Specific Scaffolds Developed for Cardiac Tissue Engineering

For cardiac tissue engineering, the ideal scaffolds should consider mixtures of different polymers to achieve the following properties (1) high porosity (large interconnected pores) to facilitate mass transport (2) bioadhesiveness to enhance cell attachment (3) structural stability to withstand both static and dynamic in-vitro cultivation (4) biodegradability to ensure tissue grafting (5) elasticity to enable the transmission of contractile forces and (6) conductivity to facilitate electrical...

Microfabrication Techniques to Generate Miniaturized Chip Components

Based on conventional microfabrication technologies (which include photolithography technology), it is now possible to generate a large variety of complex structured biochips (e.g., planar MEAs and multi-microcapillary arrays) comprising microchannels (for hydrodynamic cell and tissue positioning), interconnects, and up to thousands of microelectrodes and or field-effect transistors (FETs) for the detection of morphological and physiological alterations induced by drug and compound...

Matrices

Supporting matrices for the CCS need to be perfusable, cell-adhesive, and cell-migrational. In contrast to biohybrid implant technology (e.g., cartilage), biodegradability is irrelevant, but cell proliferation and the growth of organoids is necessary. Matrix sheets of nonwoven fibers (polyamides), hygrogels (peptides, agaroses, alginates, fibrin) and sponges (collagens) are particularly suitable for this purpose. Matrix performance is defined by the composition and structural properties on...

ExVivo Detection of Antigen Specific T Cells

Originally, the only means of detecting antigen-specific T cells was to separate the T lymphocytes from whole blood and to expose predetermined numbers of cells deposited into wells of microtiter plates to synthetic antigen plus growth stimulatory cytokines, such as interleukin-2 (IL-2) and or autologous feeder cells 50 . In order to convert this method into an assay for determining the number of antigen-specific cells, sequential dilution of T lymphocytes in 96-well plates, expansion of...

Modeling of Bioreactor Systems for Tissue Engineering

The appropriate molecular and macroscopic architecture of 3D tissue constructs is essential when producing a phenotypically appropriate tissue 8 . The exact local conditions experienced by the cells must be understood, yet in many cases the culture systems and bioreactors used for 3D tissue culture have not been optimized in this respect. Several parameters, such as perfusion rate, flow conditions, shear stress, and compression magnitude, have been varied, quite often by using a trial-and-error...

Conclusions for BBB InVitro Models

Recent advances in combinatorial chemistry and HTS for pharmacological activity have greatly expanded the number of drug candidates for the therapy of neurological diseases. Rapid screening for BBB penetration early in the drug discovery phase can streamline the quest for promising lead compounds, and provide guidance for the rational design and synthesis of novel compounds targeting the CNS. The tight barrier of the specialized endothelial cells forming lining the BBB will prevent most drugs...

An Overview on Bioreactor Design Prototyping and Process Control for Reproducible Three Dimensional Tissue Culture

Bioreactor systems play an important role in tissue engineering, as they enable reproducible and controlled changes to be made in specific environmental factors. They can also provide the technical means to perform controlled studies aimed at understanding specific biological, chemical, or physical effects. Furthermore, bioreactors allow for a safe and reproducible production of tissue constructs. For later clinical applications, the bioreactor system should be an advantageous method in terms...

Conclusions and Future Prospects

The use of human tissue is increasingly difficult due to increasing regulations. In addition, the variability of such materials limits their value for drug development and toxicology. It is to be hoped that cell-based systems will increasingly use human cell lines, many of which have qualities yet to be fully realized through the application of cell differentiation protocols and three-dimensional (3-D) cell culture methods. A number of cell differentiation protocols are available that can be...

Important Aspects for Bioreactor Design

With regard to tissue engineering, bioreactors are used for cell proliferation on a small scale (e.g., for individual patients) and on a large scale (e.g., for allogeneic therapy concepts), to generate 3D tissue constructs from isolated and proliferated cells in vitro and for direct organ-support devices 23 . These bioreactors should enable the control of environmental conditions such as oxygen tension, pH, temperature, and shear stress, as well as allowing aseptic operation (e.g., feeding and...

