Genetic therapies for xenotransplantation.
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Animals↗
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Results for “cellular engineering”
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Photoactivation of a substrate for cell adhesion under standard fluorescence microscopes.
Cell-culturing substrates where cell adhesion can be switched on by external stimuli during cell cultivation are useful scaffolds for tissue engineering, cell-based drug screening, and fundamental cellular studies. Here, we show a new strategy for photoactivation of a substrate for cell adhesion under standard fluorescence microscopes. A glass substrate chemically modified with an alkylsiloxane having a photocleavable 2-nitrobenzyl group was coated with bovine serum albumin to prevent cell adhesion. Upon irradiation under a fluorescence microscope, the protein was replaced with fibronectin, which made the irradiated region cell-adhesive. Subsequent seeding of HEK293 or COS7 cells produced patterns corresponding to the irradiated patterns. We succeeded for the first time in positioning single cells in proximity to cultivating single cells. The present method provides a general strategy for positioning single cells of same or different types at any locations on the substrate and will be useful for studying cell-cell interactions.
Animals↗
Mechanical and cellular changes during compaction of a collagen-sponge-based corneal stromal equivalent.
The need for corneas suitable for transplantation, combined with the decreasing supply, has fueled interest in the development of a corneal replacement. In this study, a collagen-sponge-based stromal equivalent, consisting of human corneal fibroblasts cultured on a type I collagen sponge, was maintained in culture for up to 21 days and characterized with respect to mechanical properties and cellular behavior. The Young's modulus of the stromal equivalent varied from 95 to 370 Pa, and its permeability varied from 5.3 x 10(-8) - 4.2 x 10(-7) m4 N(-1) s(-1). The greatest changes occurred during the first few days in culture, but the mechanical properties continued to change during the entire 21 days. Cell traction stress, determined from sponge compaction and DNA count, decreased during the compaction process with the maximum traction value the initial value of 6.6 +/- 2.9 x 10(-3) Pacm3 cell(-1). Microarray data showed that the expression level of fibronectin, decorin sulfate, collagenase, and gelatinase A was upregulated at day 14 in the sponge. This suggested that the repair fibroblast phenotype was being expressed by the fibroblasts. Additional analysis suggested that a subpopulation of cells expressed the myofibroblast phenotype.
Biocompatible Materials↗
Structural basis of RNA folding and recognition in an AMP-RNA aptamer complex.
The catalytic properties of RNA and its well known role in gene expression and regulation are the consequence of its unique solution structures. Identification of the structural determinants of ligand recognition by RNA molecules is of fundamental importance for understanding the biological functions of RNA, as well as for the rational design of RNA Sequences with specific catalytic activities. Towards this latter end, Szostak et al. used in vitro selection techniques to isolate RNA sequences ('aptamers') containing a high-affinity binding site for ATP, the universal currency of cellular energy, and then used this motif to engineer ribozymes with polynucleotide kinase activity. Here we present the solution structure, as determined by multidimensional NMR spectroscopy and molecular dynamics calculations, of both uniformly and specifically 13C-, 15N-labelled 40-mer RNA containing the ATP-binding motif complexed with AMP. The aptamer adopts an L-shaped structure with two nearly orthogonal stems, each capped proximally by a G x G mismatch pair, binding the AMP ligand at their junction in a GNRA-like motif.
Adenosine Monophosphate↗
Future management of scarring.
Future growth and development of plastic and reconstructive surgery will push forward on all fronts, from the prevention of injury to improvements in primary and secondary wound closure, healing, surgical planning, instrumentation, and techniques. Current technology is on the brink of promoting rapid healing and preventing scar formation at the cellular level by affecting the healing process. Tissue engineering has the potential of creating new tissue with the potential to closely approximate missing or damaged tissue from a biopsy of the original. The next generation of reconstructive surgeons may approach traumatic wounds in a completely different fashion, with computers, growth factors, and cell cultures as opposed to a scalpel and suture.
Cicatrix↗
[Advantages of biomatrices in chondrogenesis of pluripotent mesenchymal stem].
