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At least 19 recordsLinked to original sources
PubMed1991

Cellular engineering.

Cellular engineering applies the principles and methods of engineering to the problems of cell and molecular biology of both a basic and applied nature. As biomedical engineering has shifted from the organ and tissue level to the cellular and sub-cellular level, cellular engineering has emerged as a new area. A cornerstone of much of this activity is cell culture technology, i.e., the ability to grow living cells in the artificial environment of a laboratory. Cellular engineering includes the role of engineering in both basic cell biology research and in the making of products which use living cells, e.g., tissue engineering and bioprocess engineering. The former involves the use of living cells in the development of biological substitutes for the restoration or replacement of function, and the latter the use of living cells to manufacture a biochemical product, e.g., through the use of recombinant DNA technology. In fact, as biomedical engineering has expanded to include the cellular level, and bioprocess engineering has shifted in interest from microbial organisms to include mammalian cells, there are intellectual issues in which an interest is shared by these two formerly separate areas of engineering activity. Cellular engineering thus transcends the field of biomedical engineering.

Biomedical Engineering↗
PubMed2006

Teaching cellular engineering.

Cellular engineering is one of the fastest growing subdisciplines in the field of Biomedical Engineering. It involves the application of engineering analysis to understand and control cellular behavior, with the ultimate objective of developing novel therapeutic or diagnostic approaches for the clinic or harnessing cellular function for commercial applications. Well-educated students in this area need strong foundational knowledge in engineering science, chemistry, and cell and molecular biology. In undergraduate curricula, the challenge is to include essential engineering skills plus appropriate levels of training in chemistry and biology while satisfying accreditation-mandated breadth in engineering training. At the graduate level, educators must accommodate students with diverse backgrounds and provide them with both a state-of-the-art understanding of the life sciences and the most advanced engineering skills. Engineering curricular content should include mechanics and materials, physical chemistry, transport phenomena, and control theory. Training from faculty with appointments and research programs in the life sciences is generally recommended, and additional life science content should also be integrated within the engineering curriculum. A capstone course in cellular engineering that includes opportunities for students to have hands-on experiences with state-of-the-art laboratory techniques is highly recommended.

Benchmarking↗
PubMed2004

Kinetic analysis of RSK2 and Elk-1 interaction on the serum response element and implications for cellular engineering.

Immediate early gene activation upon mitogenic activation occurs through the serum response element (SRE), which makes the delineation of the upstream pathways a powerful means to engineer cellular responses. The malfunctioning of this system leads to a variety of disorders, ranging from neurological disorders such as Coffin-Lowry syndrome (RSK2 mutations) to cancer (c-fos mutations). We therefore investigated the SRE activation mechanism in a typical mammalian cell. Mitogenic signaling uses the mitogen-activated protein kinase (MAPK) module through increased binding of the ternary complex factor (TCF), such as Elk-1, to the promoter DNA (the SRE element) and subsequent transcriptional activation, as well as through activation of a histone kinase, such as the MAPK-activated protein kinase (MAPKAP-K) ribosomal S6 kinase (RSK2). This computational model uses the biochemical simulation environment GEPASI 3.30 to investigate three major models of interaction for Elk-1 and RSK2, and to study the effect of histone acetyl transferase (HAT) recruitment in each of these models on the local chromatin modifications in the presence and absence of MAPK activation. We show that the quickest response on the chromatin can be achieved in the presence of a preformed complex of RSK2, Elk-1 and HAT, with HAT being activated upon dissociation from the complex upon activation of the MAPK cascade. This study presents critical components in the pathway that can be targeted for engineering of specific inhibitors or activators of the system.

Acetyltransferases↗
PubMed2003

Proteome profiling and its use in metabolic and cellular engineering.

Proteome profiling of microorganisms makes it possible to generate valuable knowledge that can be used for the development of metabolic and cellular engineering strategies, which consequently are used to enhance the yield and productivity of native or foreign bioproducts and to modify cellular properties to improve mid-stream and down-stream processes. Advances in two-dimensional gel electrophoresis technology combined with mass spectrometry allow the creation of global scale proteome contents which can be used to elucidate valuable information on the dynamics of the metabolic, signaling and regulatory networks apart from understanding the physiological changes. In this paper, we review the approaches of exploiting the proteome profiling results to the development of the strategies for the metabolic and cellular engineering of microorganisms.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2003

Cellular engineering of HSV-tk transduced, expanded T lymphocytes for graft-versus-host disease management.

