Showing papers in "Advances in Biochemical Engineering \/ Biotechnology in 2012"

Adipose-Derived Mesenchymal Stem Cells: Biology and Potential Applications

Abstract: Adipose tissue is derived from the mesoderm during embryonic development and is present in every mammalian species, located throughout the body. Adipose tissue serves as an endocrine organ, functioning to maintain energy metabolism through the storage of lipids. While two types of adipose tissue exist (brown and white), white adipose yields the commonly studied adipose-derived stem cells (ASCs). Adipose-derived stem cells provide a promising future in the field of tissue engineering and regenerative medicine. Due to their wide availability and ability to differentiate into other tissue types of the mesoderm—including bone, cartilage, muscle, and adipose—ASCs may serve a wide variety of applications. Adipose stem cells have been utilized in studies addressing osteoarthritis, diabetes mellitus, heart disease, and soft tissue regeneration and reconstruction after mastectomy and facial repair. Various delivery systems and scaffolds to incorporate adipose stem cells have also been established. Adipose stem cells have been studied in vitro and in vivo. Much information in vitro has been obtained on adipose stem cell potency and biology as a function of donor gender, body mass index, and anatomical location. Further in vitro studies have examined the various cell populations within the heterogeneous population within the stromal vascular fraction (SVF) from which ASCs are obtained. While many animal models are used to investigate adipose tissue, preclinical in vivo experiments are most widely conducted in the mouse model. Common analyses of animal studies utilizing ASCs include pre-labeling cells and immunostaining cells.

Automatic control of bioprocesses.

Abstract: In this chapter, different approaches for open-loop and closed-loop control applied in bioprocess automation are discussed. Although in recent years many contributions dealing with closed-loop control have been published, only a minority were actually applied in real bioprocesses, the majority being simulations. As a result of the diversity of bioprocess requirements, a single control algorithm cannot be applied in all cases; rather, different approaches are necessary. Most publications combine different closed-loop control techniques to construct hybrid systems. These systems are supposed to combine the advantages of each approach into a well-performing control strategy. The majority of applications are soft sensors in combination with a proportional-integral-derivative (PID) controller. The fact that soft sensors have become this importance for control purposes demonstrates the lack of direct measurements or their large additional expense for robust and reliable online measurement systems. The importance of model predictive control is increasing; however, reliable and robust process models are required, as well as very powerful computers to address the computational needs. The lack of theoretical bioprocess models is compensated by hybrid systems combining theoretical models, fuzzy logic, and/or artificial neural network methodology. Although many authors suggest a possible transfer of their presented control application to other bioprocesses, the algorithms are mostly specialized to certain organisms or certain cultivation conditions as well as to a specific measurement system.

Cellulolytic Enzyme Production and Enzymatic Hydrolysis for Second-Generation Bioethanol Production

Abstract: Second-generation bioethanol made from lignocellulosic biomass is considered one of the most promising biofuels. However, the enzymatic hydrolysis of the cellulose component to liberate glucose for ethanol fermentation is one of the major barriers for the process to be economically competitive because of the recalcitrance of feedstock. In this chapter, the progress on the understanding of the mechanisms of lignocellulose degradation, as well as the identification and optimization of fungal cellulases, cellulolytic strains, and cellulase production is reviewed. The physiologic functions and enzymatic mechanisms of two groups of enzymes involved in lignocellulose degradation, cellulases and hemicellulases, are discussed, and the synergism of the cellulase components during lignocellulose degradation is addressed. Furthermore, the methods for screening filamentous fungal strains capable of degrading lignocellulose are evaluated and the production of cellulases by these fungal strains is discussed. Aside from traditional mutagenesis for improving the secretion level and enzymatic activities of cellulases from filamentous fungal species, genetic engineering of strains and protein engineering on cellulase molecules are also highlighted.

A highly versatile microscope imaging technology platform for the multiplex real-time detection of biomolecules and autoimmune antibodies.