AIM: The autologous in vitro expansion of chondrocytes is a new method for the treatment of localized cartilage defect zones in humans. In the past several investigators have shown the occurrence of mesenchymal stem cells (MSC) in human bone marrow, periosteum and somite mesoderm. Moreover it has been shown that these progenitor cells are able to differentiate into chondral tissue under special in vitro conditions. The following study shows current possibilities and borders of a chondrogenetic stem cell therapy. Furthermore advantages and disadvantages of different cellular biomatrix carriers are described, cartilage tissue engineering-related problems are discussed and possible solutions were pointed out. METHODS: A literature investigation served for evaluation of the present clinical and experimental data. Furthermore our own cell culture experiences were considered. RESULTS: Until now there exists no clinical concept using the potential of MSC for cartilage tissue engineering. Reasons are the lack of biomechanical and histological stability and handling problems of the cultivated cartilage tissue, especially the difficulties to fix and secure the transplant in the cartilage defect zones in situ. CONCLUSION: The systematic investigation of biomatrices by chondrogenic progenitor cell culture systems may lead to important data for the evaluation of the chondrogenic potency biomatrices.
Cell Differentiation↗
Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors.
ATP-gated P2X(2) receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X(2) receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X(2)-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X(2)-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X(2)-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X(2) receptor distribution on the seconds time scale.
Adenosine Triphosphate↗
Increased prolyl 4-hydroxylase domain proteins compensate for decreased oxygen levels. Evidence for an autoregulatory oxygen-sensing system.
Prolyl 4-hydroxylase domain (PHD) proteins are oxygen-dependent enzymes that hydroxylate hypoxia-inducible transcription factor (HIF) alpha-subunits, leading to their subsequent ubiquitination and degradation. Paradoxically, the expression of two family members (PHD2 and PHD3) is induced in hypoxic cell culture despite the reduced availability of the oxygen co-substrate, and it has been suggested that they become functionally relevant following re-oxygenation to rapidly terminate the HIF response. Here we show that PHDs are also induced in hypoxic mice in vivo, albeit in a tissue-specific manner. As demonstrated under chronically hypoxic conditions in vitro, PHD2 and PHD3 show a transient maximum but remain up-regulated over more than 10 days, suggesting a feedback down-regulation of HIF-1alpha which then levels off at a novel set point. Indeed, hypoxic induction of PHD2 and PHD3 is paralleled by the attenuation of endogenous HIF-1alpha. Using an engineered oxygen-sensitive reporter gene in a cellular background lacking endogenous HIF-1alpha and hence inducible PHD expression, we could show that increased exogenous PHD levels can compensate for a wide range of hypoxic conditions. Similar data were obtained in a reconstituted cell-free system in vitro. In summary, these results suggest that due to their high O2 Km values, PHDs have optimal oxygen-sensing properties under all physiologically relevant oxygen concentrations; increased PHDs play a functional role even under oxygen-deprived conditions, allowing the HIF system to adapt to a novel oxygen threshold and to respond to another hypoxic insult. Furthermore, such an autoregulatory oxygen-sensing system would explain how a single mechanism works in a wide variety of differently oxygenated tissues.
Animals↗
Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor.
We have previously described the design and operation of a microfabricated bioreactor that supports perfused 3D culture of liver cells and facilitates evolution of tissue-like morphological structures. Here, we describe the functional viability of cells maintained in this microarray bioreactor and examine the influence of different seeding protocols on the evolution of structure and function in comparison with static culture. Primary rat hepatocytes were seeded into the perfusion reactors either as single-cell suspensions immediately after isolation or as spheroidal aggregates formed over a 2- to 3-day period. Initial studies in which cells were cultured for 7 days postisolation revealed significantly greater functional activity and morphological stability of cells that were preaggregated for up to 3 days before seeding in the reactor, compared with direct seeding of single cells. Total albumin secretion and urea genesis rates in single-cell reactor cultures declined significantly during this initial culture period while remaining constant in preaggregated reactor cultures. Longer term studies indicate that rates of albumin secretion and urea genesis are maintained at constant levels through 15 days postisolation. These metabolic rates are an order of magnitude higher than observed for the same preaggregated structures cultured statically with comparable medium ratio and exchange conditions. The metabolic function data are supported by light microscopy images showing viable tissue structures, and electron microscopy images that reveal tight junctions, glycogen storage, and bile canaliculi.
