Non-invasive monitoring of tissue-engineered pancreatic constructs by NMR techniques.
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Animals↗
Biomedical subjects
Athanassios Sambanis
Publications and source records attributed to Athanassios Sambanis.
Proteasome modulating agents induce rAAV2-mediated transgene expression in human intestinal epithelial cells.
Intestinal gene transfer offers promise as a therapeutic option for treatment of both intestinal and non-intestinal diseases. Recombinant adeno-associated virus serotype 2, rAAV2, based vectors have been utilized to transduce lung epithelial cells in culture and in human subjects. rAAV2 transduction of intestinal epithelial cells, however, is limited both in culture and in vivo. Proteasome-inhibiting agents have recently been shown to enhance rAAV2-mediated transgene expression in airway epithelial cells. We hypothesized that similar inhibition of proteasome-related cellular processes can function to induce rAAV2 transduction of intestinal epithelial cells. Our results demonstrate that combined treatment with proteasome-modulating agents MG101 (N-acetyl-L-leucyl-L-leucyl-L-norleucine) and Doxorubicin synergistically induces rAAV2-mediated luciferase transgene expression by >400-fold in undifferentiated Caco-2 cells. In differentiated Caco-2 monolayers, treatment with MG101 and Doxorubicin induces transduction preferentially from the basolateral cell surface. In addition to Caco-2 cells, treatment with MG101 and Doxorubicin also results in enhanced rAAV2 transduction of HT-29, T84, and HCT-116 human intestinal epithelial cell lines. We conclude that MG101 and Doxorubicin mediate generic effects on intestinal epithelial cells that result in enhanced rAAV2 transduction. Use of proteasome-modulating agents to enhance viral transduction may facilitate the development of more efficient intestinal gene transfer protocols.
Caco-2 Cells↗
Viscoelastic testing methodologies for tissue engineered blood vessels.
In order to function in vivo, tissue engineered blood vessels (TEBVs) must encumber pulsatile blood flow and withstand hemodynamic pressures for long periods of time. To date TEBV mechanical assessment has typically relied on single time point burst and/or uniaxial tensile testing to gauge the strengths of the constructs. This study extends this analysis to include creep and stepwise stress relaxation viscoelastic testing methodologies. TEBV models exhibiting diverse mechanical behaviors as a result of different architectures ranging from reconstituted collagen gels to hybrid constructs reinforced with either untreated or glutaraldhyde-crosslinked collagen supports were evaluated after 8 and 23 days of in vitro culturing. Data were modeled using three and four-parameter linear viscoelastic mathematical representations and compared to porcine carotid arteries. While glutaraldhyde-treated hybrid TEBVs exhibited the largest overall strengths and toughness, uncrosslinked hybrid samples exhibited time-dependent behaviors most similar to native arteries. These findings emphasize the importance of viscoelastic characterization when evaluating the mechanical performance of TEBVs. Limits of testing methods and modeling systems are presented and discussed.
Animals↗
Effects of cryopreservation on cell viability and insulin secretion in a model tissue-engineered pancreatic substitute (TEPS).
The use of encapsulated insulin-secreting cells constitutes a promising approach towards the treatment of insulin-dependent diabetes. However, long- term storage for off-the-shelf availability still remains an issue, which can be addressed by cryopreservation. This study investigated cryopreservation of a model tissue-engineered pancreatic substitute by two ice-free cryopreservation (vitrification) solutions (designated VS55 and PEG400) in comparison to a conventional freezing protocol. The model substitute consisted of insulin-secreting mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cell viability and static insulin secretion from the thawed cryopreserved groups were characterized and compared against fresh controls. Cell viability tests using alamarBlue showed that, compared to the fresh groups, the VS55 had the highest viability (p < 0.05), followed by both the PEG400 (p < 0.001) and the frozen groups (p < 0.001). In response to a square wave of glucose, the static insulin secretion data showed that the VS55 and PEG400 groups had similar induction levels against the fresh group, whereas the frozen group had the poorest secretion rate. Cryosubstitution of capsules showed ice formation in the frozen group but no ice in the vitrified groups. Microscopic observations revealed holes and/or tears within beads subjected to freezing, whereas no such abnormalities were detected in the vitrified samples. Overall, vitrification was found to be a promising preservation procedure for this encapsulated cell system.