Abstract: The analysis of different biomolecules is of prime importance for life science research and medical diagnostics. Due to the discovery of new molecules and new emerging bioanalytical problems, there is an ongoing demand for a technology platform that provides a broad range of assays with a user-friendly flexibility and rapid adaptability to new applications. Here we describe a highly versatile microscopy platform, VideoScan, for the rapid and simultaneous analysis of various assay formats based on fluorescence microscopic detection. The technological design is equally suitable for assays in solution, microbead-based assays and cell pattern recognition. The multiplex real-time capability for tracking of changes under dynamic heating conditions makes it a useful tool for PCR applications and nucleic acid hybridization, enabling kinetic data acquisition impossible to obtain by other technologies using endpoint detection. The paper discusses the technological principle of the platform regarding data acquisition and processing. Microbead-based and solution applications for the detection of diverse biomolecules, including antigens, antibodies, peptides, oligonucleotides and amplicons in small reaction volumes, are presented together with a high-content detection of autoimmune antibodies using a HEp-2 cell assay. Its adaptiveness and versatility gives VideoScan a competitive edge over other bioanalytical technologies.

Expansion of Mesenchymal Stem/Stromal cells under xenogenic-free culture conditions.

Abstract: Mesenchymal Stem/Stromal cells (MSCs) are increasingly applied in cell-based regenerative medicine. To yield clinically relevant cell doses, ex vivo expansion of MSCs is required to be compliant with good manufacturing practice (GMP) guidelines. A lack of standardization and harmonization seems to hamper rapid progress in the translational phase. Most protocols still use fetal bovine serum (FBS) to expand MSCs. However, the high lot-to-lot variability, risk of contamination and immunization call for xenogenic-free culture conditions. Chemically defined media are the ultimate achievement in terms of standardization. These media, however, need to maintain all key cellular and therapy-relevant features of MSCs. Because of the numerous constituents of FBS, the development of such chemically defined media with an optimal composition of the few essential factors is only beginning. Meanwhile, various human blood-derived components are under investigation, including human plasma, human serum, human umbilical cord blood serum and human platelet derivatives such as platelet lysate.

MSC and Tumors: Homing, Differentiation, and Secretion Influence Therapeutic Potential

Abstract: Mesenchymal stromal/stem cells (MSC) are adult multipotent progenitors with fibroblast-like morphology able to differentiate into adipocytic, osteogenic, chondrogenic, and myogenic lineages. Due to these properties, MSC have been studied and introduced as therapeutics in regenerative medicine. Preliminary studies have also shown a possible involvement of MSC as precursors of cellular elements within tumor microenvironments, in particular tumor-associated fibroblasts (TAF). Among a number of different possible origins, TAF may originate from a pool of circulating progenitors from bone marrow or adipose tissue-derived MSC. There is growing evidence to corroborate that cells immunophenotypically defined as MSC are able to reside as TAF influencing the tumor microenvironment in a potentially bi-phasic and obscure manner: either promoting or inhibiting growth depending on tumor context and MSC sources. Here we focus on relationships between the tumor microenvironment, cancer cells, and MSC, analyzing their diverse ability to influence neoplastic development. Associated activities include MSC homing driven by the secretion of various mediators, differentiation towards TAF phenotypes, and reciprocal interactions with the tumor cells. These are reviewed here with the aim of understanding the biological functions of MSC that can be exploited for innovative cancer therapy.

RNA Aptamers: A Review of Recent Trends and Applications

Abstract: RNA aptamers, small oligonucleotides derived by an in-vitro selection process called SELEX (Systematic Evolution of Ligands by EXperimental enrichment), are important candidates for therapeutic and diagnostic applications. RNA aptamers have high affinity and specificity for their target molecules. In this review, we describe methods for generating RNA aptamers (the SELEX technique and modified SELEX processes) and therapeutic applications for diseases such as neovascular age-related macular degeneration (AMD), inflammatory diseases, and obesity. We also analyze the social networks among researchers and organizations (universities, research institutes, firms, etc.) that are active in the pursuit of aptamer-based therapeutic approaches. This study provides relevant information on recent research trends in RNA aptamers.

