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At least 595 records · Page 33Linked to original sources

PubMed1999

Enhancing therapeutic glycoprotein production in Chinese hamster ovary cells by metabolic engineering endogenous gene control with antisense DNA and gene targeting.

Recombinant glycoprotein therapeutics have proven to be invaluable pharmaceuticals for the treatment of chronic and life-threatening diseases. Although these molecules are extraordinarily efficacious, many diseases have high dosage requirements of several hundred milligrams of protein for each administration. Multiple doses at this level are often required for treatment. One of the major challenges currently facing the biotechnology industry is the development of large-scale, cost-effective production and manufacturing processes of these biologically synthesized molecules. Metabolic engineering of animal cell expression hosts promises to address this challenge by substantially enhancing recombinant protein quality, productivity, and biological activity. In this report, we describe a novel approach to metabolic engineering in Chinese hamster ovary cells by control of endogenous gene expression. Analysis of the advantages and limitations of using antisense DNA and gene targeting as a means of control are discussed and several gene candidates for regulation with these techniques are identified. Practical considerations for using these technologies to reduce the levels of the CHO cell sialidase (Warner et al., Glycobiology, 3, 455-463, 1993) as a model gene system for regulation are also presented.

Animals↗

PubMed2000

Neuroma: a donor-age independent source of human Schwann cells for tissue engineered nerve grafts.

Schwann cells are used in combination with biological matrices as tissue engineered nerve grafts in animal models offering a new therapeutic approach for treatment of lesions of the peripheral nervous system. A high yield of human Schwann cells from adult donors is only achieved by pharmacological stimulation, which should, however, be avoided in clinical therapy. Here, we establish cultures of activated human Schwann cells which were isolated from peripheral nerve neuroma which developed after a median nerve lesion. To allow nerve reconstruction neuroma have to be resected. Such neuroma tissue is virtually predegenerated and shows activation of Schwann cells, implying good adherence and high mitotic activity. This allows, irrespective of donor age, growing within a short time period and without any pharmacological treatment.

Adult↗

PubMed2002

Proliferating cells versus differentiated cells in tissue engineering.

The efficiency of cell or tissue cultures is usually judged by how quickly confluence is reached within a Petri dish or on a scaffold. Growth factors and fetal bovine serum are employed to drive cultured cells from one mitosis to the next as quickly as possible. The tissue specific interphase is extremely short under these conditions, so that the degree of differentiation desired in tissue engineering cannot be achieved. To reach the goal of functional differentiation in vitro mitosis and interphase must be separated experimentally and tailored to the specific requirements of the cell-type used. This could be achieved by a three step concept for tissue-engineering in vitro as we present here. The expansion phase is followed by a phase in which tissue differentiation is initiated. The final phase serves to express and maintain histotypical differentiation of the generated tissue.

Animals↗

PubMed2001

The amniotic fluid as a source of cells for fetal tissue engineering.

PURPOSE: This study was aimed at determining whether fetal tissue constructs can be engineered from cells normally found in the amniotic fluid. METHODS: A subpopulation of morphologically distinct cells was isolated mechanically from the amniotic fluid of pregnant ewes (n = 5) and expanded selectively. Its lineage was determined by immunofluorescent staining against multiple intermediate filaments and surface antigens. Proliferation rates were determined by both oxidation and total DNA assays and compared with immunocytochemically identical adult and fetal sheep cells. Statistical analysis was by analysis of variance for repeated measures (ANOVA). After expansion, the amniocytes were seeded onto a polyglycolic acid polymer/poly-4-hydroxybutyrate scaffold. The resulting construct was analyzed by both optical and scanning electron microscopy. RESULTS: The immunocytochemical profile of expanded amniocytes was consistent with a mesenchymal, fibroblast/myofibroblast cell lineage. These cells proliferated significantly faster than comparable fetal and adult cells in culture. Amniocyte construct analysis showed dense, confluent layers of cells firmly attached to the scaffold, with no evidence of cell death. CONCLUSIONS: (1) Subpopulations of fetal mesenchymal cells can be isolated consistently from the amniotic fluid. (2) Mesenchymal amniocytes proliferate more rapidly in vitro than comparable fetal and adult cells. (3) Mesenchymal amniocytes attach firmly to polyglycolic acid polymer. The amniotic fluid can be a reliable and practical source of cells for the engineering of select fetal tissue constructs.

Amniotic Fluid↗

PubMed2002

Microencapsulation of cells producing therapeutic proteins: optimizing cell growth and secretion.

