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PubMedDate not supplied

Constrained cell recognition peptides engineered into streptavidin.

Streptavidin is widely used as an adaptor molecule in diagnostics, separations, and laboratory assay applications. We have here engineered cell adhesive peptides into the three-dimensional scaffolding of streptavidin to convert streptavidin into a functional protein. The mutations did not alter refolding or tetramer assembly and the slow biotin dissociation rate of wild-type streptavidin was retained. The peptide targets were hexapeptide sequences derived from osteopontin and fibronectin that contain the RGD cell adhesion sequence. Cell binding assays directly demonstrated that rat aortic endothelial cells and human melanoma cells adhered to surfaces coated with either of the two RGD streptavidin mutants in a dose-dependent fashion. Wild-type streptavidin displayed no significant cell binding activity. Inhibition studies with soluble RGD peptides confirmed that the cell adhesion was RGD mediated. Further inhibition studies with antibodies directed against alphavbeta3 demonstrated that the RGD-streptavidin interaction was primarily mediated by this integrin with melanoma cells. These results demonstrate that peptide recognition sequences can be engineered into accessible surface regions of streptavidin without disrupting biotin binding properties. This approach to introducing secondary functional activities into streptavidin may improve streptavidin's utility in existing applications or provide new technology opportunities.

Amino Acid Sequence↗
PubMed2005

Aqueous sol-gel encapsulation of genetically engineered Moraxella spp. cells for the detection of organophosphates.

An all-aqueous sol-gel method for encapsulation of bacterial cells in porous silicate matrices towards the development of a biosensor is described. The sol-gel encapsulation of cells is achieved at room temperature and neutral pH. Furthermore, use of sodium silicate as precursor avoids generation of alcohol that can be detrimental to cells in contrast to the traditional alkoxide sol-gel encapsulation process. Moraxella spp. cells engineered to express recombinant organophosphorus hydrolase (OPH) on the cell surface were encapsulated and OPH enzymatic activity was measured for paraoxon hydrolysis. Kinetic parameters (Km and Vmax) as well as pH behavior of surface-expressed OPH were determined to evaluate the effect of encapsulation. Cells encapsulated by the sodium silicate method displayed higher activity retention compared to those by the traditional alkoxide process. Time-course studies over a 2-month period indicate that immobilization through the sodium silicate process led to a reduction in activity of approximately 5% as compared to approximately 30% activity reduction in case of free cells in buffer indicating that immobilization leads to stabilization, a key parameter in biosensor development.

Biosensing Techniques↗
PubMed1994

Genotoxicity assessment of aromatic amines and amides in genetically engineered V79 cells.

A genetically engineered V79 cell line expressing rat CYP1A2 and another cell line expressing rat CYP1A2 as well as endogenous acetyltransferase activity, as well as CYP-deficient parental V79 cell lines, were used to assess the genotoxicity of the aromatic amines and amides 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, 4-acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline, with chromosomal aberrations and sister chromatid exchanges as the end-points. None of the test compounds showed a clear effect on the frequency of chromosomal aberrations in any cell line used. Sister chromatid exchanges, however, were induced by 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene in the CYP1A2-proficient cells, but not in the CYP1A2-deficient cells. The presence of acetyltransferase activity enhanced the effect of 2-aminoanthracene, 2-aminofluorene and 2-acetylaminofluorene. 4-Acetylaminofluorene and 2-amino-3-methylimidazo[4,5-f]quinoline did not induce sister chromatid exchanges in the investigated cell lines. The use of cell lines with defined metabolic capabilities seems to be a valuable tool to study specific metabolic pathways important in the activation of procarcinogens.

2-Acetylaminofluorene↗
PubMed2000

Targeting antigen-specific T cells by genetically engineered antigen presenting cells. A strategy for specific immunotherapy of autoimmune disease.

We describe a strategy for specific immunotherapy of autoimmune disease based on targeting the antigen-specific T cells in an experimental model of myasthenia gravis. To address the problem of heterogeneity of the T cell repertoire, we have genetically engineered antigen presenting cells (APCs) to process and present epitopes of the autoantigen, acetylcholine receptor (AChR), to the entire spectrum of AChR-specific syngeneic T cells. APCs derived from BALB/c mice were stably transfected with cDNA for the key immunogenic domain of the AChR alpha-subunit, flanked by sequences of the lysosome-associated membrane protein (LAMP) that direct APCs to process and present the antigen via the MHC Class II pathway. Transfected APCs strongly stimulated AChR-specific T cells from BALB/c mice. Fas ligand, or antibody to Fas, abrogated the T cell response, by inducing apoptosis of the APC-stimulated T cells. The new results of this investigation are (1) that autoreactive T cells can be effectively targeted by autologous APCs that are engineered to present the relevant autoantigen, and (2) that these specifically targeted and activated T cells can be profoundly inhibited by agents that trigger the Fas-mediated apoptosis pathway. The present findings suggest that engineering APCs for simultaneous presentation of the autoantigen and delivery of FasL will provide a powerful strategy for the elimination of autoreactive T cells.

