrolysis of flour suspensions; and nitrogen/micronutrient enhancement via fungal cell autolysis. Preliminary costings show that the operating cost of the process depends on plant capacity, cereal market price, presence and market value of added-value by-products, labour costs, and mode of processing (batch or continuous).

The risk of xenozoonosis infections poses the greatest obstacle against the clinical application of a hybrid artificial liver support system (HALSS). Primary human hepatocytes are an ideal source for HALSS, but the shortage of human livers available for hepatocyte isolation limits this modality. To resolve this issue, we previously demonstrated the upregulation of hepatocyte-specific function by spheroid formation in polyurethane foam and by culturing with the histone deacetylase inhibitor, trichostatin A (TSA), in a human hepatoma cell line (Huh 7). In this article we analyze the gene expression profile using cDNA microarray (1281 genes) in spheroid formation or culturing with TSA in Huh 7 to determine the target genes in hepatocyte differentiation. In both the spheroid formation and in the culture with TSA, the Oct-3/4 transcription factor was upregulated more thantwofold, while the early growth response-1 (EGR-1) transactivator was downregulated less than 0.5-fold. These results indicate that expressions of Oct-3/4 and EGR-1 may be key factors in the induction of hepatocyte differentiation in Huh 7.

A variety of whole-cell-based biosensors has been developed using numerous native and recombinant biosensing cells. The use of reporter genes, for example bacterial luciferase and gfp, to monitor gene expression is discussed in terms of each reporters' benefits and disadvantages, including their possible use on-line, their sensitivity, the need for extra substrate, etc. All biosensing cells in use can be classified into two groups in terms of their biosensing mechanisms--constitutive expression and stress- or chemical-specific inducible expression. In this review several examples of each are presented and discussed. The use of recombinant whole-cell biosensors in the field requires three components--biosensing cells, a measurement device, and a signal-transducing apparatus, the last two depending on the first and the final applications of the system. The use of different immobilization techniques in several studies to maintain the cells and their viability is also discussed, in particular their use in the development of both high-throughput and chip-based biosensing systems. Finally the application of whole-cell-based biosensors to different environmental media, such as water, soil, and atmospheric monitoring is discussed; particular attention is given to their use for detection of various stressors, including dioxins, endocrine-disrupting chemicals, and ionizing radiation.

The major task of modern cell biology is to identify the function and relation of the many different gene products, discovered by genomics and proteomics approaches, in the context of the living cell. To achieve this goal, an increasing toolbox of custom-designed biosensors based on fluorescent labels is available to study the molecular activities of the cellular machinery. An overview of the current status of the young field of molecular-cellular physiology is presented that includes the application of fluorescent labels in the design of biosensors and the major detection schemes used to extract their sensing information. In particular, the use of the photophysical phenomenon of Förster resonance energy transfer (FRET) as a powerful indicator of cellular biochemical events is discussed. In addition, we will point out the challenges and directions of the field and project the short-term future for the application of fluorescence-based biosensors in biology.

Changes in the composition of media used for human in vitro fertilization and embryo culture are reviewed, and their impact on improved pregnancy rates is discussed. It is concluded that changes to the formulation of culture media for assisted reproductive technologies over the past decade have played a significant role in improving outcomes from assisted reproductive technologies procedures involving the culture of embryos.

Plant cells catalyze multiple-step reactions of secondary metabolite biosynthesis, and selectively synthesize chiral compounds with polycyclic structures. Taking advantage of this characteristic, we studied the production of the anticancer drug paclitaxel, which is currently produced in limited supply. Callus culture investigations indicate that woody plant medium supplemented with 10(-5) mol L(-1) 1-naphthylacetic acid and without the NH4+ -type ion is the best condition for growth of the callus. The accumulation of paclitaxel and related taxanes in Taxus plants is thought to be a biological response to specific external stimuli. Several signal transducers were screened; taxane biosynthesis was strongly promoted by methyl jasmonate (MeJA) and silver thiosulfate (STS) as an anti-ethylene compound. Of ten taxane-type diterpenoids isolated from T. baccata suspension-cultured cells treated with MeJA, five have a phenylisoserine side-chain at the C-13 position of the taxane skeleton. Time-course analysis revealed two regulatory steps in taxane biosynthesis: the taxane-ring formation step and the acylation step of the C-13 position. Methyl jasmonate promoted the formation of the taxane-ring. The production of paclitaxel reached a maximum level of 295 mg L(-1) in a large-scale culture of T. x media cells using a two-stage process.

Human embryonic stem (ES) cells provide a unique model and an important resource to analyze early hematopoietic development. Other systems to study mammalian hematopoiesis include mouse ES cells, dissection of timed mouse embryos, or use of human postnatal hematopoietic tissue typically isolated from bone marrow or umbilical cord blood. All these models have particular strengths and weaknesses. The extensive studies on murine hematopoiesis provide a basis for work on the human developmental system. Since there are likely some important species differences, use of human ES cells now provides an optimal means to evaluate basic cellular and molecular mechanisms that regulate the beginning stages of human blood development, prior to derivation of hematopoietic stem cells (HSCs). Eventually, research on human ES cells may provide an alternative source of HSCs and other blood products for hematopoietic cell transplantation or other cellular therapies.

This perspective summarizes several important advances in hematopoietic stem cell (HSC) biology in the past few years and places these advances in the context of future directions in stem cell research. The potential utility of stem cells for gene therapy, tissue engineering, and the treatment of neurological and other forms of disease is simply too significant to ignore, and yet our knowledge and ability to deliver these forms of therapy in a safe and efficacious manner will require additional advances in the understanding of the basic biology of stem cells.

Cryogels are gel matrices that are formed in moderately frozen solutions of monomeric or polymeric precursors. Cryogels typically have interconnected macropores (or supermacropores), allowing unhindered diffusion of solutes of practically any size, as well as mass transport of nano- and even microparticles. The unique structure of cryogels, in combination with their osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of biological nanoparticles (plasmids, viruses, cell organelles) and even whole cells. Polymeric cryogels are efficient carriers for the immobilization of biomolecules and cells.