ObjectiveAdipose tissue derived stem cells (ADSCs) transplantation has recently gained widespread enthusiasm, particularly in the perspective to use them as potential alternative cell sources for hepatocytes in cell based therapy, mainly because of their capability of hepatogenic differentiation in vitro and in vivo. But some challenges remain to be addressed, including whether ADSCs can be provided effectively to the target organ and whether subsequent proliferation of transplanted cells can be achieved. To date, intrasplenic injection is the conventional method to deliver ADSCs into the liver; however, a number of donor cells retained in the spleen has been reported. In this study, our objective is to evaluate a novel route to transplant ADSCs specifically to the liver. We aimed to test the feasibility of in situ transplantation of ADSCs by injecting bioencapsulated ADSCs into the liver in mouse model.MethodsThe ADSCs isolated from human alpha 1 antitrypsin (M-hAAT) transgenic mice were used to allow delivered ADSCs be readily identified in the liver of recipient mice, and alginate was selected as a cell carrier. We first evaluated whether alginate microspheres are implantable into the liver tissue by injection and whether ADSCs could migrate from alginate microspheres (study one). Once proven, we then examined the in vivo fate of ADSCs loaded microspheres in the liver. Specifically, we evaluated whether transplanted, undifferentiated ASDCs could be induced by the local microenvironment toward hepatogenic differentiation and the distribution of surviving ADSCs in major tissue organs (study two).ResultsOur results indicated ADSCs loaded alginate microspheres were implantable into the liver. Both degraded and residual alginate microspheres were observed in the liver up to three weeks. The viable ADSCs were detectable surrounding degraded and residual alginate microspheres in the liver and other major organs such as bone marrow and the lungs. Importantly, transplanted ADSCs underwent hepatogenic differentiation to become cells expressing albumin in the liver. These findings improve our understanding of the interplay between ADSCs (donor cells), alginate (biomaterial), and local microenvironment in a hepatectomized mouse model, and might improve the strategy of in situ transplantation of ADSCs in treating liver diseases.

The metabolism of glycine into glutathione was monitored noninvasively in vivo in intact rat mammary adenocarcinomas (R3230Ac) by MRI and MRS. Metabolism was tracked by following the isotope label from intravenously infused [2‐13C]‐glycine into the glycinyl residue of glutathione. Signals from [2‐13C]‐glycine and γ‐glutamylcysteinyl‐[2‐13C]‐glycine (13C‐glutathione) were detected by nonlocalized 13C spectroscopy, as these resonances are distinct from background signals. In addition, using spectroscopic imaging methods, heterogeneity in the in vivo tumor distribution of glutathione was observed. In vivo spectroscopy also detected isotope incorporation from [2‐13C]‐glycine into both the 2‐ and 3‐carbons of serine. Analyses of tumor tissue extracts showed single‐ and multiple‐label incorporation from [2‐13C]‐glycine into serine from metabolism through the serine hydroxymethyltransferase and glycine cleavage system pathways. Mass spectrometric analysis of extracts also showed that isotope‐labeled serine is further metabolized via the trans‐sulfuration pathway, as 13C isotope labels appear in both the glycinyl and cysteinyl residues of glutathione. Our studies demonstrate the use of MRI and MRS for the monitoring of tumor metabolic processes central to oxidative stress defense. Copyright © 2011 John Wiley & Sons, Ltd.

Introduction Glutathione (GSH) is a tripeptide found in almost all tissues, where it acts as an antioxidant and forms part of the cellular defences against oxidative stress. Cancerous cells exhibit highly reduced intracellular environments, characterized by high levels of reduced GSH compared to its oxidized disulfide counterpart (1). Higher levels of GSH and its associated enzymes appear to play a significant role in therapy-resistance (2) and reduced overall survival (3). We’ve previously used C MRSI to image GSH distribution in rat fibrosarcoma tumours, using C-glycine infusion to introduce a C label into GSH and thus provide a biomarker synthesis rate and concentration. The fibrosarcoma studies demonstrated high GSH levels (>2 μmol/gram-tissue). We have extended our studies to the noninvasive detection of GSH in a rat mammary R3230Ac adenocarcinoma, allowing us to test our ability to detect and image GSH distribution in tumours with lower average levels of GSH, and also to probe the metabolic fate of C-labelled glycine via serine hydroxymethyltransferase and other pathways.