Engineering donor T lymphocytes with inducible 'suicide genes', such as herpes simplex virus thymidine kinase, has potential to improve safety and efficacy in allogeneic transplantation by facilitating management of graft-versus-host disease. Elective administration of a relatively nontoxic pro-drug would induce in vivo negative selection of engineered lymphocytes specifically, sparing other donor hematopoietic cells. The engineered cells must retain immunologic function, and undergo negative selection in response to clinically attainable plasma concentrations of pro-drug. The cell engineering process itself, typically involving activation, transduction, ex vivo expansion, and selection, must produce clinically useful numbers of genetically modified cells at high purity. We discuss development of a cellular engineering manufacturing process that yields transduced, expanded T lymphocytes meeting these requirements.

Adoptive Transfer↗
PubMed2004

Cellular engineering in a minimal microbe: structure and assembly of the terminal organelle of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a minimal microbe with respect to cell envelope composition, biosynthetic and regulatory capabilities and genome size, yet it possesses a remarkably complex, multifunctional terminal organelle. This membrane-bound extension of the mycoplasma cell is defined by the presence of an electron-dense core that appears as paired, parallel bars oriented longitudinally and enlarging at the distal end to form a terminal button. Most non-cytadhering mutants of M. pneumoniae isolated to date exhibit defects in the architecture of the terminal organelle. Detailed characterization of those mutants has revealed the identities of many component proteins of the terminal organelle as well as the likely order in which some of those components are required. Additional questions regarding the composition of the electron-dense core, the means by which the terminal organelle is duplicated during cell division and the manner in which this process is regulated remain to be answered. Thus, it seems that there is much to be learned about cellular engineering and spatial regulation in these 'simple' cell wall-less bacteria.

Adhesins, Bacterial↗
PubMed2006

Characterization of cord blood natural killer and lymphokine activated killer lymphocytes following ex vivo cellular engineering.

Cord blood (CB) natural killer (NK) and lymphokine-activated killer (LAK) cytotoxic cells are poorly characterized but might be used to treat minimal residual and/or recurrent malignant disease. Currently, there is no mechanism to use CB for adoptive cancer cellular immunotherapy after CB transplantation (CBT). Recognizing this as a deficiency, we hypothesized that CB aliquots could be engineered ex vivo for potential donor lymphocyte infusion after CBT. Cryopreserved CB aliquots were thawed, depleted of monocytes, and cultured in serum-free medium alone or serum-free medium with anti-CD3 and interleukins 2, 7, and 12 combined with antibody/cytokines for 48 hours. Immunophenotyping, cytotoxicity, and proliferation were evaluated. A significant expansion of CD3+ was seen, in addition to increases in lymphocyte subsets of CD8+, CD8+/CD25+, and CD3+/45RO+ versus medium alone. A significant enhancement of CD3 proliferation (P<.001), NK cytotoxicity, NK subset expansion, LAK cytotoxicity, and T-helper 1 subset expansion was also demonstrated. Significant enrichment was seen in NK CD16+/CD56+bright, CD16+/CD56+dim, CD56+bright and CD56+dim/KIR3DL1+, CD56+bright and CD56+dim/KIR2DL1+, CD56+bright and CD56+dim/KIR2DL2+ and CD94+/NKG2a+ subsets. These increases in CB NK subsets were in part secondary to augmentation of cell survival. Further, survival of NOD-SCID mice xenografted with human K562 cells and treated with CB cells expanded with antibody/cytokines was significantly higher than that in animals that received no treatment (phosphate buffered saline) and those that were treated with CB ex vivo expanded in medium alone (P<.005, respectively). These data suggest that cryopreserved CB cells could be ex vivo engineered for potential use as adoptive cancer cellular immunotherapy for donor lymphocyte infusion after CBT.

Animals↗
PubMed2002

Engineering cellular microenvironments to improve cell-based drug testing.

Recent progress in the biology of cell adhesion is enabling cell culture models to better reproduce in vivo functions. Cues from adhesion to extracellular matrix and neighboring cells are important regulators of cell behaviors. The recent adaptation of semiconductor tools to spatially organize cells and their adhesions has enhanced our ability to engineer cell functions ex vivo. By using these tools to create more in vivo-like cultures, cell-based drug discovery and target validation could be improved. This review explores the biological advances made by these microfabrication tools and discusses how they could enable high-throughput cell-based assays.

Animals↗
PubMed2003

Engineered cellular response to scaffold architecture in a rabbit trephine defect.