Potential for Neural Differentiation of Mesenchymal Stem Cells

Abstract: Adult human stem cells have gained progressive interest as a promising source of autologous cells to be used as therapeutic vehicles. Particularly, mesenchymal stem cells (MSCs) represent a great tool in regenerative medicine because of their ability to differentiate into a variety of specialized cells. Among adult tissues in which MSCs are resident, adipose tissue has shown clear advantages over other sources of MSCs (ease of surgical access, availability, and isolation), making adipose tissue the ideal large-scale source for research on clinical applications. Stem cells derived from the adipose tissue (adipose-derived stem cells = ADSCs) possess a great and unique regenerative potential: they are self-renewing and can differentiate along several mesenchymal tissue lineages (adipocytes, osteoblasts, myocytes, chondrocytes, endothelial cells, and cardiomyocytes), among which neuronal-like cells gained particular interest. In view of the promising clinical applications in tissue regeneration, research has been conducted towards the creation of a successful protocol for achieving cells with a well-defined neural phenotype from adipose tissue. The promising results obtained open new scenarios for innovative approaches for a cell-based treatment of neurological degenerative disorders.

CHO glycosylation mutants as potential host cells to produce therapeutic proteins with enhanced efficacy.

Abstract: CHO glycosylation mutants, pioneered by Stanley and co-workers, have proven to be valuable tools in glycobiology and biopharmaceutical research. Here we aim to provide a summary of our efforts to isolate industrially applicable CHO glycosylation mutants, termed CHO-gmt cells, using cytotoxic lectins and zinc-finger nuclease technology. The genetic defects in the glycosylation machinery in these cells lead to the production of recombinant glycoproteins with consistent and unique glycan structures. In addition, these mutant cells can be easily adapted to serum-free medium in suspension cultures, the condition used by the biotech industry for large-scale production of recombinant therapeutics. In light of the critical impact of glycosylation on biopharmaceutical performances, namely, safety and efficacy, the CHO-gmt lines have enormous potential in producing glycoprotein therapeutics with optimal glycosylation profiles, thus, representing a panel of ideal host cell lines for producing recombinant biopharmaceuticals with improved safety profiles and enhanced efficacy.

Cell-Free Biosystems for Biomanufacturing

Abstract: Although cell-free biosystems have been used as a tool for investigating fundamental aspects of biological systems for more than 100 years, they are becoming an emerging biomanufacturing platform in the production of low-value biocommodities (e.g., H(2), ethanol, and isobutanol), fine chemicals, and high-value protein and carbohydrate drugs and their precursors. Here we would like to define the cell-free biosystems containing more than three catalytic components in a single reaction vessel, which although different from one-, two-, or three-enzyme biocatalysis can be regarded as a straightforward extension of multienzymatic biocatalysis. In this chapter, we compare the advantages and disadvantages of cell-free biosystems versus living organisms, briefly review the history of cell-free biosystems, highlight a few examples, analyze any remaining obstacles to the scale-up of cell-free biosystems, and suggest potential solutions. Cell-free biosystems could become a disruptive technology to microbial fermentation, especially in the production of high-impact low-value biocommodities mainly due to the very high product yields and potentially low production costs.

Automated measurement and monitoring of bioprocesses: key elements of the M(3)C strategy.

Abstract: The state-of-routine monitoring items established in the bioprocess industry as well as some important state-of-the-art methods are briefly described and the potential pitfalls discussed. Among those are physical and chemical variables such as temperature, pressure, weight, volume, mass and volumetric flow rates, pH, redox potential, gas partial pressures in the liquid and molar fractions in the gas phase, infrared spectral analysis of the liquid phase, and calorimetry over an entire reactor. Classical as well as new optical versions are addressed. Biomass and bio-activity monitoring (as opposed to "measurement") via turbidity, permittivity, in situ microscopy, and fluorescence are critically analyzed. Some new(er) instrumental analytical tools, interfaced to bioprocesses, are explained. Among those are chromatographic methods, mass spectrometry, flow and sequential injection analyses, field flow fractionation, capillary electrophoresis, and flow cytometry. This chapter surveys the principles of monitoring rather than compiling instruments.

