Ioannis Constantinidis

The effects of alginate composition on encapsulated βTC3 cells

The effects of alginate composition on the growth of murine insulinoma βTC3 cells encapsulated in alginate/poly-l-lysine/alginate (APA) beads, and on the overall metabolic and secretory characteristics of the encapsulated cell system, were investigated for four different types of alginate. Two of the alginates used had a high guluronic acid content (73% in guluronic acid residues) with varying molecular weight, while the other two had a high mannuronic acid content (68% in mannuronic acid residues) with varying molecular weight. Each composition was tested using two different polymer concentrations. Our data show that βTC3 cells encapsulated in alginates with a high guluronic acid content experienced a transient hindrance in their metabolic and secretory activity because of growth inhibition. Conversely, βTC3 cells encapsulated in alginates with a high mannuronic acid content experienced a rapid increase in metabolic and secretory activity as a result of rapid cell growth. Our data also demonstrate that an increase in either molecular weight or concentration of high mannuronic acid alginates did not alter the behavior of the encapsulated βTC3 cells. Conversely, an increase in molecular weight and concentration of high guluronic acid alginates prolonged the hindrance of glucose metabolism, insulin secretion and cell growth. These observations can be best interpreted by changes in the microstructure of the alginate matrix, i.e., interaction between the contiguous guluronic acid residues and the Ca2+ ions, as a result of the different compositions.

Effects of alginate composition on the metabolic, secretory, and growth characteristics of entrapped βTC3 mouse insulinoma cells

The effects of alginate composition on cell growth as well as the metabolic and secretory profile of transformed beta-cells entrapped in alginate/poly-L-lysine/alginate (APA) solid beads were investigated following entrapment of beta TC3 mouse insulinoma cells in alginate composed of either high mannuronic acid or high guluronic acid residues. Entrapped cultures were maintained in spinner flasks for 40-60 days. The pattern of cell growth and the overall rates of glucose consumption and insulin secretion were investigated. Cultures of beta TC3 cells entrapped in alginate composed predominantly of mannuronic acid units (77%) displayed a linear increase in the rates of glucose consumption and insulin secretion concomitant with an increase in cell population in the periphery of the beads. Conversely, cultures of beta TC3 cells entrapped in alginate composed predominantly of high guluronic acid units (69%) displayed a decrease in the rates of glucose consumption and insulin secretion during the first three weeks of culture, followed by a rapid recovery that surpassed the initial rates by day 40. This biphasic pattern was concomitant to a decrease in viable cells during the first three weeks as ascertained by histology, followed by an increase in cell proliferation. Cell growth in high guluronic acid alginate took place at random locations throughout the solid bead and not in the periphery, as was the case in high mannuronic acid alginate preparations. Possible reasons for these differences and the significance of these findings in the context of a bioartificial pancreas composed of APA entrapped transformed cells are discussed.

Novel synthesis of cerium oxide nanoparticles for free radical scavenging

AIMS The aim of this article is to present a novel synthetic route to form CeO(2) nanoparticles that protects against the detrimental influence of oxidative stress in mammalian cells. METHODS The noncytotoxic surfactant lecithin was used to synthesize CeO(2) nanoparticles and the products were colloidally stabilized in a biocompatible tri-sodium citrate buffer. These nanoparticles were delivered into murine insulinoma betaTC-tet cells, and intracellular free radical concentrations responding to exposure to hydroquinone were measured in a variety of extracellular CeO(2) concentrations. RESULTS Well-dispersed, highly crystallized CeO(2) nanoparticles of 3.7 nm in size were achieved that are chemically and colloidally stable in Dulbeccos modified Eagles medium for extended periods of time. Treating betaTC-tet cells with these nanoparticles alleviated detrimental intracellular free radical levels down to the primary level. CONCLUSION CeO(2) nanoparticles synthesized from this route are demonstrated to be effective free radical scavengers within betaTC-tet cells. Furthermore, it is shown that CeO(2) nanoparticles provide an effective means to improve cellular survival in settings wherein cell loss due to oxidative stress limits native function.

Development of a bioartificial pancreas: II. Effects of oxygen on long‐term entrapped βTC3 cell cultures