Human Cell Supply

Rather than collecting differentiated tissues from humans to reassemble each and every relevant sub-organoid structure in vitro, the expansion and differentiation of the stem cells of a given donor with a known genotype into the relevant cell types for the in-vitro assembly of micro-organoids should be the preferred strategy. The expansion and differentiation capabilities of human stem cells for the generation of histotypic micro-organoids of each and every organ is illustrated in Figure 11.1....

Viruses and Host Cells in Drug Tests

Screens for drugs against targets that consist of several subunits, or that assemble into linked pathways, require the generation of designer cells with multiple expression cassettes. Although complicated in the generation, the advantage of the established assay is isolation of the target from overlapping biochemical pathways in the native context and the opportunity for robust and fast quantification of drug effects via coupled reporter genes. Desired screen complexity that can be solved with...

One Hundred Years of InVitro Culture

Over the past 100 years, scientists have been trying to culture human tissue in vitro, both to gain knowledge and to develop new medicines. Surprisingly, as early as 1912 Alexis Carrel from the Rockefeller Institute for Medical Research in New York published in an article On the permanent life of tissues outside of the organism, that some in-vitro cultures could be maintained in active life for fifty, fifty-five, and even for sixty days. These results showed that the early death of tissues...

The Liver

The liver is a complex soft organ composed of parenchymal cells, such as hepatocytes, hepatocyte precursor cells (oval cells or Ito cells), stellate cells, Kupffer cells, epithelial cells, sinusoidal epithelial cells, biliary epithelial cells, and fibroblasts 67 . Liver cells are mitotically quiescent, although they are all capable of undergoing division, for example after partial resection of the liver. Hepatocytes are instrumental in detoxification and drug metabolism, involving a large group...

Conclusions

An overall comparison of different culture methods clearly shows the advantages of 3D tissue constructs for drug screening. Bioreactors can provide a better process control by taking into account the different demands of cells during cultivation. Furthermore, they can provide the technical means of performing controlled studies aimed at understanding specific biological, chemical, or physical effects, or for drug screening. Moreover, bioreactors enable a safe and reproducible production of...

Outlook

In the past, technical systems capable of orchestrating all relevant parameters into functional micro-organoid cultures with balanced homeostasis in vitro seemed to be a dream. However, the various chapters of this book have addressed recent advancements in different research areas which, once they can be synchronized into comprehensive interdisciplinary development programs, will overcome the remaining challenges and hurdles. Moreover, when the dream becomes reality, broad applications in...

The Design of Cell Culture Systems and Bioreactors

The aim in designing any drug-testing bioreactor should be to adhere as much as possible to the architecture and homeostasis of the respective sub-organoid structure in man. Thus, the development of biomaterials and shapes that emulate the extracellular habitat and architecture are mandatory. Due to the fact that Nature miniaturizes these functionally self-reliant sub-organoids in the body (often to a volume of less than 1 mm ), bioreactors should employ this possibility with regard to...

Designed Host Cells Combined

Yet further increased complexity is realized in assays that rely on mixtures of different designed cells or cell types 83 . Although these assays are complicated, they yield insights into molecular mechanisms at the cell membrane, such as fusion events that are required for enveloped viruses to enter the target cell. Interactions at lipid bilayers with trans-membrane or integral proteins are difficult to simulate without cellular in-vitro systems. If the fusion event can be quantified, a screen...

Culture Systems and Bioreactors Used in Tissue Engineering

An overview of the culture systems and bioreactors used for the engineering of 3D tissue constructs, including cell maintenance, proliferation, and tissue formation, is illustrated schematically in Figure 2.1a-c. Culture systems developed for the monolayer culture of adherent cells (T-flasks, Petri dishes, multiwell plates) are normally used for cell maintenance and proliferation. These systems allow for sterile handling procedures and are easy to use, disposable, and inexpensive 46 . By...

Conclusions and Novel Trends in Liver Tissue Engineering

Until now, the development of liver tissue engineering has focused primarily on generating functional liver constructs for in-vivo use (replacement of diseased liver), or on the engineering of bioartificial hybrid BAL systems for detoxification. Novel types of scaffold that have been designed more recently for hepatic constructs, are well suited not only for implantation but also as matrices for engineering liver tissue-equivalents for basic and applied research, including in-vitro models for...

