Cell processing engineering for ex-vivo expansion of hematopoietic cells.
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The cell processing engineering for ex vivo expansion of hematopoietic cells is reviewed. All hematopoietic cells of different lineages and/or at various stages of differentiation are derived from the same precursor, pluripotent hematopoietic stem cells. Bone marrow stromal cells promote and regulate the self-renewal, commitment, differentiation, and proliferation of stem cells and progenitors through their secreted extracellular matrices and cytokine environment in the hematopoietic microenvironment. Although stroma-mediated hematopoiesis has been studied in vitro using the Dexter culture system in tissue culture flasks, hematopoiesis in the Dexter culture system is almost limited to a granulocyte lineage and the system could not expand primitive cells. The addition of large amounts of cytokines to the culture of hematopoietic cells enabled their expansion, but is too expensive. Some clonal stromal cell lines have been established from the Dexter culture of murine bone marrow cells in order to simplify and stimulate the ex vivo expansion of hematopoietic cells. In order to solve the problem regarding the usage of exogeneic stromal cell lines, a novel membrane-separated coculture system, in which stromal cells adhere onto the lower surface of a porous membrane and hematopoietic cells are incubated on the upper surface of the membrane, was proposed. In order to mimic the contact between stromal and hematopoietic cells in vivo in the bone marrow, several types of three-dimensional (3-D) culture of hematopoietic cells were developed. The 3-D coculture of hematopoietic cells with spatial development of stromal cells in nonwoven fabrics enabled the expansion of progenitors without cytokine addition. Progenitors in cord blood mononucleated cells were also successfully expanded without the addition in the 3-D coculture with primary human bone marrow stromal cells in 3-D. Heparin addition to the 3-D coculture and coating the nonwoven fabrics with N-(O-beta-(6-O-sulfogalactopyranosyl)-6-oxyhexyl)-3,5-bis(dodecyloxy)-benzamide further increased the number of progenitors. (+info)
Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues.
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Recently, the field of tissue engineering has progressed rapidly, but poor vascularization remains a major obstacle in bioengineering cell-dense tissues, limiting the viable size of constructs due to hypoxia, nutrient insufficiency, and waste accumulation. Therefore, new technologies for fabricating functional tissues with a well-organized vasculature are required. In the present study, neonatal rat cardiomyocytes were harvested as intact sheets from temperature-responsive culture dishes and stacked into cell-dense myocardial tissues. However, the thickness limit for layered cell sheets in subcutaneous tissue was approximately 80 microm (3 layers). To overcome this limitation, repeated transplantation of triple-layer grafts was performed at 1, 2, or 3 day intervals. The two overlaid grafts completely synchronized and the whole tissues survived without necrosis in the 1 or 2 day interval cases. Multistep transplantation also created approximately 1 mm thick myocardium with a well-organized microvascular network. Furthermore, functional multilayer grafts fabricated over a surgically connectable artery and vein revealed complete graft perfusion via the vessels and ectopic transplantation of the grafts was successfully performed using direct vessel anastomoses. These cultured cell sheet integration methods overcome long-standing barriers to producing thick, vascularized tissues, revealing a possible solution for the clinical repair of various damaged organs, including the impaired myocardium. (+info)
Stem cells: the next therapeutic frontier.
(19/58)
Cell therapy is one of the most exciting fields in translational medicine. It stands at the intersection of a variety of rapidly developing scientific disciplines: stem cell biology, immunology, tissue engineering, molecular biology, biomaterials, transplantation biology, regenerative medicine, and clinical research. Cell-based therapy may develop into a new therapeutic platform to treat a vast array of clinical disorders. Blood transfusions and bone marrow transplantation are prime examples of the successful application of cell-based therapeutics; but recent advances in cellular and molecular biology have expanded the potential applications of this approach. Although recombinant genetic engineering to produce a variety of therapeutics such as human erythropoietin and insulin has proven successful, these treatments are unable to completely correct or reverse disease states, because most common disease processes are not due to the deficiency of a single protein but develop due to alterations in the complex interactions of a variety of cell components. In these complex situations, cell-based therapy may be a more successful strategy by providing a dynamic, interactive, and individualized therapeutic approach that responds to the pathophysiological condition of the patient. In this regard, cells may provide innovative methods for drug delivery of biologics, immunotherapy, and tissue regenerative or replacement engineering (1,2). The translation of this discipline to medical practice has tremendous potential, but in many applications technological issues need to be overcome. Since many cell-based indications are already being evaluated in the clinic, the field appears to be on the threshold of a number of successes. This review will focus on our group's use of human stem/progenitor cells in the treatment of acute and chronic renal failure as extensions to the current successful renal substitution processes of hemodialysis and hemofiltration. (+info)
Similarities and differences between induced organ regeneration in adults and early foetal regeneration.
(20/58)
At least three organs (skin, peripheral nerves and the conjunctiva) have been induced to regenerate partially in adults following application of porous, degradable scaffolds with highly specific structure (templates). Templates blocked contraction and scar formation by inducing a reduction in the density of contractile fibroblasts (probably myofibroblasts) and by preventing these cells to organize themselves appropriately in the wound. In contrast, during early foetal healing, myofibroblasts were absent and wounds did not close by contraction but rather by spontaneous regeneration. The adult regenerative process has so far led to imperfect recovery of the physiological anatomy of skin (skin appendages were missing), while early foetal healing has led to apparently complete restoration. Furthermore, the mechanism of the adult regenerative process involves thwarting of myofibroblast function while, during early foetal healing, differentiation of myofibroblasts has not yet occurred. The data suggest that induced organ regeneration in the adult is the result of partial reversion to early foetal healing. If so, the adult may conceal a foetal response that may be subject to activation following application of highly active scaffolds or of other substances or cells. (+info)
Inductive tissue engineering with protein and DNA-releasing scaffolds.