Animals↗
Magnetically labeled insulin-secreting cells.
Iron oxide nanoparticles have been shown to magnetically label cells in order to visualize them in vivo via MR imaging. This technology has yet to be implemented in insulin secreting cells, thus it is not known whether the presence of these nanoparticles in the cytoplasm of the cells affects insulin secretion. This study investigates the effectiveness and consequence of labeling mouse insulinoma betaTC3 and betaTC-tet cells with monocrystalline iron oxide nanoparticles (MION). Our data show that MION can be internalized in both betaTC3 and betaTC-tet cells following a 24h exposure to 0.02mg/ml MION solution. The metabolic and secretory activities of both MION-labeled cell lines were statistically indistinguishable from sham treatment. Furthermore, cell viability and apoptosis remained constant throughout the cell's exposure to MION. Finally, MR images demonstrated significant contrast between labeled and sham-treated cells. Thus, labeling murine insulinoma cell lines with magnetic iron oxide nanoparticles does not hinder their insulin secretion, while it provides MR imaging contrast.
Animals↗
Differential rAAV2 transduction efficiencies and insulin secretion profiles in pure and co-culture models of human enteroendocrine L-cells and enterocytes.
BACKGROUND: Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than insulin injections. Previously, we developed an engineered human enteroendocrine L-cell model for regulated insulin release via recombinant adeno-associated virus serotype 2, or rAAV2, transduction. The aim of this study was to evaluate the efficiency and selectivity of rAAV2-mediated insulin gene delivery to enteroendocrine L-cells in co-culture with a prevailing number of enterocytes, which are the predominant cell type in intestinal epithelium. METHODS: We tested rAAV2 transduction in pure and co-culture models of human cell lines of enterocytes (Caco-2 and T84 cell lines) and enteroendocrine L-cells (NCI-H716 cell line). Non-viral, chemical-mediated transfection was used as a control. Transduced and transfected co-cultures were subjected to insulin secretion studies. RESULTS: In pure cultures, rAAV2 exhibited a low transduction efficiency towards both Caco-2 and T84 enterocytes, as opposed to a strong reporter expression in permissive NCI-H716 L-cells. In co-cultures of NCI-H716 L-cells and Caco-2 or T84 enterocytes, rAAV2 exhibited differential transduction efficiency with a strong preference towards NCI-H716 L-cells. The rAAV2-transduced co-culture achieved regulated insulin release against stimulation, whereas the chemically transfected co-culture failed to respond. CONCLUSIONS: This study demonstrated that rAAV2-mediated insulin gene transfer can differentiate human intestinal cell types in vitro, in particular enterocyte and enteroendocrine L-cell lines. We consider the AAV2 vector a useful tool in developing enteroendocrine L-cell-specific insulin gene delivery for IDD treatment, in terms of AAV2 avoiding enterocytes and targeting selectively L-cells.
Cell Line↗
The role of the CaCl2-guluronic acid interaction on alginate encapsulated betaTC3 cells.
Previously we demonstrated that alginate composition has a significant effect on the growth of encapsulated betaTC3 cells and consequently on the overall metabolic and secretory activities of the encapsulated cultures. Based on these results we postulated that the mechanical properties of alginate were not responsible for the observed effects but rather, changes in the strength of the alginate gel network caused by changes in the number of alginate strands held together in the "egg-box" model are responsible for the observed effects. In this study we address this hypothesis with a series of experiments in which the strength of this interaction is manipulated by varying the calcium concentration either at the time of gelation or during culture maintenance. Our data show that increasing the concentration of the CaCl2 solution used at the time of gelation, thus increasing the strength of the alginate gel network, impedes the growth characteristics of betaTC3 cells encapsulated in a high guluronic acid content alginate. This effect is amplified by maintaining a constant supply of calcium ions during culture thus sustaining the interaction between guluronic acid residues and calcium ions. However, preparations of betaTC3 cells encapsulated in an alginate with high mannuronic acid content are not affected by changes in CaCl2 concentration due to the low percentage of consecutive guluronic acid residues. Therefore, the present data show that the strength of the alginate gel network is an important factor that influences the growth characteristics of encapsulated cell preparations.