Fate of intravenously injected mesenchymal stem cells and significance for clinical application

Abstract: Mesenchymal stromal cells (MSCs) have initially been characterized as a fibroblastlike cell population that can be expanded readily in vitro, and is able to support hematopoiesis in vitro and in vivo. By serendipity it was discovered that MSCs can also be administered into the bloodstream. This mode of application formed a major breakthrough in the clinical use of MSCs, because MSC transplantation was found to cure severe immune hyperactivation states such as graft-versus-host disease after allogeneic bone marrow transplantation, or bacterial sepsis. However, MSCs were found difficult to trace and consensus to date is lacking in the scientific community as to where transplanted MSCs end up in the body and which major principles are responsible for the therapeutic effects of MSCs. This chapter gives an overview of the current knowledge on interactions of freshly transplanted MSCs with the cells in the blood stream and the vessel wall, with major organs such as lung, liver, gut, and spleen, and discusses the limitations of the methodologies used to trace transplanted MSCs. The findings will be put into perspective on how therapeutically applied, culture-expanded MSCs may exert beneficial effects.

Adult Mesenchymal Stem Cells Explored in the Dental Field

Abstract: During the last decade it was realized that stem cell-based therapies hold an enormous therapeutic potential, improving the life of patients with conditions ranging from neurodegenerative and traumatic diseases to regenerative medicine requiring replacement of complex structures such as bones and teeth. Based on their ability to regenerate and/or repair damaged tissue and eventually restore organ function, multiple types of stem/progenitor cells have been discovered. In the field of periodontal regeneration and tooth engineering, several types of adult multipotent mesenchymal stem cells from various sources are currently being investigated. These include the bone marrow stromal stem cells (BMSSCs), adipose-derived stromal cells (ADSCs), dental pulp stem cells (DPSCs), dental follicle stem cells (DFSCs), stem cells from human exfoliated deciduous teeth (SHEDs), stem cells from the apical papilla (SCAP), periodontal ligament stem cells (PDLSCs), alveolar bone proper-derived stem cells, and gingival stem cells. The potential of these different MSCs as precursors for regenerative purposes in the dental field is discussed in this chapter.

Interactions Between Mesenchymal Stem Cells and Dendritic Cells

Abstract: Mesenchymal stem or stromal cells (MSC) are considered a promising new therapeutic strategy for the treatment of several pathological conditions. Due to their immunomodulatory properties, they are currently employed in clinical trials aimed at preventing or treating steroid-resistant acute graft-versus-host disease (GvHD), a frequent complication of allogeneic hematopoietic stem cell transplantation (HSCT). In addition, the use of MSC has been proposed for the treatment of autoimmune diseases. A number of recent studies have focused on the influence of MSC on dendritic cell (DC) function. DCs play a critical role in initiating and regulating immune responses by promoting antigen-specific T cell activation. Moreover, they are involved in efficient cross-talk with different cells of the innate immune system. DC are the most effective antigen-presenting cells and prime naive T cells to initiate adaptive immune responses including those against allogeneic cells or self-antigens. Thus, alteration of DC generation or function may greatly contribute to the inhibition of T cell responses. In this context, MSC were shown to interfere with DC maturation from monocytes or CD34(+) hemopoietic precursors thus further confirming their role in immune regulation and their usefulness in cell-based therapies.

Multivariate Data Analysis for Advancing the Interpretation of Bioprocess Measurement and Monitoring Data

Abstract: The advances in measurement techniques, the growing use of high-throughput screening and the exploitation of ‘omics’ measurements in bioprocess development and monitoring increase the need for effective data pre-processing and interpretation. The multi-dimensional character of the data requires the application of advanced multivariate data analysis (MVDA) tools. An overview of both linear and non-linear MVDA tools most frequently used in bioprocess data analysis is presented. These include principal component analysis (PCA), partial least squares (PLS) and their variants as well as various types of artificial neural networks (ANNs). A brief description of the basic principles of each of the techniques is given with emphasis on the possible application areas within bioprocessing and relevant examples.