Cancer cells exhibit altered glucose metabolism characterized by a preference for aerobic glycolysis or the Warburg effect, and the cells resist matrix detachment-induced apoptosis, which is called anoikis, a barrier to metastasis. It remains largely unclear whether tumor metabolism influences anoikis and metastasis. Here we show that when detached from the matrix, untransformed mammary epithelial cells undergo metabolic reprogramming by markedly upregulating pyruvate dehydrogenase (PDH) kinase 4 (PDK4) through estrogen-related receptor gamma (ERRγ), thereby inhibiting PDH and attenuating the flux of glycolytic carbon into mitochondrial oxidation. To decipher the significance of this metabolic response, we found that depletion of PDK4 or activation of PDH increased mitochondrial respiration and oxidative stress in suspended cells, resulting in heightened anoikis. Conversely, overexpression of PDKs prolonged survival of cells in suspension. Therefore, decreased glucose oxidation following cell detachment confers anoikis resistance. Unlike untransformed cells, most cancer cells demonstrate reduced glucose oxidation even under attached conditions, and thus they inherently possess a survival advantage when suspended. Normalization of glucose metabolism by stimulating PDH in cancer cells restores their susceptibility to anoikis and impairs their metastatic potential. These results suggest that the Warburg effect, more specifically, diminished glucose oxidation, promotes anoikis resistance and metastasis and that PDKs are potential targets for antimetastasis therapy.

Cryopreservation is important for clinical translation of tissue-engineered constructs. With respect to a pancreatic substitute, encapsulated islets or beta cells have been widely studied for the treatment of insulin-dependent diabetes mellitus. Besides cell viability loss, cryopreservation may affect the function of the remaining viable cells in a pancreatic substitute by altering fundamental processes in glucose-stimulated insulin secretion, such as pathways associated with intermediary metabolism, potentially leading to insulin-secretion defects. In this study, we used (13)C nuclear magnetic resonance (NMR) spectroscopy and isotopomer analysis to determine the effects of conventional freezing and ice-free cryopreservation (vitrification) on carbon flow through tricarboxylic acid (TCA) cycle-associated pathways in encapsulated murine insulinoma βTC-tet cells; the secretory function of the encapsulated cells postpreservation was also evaluated. Specifically, calcium alginate-encapsulated βTC-tet cells were frozen or vitrified with a cryoprotectant cocktail. Beads were warmed and (13)C labeling and extraction were performed. Insulin secretion rates were determined during basal and labeling periods and during small-scale glucose stimulation and K(+)-induced depolarization. Relative metabolic fluxes were determined from (13)C NMR spectra using a modified single pyruvate pool model with the tcaCALC modeling program. Treatments were compared with nonpreserved controls. Results showed that relative carbon flow through TCA-cycle-associated pathways was not affected by conventional freezing or vitrification. However, vitrification, but not freezing, led to impaired insulin secretion on a per viable cell basis. The reduced secretion from the Vitrified group occurred irrespective of scale and was present whether secretion was stimulated by glucose or K(+)-induced depolarization, indicating that it might be due to a defect in late-stage secretion events.

The cysteine precursor L-2-oxothiazolidine-4-carboxylate (OTZ, procysteine) can raise cysteine concentration, and thus glutathione levels, in some tissues. OTZ has therefore been proposed as a prodrug for combating oxidative stress. We have synthesized stable isotope labeled OTZ (i.e. L-2-oxo-[5- 13 C]-thiazolidine-4-carboxylate, 13 C-OTZ) and tracked its uptake and metabolism in vivo in rat brain by 13 C magnetic resonance spectroscopy. Although uptake and clearance of 13 C-OTZ was detectable in rat brain following a bolus dose by in vivo spectroscopy, no incorporation of isotope label into brain glutathione was detectable. Continuous infusion of 13 C-OTZ over 20 h, however, resulted in 13 C-label incorporation into glutathione, taurine, hypotaurine and lactate at levels sufficient for detection by in vivo magnetic resonance spectroscopy. Examination of brain tissue extracts by mass spectrometry confirmed only low levels of isotope incorporation into glutathione in rats treated with a bolus dose and much higher levels after 20 h of continuous infusion. In contrast to some previous studies, bolus administration of OTZ did not alter brain glutathione levels. Even a continuous infusion of OTZ over 20 h failed to raise brain glutathione levels. These studies demonstrate the utility of in vivo magnetic resonance for non-invasive monitoring of antioxidant uptake and metabolism in intact brain. These types of experiments can be used to evaluate the efficacy of various interventions for maintenance of brain glutathione.

