Biomedical subjects

A Sambanis

Publications and source records attributed to A Sambanis.

31 records · Page 2Linked to original sources

PubMed1996

Engineering a normally responsive bioartificial pancreas based on glucose-hypersensitive cells.

An implantable bioartificial pancreas is promising for diabetes treatment; transformed cells may allow the fabrication of these tissue constructs at a medically relevant scale. However, transformed cells are generally glucose hypersensitive, i.e. insulin secretion is near maximal at much lower than physiological glucose levels. The development of a normally responsive construct based on hypersensitive cells was examined. Two systems of immobilized cells and glucose-consuming enzyme were considered: cells surrounded by the enzyme, and cells coimmobilized with the enzyme. Model simulations showed that, with properly chosen parameter values, the first system can mimic the response of normal islets.

Blood Glucose↗

PubMed1995

Oxygen consumption rates of free and alginate-entrapped beta TC3 mouse insulinoma cells.

Oxygen consumption rates of beta TC3 mouse insulinoma cells were measured in three different cell culture systems: monolayer, freshly trypsinized cell suspension, and trypsinized cells entrapped in alginate-poly-L-lysine-alginate (APA) polymer beads. The oxygen consumption rate for cells in the APA beads was similar to the rate for freshly trypsinized cells, indicating that cells can be entrapped without affecting their oxygen consumption rate. The cells in monolayers consumed oxygen at a higher rate than freshly trypsinized cells or cells in APA beads, suggesting that trypsinization may lower oxygen consumption capability. In addition, the oxygen consumption of beta TC3s during glucose-induced insulin secretion was not significantly different from the basal rate, in contrast to the increased oxygen demand of actively secreting islets. Our results suggest that beta TC3s may be better suited than islets for use in a bioartificial pancreas due to their stable oxygen requirements.

Animals↗

PubMed1994

Modeling of cell culture processes.

Models of cell processes can be particularly useful in simulating, optimizing and controlling cell culture systems. Models reported in the literature are of various degrees of biological structure and mathematical complexity and describe cell growth, death, metabolism, and product formation, alone or in combination with each other. This paper reviews these modeling efforts, discusses their results, potential and limitations, and identifies areas where future modeling studies may be especially valuable.

Animals↗

PubMed1994

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 beta TC3 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 to 1H and 31P, 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 acquisitions of 1H images and 31P 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.

Alginates↗

PubMed1994

Ammonium selectively inhibits the regulated pathway of protein secretion in two endocrine cell lines.

The effect of ammonium on protein processing and secretion from mouse insulinoma beta TC3 cells and recombinant mouse pituitary AtT-20 cells was investigated. Protein stored in granules of the regulated secretion pathway was discharged from cells with secretagogues, and the addition of new protein was encouraged by recharging cells in serum-containing medium with or without added ammonium. Ammonium at 6 mM inhibits the addition of insulin-related peptides to intracellular stores in both cell lines; it is not clear whether this effect is dose dependent for concentrations between 0 and 6 mM. There is a slight increase in insulin-related proteins secreted during recharging of cells in the presence of ammonium. Using reverse-phase high-performance liquid chromatography to separate proinsulin from insulin, we found that extracts from beta TC3 cells recharged in the presence of 6 mM ammonium contain significantly less insulin than control cells recharged in the absence of ammonium.

Ammonium Chloride↗

PubMed1993

Cultivation of recombinant, insulin-secreting AtT-20 cells as free and entrapped spheroids.

Animal cells from endocrine glands have potential applications in bioprocessing, for the production of hormones, enzymes, possibly also recombinant proteins, and in tissue engineering, for the development of immunoisolated, implantable devices for long-term treatment of endocrine disorders. Immunoisolation can be achieved by surrounding cells with a biocompatible polymer which allows diffusion of nutrients and metabolites, including hormones, but excludes higher molecular weight antibodies and cytotoxic cells. Primary hormone-secreting cells cannot be effectively amplified in culture, so the large-scale application of implantable systems based on such cells is limited by cell availability. In this study, we conducted an initial assessment of the feasibility of using transformed, continuous cell lines in immunoisolated devices. The model system employed consisted of mouse pituitary tumor AtT-20 cells which secrete recombinant proinsulin and an insulin-like peptide and exhibit a high growth potential. Cells were cultivated as spheroids in spinner flasks and entrapped as such in alginate/polylysine/alginate beads. Free and entrapped spheroids were propagated in fed-batch, suspension cultures. Entrapment did not significantly affect spheroid metabolism or basal secretion. Entrapped spheroids did not increase in size or number and maintained roughly constant metabolic and basal secretory activities over a 15-day period. Free spheroids in suspension increased in size during the same period, but also maintained constant metabolism and basal secretion, apparently because of a concomitant increase in hypoxic and/or necrotic cells. The potential of using continuous cell lines in the development of bioartificial endocrine organs is discussed.