Impacts of Quorum Sensing on Microbial Metabolism and Human Health

Abstract: Bacteria were considered to be lonely ‘mutes’ for hundreds of years. However, recently it was found that bacteria usually coordinate their behaviors at the population level by producing (speaking), sensing (listening), and responding to small signal molecules. This so-called quorum sensing (QS) regulation enables bacteria to live in a ‘society’ with cell–cell communication and controls many important bacterial behaviors. In this chapter, QS systems and their signal molecules for Gram-negative and Gram-positive bacteria are introduced. Most interestingly, QS regulates the important bacterial behaviors such as metabolism and pathogenesis. QS-regulated microbial metabolism includes antibiotic synthesis, pollutant biodegradation, and bioenergy production, which are very relevant to human health. QS is also well-known for its involvement in bacterial pathogenesis, such as iin nfections by Pseudomonas aeruginosa and Staphylococcus aureus. Novel disease diagnosis strategies and antimicrobial agents have also been developed based on QS regulation on bacterial infections. In addition, to meet the requirements for the detection/quantification of QS signaling molecules for research and application, different biosensors have been constructed, which will also be reviewed here. QS regulation is essential to bacterial survival and important to human health. A better understanding of QS could lead better control/manipulation of bacteria, thus making them more helpful to people.

Potential for osteogenic and chondrogenic differentiation of MSC.

Abstract: The introduction of mesenchymal stem cells (MSC) into the field of tissue engineering for bone and cartilage repair is a promising development, since these cells can be expanded ex vivo to clinically relevant numbers and, after expansion, retain their ability to differentiate into different cell lineages. Mesenchymal stem cells isolated from various tissues have been intensively studied and characterized by many research groups. To obtain functionally active differentiated tissue, tissue engineered constructs are cultivated in vitro statically or dynamically in bioreactors under controlled conditions. These conditions include special cell culture media, addition of signalling molecules, various physical and chemical factors and the application of different mechanical stimuli. Oxygen concentration in the culture environment is also a significant factor which influences MSC proliferation, stemness and differentiation capacity. Knowledge of the different aspects which affect MSC differentiation in vivo and in vitro will help researchers to achieve directed cell fate without the addition of differentiation agents in concentrations above the physiological range.

Applying Mechanistic Models in Bioprocess Development

Abstract: The available knowledge on the mechanisms of a bioprocess system is central to process analytical technology. In this respect, mechanistic modeling has gained renewed attention, since a mechanistic model can provide an excellent summary of available process knowledge. Such a model therefore incorporates process-relevant input (critical process variables)-output (product concentration and product quality attributes) relations. The model therefore has great value in planning experiments, or in determining which critical process variables need to be monitored and controlled tightly. Mechanistic models should be combined with proper model analysis tools, such as uncertainty and sensitivity analysis. When assuming distributed inputs, the resulting uncertainty in the model outputs can be decomposed using sensitivity analysis to determine which input parameters are responsible for the major part of the output uncertainty. Such information can be used as guidance for experimental work; i.e., only parameters with a significant influence on model outputs need to be determined experimentally. The use of mechanistic models and model analysis tools is demonstrated in this chapter. As a practical case study, experimental data from Saccharomyces cerevisiae fermentations are used. The data are described with the well-known model of Sonnleitner and Kappeli (Biotechnol Bioeng 28: 927-937, 1986) and the model is analyzed further. The methods used are generic, and can be transferred easily to other, more complex case studies as well. (Less)