Noninvasive in vivo monitoring of tissue implants provides important correlations between construct function and the observed physiologic effects. As oxygen is a key parameter affecting cell and tissue function, we established a monitoring method that utilizes 19 F nuclear magnetic resonance (NMR) spectroscopy, with perfluorocarbons (PFCs) as oxygen concentration markers, to noninvasively monitor dissolved oxygen concentration (DO) in tissue engineered implants. Specifically, we developed a dual PFC method capable of simultaneously measuring DO within a tissue construct and its surrounding environment, as the latter varies among animals and with physiologic conditions. In vitro studies using an NMR‐compatible bioreactor demonstrated the feasibility of this method to monitor the DO within alginate beads containing metabolically active murine insulinoma βTC‐tet cells, relative to the DO in the culture medium, under perfusion and static conditions. The DO profiles obtained under static conditions were supported by mathematical simulations of the system. In vivo, the dual PFC method was successful in tracking the oxygenation state of entrapped βTC‐tet cells and the surrounding peritoneal DO over 16 days in normal mice. DO measurements correlated well with the extent of cell growth and host cell attachment examined postexplantation. The peritoneal oxygen environment was found to be variable and hypoxic, and significantly lower in the presence of metabolically active cells. The significance of the dual PFC system in providing critical DO measurements for entrapped cells and other tissue constructs, in vitro and in vivo, is discussed. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Developing a method to noninvasively monitor tissue-engineered constructs is critical for the optimization of construct design and for assessing therapeutic efficacy. For this purpose, NMR is a powerful technique that can be used to obtain both images and spectroscopic data. But the inherent sensitivity of NMR limits the observation of a bioartificial construct with current NMR surface coil technology. In this study, we address this limitation through the development of an inductively coupled, implantable coil system, demonstrate its use at high field (11.1 T), and investigate the use of this coil system for monitoring a bioartificial construct in vitro and in vivo. The results establish that large gains in signal to noise can be obtained with this coil system over that obtainable with a surface coil. This coil system provides a means to quantitatively analyze the structure and function of implanted bioartificial organs. Magn Reson Med 63:998-1006, 2010. (C) 2010 Wiley-Liss, Inc.

Due to the high solubility of oxygen in perfluorocarbons (PFCs), these compounds have been explored for improved cell and tissue oxygenation. The goal of this study is to investigate the effects of a PFC emulsion on cellular growth and function in a tissue engineered construct. A perfluorotributylamine (PFTBA) emulsion was co-encapsulated at 10vol% with mouse βTC-tet insulinoma cells in calcium alginate beads and cultured under normoxic and severely hypoxic conditions. The number of metabolically active cells and the induced insulin secretion rate were measured over time for up to 16 days. Results showed no significant effect of PFTBA relative to the PFTBA-free control. The alginate–PFC-cell system was also modeled mathematically, and simulations tracked the number of viable cells over time under the same conditions used experimentally. Simulations revealed only a small, likely experimentally undetectable difference in cell density between the PFC-containing and PFC-free control beads. It is concluded that PFTBA up to 10vol% has no significant effect on the growth and function of encapsulated βTC-tet cells under normoxic and hypoxic conditions.

Introduction Cryopreservation is essential for bringing tissue engineered constructs from the benchtop to the clinic. Two main methods of cryopreservation that may be used are conventional freezing and vitrification (ice-free cryopreservation). For a pancreatic substitute containing beta cells, metabolism is key to construct function (glucose-stimulated insulin secretion). However, little is known about the effects of cryopreservation on intermediary metabolism. We hypothesize that cryopreservation will significantly affect intermediary metabolism in a pancreatic substitute containing encapsulated murine insulinomas. To investigate this, we have used C nuclear magnetic resonance (NMR) and isotopomer analysis. By exposing encapsulated cells to C-glucose and analyzing steady-state, cell-derived Cglutamate isotopomer patterns with metabolic models in the tcaCALC software [1], this method allows for a quantitative measure of metabolism postcryopreservation. Materials and Methods βTC-tet cells were encapsulated at a density of 7x10 cells/ml in 2% LVM alginate (FMC Biopolymer, Philadelphia, PA). Studies were first performed with fresh beads to determine isotopomeric steady-state. Beads were conventionally frozen using 10% dimethylsulfoxide (DMSO) in fully supplemented DMEM and vitrified using the DPS cryoprotectant cocktail (3 M 1,2 propanediol, 3 M DMSO, and 0.5 M sucrose). For C labeling and extraction, beads were exposed to basal medium without glucose for 1 hour prior to labeling medium containing C-glucose (Cambridge Isotopes, Andover, MA) and subsequent perchloric acid extraction [2]. After C NMR spectroscopy on extracts, glutamate C2, C3, and C4 multiplet peak areas were determined using line-fitting. Isotopomer analyis was performed using metabolic models in tcaCALC. The tcaSIM software was used to simulate spectra based on tcaCALCderived results. Statistics were performed using a one-way ANOVA in the General Linear Model in Minitab. Results Isotopomeric steady-state for encapsulated cells was reached by six hours (data not shown). Analysis with tcaCALC indicated that the modified single pyruvate pool model with a second non-pyruvate carboxylase anaplerotic entrance [2,3] to the TCA cycle fit the experimental data better than the standard model of glucose metabolism (p<0.05) (data not shown). Simulated resonances also indicate that the modified model is a good representation of the experimental data (Figure 1). Using this model, initial results from tcaCALC (Table 1) show that cells in both vitrified and frozen beads may have lower pyruvate carboxylase activity and flux through glycolysis compared to those in fresh beads. Also, cells in frozen beads may have a lower flux through a second non-pyruvate carboxylase anaplerotic entrance. However, further experiments with the frozen group need to be performed to determine statistical significance. Current studies are focusing on analyzing insulin secretory response in beads post-cryopreservation and correlating it with metabolic fluxes.