Albumins↗
Problems in the differential diagnosis of choroidal nevi and malignant melanomas. The XXXIII Edward Jackson Memorial Lecture.
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Adolescent↗
Herpes simplex-1 virus thymidine kinase gene is unable to completely eliminate live, nonimmunogenic tumor cell vaccines.
Recent experiments with genetically engineered tumors have generated renewed interest in active cellular immunotherapy as a cancer treatment modality. In order to consider the use of live tumor cells for immunotherapy in human cancer patients, it will be important to ensure that these cells do not themselves produce morbidity in the event the immune system fails to eliminate them. Toward this end, we have examined a strategy for eliminating genetically manipulated nonimmunogenic tumors in vivo. When B16F10 melanoma cells were transfected with the Herpes simplex virus 1 thymidine kinase (HSV-TK) gene, cells were rendered susceptible to killing by the nucleoside analogs acyclovir (ACV) and ganciclovir (GCV). B16-HSV-TK+ tumors established in C57BL6 mice were successfully "suicided" in vivo when GCV was administered by continuous infusion. However, late recurrences were observed even after 1 month of continuous GCV treatment. In vivo growth kinetics suggested that the recurrences resulted from a tiny number (< 20) of cells that had survived the GCV treatment. Interestingly, recurrent tumors were as sensitive to GCV as the parental B16-HSV-TK+ line. While these results demonstrate potential feasibility of the suicide gene strategy for active immunotherapy with live tumor cells, they also illustrate that approaches dependent on the intracellular generation of cell cycle-dependent toxins may fail to eliminate small numbers of cells that temporarily exit cell cycle or that are pharmacologically sequestered.
Acyclovir↗
Enabling computer models of the heart for high-performance computers and the grid.
Although it is now feasible to compute multi-cellular models of the heart on a personal desktop or laptop computer, it is not feasible to undertake the detailed sweeps of high-dimensional parameter spaces required if we are to undertake in silico experimentation of the complex processes that constitute heart disease. For this research, modelling requirements move rapidly beyond the limit of commodity computers' resource both in terms of their memory footprint and the speed of calculation, so that multi-processor architectures must be considered. In addition, as such models have become more mature and have been validated against experimental data, there is increasing pressure for experimentalists to be able to make use of these models themselves as a key tool for hypothesis formulation and in planning future experimental studies to test those hypotheses. This paper discusses our initial experiences in a large-scale project (the Integrative Biology (IB) e-Science project) aimed at meeting these dual aims. We begin by putting the research in context by describing in outline the overall aims of the IB project, in particular focusing on the challenge of enabling novice users to make full use of high-performance resources without the need to gain detailed technical expertise in computing. We then discuss our experience of adapting one particular heart modelling package, Cellular Open Resource, and show how the solving engine of this code was dissected from the rest of the package, ported to C++ and parallelized using the Message-Passing Interface. We show that good parallel efficiency and realistic memory reduction can be achieved on simple geometries. We conclude by discussing lessons learnt in this process.
Action Potentials↗
Stability diagram of a few-electron triple dot.
Individual and coupled quantum dots containing one or two electrons have been realized and are regarded as components for future quantum information circuits. In this Letter we map out experimentally the stability diagram of the few-electron triple dot system, the electron configuration map as a function of the external tuning parameters, and reveal experimentally for the first time the existence of quadruple points, a signature of the three dots being in resonance. In the vicinity of these quadruple points we observe a duplication of charge transfer transitions related to charge and spin reconfigurations triggered by changes in the total electron occupation number. The experimental results are largely reproduced by equivalent circuit analysis and Hubbard models. Our results are relevant for future quantum mechanical engineering applications within both quantum information and quantum cellular automata architectures.
Journal Article↗
Medical devices of human origin.
The introduction of the concepts of tissue engineering into medical practice especially those involving human cellular and tissue-based products has necessitated a reappraisal of some of the principles of manufacturing and quality control. This article addresses some of these issues.
Biological Products↗
Formation of human fibroblast aggregates (spheroids) by rotational culture.