Lung Tissue Engineering The Current State of Play

The biological function of the lung is to facilitate exchange of oxygen and carbon dioxide between the cardiovascular circulation and the environment. The lung is a physiologically and anatomically complex vital organ that is hallmarked by a compartmentalized tissue architecture. It is convenient to divide the lung into two anatomically and functionally distinct units, namely the proximal and distal airways. The proximal airways (and proximal pulmonary arteries) are smooth muscle-lined conduits...

Defined Viral and Cellular Pathways and Designed Host Cells

In a related system with the stably integrated Tat-responsive reporter cassette, the activity ofAZT was further characterized by transfection of an expression plasmid for Tat in the presence or absence of AZT 65 . Such a system where isolated viral events are probed in cells allows drug screens without the hazards ofviable virus, and this may be advantageous for more dangerous pathogens than HIV. However, as AZT interferes with reverse transcription the reporter signal strength was not...

Cell Lines and Primary Tissues for InVitro Evaluation ofVaccine Efficacy 153

6.2 Measurement of Antigen Expression 155 6.3 Post-Vaccination Testing 158 6.3.1 Ex-Vivo Detection of Antigen-Specific T Cells 160 6.3.1.2 Cytokine Capture Assay and Intracellular Cytokine Staining 162 6.3.1.3 Measurement of T-Cell Cytotoxicity 163 6.3.2 Current Knowledge on T-Cell Responses in Vaccine Trials 165 6.4 Future Directions 167 References 168

The Operation of Bioreactors

Bioreactors allow for different process strategies including batch, fed-batch, or continuous cultivation (Figs. 2.2 and 2.3). Continuous perfusion, in particular, enables cultivation to be carried out under constant and controlled environmental conditions 44, 51, 68, 69, 73, 74 . Martin et al. 7 summarized some of the effects of direct perfusion on tissue-specific properties such as growth, differentiation and mineralized matrix deposition by bone cells, the proliferation of human oral...

Suitability and Limitations of Primary Cells as Physiologic Models

Growing numbers of hits from primary high-throughput discovery programs generate the need for early, fast and robust lead candidate identification. In order to avoid creating a bottleneck at this stage, decisions on compound selection rejection must be made before animal testing, with the required throughput and minimal substance use. For the accurate prediction of compound specificity and related toxicity, these assays must rely on physiological models that clearly reflect the situation in the...

Intracellular Recording of Electroactive Cells Chip Based Automated Patch Clamp Recording

Fundamental cellular processes, such as neuronal signaling and the contraction of heart and skeletal muscle cells, are regulated by ion channels. A number of diseases which are caused by pathology of ion channels - so-called channelopathies 35 - originate from the loss or dysfunction of ion channels. Some well-known channelopathies affect the central nervous system (episodic ataxias, familial hemiplegic migraine and inherited epilepsies), skeletal muscles (myotonias, periodic paralyses,...

Measurement of TCell Cytotoxicity

Cytotoxic Assays Ctl

Cytotoxicity, which has been shown to be important in clearing many viral infections 70-72 , may be a more reliable indicator of the effector function of antigen-specific CD8+ T cells than cytokine synthesis. It could be important, for example, in controlling HIV-1 pathogenesis, though this has recently been questioned as HIV-1 antigen-specific CD8+ T cells appear to kill infected cells at a slow rate in vivo 43 . As previously alluded to, measuring IFN-y-secreting CD8+ T cells does not provide...

Alternative Methods Used in the Development and Safety Testing of Drugs Biologicals and Medical Devices Table 101

As outlined above, the number of experimental animals used by the drug industry in Germany has decreased during the past 25 years by more than 90 (see Fig. 10.2). This development is typical for the international drug industry, and is due to a general change of models that are used in the life sciences. Research in pharmacology is no longer centered around animal models of disease but rather is focused on molecular models. Biochemical pharmacology has been replaced by molecular pharmacology,...