Alginates↗
Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer.
Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include intestinal enteroendocrine cells genetically engineered to secrete insulin in response to physiologic stimuli; responsiveness occurs at the exocytosis level to regulate the acute release of recombinant insulin. In this work, we established a human cellular model to demonstrate that meat hydrolysate can simultaneously stimulate glucagon-like peptide-1 (GLP-1, an enteroendocrine cell-derived incretin hormone) and recombinant insulin secretion from the engineered human NCI-H716 intestinal cell line. Cells were genetically modified using the recombinant adeno-associated virus (rAAV)-mediated insulin gene transfer. Recombinant cells were then differentiated to display endocrine features, in particular the formation of granule-like compartments. A fusion protein of insulin and enhanced green fluorescence protein (EGFP) was designed to reveal the compartments of localization of the fusion protein and assess its co-localization with endogenous GLP-1. Our work provides a unique human cellular model for regulated insulin release through genetic engineering of GLP-1-secreting intestinal cells, which is expected to be useful for cell-based therapies of IDD.
Cell Line↗
Preproinsulin mRNA engineering and its application to the regulation of insulin secretion from human hepatomas.
Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than daily insulin injections. Promising cells include non-beta cells genetically engineered to secrete insulin in response to physiologic cues; responsiveness can be introduced at the transcriptional level to regulate preproinsulin (PPI) mRNA biosynthesis. However, these cells exhibit sluggish secretion dynamics, which is not appropriate for achieving euglycemia in higher animals and, eventually, humans. In this work, we have engineered the PPI mRNA so as to destabilize it through nonsense-mediated mRNA decay (NMD). When expressed under transcriptional regulation in HepG2 hepatomas, the engineered PPI mRNA level and of the insulin secretion rate declined faster upon switching off transcription, compared to the one-copy non-engineered control. Our work provides a simple and straightforward method to improve the dynamics of transcriptionally regulated insulin secretion, which can be a useful tool in developing cell-based therapies for IDD.
Base Sequence↗
A biological hybrid model for collagen-based tissue engineered vascular constructs.
Various approaches to tissue engineering a small diameter blood vessel have historically relied upon extended culturing periods and/or synthetic materials to create mechanical properties suitable to withstand the hemodynamic stresses of the vasculature. In this work, we present the concept of a construct-sleeve hybrid (CSH) graft, which uses a biological support to provide temporary reinforcement while cell-mediated remodeling of the construct occurs. Support sleeves were fabricated from Type I collagen gels and crosslinked with glutaraldehyde, ultraviolet, or dehydrothermal treatments. Uniaxial tensile testing of acellular sleeves revealed increased stiffness moduli and tensile stresses with crosslinking treatments. A second collagen layer containing cells was molded about the sleeve to create a CSH. After in vitro culture, CHSs with uncrosslinked (UnXL) and glutaraldehyde treated (Glut) sleeves exhibited significant increases in mechanical strength (20.4-fold and 121-fold increases in ultimate stress, respectively) compared to unreinforced control constructs. Burst testing produced similar findings with peak pressures of 100 and 650mmHg in the UnXL and Glut CSHs, respectively. Construct compaction, cell viability, and histological examination demonstrated that the function of most cells remained unimpaired with the incorporation of the biological support sleeve.
Biomedical Engineering↗
Encapsulated islets in diabetes treatment.