Tight control of pore architecture in porous scaffolds for bone repair is critical for a fully elucidated tissue response. Solid freeform fabrication (SFF) enables construction of scaffolds with tightly controlled pore architecture. Four types of porous scaffolds were constructed using SFF and evaluated in an 8-mm rabbit trephine defect at 8 and 16 weeks (n = 6): a lactide/glycolide (50:50) copolymer scaffold with 20% w/w tri-calcium phosphate and random porous architecture (Group 1); another identical design made from poly(desaminotyrosyl-tyrosine ethyl ester carbonate) [poly(DTE carbonate)], a tyrosine-derived pseudo-polyamino acid (Group 2); and two poly(DTE carbonate) scaffolds containing 500 microm pores separated by 500-microm thick walls, one type with solid walls (Group 3), and one type with microporous walls (Group 4). A commercially available coralline scaffold (Interpore) with a 486-microm average pore size and empty defects were used as controls. There was no significant difference in the overall amount of bone ingrowth in any of the devices, as found by radiographic analysis, but patterns of bone formation matched the morphology of the scaffold. These results suggest that controlled scaffold architecture can be superimposed on biomaterial composition to design and construct scaffolds with improved fill time.

Animals↗
PubMed2004

Treatment of advanced Ewing tumors by combined radiochemotherapy and engineered cellular transplants.

This review will focus primarily on own recent work on the treatment of advanced Ewing tumors (AETs) and will attempt, in addition, to give a comprehensive overview of novel developments. The field under review has been shaped by investigators from both Europe and the United States of America in a scientific debate evolving over more than a decade at the meetings of the International Society of Pediatric Oncology and other scientific meetings. In the light of this debate, most oncologists will agree that patients with AETs are facing the worst prognosis of all patients with this disease and include both: (i) patients with primary metastatic disease with the worst prognosis as well as (ii) patients with relapse with the worst prognosis. The contributions of various investigators have lead to the identification of specific risk stratification criteria to overcome the heterogeneity of patients within the conventionally defined clinical stages of localized metastatic and relapsed disease. This review will address the following issues of treatment of AETs: (i) a definition of AET; (ii) risks and benefits of allogeneic vs. autologous stem cell transplantation; (iii) the role of total body irradiation; (iv) the number of involved bones as a risk factor in multifocal bone disease in AET; (v) the development of immunogene therapy in AET; (vi) the matching of radiochemo- and immunotherapy in AET; (vii) the future perspective of functional genomics and targeted therapy.

Animals↗
PubMed2003

Surface functionalization and grafting of heparin and/or RGD by an aqueous-based process to a poly(carbonate-urea)urethane cardiovascular graft for cellular engineering applications.

An aqueous-based process is reported for surface functionalization and grafting of anticoagulant and cell attachment moieties, such as heparin and/or arginine-glycine- aspartate (RGD) onto the lumenal surface of a prefabricated cardiovascular graft (5 mm i.d.) made of poly(carbonate- urea)urethane (MyoLink). It is a three-stage process, all aqueous: (1) hydroxylation using an azobis compound, particularly 2,2'-azobis(2-methylpropionamidine)dihydrochloride, which abstracts hydrogen via an electron transfer process from the polyurethane surface (strong oxygen purging); (2) grafting using the as-generated hydroxide groups to allow attachment of an acrylamide monomer using a conventional ceric ion technique (strong nitrogen purging); and (3) moiety attachment, preactivated with [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] in acidic solution. The technique was validated by attaching heparin and RGD/heparin to the MyoLink polymer. Following bonding, the graft segments were exposed to prolonged physiologic shear force in a flow circuit (10 h). The grafts first were analyzed by X-ray photoelectron spectroscopy (XPS) to determine the degree of attachment of the moieties and then by materials methods to assess whether any degradation of the graft material itself had occurred since polyurethanes with carbonate amorphous segments are readily susceptible to hydrolytic degradation following functionalization processes. XPS showed the moieties were present on the surface at a concentration of 10%. The S2p(3/2) states of sulfur indicated that there were high degrees of ionic covalent bonding, indicating high degrees of moiety bioactivity. Heparin was found to be present from the sulfur signal, namely NSO(3). RGD was found to be present from the nitrogen signal present at the binding energy of 399 eV. Macroscopic analysis and ESEM showed no signs of polyurethane degradation or small protuberances indicative of microgel formation. Quality control (QC) showed that the internal diameters and wall thicknesses of all the respective grafts postbonding remained within normal batch release limits (5 +/- 0.1mm, i.d.; 0.9 +/- 0.05 mm, wall thickness). Gel permeation chromatography (GPC) showed there were no statistical differences between the control, which was nonbonded (MN 45,300, MW 98,500, D 2.17) and all of the bonded samples, respectively (MN 41,800, MW 104,000, D 2.45). Radial tensile strength (RTS) analysis also showed that all of the respective samples postbonding (1.48N/mm) remained within batch release specifications (>1N/mm). A simple aqueous polymer surface functionalization and grafting technique has been developed for covalent bonding of anticoagulant and cell-attachment moieties onto poly(carbonate-urea)urethane(s) and has been validated by surface and materials analyses. The moieties were attached uniformly and were bioactive at a high surface density. No degradation in terms of a loss in mechanical properties was evident following bonding of the polyurethane.