The Inhalation of Air Contaminants

Air contaminants are exogenous substances in indoor or outdoor air, and include both particulates and gaseous contaminants that may cause adverse health effects in humans or animals. In addition, they may affect plant life and impact on the global environment by changing the atmosphere of the Earth 5 . Various physical, chemical and dynamic processes may generate air pollution leading to the emission of gases, particulates, or mixtures of these, into the atmosphere 6 . Whilst major attempts...

Scaffolds for Liver Tissue Engineering

In general, either mixed cultures of primary isolated cells 68 or freshly isolated purified primary hepatocytes are applied to the scaffold 69 . In order to generate a functional liver construct, the liver cells, once seeded onto the scaffolds, must migrate into the scaffold interior, expand, and populate the new tissue constructs. To facilitate these processes the scaffolds must provide a biocompatible surface suitable for cell recognition and adherence. Following attachment, the cells must...

InVitro BBB Models Cells and Devices

A minimal approach to an in-vitro BBB model focuses mainly on brain microvascular ECs (see Table 1.3). For the past three decades, capillaries have been prepared from the brains of various species of embryonic or adult animals. Brain ECs have been isolated and primary ECs were cultured in vitro. The disadvantages of using primary cultures include the extensive use of animals, tedious, complicated protocols for cell isolation, slow cell proliferation rates, the limitation in passaging these...

References

Casarett and Doull's Toxicology the Basic Science of Poisons, 6th edition. McGraw-Hill, New York. 2 Greenberg, M. I., Hamilton, R. J., Phillips, S. D., McCluskey, G. J. (Eds.) (2003). Occupational, Industrial, and Environmental Toxicology, 2nd edition. Mosby, Inc., Philadelphia, Pennsylvania. 3 Johnson, D. L., Swift, D. L. (1997). Sampling and sizing particles. In Dinardi, S. R. (Ed.), The Occupational Environment - Its evaluation and Control. American Industrial...

Selection of the Most Suitable Cell Line

A selected cell line for HTS (Fig. 5.1) must express the desired function in the correct signal context with a stable and reproducible signal window. The different cell types from which to choose have different advantages primary cells best resemble the physiological situation 3 , but cultivation and available cell numbers are critical issues, even when using techniques such as conditional immortalization 4 or stem cell differentiation. A large panel of easy-to-cultivate immortalized cell lines...

Current Knowledge on TCell Responses in Vaccine Trials

Knowledge gained from analyzing T-cell responses in natural infections, as in HIV-1 infection previously described (above), has provided valuable insights into what is needed for vaccine design and vaccination schedules. Today, novel technological advances are being made, leading in turn to improved ways of measuring CD8+ T-cell responses. Increasingly, these responses are being tested for in animal models and clinical trials of new vaccines. Against HIV infection, much of the recent effort to...

The BBB a Neurovascular Physiological Unit The Concept

The BBB, which separates the blood from the brain, and vice versa, was first described in 1885 by Paul Ehrlich and confirmed later in 1909 by Edwin Goldman. Both investigators showed that, following intravenous injection, trypan blue (an albumin-binding dye) was dispersed throughout the whole body, except for the brain. Conversely, injection of this dye into the brain subarachnoidal space selectively stained only the brain. Reese and Karnovsky 161 , Brightman and Reese 162 and others,...

Regulatory Acceptance ofthe Successfully Validated 3T3 NRU InVitro Phototoxicity Test

As no standard guidelines for testing photoirritation potential, either in vivo or in vitro, had been accepted for regulatory purposes at the international level by the OECD, in 1991 the European Commission EC and the European Cosmetics, Toiletry and Perfumery Association COLIPA established a joint program to develop and validate in-vitro photoirritation tests. In the first phase of the study, which was funded by DG XI of the EC and co-ordinated by ZEBET, in-vitro phototoxicity tests...

Elispot Assay

Enzyme Linked Immunospot Assay

This assay depends on T cells responding specifically to antigen by the synthesis and secretion of cytokines, particularly IFN-y. The principle of the ELISPOT assay is that secreted IFN-y from stimulated antigen-specific T cells is captured by an anti-IFN-y monoclonal antibody immobilized on a nitrocellulose membrane. The captured IFN-y is then detected by a second anti-IFN-y antibody conjugated to an enzyme cytokine spots on the membrane which represent individual IFN-y-secreting T cells are...