Microencapsulation of genetically engineered cells may have important applications as delivery systems for therapeutic proteins. However, optimization of the microcapsules with regard to mechanical stability, cell growth, and secretion of proteins is necessary in order to evaluate the future use of this delivery technology. We have explored the growth, survival, and secretion of therapeutic proteins from 293-EBNA cells producing endostatin (293 endo cells) and JJN3 myeloma cells producing hepatocyte growth factor (HGF) that have been embedded in various types of alginate capsules. Parameters that affect capsule integrity such as homogenous and inhomogenous gel cores and addition of an outer poly-L-lysine (PLL)-alginate coating were evaluated in relation to cell functions. When cells were encapsulated, the PLL layer was found to be absolutely required for the capsule integrity. The JJN3 and 293 endo cells displayed completely different growth and distribution patterns of live and dead cells within the microcapsules, as shown by 3D pictures reconstructed from images taken with confocal laser scanning microscopy (CLSM). Encapsulated JJN3 cells showed a bell-shaped growth and HGF secretion curve over a time period of 5 months. The 293 endo cells reached a plateau phase in growth after 23 days postencapsulation; however, after around 30 days a fraction of the microcapsules started to disintegrate. Microcapsule disintegration occurred with time irrespective of capsule and cell type, showing that alginate microcapsules possessing relatively high gel strength are not strong enough to keep proliferating cells within the microcapsules for prolonged time periods. Although this study shows that the stability of an alginate-based cell factory can be increased by a PLL-alginate coating, further improvement is necessary with regard to capsule integrity as well as controlling the cell growth before this technology can be used for therapy.

Alginates↗

PubMed2006

Natural polymers for gene delivery and tissue engineering.

Although the field of gene delivery is dominated by viral vectors and synthetic polymeric or lipid gene carriers, natural polymers offer distinct advantages and may help advance the field of non-viral gene therapy. Natural polymers, such as chitosan, have been successful in oral and nasal delivery due to their mucoadhesive properties. Collagen has broad utility as gene activated matrices, capable of delivering large quantities of DNA in a direct, localized manner. Most natural polymers contain reactive sites amenable for ligand conjugation, cross-linking, and other modifications that can render the polymer tailored for a range of clinical applications. Natural polymers also often possess good cytocompatibility, making them popular choices for tissue engineering scaffolding applications. The marriage of gene therapy and tissue engineering exploits the power of genetic cell engineering to provide the biochemical signals to influence proliferation or differentiation of cells. Natural polymers with their ability to serve as gene carriers and tissue engineering scaffolds are poised to play an important role in the field of regenerative medicine. This review highlights the past and present research on various applications of natural polymers as particulate and matrix delivery vehicles for gene delivery.

Animals↗

PubMed2005

Role of nutrient supply on cell growth in bioreactor design for tissue engineering of hematopoietic cells.

In the present study, a dynamic mathematical model for the growth of granulocyte progenitor cells in the hematopoietic process is developed based on the principles of diffusion and chemical reaction. This model simulates granulocyte progenitor cell growth and oxygen consumption in a three-dimensional (3-D) perfusion bioreactor. Material balances on cells are coupled to the nutrient balances in 3-D matrices to determine the effects of transport limitations on cell growth. The method of volume averaging is used to formulate the material balances for the cells and the nutrients in the porous matrix containing the cells. All model parameters are obtained from the literature. The maximum cell volume fraction reached when oxygen is depleted in the cell layer at 15 days and is nearly 0.63, corresponding to a cell density of 2.25 x 10(8) cells/mL. The substrate inhibition kinetics for cell growth lead to complex effects with respect to the roles of oxygen concentration and supply by convection and diffusion on cell growth. Variation in the height of the liquid layer above the cell matrix where nutrient supply is introduced affected the relative and absolute amounts of oxygen supply by hydrodynamic flow and by diffusion across a gas permeable FEP membrane. Mass transfer restrictions of the FEP membrane are considerable, and the supply of oxygen by convection is essential to achieve higher levels of cell growth. A maximum growth rate occurs at a specific flow rate. For flow rates higher than this optimal, the high oxygen concentration led to growth inhibition and for lower flow rates growth limitations occur due to insufficient oxygen supply. Because of the nonlinear effects of the autocatalytic substrate inhibition growth kinetics coupled to the convective transport, the rate of growth at this optimal flow rate is higher than that in a corresponding well-mixed reactor where oxygen concentration is set at the maximum indicated by the inhibitory kinetics.