Tissue-engineered pancreatic constructs based on immunoisolated, insulin-secreting cells are promising in providing an effective, relatively inexpensive, long-term treatment for type I (insulin-dependent) diabetes. An in vitro characterization of construct function under conditions mimicking the in vivo environment is essential prior to any extensive animal experimentation. Encapsulated cells may experience hypoxic conditions postimplantation as a result of one or more of the following: the design of the construct; the environment at the implantation site; or the development of fibrosis around the construct. In this work, we studied the effects of 3- and 4-day-long hypoxic episodes on the metabolic and secretory activities and on the levels of intracellular metabolites detectable by phosphorus-31 nuclear magnetic resonance ((31)P NMR) of alginate/poly-L-lysine/alginate entrapped betaTC3 mouse insulinomas continuously perfused with culture medium. Results show that, upon decreasing the oxygen concentration in the surrounding medium, the encapsulated cell system reached a new, lower metabolic and secretory state. Hypoxia drove the cells to a more anaerobic glycolytic metabolism, increased the rates of glucose consumption (GCR) and lactate production (LPR), and reduced the rates of oxygen consumption (OCR) and insulin secretion (ISR). Furthermore, hypoxia reduced the levels of intracellular nucleotide triphosphates (NTP) and phosphorylcholine (PC) and caused a rapid transient increase in inorganic phosphate (P(i)). Upon restoration of the oxygen concentration in the perfusion medium, all parameters returned to their prehypoxic levels within 2 to 3 days following either gradual unidirectional changes (ISR, NTP, PC) or more complicated dynamic patterns (OCR, GCR, LPR). A further increase in oxygen concentration in the perfusion medium drove OCR, ISR, NTP, PC, and P(i) to new, higher levels. It is concluded that (31)P NMR spectroscopy can be used for the prolonged noninvasive monitoring of the bioenergetic changes of encapsulated betaTC3 cells occurring with changes in oxygen tension. The data also indicate that the oxygen-dependent states might be related to the total number of viable, metabolically active cells supported by the particular oxygen level to which the system is exposed. These findings have significant implications in developing and non-invasively monitoring a tissue-engineered bioartificial pancreas based on transformed beta cells, as well as in understanding the biochemical events pertaining to insulin secretion from betaTC3 insulinomas.

Development of a bioartificial pancreas: I. Long‐term propagation and basal and induced secretion from entrapped βTC3 cell cultures

Bioartificial pancreatic constructs based on immunoisolated, insulin-secreting cells have the potential for providing effective, long-term treatment of type I (insulin-dependent) diabetes. Use of insulinoma cells, which can be amplified in culture, relaxes the tissue availability limitation that exists with normal pancreatic islet transplantations. We have adopted mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads as our model system for a bioartificial pancreas, and we have characterized the effects of long-term propagation and of glucose concentration step changes on the bioenergetic status and on the metabolic and secretory activities of the entrapped cells. Cell bioenergetics were evaluated nonivasively by phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy, and metabolic and secretory parameters by assaying cell culture medium. Data indicate that net cell growth occurred between days 3 and 10 of the experiment, resulting in an approximate doubling of the overall metabolic and secretory rates and of the intracellular metabolite levels. Concurrently, a reorganization of cell distribution within the beads was observed. Following this growth period, the measured metabolic and secretory parameters remained constant with time. During glucose step changes in the perfusion medium from a high concentration of 12 to 15 mM to 0 mM for 4.5 h to the same high glucose concentration, the oxygen consumption rate was not affected, whereas insulin secretion was always glucose-responsive. Intracellular nucleotide triphosphates did not change during 0 mM glucose episodes performed early in culture history, but they declined by 20% during episodes performed later in the experiment. It is concluded that the system of APA-entrapped betaTC3 cells exhibits several of the desirable characteristics of a bioartificial pancreas device, and that a correlation between ATP and the rate of insulin secretion from betaTC3 cells exists for only a domain of culture conditions. These findings have significant implications in tissue engineering a long-term functional bioartificial endocrine pancreas, in developing noninvasive methods for assessing construct function postimplantation, and in the biochemical processes associated with insulin secretion.

Effects of oxygen on metabolic and secretory activities of βTC3 cells

Abstract We have investigated the rates of glucose consumption, lactate production and insulin secretion by mouse insulinoma βTC3 cells exposed to high glucose and oxygen concentrations in the range of 132 mmHg (normoxia) to 0 mmHg (anoxia). The rates of glucose consumption and lactate production, and the yield of lactate on glucose were 6.4 ± 0.2 nmol/h − 10 5 cells, 7.7 ± 0.5 nmol/h − 10 5 cells, and 1.2 ± 0.1 respectively, at oxygen concentrations between 132-25 mmHg. These values increased gradually as the oxygen concentration was reduced below 25 mmHg, reaching a maximum value of 12.8 ± 0.4, 23.8 ± 1.1, 1.9 ± 0.1 respectively, at complete anoxia. Insulin secretion remained constant at 360 ± 24 pmol/h − 10 8 cells at oxygen concentrations between 132-7 mmHg, but was inhibited at lower oxygen concentrations, dropping to 96 ± 24 pmol/h − 10 8 cells at 0 mmHg. The rate of insulin secretion in the presence of high glucose under anoxia was significantly higher than the rate of basal secretion (28.2 ± 3.0 pmol/h − 10 8 cells) at normoxia. The secretory properties of βTC3 cells at low oxygen concentrations may have implications in the development of a diffusion-based bioartificial tissue constructs for the long-term treatment of Insulin Dependent Diabetes Mellitus.