Mesenchymal Stem Cell Therapy and Lung Diseases

Abstract: Mesenchymal stem cells (MSCs), a distinct population of adult stem cells, have amassed significant interest from both medical and scientific communities. An inherent multipotent differentiation potential offers a cell therapy option for various diseases, including those of the musculoskeletal, neuronal, cardiovascular and pulmonary systems. MSCs also secrete an array of paracrine factors implicated in the mitigation of pathological conditions through anti-inflammatory, anti-apoptotic and immunomodulatory mechanisms. The safety and efficacy of MSCs in human application have been confirmed through small- and large-scale clinical trials. However, achieving the optimal clinical benefit from MSC-mediated regenerative therapy approaches is entirely dependent upon adequate understanding of their healing/regeneration mechanisms and selection of appropriate clinical conditions. MSC-mediated acute alveolar injury repair. A cartoon depiction of an injured alveolus with associated inflammation and AEC apoptosis. Proposed routes of MSC delivery into injured alveoli could be by either intratracheal or intravenous routes, for instance. Following delivery a proposed mechanism of MSC action is to inhibit/reduce alveolar inflammation by abrogation of IL-1_-depenedent Tlymphocyte proliferation and suppression of TNF-_ secretion via macrophage activation following on from stimulation by MSC-secreted IL-1 receptor antagonist (IL-1RN). The inflammatory environment also stimulates MSC to secrete prostaglandin-E2 (PGE2) which can stimulate activated macrophages to secrete the anti-inflammatory cytokine IL-10. Inhibition of AEC apoptosis following injury can also be promoted via MSC stimulated up-regulation of the anti-apoptotic Bcl-2 gene. MSC-secreted KGF can stimulate AECII proliferation and migration propagating alveolar epithelial restitution. Alveolar structural engraftment of MSC is a rare event.

Dissecting paracrine effectors for mesenchymal stem cells.

Abstract: There has been increasing interest in the application of mesenchymal stem cells (MSCs) in regenerative medicine in recent years. In this context, the beneficial effects of MSCs have been ascribed mainly to a paracrine action rather than to direct replacement of the injured tissue. Indeed, MSCs produce a great variety of trophic and immunomodulatory factors. In this chapter, we provide an overview of growth factors and chemokines involved in stimulation of cell proliferation, inhibition of apoptosis, enhancement of angiogenesis, and suppression of inflammatory and immune response. In addition, we discuss the emerging role of the extracellular vesicles released from MSCs as possible paracrine mediators.

New Cell-Based Therapy Paradigm: Induction of Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells into Pro-Inflammatory MSC1 and Anti-inflammatory MSC2 Phenotypes.

Abstract: Cell-based therapies (CBTs) are quickly taking hold as a revolutionary new approach to treat many human diseases. Among the cells used in these treatments, multipotent mesenchymal stromal cells, also often and imprecisely termed mesenchymal stem cells (MSC), are widely used because they are considered clinically safe, unique in their immune-modulating capabilities, easily obtained from adult tissues, and quickly expanded as well as stored. However, despite these established advantages, there are limiting factors to employing MSCs in these therapeutic strategies. Foremost is the lack of a general consensus on a definition of these cells, marring efforts to prepare homogeneous lots and more importantly complicating their in vitro and in vivo investigation. Furthermore, although one of the most profound clinical effects of MSC intravenous administration is the modulation of host immune responses, no adequate ex vivo assays exist to consistently predict the therapeutic effect of each MSC lot in the treated patient. Until these issues are addressed, this very promising and safe new therapeutic approach cannot be used to its full advantage. However, these confounding issues do present exciting opportunities. The first is an opportunity to discover unknown aspects of host immune responses because the unique effect driven by MSC infusion on a patient's immunity has not yet been identified. In addition, there is an opportunity to develop methods, tests, and tools to better define MSCs and MSC-based therapy and provide consistency in preparation and effect. To this end, my laboratory recently developed a new approach to induce uniform pro-inflammatory MSC1 and anti-inflammatory MSC2 phenotypes from bone marrow-derived MSC preparations. I anticipate that MSC1 and MSC2 provide convenient tools with which to address some of these limitations and will help advance safe and effective CBTs for human disease.