Bioreactors↗

PubMed2001

Local endostatin treatment of gliomas administered by microencapsulated producer cells.

We describe a technique for the treatment of malignant brain tumors based on local delivery of the anti-angiogenic protein endostatin from genetically engineered cells encapsulated in ultrapure sodium alginate. Alginate consists of L-guluronic and D-mannuronic acid, which in the presence of divalent cations forms an extended gel network, in which cells reside and remain immunoisolated, when implanted into the rat brain. Here, we show that endostatin-transfected cells encapsulated in alginate maintain endostatin secretion for at least four months after intracerebral implantation in rats. During the implantation period 70% of the encapsulated cells remained viable, as opposed to 85% in in vitro-cultured capsules. Rats that received transplants of BT4C glioma cells, together with endostatin-producing capsules (0.2 microg/ml per capsule), survived 84% longer than the controls. The endostatin released from the capsules led to an induction of apoptosis, hypoxia, and large necrotic avascular areas within 77% of the treated tumors, whereas all the controls were negative. The encapsulation technique may be used for many different cell lines engineered to potentially interfere with the complex microenvironment in which tumor and normal cells reside. The present work may thus provide the basis for new therapeutic approaches toward brain tumors.

Alginates↗

PubMed2005

Enforced expression of cytosolic 5'-nucleotidase I confers resistance to nucleoside analogues in vitro but systemic chemotherapy toxicity precludes in vivo selection.

PURPOSE: Retroviral transfer of cDNA sequences that confer drug resistance can be used to protect against chemotherapy-induced hematopoietic toxicity and for the selective expansion of gene-modified cells. To successfully expand genetically engineered cells in vivo, an appropriate balance must be achieved between systemic toxicity induced by the selecting agent and the expansion of modified cells. METHOD: In this study, we investigate retroviral transfer of cytosolic 5'-nucleotidase I (cN-I) for protection and selection of gene-modified cells when treated with 2-chloro-2'-deoxyadenosine (2-CdA) and 5-fluorouracil (5-FU) alone and in combination. We also attempt to design a treatment strategy for the potential in vivo selection of cN-I-modified cells by administering 5-FU to mice prior to 2-CdA treatment. RESULTS: Our results show that cN-I can be transferred by recombinant retroviruses, and that enforced expression of cN-I protects murine fibroblast and hematopoietic progenitor cells from the cytotoxic effects of 2-CdA and/or 5-FU. Furthermore, we show that the combined administration of 5-FU and 2-CdA potentiates hematopoietic stem cell toxicity. However, the treatment also results in severe myelosuppression. CONCLUSION: These results show that while cN-I provides both protective and selective benefits to gene-modified cells in vitro, selection requires a treatment strategy that is likely too toxic to consider cN-I as an in vivo selectable marker.

5'-Nucleotidase↗

PubMed2004

Cell response to nano-islands produced by polymer demixing: a brief review.

This review looks at the present literature available regarding cell response to nano-islands produced by nanotopography. Polymer demixing is a chemical method of fabricating large areas of nanotopography quickly and cheaply, making it ideal for cell testing and thus allowing it to be one of the first well-researched methods in cell engineering. The review shows that cells respond strongly to the islands (cell types observed include endothelial cells, fibroblasts, osteoblasts, leucocytes and platelets). Such changes include differences in adhesion, growth, gene expression and morphology.

Journal Article↗

PubMed1997

Mast cells.

Mast cells are found resident in tissues throughout the body, particularly in association with structures such as blood vessels and nerves, and in proximity to surfaces that interface the external environment. Mast cells are bone marrow-derived and particularly depend upon stem cell factor for their survival. Mast cells express a variety of phenotypic features within tissues as determined by the local environment. Withdrawal of required growth factors results in mast cell apoptosis. Mast cells appear to be highly engineered cells with multiple critical biological functions. They may be activated by a number of stimuli that are both Fc epsilon RI dependent and Fc epsilon RI independent. Activation through various receptors leads to distinct signaling pathways. After activation, mast cells may immediately extrude granule-associated mediators and generate lipid-derived substances that induce immediate allergic inflammation. Mast cell activation may also be followed by the synthesis of chemokines and cytokines. Cytokine and chemokine secretion, which occurs hours later, may contribute to chronic inflammation. Biological functions of mast cells appear to include a role in innate immunity, involvement in host defense mechanisms against parasitic infestations, immunomodulation of the immune system, and tissue repair and angiogenesis.