NMR properties of alginate microbeads

Alginates are a family of unbranched polysaccharides with properties that vary widely depending on their composition. In the presence of multivalent cations (frequently Ca2+), alginates form a gel. Consequently, alginates have been used to encapsulate a variety of biological materials, including cells. In this study, we present NMR relaxation and diffusion data from alginate microbeads with similar size and properties to those used in the development of a bioartificial pancreas. Our data demonstrate that the transverse relaxation time (T2) of water within the gel depends on the guluronic acid content of the alginate, whereas the longitudinal relaxation time (T1) and the apparent diffusion coefficient of water do not. Our data further suggest that the diffusion of Ca2+ ions is hindered by the presence of a poly-L-lysine layer, a layer commonly added to provide mechanical support to the beads and immunoprotection to the encapsulated cells in the event of implantation. The impact of these data on our understanding of the role of alginate gels in the development of a bioartificial pancreas is discussed.

Towards the development of a bioartificial pancreas: effects of poly-L-lysine on alginate beads with BTC3 cells.

A bioartificial tissue construct that consists of insulin-secreting cells entrapped in an alginate/poly-L-lysine (PLL) matrix offers a promising approach for the treatment of type I diabetes. Use of transformed cells has been proposed as a solution to the cell availability problem posed by islets. The growth characteristics of transformed cells in their sequestered environment and the effects of PLL on their metabolic and secretory activities have not yet been characterized. Our data demonstrate that mouse insulinoma beta TC3 cells proliferate while they are entrapped in both PLL-free and PLL-coated alginate beads. During this process, cell aggregates develop in the bead periphery, which increase in number and size with time. PLL is crucial for the long-term in vitro structural stability of beads, and it does not appear to affect the metabolic and secretory activities of entrapped beta TC3 cells. The implications of these findings in the development of a bioartificial pancreatic construct based on transformed cells are discussed.

In vivo noninvasive monitoring of a tissue engineered construct using 1H NMR spectroscopy.

Direct, noninvasive monitoring of tissue engineered substitutes containing live, functional cells would provide valuable information on dynamic changes that occur postimplantation. Such changes include remodeling both within the construct and at the interface of the implant with the surrounding host tissue, and may result in changes in the number of viable cells in the construct. This study investigated the use of 1H NMR spectroscopy in noninvasively monitoring the viable cell number within a tissue engineered construct in vivo. The construct consisted of mouse βTC3 insulinomas in a disk-shaped agarose gel, surrounded by a cell-free agarose gel layer. Localized 1H NMR spectra were acquired from within implanted constructs, and the total choline resonance was measured. Critical issues that had to be addressed in accurately quantifying total choline from the implanted cells included avoiding signal from host tissue and correcting for interfering signal from diffusing solutes. In vivo NMR measurements were correlated with MTT assays and NMR measurements performed in vitro on explanted constructs. Total choline measurements accurately and noninvasively quantified viable βTC3 cell numbers in vivo, in the range of 1 × 106 to more than 14 × 106 cells, and monitored changes in viable cell number that occurred in the same construct over time. This is the first study using NMR techniques to monitor viable cell numbers in an implanted tissue substitute. It established architectural characteristics that a construct should have to be amenable to NMR monitoring, and it set the foundation for future in vivo investigations with other tissue engineered implants.

Towards the development of a bioartificial pancreas: Immunoisolation and NMR monitoring of mouse insulinomas

A promising method for diabetes treatment is the implantation of immunoisolated cells secreting insulin in response to glucose. Cell availability limits the application of this approach at a medically-relevant scale. We explore the use of transformed cells that can be grown to large homogeneous populations in developing artificial pancreatic tissues. We also investigate the use of NMR in evaluating, non-invasively, cellular bioenergetics in the tissue environment. The system employed in this study consisted of mouse insulinoma βTC3 cells entrapped in calcium alginate/poly-L-lysine (PPL)/alginate beads. The PPL layer imposed a molecular weight cutoff of approximately 60 kDa, allowing nutrients and insulin to diffuse through but excluding high molecular weight antibodies and cytotoxic cells of the host. We fabricated a radiofrequency coil that can be double-tuned to1H and31P, and an NMR-compatible perfusion bioreactor and support circuit that can maintain cells viable during prolonged studies. The bioreactor operated differentially, was macroscopically homogeneous and allowed the acquisition of1H images and31P NMR spectra in reasonable time intervals. Results indicated that entrapment had little effect on cell viability; that insulin secretion from beads was responsive to glucose; and that the bioenergetics of perfused, entrapped cells were not grossly different from those of cells never subjected to the immobilization procedure. These findings offer promise for developing an artificial pancreatic tissue for diabetes treatment based on continuous cell lines.