Prospective isolation and characterization of human bone marrow-derived MSCs.

Abstract: There is an increasing interest in adult stem cells, especially mesenchymal stem/stromal cells (MSCs), in hematology and regenerative medicine because of the simplicity of isolation and ex vivo expansion of these cells Conventionally, MSCs are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks This isolation procedure is hampered by the unpredictable influence of secreted molecules and interactions with co-cultured hematopoietic and other unrelated cells, as well as by the arbitrarily selected removal time of non-adherent cells prior to the expansion of MSCs Finally, functionally isolated cells do not provide biological information about the starting population To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression or absence of surface markers The isolation and ex vivo expansion of these cells require an adequate quality control of the source and product Here we summarize the most frequently used markers and introduce new targets for antibody-based isolation and characterization of bone marrow-derived MSCs

Urine as a source of stem cells.

Abstract: Traditionally, clinicians and researchers have relied on a skin biopsy or blood extraction as relatively accessible supplies for in vitro cell expansion and biological studies Perhaps surprisingly, limited attention has been given to a totally noninvasive source, urine, which eliminates the discomfort associated with other procedures This may arise from the perception that urine is merely a body waste Yet, the analysis of urine is a longstanding fundamental test for diagnostic purposes and nowadays there is growing interest in using urine for detecting biomarkers In addition, recent work including ours reinforces the idea that urine contains a variety of viable cell types with relevant applications In this review, we describe those cell types and their potential uses

The Choice of Suitable Online Analytical Techniques and Data Processing for Monitoring of Bioprocesses

Abstract: With increasing pressure from regulatory authorities on industry to develop processes embracing process analytical technology (PAT) initiatives, there is a growing demand to establish reliable tools and systems capable of meeting this need. With regard to monitoring and control of bioprocesses, this need translates to a search for robust instrumentation capable of monitoring the critical process parameters in real time. The application of such technologies at all stages of the process, from the initial R&D phase to process optimisation and production, enhances process understanding and paves the way for the development of control platforms. An examination of the PAT concept and selected tools (NIR, MIR, Raman, dielectric spectroscopy and calorimetry) are presented here. A description of each tool is given, with particular emphasis on the nature of the signal produced and how these relate to measurements of biomass, metabolites and product. A description of the signal processing that is necessary to gain meaningful results from the different tools is also given, together with online data reconciliation techniques based on mass and energy balances. Many techniques such as those based on vibrational spectroscopy are of particular interest, since they are capable of monitoring several critical process parameters which are typically controlled in a bioprocess. A window of application for each of the techniques, when used in the area of bioprocessing, is suggested based on their uses and inherent limitations.

Systems metabolic engineering: the creation of microbial cell factories by rational metabolic design and evolution.

Abstract: It is widely acknowledged that in order to establish sustainable societies, production processes should shift from petrochemical-based processes to bioprocesses. Because bioconversion technologies, in which biomass resources are converted to valuable materials, are preferable to processes dependent on fossil resources, the former should be further developed. The following two approaches can be adopted to improve cellular properties and obtain high productivity and production yield of target products: (1) optimization of cellular metabolic pathways involved in various bioprocesses and (2) creation of stress-tolerant cells that can be active even under severe stress conditions in the bioprocesses. Recent progress in omics analyses has facilitated the analysis of microorganisms based on bioinformatics data for molecular breeding and bioprocess development. Systems metabolic engineering is a new area of study, and it has been defined as a methodology in which metabolic engineering and systems biology are integrated to upgrade the designability of industrially useful microorganisms. This chapter discusses multi-omics analyses and rational design methods for molecular breeding. The first is an example of the rational design of metabolic networks for target production by flux balance analysis using genome-scale metabolic models. Recent progress in the development of genome-scale metabolic models and the application of these models to the design of desirable metabolic networks is also described in this example. The second is an example of evolution engineering with omics analyses for the creation of stress-tolerant microorganisms. Long-term culture experiments to obtain the desired phenotypes and omics analyses to identify the phenotypic changes are described here.