Animals↗

PubMed2000

[Management of intractable cancer pain: from intrathecal morphine to cell allograft].

The durable effectiveness of intrathecal morphine administration is well established for the management of intractable cancer pain, after failure of systemic opioids, secondary to the persistence of non-reversible undesirable side effects. Many patients are referred to late in the disease course. This conservative method to control pain of malignant origin must not be reserved for last resort treatment for terminal patients. Intra-cerebro-ventricular morphine administration is a very effective and generally safe method for controlling intractable cancer pain. Because of the chronic implantation of an intra-ventricular catheter this method is somewhat invasive. Its indications remain a simple and effective alternative when the topography of nociceptive pain is diffuse or cephalic. In clinical practice, intrathecal and/or intra-cerebro-ventricular administration of opioids is limited by cost, the need for specialized maintenance and mechanical malfunctions if implantable drug delivery systems, or by the risk of bacterial contamination and ambulatory constraints when repeated daily injections via an intrathecal access port are used. To answer these limitations, cell therapy using intrathecal chromaffin cell allograft is a promising approach for the management of cancer pain refractory to traditional drug therapy and pain lesion surgery. The basic rationale and preclinical studies on experimental pain models have enabled starting prospective clinical trials. Prior to transplantation, handling and preparation of the chromaffin tissue is critical for allograft viability. The initial results of clinical trials with human chromaffin cell grafts from intractable cancer pain have reported long-lasting pain relief, in correlation with met-enkephalin release into the CSF. Convincing evidence will require controlled studies. The limitations of this innovative cell therapy and especially the lack of human adrenal gland availability point to the need for new sources of cells. Perspectives include xenogenic or engineered cell lines.

Analgesics, Opioid↗

PubMed2005

Efficient lentiviral transduction and improved engraftment of human bone marrow mesenchymal cells.

Human bone marrow (BM) mesenchymal stem/progenitor cells are potentially attractive targets for ex vivo gene therapy. The potential of lentiviral vectors for transducing BM mesenchymal cells was examined using a self-inactivating vector that expressed the green fluorescent protein (GFP) from an internal cytomegalovirus (CMV) promoter. This vector was compared with oncoretroviral vectors expressing GFP from the CMV promoter or a modified long-terminal repeat that had been optimized for long-term expression in stem cells. The percentage of GFP-positive cells was consistently higher following lentiviral versus oncoretroviral transduction, consistent with increased GFP mRNA levels and increased gene transfer efficiency measured by polymerase chain reaction and Southern blot analysis. In vitro GFP and FVIII expression lasted for several months post-transduction, although expression slowly declined. The transduced cells retained their stem/progenitor cell properties since they were still capable of differentiating along adipogenic and osteogenic lineages in vitro while maintaining high GFP and FVIII expression levels. Implantation of lentivirally transduced human BM mesenchymal cells using collagen scaffolds into immunodeficient mice resulted in efficient engraftment of gene-engineered cells and long-term transgene expression in vivo. These biocompatible BM mesenchymal implants represent a reversible, safe, and versatile protein delivery approach because they can be retrieved in the event of an unexpected adverse reaction or when expression of the protein of interest is no longer required. In conclusion, efficient gene delivery with lentiviral vectors in conjunction with the use of bioengineered reversible scaffolds improves the therapeutic prospects of this novel approach for gene therapy, protein delivery, or tissue engineering.

Adipogenesis↗

PubMed2005

Murine spinal fusion induced by engineered mesenchymal stem cells that conditionally express bone morphogenetic protein-2.

OBJECT: The authors hypothesized that spinal fusion can be achieved and monitored by using cell-mediated gene therapy. Mesenchymal stem cells (MSCs) genetically engineered to express recombinant human bone morphogenetic protein-2 (rhBMP-2) conditionally, were implanted into the paraspinal muscles of mice to establish spinal fusion. The goal was to demonstrate an MSC-based gene therapy platform in which controlled gene expression is used to obtain spinal fusion in a murine model. METHODS: Mesenchymal stem cells expressing the rhBMP-2 gene were injected into the paravertebral muscle in mice. Bone formation in the paraspinal region was longitudinally followed by performing micro-computerized tomography scanning, histological studies, and an analysis of osteocalcin expression to demonstrate the presence of engrafted engineered MSCs. The minimal period of rhBMP-2 expression by the engineered MSCs required to induce fusion was determined. The results of this study demonstrate that genetically engineered MSCs induce bone formation in areas adjacent to and touching the posterior elements of the spine. This newly formed bone fuses the spine, as demonstrated by radiological and histological studies. The authors demonstrate that injected cells induce active osteogenesis at the site of implantation for up to 4 weeks postinjection. They found that a 7-day induction of rhBMP-2 expression in genetically engineered MSCs was sufficient to form new bone tissue, although the quantity of this bone increased as longer expression periods were implemented. CONCLUSIONS: After their injection genetically engineered MSCs can efficiently form new bone in the paraspinal muscle of the mouse to obtain spinal fusion. The extent and quantity of this newly formed bone can be monitored by controlling the duration of rhBMP-2 gene expression.