Animals↗
PubMed2004

[Basic researches on cell adhesion and its application in tendon tissue engineering].

Cell adhesion is a basic and very important tissue in the field of tissue engineering. Fibronectin and integrins are the most important elements to cell adhesion. Some surface receptors of fibroblast can also conjugate with type I collagen in extracellular matrix (ECM) directly. Laminin receptors on the surface of fibroblast bound to laminin also play a role in cell adhesion. In this paper are reviewed a number of related articles. The structures and function of fibronectin and integrins are discussed in detail; the tendon cell's adhesion structures are also discussed. Yet, there was scarcely any paper on the effects which the preservation of tissue engineered products may have on cells' adhesion fo ECM. Therefore, researching on cell adhesion and finding a way of preservation that has no or very little adverse effect on cell adhesion is an important topic. Results from expected advanced researches on cell adhesion may probably find promising applications in the field of tissue engineering.

Cell Adhesion↗
PubMed2004

Nanoscale surface engineered living cells with extended substrate spectrum.

We report on cell surface engineering of living microorganisms by using Layer-by-Layer (LbL) technology to extend the substrate spectrum. The yeast Arxula adeninivorans LS3 (Arxula) was employed as a model organism and biological template. By using LbL technology, Arxula cells were encapsulated by polyelectrolyte and enzyme layers. The biological activity of the Arxula was retained after the encapsulation process. The polymeric capsule surrounding the Arxula provides a stable interface for surface engineering of living cells. LbL of polyelectrolytes followed by an enzyme layer of lactate oxidase were assembled. The outer enzyme layer provides an additional biological function for Arxula to convert the unfavourable substrate lactate into the favourable substrate pyruvate, thus extending the substrate spectrum of the organism. Moreover, capsule stability and enzyme conjugate stability of the surface engineered Arxula were studied.

Journal Article↗
PubMedDate not supplied

Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering.

Cell seeding of three-dimensional polymer scaffolds is the first step of the cultivation of engineered tissues in bioreactors. Seeding requirements of large scaffolds to make implants for potential clinical use include: (a) high yield, to maximize the utilization of donor cells, (b) high kinetic rate, to minimize the time in suspension for anchorage-dependent and shear-sensitive cells, and (c) high and spatially uniform distribution of attached cells, for rapid and uniform tissue regeneration. Highly porous, fibrous polyglycolic acid scaffolds, 5-10 mm in diameter and 2-5 mm thick, were seeded with bovine articular chondrocytes in well-mixed spinner flasks. Essentially, all cells attached throughout the scaffold volume within 1 day. Mixing promoted the formation of 20-32-micron diameter cell aggregates that enhanced the kinetics of cell attachment without compromising the uniformity of cell distribution. The kinetics and possible mechanisms of cell seeding were related to the formation of cell aggregates by a simple mathematical model that can be used to optimize seeding conditions for cartilage tissue engineering.

Animals↗
PubMed1999

Engineering novel cell surface receptors for virus-mediated gene transfer.

The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface.

3T3 Cells↗
PubMed2006

Cell delivery in regenerative medicine: the cell sheet engineering approach.

Recently, cell-based therapies have developed as a foundation for regenerative medicine. General approaches for cell delivery have thus far involved the use of direct injection of single cell suspensions into the target tissues. Additionally, tissue engineering with the general paradigm of seeding cells into biodegradable scaffolds has also evolved as a method for the reconstruction of various tissues and organs. With success in clinical trials, regenerative therapies using these approaches have therefore garnered significant interest and attention. As a novel alternative, we have developed cell sheet engineering using temperature-responsive culture dishes, which allows for the non-invasive harvest of cultured cells as intact sheets along with their deposited extracellular matrix. Using this approach, cell sheets can be directly transplanted to host tissues without the use of scaffolding or carrier materials, or used to create in vitro tissue constructs via the layering of individual cell sheets. In addition to simple transplantation, cell sheet engineered constructs have also been applied for alternative therapies such as endoscopic transplantation, combinatorial tissue reconstruction, and polysurgery to overcome limitations of regenerative therapies and cell delivery using conventional approaches.

Acrylic Resins↗
PubMed2005

Assimilating cell sheets and hybrid scaffolds for dermal tissue engineering.