Migratory Properties of Mesenchymal Stem Cells

Abstract: Mesenchymal stem cells raise great expectations in regenerative medicine due to their capacity to regenerate damaged tissues, thereby restoring organ tissue integrity and functionality. Even though it is not yet clear how mesenchymal stem cells are guided to injured tissue it is generally assumed that the directed migration of these cells is facilitated by the same soluble factors that also recruit immune competent cells to inflamed tissue areas. Tumor tissue represents another type of (chronically) inflamed tissue and because of that mesenchymal stem cells are highly attracted. Although some data indicate that esenchymal stem cells might have a beneficial effect on tumor growth due to anti-tumor effects the plethora of data suggest that tumor tissue recruited mesenchymal stem cells rather promote tumor growth and metastasis formation. Nonetheless, the enhanced tumor tropism of mesenchymal stem cells makes them ideal candidates for novel anti-cancer strategies. Like Trojan Horses genetically modified mesenchymal stem cells will deliver their deadly cargo, such as anti-tumor cytokines or oncolytic viruses, into cancerous tissues, thereby destroying the tumor form within. In this chapter we will summarize the current concepts of genetic modification of mesenchymal stem cells for future anti-cancer therapies.

Platform technologies for molecular diagnostics near the patient's bedside.

Abstract: It is believed Lab-on-Chip systems will become a mainstream technology within the next centuries. Especially because of new findings in molecular medicine and global trends such as the changing global population in third world countries and an ageing population in industrial countries, the need for fast and reliable diagnostics is rising tremendously. Hence, diagnostics have to become more frequently and more easily available. In this regard, technologies have to be found that enable the cost-effective production and hence an affordable price. In a joint-project between seven Fraunhofer institutes a Lab-on-Chip system was developed which consists of a credit-card-sized cartridge and a base station. The cartridges contain besides the reagents necessary for a specific assay also functionalities such as pumping or heating enabling a self-contained system without any fluidic interfaces, which tend to be error-prone. Because of the modularity of the system it is possible to integrate an optical sensor as well an electrochemical sensor into the cartridge. Hence, the system can be customized to serve the needs of the particular assays. This chapter will describe the system including generic design rules for such Lab-on-Chip systems, the development of these rules into a modular Lab-on-Chip system, the integration of biomedical assays, and the production possibility of this system.

Microfluidic technology for molecular diagnostics.

Abstract: Molecular diagnostics have helped to improve the lives of millions of patients worldwide by allowing clinicians to diagnose patients earlier as well as providing better ongoing therapies Point-of-care (POC) testing can bring these laboratory-based techniques to the patient in a home setting or to remote settings in the developing world However, despite substantial progress in the field, there still remain many challenges Progress in molecular diagnostics has benefitted greatly from microfluidic technology This chapter aims to summarise the more recent advances in microfluidic-based molecular diagnostics Sections include an introduction to microfluidic technology, the challenges of molecular diagnostics, how microfluidic advances are working to solve these issues, some alternative design approaches, and detection within these systems

Role of the EU Framework in Regulation of Stem Cell-Based Products

Abstract: The use of stem cells for therapeutic purposes is regulated by two overlapping sets of rules. If used for transplantation, stem cells are covered by the collection, traceability and technical aspects of three European directives. When the stem cells are used as part of a medicinal product, they are covered by the legislation on pharmaceutical production and marketing authorization-in particular, by Regulation 1394/2007/EC.

Knowledge Management and Process Monitoring of Pharmaceutical Processes in the Quality by Design Paradigm

Abstract: Pharmaceutical processes are complex and highly variable in nature. The complexity and variability associated with these processes result in inconsistent and sometimes unpredictable process outcomes. To deal with the complexity and understand the causes of variability in these processes, in-depth knowledge and thorough understanding of the process and the various factors affecting the process performance become critical. This makes knowledge management and process monitoring an indispensable part of the process improvement efforts for any pharmaceutical organization.