Animals↗

PubMed2006

An approachable human adult stem cell source for hard-tissue engineering.

Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/CD34+/STRO-1+ cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10(-8) mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies.

Alkaline Phosphatase↗

PubMed2002

[Potential seeding cells for cartilage tissue engineering--bone marrow stromal stem cells].

OBJECTIVE: To review the recent research progress of bone-marrow stromal stem cells (BMSCs) in the conditions of culture in vitro, chondrogenic differentiation, and the application in cartilage tissue engineering. METHODS: Recent original articles related to such aspects of BMSCs were reviewed extensively. RESULTS: BMSCs are easy to be isolated and cultivated. In the process of chondrogenesis of BMSCs, the special factors and interaction between cells are investigated extensively. BMSCs have been identified to form cartilage in vivo. One theory is the committed chondrocyte from BMSCs is only a transient stage. CONCLUSION: BMSCs are the alternative seeding cells for cartilage tissue engineering. The conditions promoting mature chondrocyte should be further investigated.

Bone Marrow Cells↗

PubMed2002

Comparison of mixed cell culture containing genetically engineered BGMK and CaCo-2 cells (Super E-Mix) with RT-PCR and conventional cell culture for the diagnosis of enterovirus meningitis.

BACKGROUND: Enteroviral meningitis has traditionally been diagnosed by cell culture. More recently, molecular techniques and shell vials with a mixture of human colon carcinoma and genetically engineered buffalo green monkey kidney cells (BGMK cells) (Super E-Mix) have been described. OBJECTIVE: We compared the results of this new cell culture technique with two reverse transcriptase polymerase chain reaction (RT-PCR) techniques and conventional cell culture to assess the accuracy of these various methods. STUDY DESIGN: 2 ml of cerebrospinal fluid (CSF) was obtained from 72 patients with suspected viral meningitis. 600 microl was used for conventional cell culture and 200 microl was inoculated to each of two Super E-Mix vials. One vial was incubated for 24 h, then stained with pan-enterovirus antibody, and the other was examined for CPE daily for 5 days and stained when positive or at the end of incubation. The final aliquot (500 microl) was centrifuged at 25,000g, and the pellet in 140 microl of supernatant was used for ribonucleic acid (RNA) extraction and tested by commercial single-step (Chemicon, Temecula, CA) and two-step (Argene, Varilhes, France) RT-PCR methods. RESULTS: Conventional culture was 51% sensitive and 100% specific. The Super E-Mix was slightly higher (sensitivity 76%, specificity 100%). The Chemicon and Argene PCR methods had a sensitivity of 93% and 88% and specificity of 90% and 97%, respectively. CONCLUSION: The Super E-Mix procedure had greater sensitivity than conventional cell culture, but RT-PCR was the most sensitive technique with this type of specimen. The RT-PCR methods performed equally.

Adolescent↗

PubMed2003

Proliferation and stratification of keratinocyte on cultured amniotic epithelial cells for tissue engineering.

Human amniotic epithelial cells (HAECs) are formed from amnioblasts, separated from the epiblast at about the 8th day after fertilization. Recent studies suggest that HAECs can produce various biologically active substances. In this study, the effects of cultured HAECs on keratinocytes were investigated. First of all, the effect of the medium conditioned by cultured HAECs on the proliferation of keratinocytes was examined. The conditioned medium significantly enhanced the proliferation (P<0.05). Next, the effect of co-culture with HAECs was also examined. The keratinocytes formed a stratified epithelium on day 7 after the start of co-culture. The cultured epithelium formed by the co-culture was five to six layers thick, could be detached by dispase treatment, and had sufficient strength as a sheet. These results suggest that HAECs will be a novel supplemental material for the tissue engineering of skin.

Journal Article↗