In the current study, we attempted to form aggregates of fibroblasts by rotationally shaking, declining fibroblast-material interactions, and augmenting cell-cell interactions. In addition, to promote cell-cell interactions, the medium was supplemented with insulin, dexamethasone, and basic fibroblast growth. Under such improved culture conditions, normal neonatal human dermal fibroblasts formed spheroidal aggregates within 1 day of rotation on a rotational shaker. The aggregates that formed had irregular shapes and were composed from only several cells after 12 h. However, they became nearly spheroidal after 24 h of shaking. The aggregates were approximately 240 microm in diameter. After 36 h of shaking, their shape became more rounded and their surfaces became smoother. No evidence of necrosis in the center of the aggregates was observed, although a small number of dead cells was scattered throughout the aggregates. After 24-36 h, aggregates of normal human fibroblasts were collected and reinoculated onto a scaffold composed of polyglycolic acid. which is used commercially as a scaffold for artificial skin, coated with collagen. The aggregates were successfully trapped to the mesh of polyglycolic acid and became attached within 24 h. Therefore, the aggregates could provide an alternative method for seeding fibroblasts to scaffold for an artificial skin, such as a mesh of polyglycolic acid.
Cell Aggregation↗
Evaluation of dynamic visco-elastic properties during cartilage regenerating process in vitro.
The dynamic visco-elastic properties of regenerated cartilage tissue were measured to evaluate its mechanical function during cultivation. Harvested chondrocytes from 4-week-old Japanese white rabbits were inoculated into fibroin sponge at a cell concentration of about 5 x 10(7) cells/ml. Dynamic visco-elasticity measurements were performed under compressive loading to evaluate the load bearing function of the articular cartilage. The dynamic modulus and the dynamic loss of the regenerated cartilage increased and the peak value of tandelta, as well as the frequency at the peak, decreased with increasing cultivation time. The pores of the fibroin sponge became filled with newly formed tissue as cultivation time increased. These changes in the visco-elastic properties of the regenerated cartilage were compared with those of a model system, ethylene propylene diene monomer sponge with interstitial fluid, and appear to be a result of increased fluid flow resistance and internal loss. We conclude that the changes in the dynamic visco-elastic properties of the regenerated cartilage were caused because of narrowing of the fluid path by synthesized extracellular matrix.
Animals↗
Organ printing: fiction or science.
Aggregates of living cells (i.e. model tissue fragments) under appropriate conditions fuse like liquid drops. According to Steinberg's differential adhesion hypothesis (DAH), this may be understood by assuming that cells are motile and tissues made of such cells possess an effective surface tension. Here we show that based on these properties three-dimensional cellular structures of prescribed shape can be constructed by a novel method: cell aggregate printing. Spherical aggregates of similar size made of cells with known adhesive properties were prepared. Aggregates were embedded into biocompatible gels. When the cellular and gel properties, as well as the symmetry of the initial configuration were appropriately adjusted the contiguous aggregates fused into ring-like organ structures. To elucidate the driving force and optimal conditions for this pattern formation, Monte Carlo simulations based on a DAH motivated model were performed. The simulations reproduced the experimentally observed cellular arrangements and revealed that the control parameter of pattern evolution is the gel-tissue interfacial tension, an experimentally accessible parameter.
Biocompatible Materials↗
[Simulation of the growth of neurosphere cultured in bioreactors].
When the size of a neurosphere cultured in vitro reaches a certain critical value, a necrotic core will appear inside the neurosphere because of the limitation of oxygen or other nutrients transport from medium to the cells in the neurasphere. Large necrotic core will greatly reduce the expansion of NSCs. The cellular automaton (CA) model is applied in this article to model the growth of NSCs in sphere state. The appearance and enlargement of the necrotic core in a neurosphere is calculated by coupling the CA model with the nutrient diffusion analysis in bioreactors. The calculation results indicate that the culture conditions, such as seeding density, the concentration of nutrients in medium and the mass transfer coefficient between a neurosphere and medium, have some effects on the appearance of the necrotic core. However, the necrotic core mainly depends on the inner diffusion. It will certainly appear if the size of the neurosphere is large enough even the outside mass transfer is in a good condition in bioreactors. Additionally, the appearance of the necrotic core resulting from the shortage of oxygen is earlier than that caused by the limitation of glucose. And the growth of the necrotic core is very fast after its appearance, and the whole neurosphere may become necrotic. The model developed with cellular automaton and mass transfer is a good qualitative representation of NSCs growth in bioreactors.
Bioreactors↗