Trends in Tissue Engineering Realistic InVitro BBB Pharmacological Models

The ECM is crucial for the culture of brain microcapillary ECs and the establishment of tight junctions 204 . In most in-vitro BBB models the surface of the transwell inserts is coated with rat tail type I collagen, to support and promote EC monolayer formation 205-207 . However, other ECM molecules, such as type IV collagen, fibronectin and laminin, either alone or in combination, were also shown significantly to elevate the TEER of low-resistance porcine brain endothelial monolayers 171 ....

Potential of Alveolar Tissue Models as Disease Models in Pharmaceutical Sciences

Engineered alveolar tissue models that recapitulate physiological and pharmacological characteristics of native distal lung tissue would be invaluable research tools, with broad applications as pharmacological models of normal and pathological tissues, and novel venues for investigation of pulmonary toxicology and infectivity. The refinement of traditional 2D models used to investigate drug permeability in lung tissue 142, 154 will result in a more physiologically relevant model to study drug...

Cytokine Capture Assay and Intracellular Cytokine Staining

Brefeldin Ics

In addition to the measurement of secreted cytokines, other methods aim to measure cell-associated cytokines, either in a cytokine capture assay CCA where cytokines e.g., IFN-y, IL-4 are detected on the cell surface Fig. 6.2 66 , or more commonly by intracellular cytokine staining ICS , where cytokine secretion is blocked by drugs such as brefeldin A or monensin, and cytokine accumulates in the cytoplasm Fig. 6.3 51, 67, 68 . For both methods, fluorescence-conjugated antibodies are used to bind...

Impedance Spectroscopy Screening the Cellular Parameters of Electrophysiological Inactive Cells

Adherent Cell Culture

Impedance spectroscopy, which is also known as cellular dielectric spectroscopy CDS or electric impedance spectroscopy EIS , can be used to measure frequency-dependent changes in the passive electrical properties of single cells or complex tissues by applying defined alternate currents. The bioimpedance of single cells or complex tissues, when combined with a working and counter electrode, is determined by different cellular parameters such as the electric resistance and capacitance of the sub...

The Artificial Immune System

Artificial Lymph Nodes

In vivo, immunogenicity and effects of immunotoxicity are localized in primary immune organs such as bone marrow or thymus, or secondary immune organs such as the lymph nodes and spleen, and spread into the peripheral tissues of the body, such as the skin and gut 106 . Tissue engineering approaches can be used to remodel cascades of cellular interactions in vitro. Recently, ex-vivo-generated and cultivated lymphatic cells have been used for immune cell therapies e.g., adoptive immune responses,...

Bidez P. R. Li S. Macdiarmid A. G. Venancio E. C. Wei Y. Lelkes P. I. J. Biomater. Sci. Polym. 2006 17 199-212

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Extracellular Recording of Electrically Excitable Cells Multiple Site Recording of Field Potentials by MEAs

In general, for extracellular recording of field potentials or low-frequency potentials from electrogenic cells, glass and silicon-based MMEAs with embedded passive metal electrodes 45, 46 or integrated FETs are commonly used 7, 29, 47 . Although, in general, intracellular recording techniques are more sensitive than extracellular recordings, the latter - when carried out with MEAs - have several remarkable advantages. For example, cells cultured on substrate-integrated electrodes allow the...

InVitro Models for Drug Discovery and Toxicity Testing

Philip Lazarovici, Mengyan Li, Anat Perets, MarkJ. Mondrinos, Shimon Lecht, Christopher D. Koharski, Paul R. Bidez III, Christine M. Finck, and Peter I. Lelkes 1.2 Intelligent Biomaterials and Scaffolds for Tissue Engineering 4 1.3 Fabrication of Scaffolds for Tissue Engineering 7 1.3.2 Controlled Lyophilization 9 1.4 Progress and Achievements in Liver Tissue Engineering 11 1.4.2 Scaffolds for Liver Tissue Engineering 12 1.4.3 Pharmaceutical Applications of Tissue-Engineered Liver Models 15...