Chromatography, Gel↗
PubMed2001

Cellular engineering of vascular bypass grafts: role of chemical coatings for enhancing endothelial cell attachment.

Surgical treatment of vascular disease has become common. The use of synthetic materials is limited to grafts larger than 5-6mm, because of the frequency of occlusion observed with small-diameter prosthetics. An alternative would be a hybrid or tissue-engineered graft with the surface coated with a monolayer of the patient's own cells. Currently, to be effective, high-density seeding regimens have to be undertaken. This is because endothelial cells (ECs) are washed off the graft lumen once exposed to physiological blood flow. EC attachment has been shown to be significantly improved by pre-coating with substances known to attach ECs selectively. The review examines the various types of coating and bonding technology used to date to enhance endothelial cell attachment onto the surface of prosthetic vascular bypass grafts.

Biocompatible Materials↗
PubMed2004

Cellular engineering of conduits for coronary and lower limb bypass surgery: role of cell attachment peptides and pre-conditioning in optimising smooth muscle cells (SMC) adherence to compliant poly(carbonate-urea)urethane (MyoLink) scaffolds.

OBJECTIVE: We are developing a hybrid arterial bypass graft of compliant poly(carbonate-urea)urethane (MyoLink), endothelial and smooth muscle cells (SMCs). To enhance adhesion of SMCs we assessed various attachment factors and the effect of pre-conditioning on cell retention. METHODS: MyoLink segments were coated with either RGD, superfibronectin, fibronectin, fibronectin-like engineered polymer protein (FEPP), FEPP plus or type 1 collagen overnight. (111)Indium-radiolabelled SMCs were placed onto MyoLink segments for 48 h before being aspirated, then lavaged off. All grafts, aspirates and lavages were counted in a gamma counter. SMC viability on the MyoLink segments was also assessed for viability using the Alamar blue redox assay. Separately, MyoLink grafts lined with radiolabelled SMCs were divided into a pre-conditioned group, exposed to subarterial pulsatile flow whilst another group were held in static culture. After 1-week, grafts were exposed to arterial pulsatile flow whilst radioactivity was assessed using a gamma camera. RESULTS: Only FEPP plus significantly enhanced SMC attachment: mean of 32+/-6% cell attachment compared to 21+/-5% for uncoated control. Cell viability was enhanced by all attachment factors except fibronectin. Pre-conditioning was shown to significantly enhance the retention of SMCs onto the MyoLink once exposed to pulsatile arterial flow: the final attachment was 57+/-7% for the static and 76+/-7% for the pre-conditioned group. CONCLUSIONS: FEPP plus enhances SMC attachment to MyoLink. We believe this is because of its repeating sequences of RGD and its positive charge. Pre-conditioning enhances the retention of SMCs to MyoLink once exposed to pulsatile arterial flow.

Blood Vessel Prosthesis↗
PubMed2003

Cellular engineering of ventricular adult rat cardiomyocytes.

OBJECTIVE: Preparation of viable cultured adult cardiomyocytes (vARCs) is a prerequisite for cell-based transplantation and tissue engineering. Ectopic gene expression is important in this context. Here, we present an in vitro cell replating strategy using Accutase for cultured vARCs, allowing ectopic gene expression. METHODS: Cultured vARCs from 6- to 8-week-old rats were used. Transfections with EGFP (enhanced green fluorescent protein) constructs, Mlc-3f-EGFP or alpha-actinin-EGFP were performed using adenovirus-enhanced transferrin-mediated infection (AVET). Accutase (PAA Laboratories, Linz, Austria) was used for the detachment of cultured cells. Immunohistochemical analysis, together with confocal laser microscopy was used for structural analysis of the cells. RESULTS: Cultured vARCs could be detached with a high yield (40 to 60%) from primary cultures using Accutase. The cultivation period plays an important role in the yield of viable cells. Resultant replated vARCs (rep-vARCs) rapidly (1-2 h) acquired a rounded up shape without degradation of their contractile apparatus, which is in contrast to the rod-shaped freshly isolated vARCs (fi-vARCs). The detached cells survived passage through a narrow syringe needle. After seeding, detached cells rapidly attached to various substrates, increased their content of the contractile apparatus, and formed cell-cell contacts within 3 days after reseeding. The detached cells survived passage through a narrow syringe needle. The high recovery of cells after replating enabled the use of the AVET system for gene delivery. AVET is free of infectious particles and does not lead to expression of viral proteins. Transfection of vARCs prior to detachment had a small effect on cell recovery and ectopically synthesized proteins were properly localized after replating. CONCLUSIONS: Detachment of cultured vARCs using Accutase is well compatible with ectopic gene expression and yields a viable transgenic population of vARCs that eventually may be suitable as transgenic cardiomyocyte grafts.

Actinin↗