Alginates↗

PubMed1992

Use of regulated secretion in protein production from animal cells: an overview.

Traditional industrial cell culture processes require extensive downstream product refining due to low product titer and purity in the spent growth medium. A controlled secretion process incorporating cells derived from endocrine or exocrine organs could potentially alleviate this processing burden by dynamically decoupling product recovery from cell growth and product biosynthesis. In addition, such specialized secretory cells may be uniquely capable of performing desirable post-translational processing of the secretory product. We briefly review the biology of regulated protein secretion as well as the biology and biochemistry of the signal transduction mechanisms by which regulated systems respond to environmental stimuli. Drawing on these and other basic principles from cell biology and bioengineering, we describe the important features of a controlled secretion process. Among other issues we discuss the choice of cell lines, expression systems, cell culture methods, and bioreactor configurations. We extensively analyze the kinetics of regulated secretion in the context of a controlled secretion process. This discussion is illustrated with experimental results from two model cell lines, recombinant AtT-20 and beta TC3, expressing recombinant human endocrine hormones or native murine insulin respectively.

Animals↗

PubMed1990

Multiple episodes of induced secretion of human growth hormone from recombinant AtT-20 cells.

Recombinant AtT-20 cells expressing human growth hormone (hGH) secreted the hormone at a constant, basal rate of 0.3-0.5 ng/10(5) cells-hour when exposed to medium without secretagogues. When triggered with 8 bromo-cyclic AMP, cells secreted hGH at an initial rate of 1.7 ng/10(5) cells-hour while intracellular hGH declined sharply. Upon extended exposure to secretagogue, secretion decreased gradually to the basal rate and intracellular hGH stabilized at a value 40% the initial. In cells switched from secretion to growth medium, the total rate of hGH accumulation intracellularly and in medium was 2.2 times that observed with cells never exposed to secretagogue; however, only a fraction of the hormone was stored intracellularly and the rest was secreted. When cells were exposed alternately to growth and secretion medium, induced cells secreted at rates at least two times higher than uninduced controls during the first five cycles. The induced response deteriorated with time, however, in parallel with outgrowth of attached cells by foci of round cells, and by the eighth cycle induced secretion did not occur. Operational modifications that may improve the performance of cycling schemes are discussed.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMedDate not supplied

Vitrification of tissue engineered pancreatic substitute.

Despite significant advances, some critical issues remain for the long-term storage of an engineered pancreas. In this study we employed a tissue engineered pancreatic substitute model-insulin-secreting betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate beads-to demonstrate that a prototype vitrification method can prevent ice formation and maintain cell viability/function. The results showed that the structure of the frozen samples was distorted by ice crystals throughout the matrix. In marked contrast, the vitrified samples appeared to be free of ice. Morphologic studies demonstrated extensive fractures and vacuolation in frozen specimens while there were no fractures in vitrified TEPSs. Both vitrified and frozen constructs showed some vacuolization compared to the control samples. Frozen beads showed a significantly decreased viability compared to fresh controls and the VS55 group (P < .001). There was no significant difference between the vitrified and fresh samples. Vitrification using the VS55 protocol shows similar viability and secretion properties to the control group of fresh beads. Vitrification using the PEG 400 protocol resulted in slightly lower viability and secretion properties relative to the control group; conventional freezing resulted in even significantly lower viability and secretion properties. These results combine to demonstrate feasibility of vitrification as a storage method for a tissue engineered pancreas.

Animals↗

PubMedDate not supplied

Tissue engineering of a bioartificial pancreas: modeling the cell environment and device function.