Cell sheets can be used to produce neo-tissue with mature extracellular matrix. However, extensive contraction of cell sheets remains a problem. We devised a technique to overcome this problem and applied it to tissue engineer a dermal construct. Human dermal fibroblasts were cultured with poly(lactic-co-glycolic acid)-collagen meshes and collagen-hyaluronic acid foams. Resulting cell sheets were folded over the scaffolds to form dermal constructs. Human keratinocytes were cultured on these dermal constructs to assess their ability to support bilayered skin regeneration. Dermal constructs produced with collagen-hyaluronic acid foams showed minimal contraction, while those with poly(lactic-co-glycolic acid)-collagen meshes curled up. Cell proliferation and metabolic activity profiles were characterized with PicoGreen and AlamarBlue assays, respectively. Fluorescent labeling showed high cell viability and F-actin expression within the constructs. Collagen deposition was detected by immunocytochemistry and electron microscopy. Transforming Growth Factor-alpha and beta1, Keratinocyte Growth Factor and Vascular Endothelial Growth Factor were produced at various stages of culture, measured by RT-PCR and ELISA. These results indicated that assimilating cell sheets with mechanically stable scaffolds could produce viable dermal-like constructs that do not contract. Repeated enzymatic treatment cycles for cell expansion is unnecessary, while the issue of poor cell seeding efficiency in scaffolds is eliminated.

Actins↗
PubMed1995

Genetically engineered mammalian cells and applications.

In general, cells genetically engineered for stable and defined expression of xenobiotic-metabolizing enzymes are useful tools whenever a metabolism-related problem in toxicology and pharmacology is to be solved. It is the genetic and phenotypic nature of a given cell that determines its applicability. Mammalian cells have useful characteristics not given in bacterial, yeast or insect cells, which also may express xenobiotic-metabolizing enzymes. It is the problem to be solved and the question to be answered which determine the optimal choice for the best-suited expression system. There may even be subtle differences between mammalian cells of different species and organ origin, which might play a role in choosing a mammalian expression system. Thus, the level and specificity of the xenobiotic-metabolizing enzyme, the experimental testing conditions, and the biological endpoints present in a chosen cell are the most important criteria to be observed in the application of the mammalian expression systems.

Animals↗
PubMed2005

Stability and homogeneity of transgene expression in isogenic cells.

Genetically engineered cells are an important tool not only for basic research applications, but also for biotechnology and molecular medicine. Among the issues yet to be solved for this technology are the consequences of randomly integrating DNA into a cell's genome. The problems encountered range from unpredictable expression levels to safety concerns. Recombinase-mediated chromosome engineering is a popular tool for generating stably transfected isogenic cell lines. Using this approach, single-copy integration of foreign DNA fragments can be achieved at predetermined chromosomal loci in the genome. We used such a technology based on the Flp/Flp recombinase target (FRT) recombination system in human 293 cells for comparative promoter studies. The expected integration patterns were obtained with high frequency. In contrast, the phenotypic characterization of expression of the integrated reporter transgene showed remarkable differences between isogenic cell lines, ranging from homogenous expression to mosaic to even complete expression silencing. As long as cell clones with homogenous expression were kept under selective conditions, their expression characteristics could be maintained over a long period. However, this desirable phenotype was progressively lost upon withdrawal of selective pressure. These results were also reflected by the expression instability of the reporter cassette originally inserted in the recipient cell line. Thus, selection for appropriate integration events that ensure reproducible and long-term gene expression has to go beyond the verification of isogenicity.

Cell Line↗
PubMed2026

A modular γδ TCR-T platform combining KRAS pMHC targeting with re-dosable mRNA engager redirection.

Solid tumors often evade TCR-engineered αβ T cells when antigen expression varies or when the restricting Human Leukocyte Antigen (HLA) allele is lost. γδ T cells, in contrast, detect cellular dysregulation through non-peptide/Major Histocompatibility Complex (MHC) cues, including phosphoantigens and stress ligands, and can be developed as allogeneic therapies. Although intratumoral γδ T cell signatures are associated with improved outcome across cancers, γδ recognition itself is broad and still selected within the thymus just as αβ T cell receptors (TCRs) are. It does not, however, anchor specificity to a defined driver-mutation pMHC epitope. We therefore asked whether a high-affinity, co-receptor-independent αβ TCR could graft oncogenic-driver specificity onto γδ T cells while leaving the endogenous γδ TCR intact. We knocked the KRASG12V/HLA-A*11:01 TCR A11v into primary human γδ T cells. Engineered cells co-expressed the transgenic αβ TCR and the endogenous γδ TCR and lysed KRASG12V/HLA-A*11:01+ tumor cells in vitro and in vivo. To cover potential resistance through loss of HLA-A*11:01, we delivered an mRNA lipid nanoparticle (LNP) encoding a secreted mesothelin×CD3 (M5) bispecific T cell engager (TCE). LNP-M5 produced circulating TCE that redirected γδ A11v T cells and polyclonal bystander T cells to kill mesothelin+ targets, accompanied by development of higher γδ A11v T cell counts in vivo. In humanized mice bearing mixed HLA-A*11:01+ and HLA-A*11:01 - KRASG12V tumors, γδ A11v T cells produced transient control, whereas adding LNP-M5 yielded complete responses and prolonged survival. Thus, this two-part therapy couples invariant driver targeting to tunable redirection and addresses loss of the restricting HLA allele, a central escape route for TCR-based therapy. It provides an off-the-shelf reagent to enable KRAS-anchored treatment with the ability to redeliver the reagent.