Noninvasive monitoring of tissue‐engineered constructs is an important component in optimizing construct design and assessing therapeutic efficacy. In recent years, cellular and molecular imaging initiatives have spurred the use of iron oxide‐based contrast agents in the field of NMR imaging. Although their use in medical research has been widespread, their application in tissue engineering has been limited. In this study, the utility of monocrystalline iron oxide nanoparticles (MIONs) as an NMR contrast agent was evaluated for βTC‐tet cells encapsulated within alginate/poly‐L‐lysine/alginate (APA) microbeads. The constructs were labeled with MIONs in two different ways: 1) MION‐labeled βTC‐tet cells were encapsulated in APA beads (i.e., intracellular compartment), and 2) MION particles were suspended in the alginate solution prior to encapsulation so that the alginate matrix was labeled with MIONs instead of the cells (i.e., extracellular compartment). The data show that although the location of cells can be identified within APA beads, cell growth or rearrangement within these constructs cannot be effectively monitored, regardless of the location of MION compartmentalization. The advantages and disadvantages of these techniques and their potential use in tissue engineering are discussed. Magn Reson Med 61:282–290, 2009. © 2009 Wiley‐Liss, Inc.

Defining mechanisms and enzymatic paths critical to cellular function (e.g., secretion) of endocrine cells is a key research goal that can lead toward novel avenues of therapeutic intervention for a variety of disorders. 13C NMR spectroscopy and isotopomer analysis of cell extracts are excellent tools to quantitatively assess metabolism through intermediate labeling and estimate carbon entry to the TCA cycle. Discussed are: cell lines and in vitro culturing; extraction of intracellular material; NMR spectroscopy of the extract; isotopomeric analysis and modeling to obtain relative metabolic fluxes to the TCA cycle. This paper describes issues related to the application of NMR spectroscopic techniques on cell line extracts. Included are results of two studies that illustrate considerations that must be taken when performing analogous studies on neuroendocrine tissue: one involving the effect of media composition on cell behavior and isotopomer labeling; the second looking at effects of applying different metabolic models to 13C data and inferences that may be drawn. NMR isotopomeric analysis is a powerful technique that may be applied to better understand endocrine cell function.

In this report, we present data to demonstrate the utility of (1)H MR microscopy to non-invasively examine alginate/poly-l-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly-l-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6+/-6.2 microm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 min). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T(2) relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T(2) over the duration of the experiment, while ADC was unaffected. This decrease in T(2) is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T(2) was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T(2) or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions.

Aims: The aim of this article is to present a novel synthetic route to form CeO2 nanoparticles that protects against the detrimental influence of oxidative stress in mammalian cells. Methods: The noncytotoxic surfactant lecithin was used to synthesize CeO2 nanoparticles and the products were colloidally stabilized in a biocompatible tri-sodium citrate buffer. These nanoparticles were delivered into murine insulinoma beta TC-tet cells, and intracellular free radical concentrations responding to exposure to hydroquinone were measured in a variety of extracellular CeO2 concentrations. Results: Well-dispersed, highly crystallized CeO2 nanoparticles of 3.7 nm in size were achieved that are chemically and colloidally stable in Dulbecco's modified Eagle's medium for extended periods of time. Treating beta TC-tet cells with these nanoparticles alleviated detrimental intracellular free radical levels down to the primary level. Conclusion: CeO2 nanoparticles synthesized from this route are demonstrated to be effective free radical scavengers within beta TC-tet cells. Furthermore, it is shown that CeO2 nanoparticles provide an effective means to improve cellular survival in settings wherein cell loss due to oxidative stress limits native function.