Cell-based implantable artificial tissues are most promising for the long-term treatment of endocrine diseases, such as diabetes. One type of a bioartificial pancreas device consists of calcium alginate microbeads containing insulin-secreting cells and is surrounded by a poly(L-lysine) (PLL) membrane. The membrane is semipermeable, allowing cellular nutrients and metabolites to diffuse through but excluding the antibodies and cytotoxic cells of the host, thus immunoprotecting the cells. The device can be modeled by writing the equations for diffusion of nutrients and metabolites through the polymer and for consumption of the former and production of the latter by the cells. In this paper, we describe the construction and analysis of such a model for alginate/PLL microbeads with insulin-secreting recombinant mouse pituitary AtT-20 and mouse insulinoma beta TC3 cells. Entrapped AtT-20 cells are a simplified model system, whereas microbeads with beta TC3 cells constitute a realistic artificial pancreatic device. Effective diffusivities of key compounds through the polymer with entrapped, inactivated AtT-20 spheroids were measured first. The kinetics of glucose and oxygen consumption and insulin secretion were modeled next, and the equations for diffusion and reaction were then combined to describe the entire system. The model was used to compute nutrient and metabolite concentration profiles in beads and the bead secretory response for different bead sizes and cell loadings. The size and loading necessary for the cells to be well nourished and for the beads to be rapidly responsive to step-ups and step-downs of secretion stimuli were evaluated. It was shown that if the cells are hypersensitive to glucose, i.e., they do not shut off secretion at the physiological glucose threshold but at a lower one, so are the microbeads. This work demonstrates the usefulness of mechanistic models with representative parameter values in optimizing the design of artificial tissues and in characterizing aspects of their behavior that are of importance for restoring in vivo function.

Animals↗

PubMedDate not supplied

Noninvasive measurement of viable cell number in tissue-engineered constructs in vitro, using 1H nuclear magnetic resonance spectroscopy.

Noninvasive monitoring of tissue-engineered constructs is of critical importance for accurate characterization of constructs and their remodeling in vitro and in vivo. This study investigated the utility of (1)H NMR spectroscopy to noninvasively quantify viable cell number in tissue-engineered substitutes in vitro. Agarose disk-shaped constructs containing betaTC3 cells were employed as the model tissue-engineered system. Two construct prototypes containing different initial cell numbers were monitored by localized, water-suppressed 1H NMR spectroscopy over the course of 13 days. (1)H NMR measurements of the total choline resonance at 3.2 ppm were compared with results from the traditional cell viability assay MTT and with insulin secretion rates. Results show a strong linear correlation between total choline and MTT (R (2) = 0.86), and between total choline and insulin secretion rate (R (2) = 0.90). Overall, this study found noninvasive measurement of total choline to be an accurate and nondestructive assay for monitoring viable betaTC3 cell numbers in tissue-engineered constructs. The applicability of this method to in vivo monitoring is also discussed.

Algorithms↗

PubMedDate not supplied

Effects of short-term hypoxia on a transformed cell-based bioartificial pancreatic construct.

Hypoxia is an adverse condition that can jeopardize the function of a bioartificial pancreatic construct. In this study we have investigated the effects of short-term hypoxic exposure (up to 24 h) on the bioenergetic status, metabolism, and insulin secretion of perfused pancreatic constructs composed of alginate/poly-L-lysine/alginate (APA) encapsulated mouse insulinoma betaTC3 cells. The bioenergetic status of the encapsulated cells was monitored noninvasively with the aid of 31P NMR spectroscopy, while glucose, lactate, and insulin concentrations were measured with off-line assays from media samples removed from the perfusion loop. Our results demonstrate that in freshly prepared constructs insulin secretion was not affected by the hypoxic conditions, although intracellular ATP concentration decreased and glucose consumption increased. Alternatively, in constructs that were maintained in our perfusion system for at least 10 days, identical hypoxic conditions resulted in a decreased insulin secretion concomitant to a decreased intracellular ATP concentration and increased glucose consumption. These results suggest that the effects of hypoxia on a transformed cell-based pancreatic construct are not constant throughout the duration of an in vitro culture. The observed differences are attributed to the significant cell growth and rearrangement that occurs with time during an in vitro culture of the constructs.

Adenosine Triphosphate↗

PubMedDate not supplied

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.

Animals↗