Humans↗
PubMed2006

Transplantation of tissue-engineered epithelial cell sheets after excimer laser photoablation reduces postoperative corneal haze.

PURPOSE: To apply tissue-engineered cell sheet transplantation after excimer laser keratectomy as a novel approach for the reduction of postoperative corneal haze. METHODS: Limbal biopsy specimens were obtained, and epithelial cells were cultured on temperature-responsive culture inserts without the use of feeder cells. Laser keratectomy (7.0-mm ablation zone and 160-microm depth) was performed in the contralateral eye, and autologous epithelial cell sheets were transplanted to the ablated corneal stroma. Transplant and control group eyes were assessed by slit lamp biomicroscopy, and corneal haze was scored in a masked fashion, according to the Fantes grading scale. For further examination histologic and immunohistochemical analyses were performed. RESULTS: Tissue-engineered cell sheets produced stable attachment to the laser-ablated sites, resulting in epithelialization, 5 minutes after transplantation. Conversely, control corneas required 3 to 5 days for complete re-epithelialization. At both 1 and 2 months after surgery, corneal haze was significantly inhibited in the transplant group. Histologic analyses showed that the number of keratocytes undergoing apoptosis was decreased in the transplant group at 3 days after surgery. Similarly, the expression of both collagen III and alpha-smooth muscle actin, which may enhance corneal haze, were diminished in the transplant group at 2 months. CONCLUSIONS: The transplantation of tissue-engineered epithelial cell sheets can successfully prevent the development of corneal haze after excimer laser keratectomy.

Actins↗
PubMed2005

Cell processing engineering for ex-vivo expansion of hematopoietic cells.

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.

Animals↗
PubMed2005

[Newly progress in applications of Saccharomyces cerevisiae cell-surface engineering].

Saccharomyces cerevisiae cell-surface engineering is a newly genetic expressing system for fusion heterologous gene. With its ability of post-translational modification, and the convenience to identify and screen the expressed products, it is undergoing a marvelous progress in its newly applications, which include using as whole-cell biocatalyst, antigen/antibody library, bioadsorbent, biosensor, combinatory protein library, immunoassays and affinity purification. It performs a more and more important role in exploring the function and application of protein molecules.

Biosensing Techniques↗
PubMed2005

Interleukin 7-engineered stromal cells: a new approach for hastening naive T cell recruitment.

In this study we determined whether human stromal cells could be engineered with a retroviral vector carrying the interleukin 7 (IL-7) gene and investigated the effects on T cells in vitro and in vivo in a murine model. Transduced mesenchymal cells strongly express CD90 (98.15%), CD105 (87.6%), and STRO-1 (86.7%). IL-7 production was 16.37 (+/-2 SD) pg/ml, which remained stable for 60 days. In vitro-immunoselected naive T cells maintained the CD45RA+ CD45RO- naive phenotype (4.2 times more than controls) after 7 days of culture with IL-7-engineered stromal cells. The apoptosis rate (4.7%) of the naive T cells cultured with transduced stromal cells overlapped with that of freshly isolated cells. Immunohistological analysis detected stromal cells in bone marrow, spleen, and thymus. Cotransplantation of IL-7-engineered stromal cells with CD34+ cells improved engraftment in terms of CD45+ cells and significantly increased the CD3+ cell count in peripheral blood, bone marrow, and spleen. These data demonstrate the following: (1) human stromal cells can be transduced, generating a normal layer; (2) transduced stromal cells in vitro maintain the naive T cell phenotype; and (3) IL-7-transduced stromal cells in vivo home to lymphoid organs and produce sufficient IL-7 in loco, supporting T cell development in a cotransplantation model. Because of their efficient cytokine production and homing, IL-7-engineered stromal cells might be an ideal vehicle to hasten immunological reconstitution in T cell-depleted hosts.

Animals↗