(21/58)
Cellular differentiation, organization, proliferation and apoptosis are determined by a combination of an intrinsic genetic program, matrix/substrate interactions, and extracellular cues received from the local microenvironment. These molecular cues come in the form of soluble (e.g. cytokines) and insoluble (e.g. ECM proteins) factors, as well as signals from surrounding cells that can promote specific cellular processes leading to tissue formation or regeneration. Recent developments in the field of tissue engineering have employed biomaterials to present these cues, providing powerful tools to investigate the cellular processes involved in tissue development, or to devise therapeutic strategies based on cell replacement or tissue regeneration. These inductive scaffolds utilize natural and/or synthetic biomaterials fabricated into three-dimensional structures. This review summarizes the use of scaffolds in the dual role of structural support for cell growth and vehicle for controlled release of tissue inductive factors, or DNA encoding for these factors. The confluence of molecular and cell biology, materials science and engineering provides the tools to create controllable microenvironments that mimic natural developmental processes and direct tissue formation for experimental and therapeutic applications. (+info)
Bioartificial sinus node constructed via in vivo gene transfer of an engineered pacemaker HCN Channel reduces the dependence on electronic pacemaker in a sick-sinus syndrome model.
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BACKGROUND: The normal cardiac rhythm originates in the sinoatrial (SA) node that anatomically resides in the right atrium. Malfunction of the SA node leads to various forms of arrhythmias that necessitate the implantation of electronic pacemakers. We hypothesized that overexpression of an engineered HCN construct via somatic gene transfer offers a flexible approach for fine-tuning cardiac pacing in vivo. METHODS AND RESULTS: Using various electrophysiological and mapping techniques, we examined the effects of in situ focal expression of HCN1-DeltaDeltaDelta, the S3-S4 linker of which has been shortened to favor channel opening, on impulse generation and conduction. Single left ventricular cardiomyocytes isolated from guinea pig hearts preinjected with the recombinant adenovirus Ad-CMV-GFP-IRES-HCN1-DeltaDeltaDelta in vivo uniquely exhibited automaticity with a normal firing rate (237+/-12 bpm). High-resolution ex vivo optical mapping of Ad-CGI-HCN1-DeltaDeltaDelta-injected Langendorff-perfused hearts revealed the generation of spontaneous action potentials from the transduced region in the left ventricle. To evaluate the efficacy of our approach for reliable atrial pacing, we generated a porcine model of sick-sinus syndrome by guided radiofrequency ablation of the native SA node, followed by implantation of a dual-chamber electronic pacemaker to prevent bradycardia-induced hemodynamic collapse. Interestingly, focal transduction of Ad-CGI-HCN1-DeltaDeltaDelta in the left atrium of animals with sick-sinus syndrome reproducibly induced a stable, catecholamine-responsive in vivo "bioartificial node" that exhibited a physiological heart rate and was capable of reliably pacing the myocardium, substantially reducing electronic pacing. CONCLUSIONS: The results of the present study provide important functional and mechanistic insights into cardiac automaticity and have further refined an HCN gene-based therapy for correcting defects in cardiac impulse generation. (+info)
Challenges in tissue engineering.
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Almost 30 years have passed since a term 'tissue engineering' was created to represent a new concept that focuses on regeneration of neotissues from cells with the support of biomaterials and growth factors. This interdisciplinary engineering has attracted much attention as a new therapeutic means that may overcome the drawbacks involved in the current artificial organs and organ transplantation that have been also aiming at replacing lost or severely damaged tissues or organs. However, the tissues regenerated by this tissue engineering and widely applied to patients are still very limited, including skin, bone, cartilage, capillary and periodontal tissues. What are the reasons for such slow advances in clinical applications of tissue engineering? This article gives the brief overview on the current tissue engineering, covering the fundamentals and applications. The fundamentals of tissue engineering involve the cell sources, scaffolds for cell expansion and differentiation and carriers for growth factors. Animal and human trials are the major part of the applications. Based on these results, some critical problems to be resolved for the advances of tissue engineering are addressed from the engineering point of view, emphasizing the close collaboration between medical doctors and biomaterials scientists. (+info)
Cellular and matrix mechanics of bioartificial tissues during continuous cyclic stretch.
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Bioartificial tissues are useful model systems for studying cell and extra-cellular matrix mechanics. These tissues provide a 3D environment for cells and allow tissue components to be easily modified and quantified. In this study, we fabricated bioartificial tissue rings from a 1 ml solution containing one million cardiac fibroblasts and 1 mg collagen. After 8 days, rings compacted to <1% of original volume and cell number increased 2.4 fold. We initiated continuous cyclic stretching of the rings after 2, 4, or 8 days of incubation, while monitoring the tissue forces. Peak tissue force during each cycle decreased rapidly after initiating stretch, followed by further slow decline. We added 2 microM Cytochalasin-D to some rings prior to initiation of stretch to determine the force contributed by the matrix. Cell force was estimated by subtracting matrix force from tissue force. After 12 h, matrix force-strain curves were highly nonlinear. Cell force-strain curves were linear during loading and showed hysteresis indicating viscoelastic behavior. Cell stiffness increased with stretching frequency from 0.001-0.25 Hz. Cell stiffness decreased with stretch amplitude (5-25%) at 0.1 Hz. The trends in cell stiffness do not fit simple viscoelastic models previously proposed, and suggest possible strain-amplitude related changes during cyclic stretch. (+info)