Encapsulation of insulin-producing cells in semipermeable membranes has the potential to provide an effective treatment for insulin-dependent diabetes with little or no immunosuppression of the host. Improvements in alginate, a marine polysaccharide commonly used for cell encapsulation, have revived interest in this material. However, serious obstacles, including a reliable cell source and a better understanding of immune acceptance issues, remain to be addressed before a clinically applicable therapeutic procedure based on encapsulated cells becomes available.
Alginates↗
Effects of alginate composition on the growth and overall metabolic activity of betaTC3 cells.
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Alginates↗
Noninvasive monitoring of a retrievable bioartificial pancreas in vivo.
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Animals↗
Interaction of vascular smooth muscle cells with collagen-impregnated embolization coils studied with a novel quantitative in vitro model.
BACKGROUND AND PURPOSE: Modifications of aneurysm occlusion devices and other biologically active molecules may reduce the risk of recanalization by promoting vascular cell migration, adhesion, and proliferation. Our purpose was to apply in vitro methods in the qualitative and quantitative analysis of vascular smooth muscle cell (VSMC) interactions with collagen-impregnated microcoils. METHODS: The adhesion of rat aortic VSMCs to collagen fiber bundles (CFBs), nitinol coils, and collagen-impregnated nitinol coils (CINCs) was examined by using an assay consisting of monopulse exposure to increasing concentrations of rat aortic VSMCs. Exposed devices were washed and examined by using confocal fluorescence microscopy. Adhesion coefficients, which quantitatively express the cell-binding quality of a surface, were determined by using a mathematical model for cell-device interactions. RESULTS: VSMCs, attached to devices, spread out and extended cytoplasmic projections over the contact surface. Cell distribution was random on CFBs and within interloop troughs on nitinol coils. On collagen-impregnated coils, VSMCs were selectively concentrated on the collagen between coil loops. The average adhesion coefficient was 25.0 for CFBs, 8.5 for CINCs (250-microm pitch), and 6.5 for nitinol coils. Adhesion coefficient differences for the three devices were significant (P =.044). CONCLUSION: The monopulse exposure assay is a simple and reproducible in vitro test that provides qualitative information about the morphology and topography of cell-device contacts and permits quantitative measurement of the intrinsic cell-binding quality of the test device. VSMCs exposed to collagen-impregnated microcoils selectively attach to collagen. Collagen enhances the rate of VSMC adhesion to embolic devices, and the degree of enhancement correlates with the surface area constituted by collagen.
Alloys↗
Incorporation of intact elastin scaffolds in tissue-engineered collagen-based vascular grafts.
Although collagen-based tissue-engineered blood vessels (TEBVs) have many interesting properties and have been utilized to study aspects of vascular biology, these constructs are too weak to be implanted as bypass grafts for in vivo investigations. This study presents a method to incorporate organized, intact elastin into collagen-based TEBVs to form hybrid constructs that better mimic arterial physiology and exhibit improved mechanical properties. Porcine carotids were digested with a series of autoclave and chemical treatments to elicit isolated elastin scaffolds. Elastin purity was verified via immunohistochemistry and amino acid analysis. Isolated scaffolds were combined with type I collagen and either human dermal fibroblasts (HDFs) or rat smooth muscle cells (RASMs) to form an elastin hybrid TEBV. Hybrid constructs exhibited increased tensile strengths (11-fold in HDFs; 7.5-fold in RASMs) and linear stiffness moduli (4-fold in HDFs; 1.8-fold in RASMs) compared with collagen control constructs with no exogenous elastin scaffold. Viscoelastic properties of the TEBVs also improved with the addition of an ancillary elastin scaffold as determined through stepwise stress relaxation analysis. Whereas the majority of resistance to deformation in collagen control constructs stemmed from viscous fluidlike effects, elastin hybrid constructs exhibited more ideal elastic solid mechanical behavior. Thus, elastin scaffolds can help recreate the elastic properties of native arteries. Future challenges include stimulating appropriate reorganization or synthesis of the collagen matrix to provide the necessary strength and viscoelastic properties for implantation.
Animals↗