Development of a bioartificial pancreas: I. long-term propagation and basal and induced secretion from entrapped betaTC3 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.     

A theoretical study of oxygen transfer including cell necrosis for the design of a bioartificial pancreas

In an extravascular bioartificial pancreas (BAP), islet functions are probably limited by diffusive mass transfer and local consumption, leading to low oxygenation. A mathematical model based on finite elements and focusing on local oxygen transport in both the alginate core and the islets of Langerhans has been proposed to help design an efficient pancreas supply. It was possible to randomly localize islets in a hollow fiber at different densities, and the effects of hypoxia and necrosis were included in the mass transfer simulations. Thorough study of the numerical results first led to the analysis of several relevant parameters, such as necrosis factor and efficacy in terms of insulin secretion, as a way to optimize fiber geometry. The approach was then to calculate the number of islets that needed to be implanted in order to obtain a correct response in terms of insulin secretion. In most configurations, it was found to be much higher than that of ultimately functional islets, because of hypoxia and necrosis. Fiber length should thus be adjusted accordingly. Finally, we demonstrated that the compromise to be found between the reduction of the number of implanted islets and fiber length and diameter did not correspond to realistic hollow fiber systems. The alternative of using flat geometry was also envisaged with more optimistic feasibility assessments.     

Effects of pH on murine insulinoma betaTC3 cells

Confluent monolayer cultures of betaTC3 cells were exposed for 4 h to acidic, neutral, or alkaline pH media. Studies determined the impact of pH on viability, insulin secretion rate, glucose consumption rate, lactate production rate, and ATP content. Cell viability was not affected by exposure to media of different pH (>95% for all groups). Insulin release from cells exposed to acidic media (pH of 6.4) was approximately 75% higher than that from cells exposed to either neutral (pH of 7.1) or alkaline (pH of 7.8) conditions. Conversely, ATP content was significantly reduced in cultures exposed to acidic conditions, although there was no statistical difference between neutral and alkaline conditions. Glucose consumption and lactate production rates increased linearly with increasing pH.     

Development of a bioartificial nerve graft. II. Nerve regeneration in vitro

A promising alternative for the repair of peripheral nerve injuries is the bioartificial nerve graft, or BNG, comprised of a tubular conduit preseeded with Schwann cells, which are an effective substrate for enhancing nerve regeneration. The physical properties of the conduit, porosity and wall thickness, as well as the Schwann cell seeding density, were tested for their effect on axon growth using rat dorsal root ganglia. These parameters can influence the amount of nutrients and growth factors made available to the neural tissue. Results show that a greater wall thickness and lower porosities have a detrimental effect on the growth of the axons. Over a four week period, axons extended 3.2 mm for the optimum case (DeltaR = 0.82 mm, epsilon = 0.75) compared to 1.8 and 1.6 mm for a lower porosity (0.55) and a greater wall thickness (1.4 mm), respectively. A maximum in the growth rate occurs at a porosity of 75% for Schwann cell seeded conduits but not for unseeded ones. When compared to mass transfer predictions, the results suggest that, at higher porosities, more growth factors diffuse out of the conduit, while at low porosities there is competition for nutrients. Increasing the Schwann cell seeding density enhances growth but also leads to an increase in the number of axons along the length of the conduit. This is indicative of branching of the axons, which requires additional resources to maintain and can lead to painful neuroma formation. Wall thickness and porosity were found not to have any significant effect on the axon number sprouting from the dorsal root ganglia and the mean diameter (p > 0.05). Considerations need to be made, not just on the polymer used, but also on its porosity, wall thickness, and Schwann cell seeding density. These parameters can be adjusted to create a bioartificial nerve graft that provides the optimal environment for nerve growth.     

A, B, D cells and a fourth cell type in long-term cultures of fetal rat pancreas.

Whole pancreases from fetal rats of 13 days and 18 days gestation were explanted onto rayon grids and grown in organ culture. Cultures were fixed in Bouin's fluid, sectioned and stained with the fluorescent antibody techniques for glucagon and insulin, aldehyde fuchsin for B cells, pseudoisocyanin for D cells and a silver technique for the fourth cell type. The 13-day explants were fixed after 10 days in culture. A, B and D and the fourth cell type were seen, indicating that precursors of all four endocrine cell types must be present in the fetal pancreas shortly after the formation of the pancreatic bud (11 days). Further, the presence of these four cell types in the walls of tubules in these cultures indicates the tubules as the site of origin of all the endocrine tissue. The 18-day explants were collected every other day of culture from 2 to 30 days in a long-term experiment. A number of large islets with well granulated B cells was still present after 30 days of culture. The relative abundance of cell types at different stages was estimated as follows: 18-day fetal controls, A greater than B=4 greater than D; after 2 to 10 days in culture, B greater than A greater than or equal to D; after 18 to 30 days in culture, B greater than D greater than A greater than 4.     

Optimization of microencapsulation parameters: Semipermeable microcapsules as a bioartificial pancreas

An improved membrane has been developed for the microencapsulation of islets of Langerhans which protects these cells from the immune system. These requirements were accomplished through the optimization of important microencapsulation parameters and through the improved biocompatibility of a new alginate-poly-l-lysine (PLL)-alginate capsule membrane. Spherical and smooth microcapsules could be formed by utilizing a purer sodium alginate and by keeping the viscosity of the sodium alginate solution above 30 cps. The strength of the capsule membrane was enhanced by increasing the alginate-PLL reaction time as well as the PLL concentration. The permeability of the membrane [4 mum thick, 93% (w/w) water] was a function of the viscosity average molecular weight (Mv) of the PLL (Mv = 4000-4 x 10(5)) used in the encapsulation procedure. Microcapsules prepared with PLL with Mv = 1.7 x 10(4) were the least permeable, being impermeable to normal serum immunoglobulin, albumin, and haemoglobin. The microencapsulation procedure, by protecting transplanted tissue from the components of the immune system, has great clinical potential as a new form of treatment for diseases such as diabetes and liver disease.     

Establishment of a cell line from long-term primary embryonic house fly cell cultures.

Thirty days after primary cultures were made of dispersed embryonic cells of the housefly, Musca domestica, large numbers of dividing undifferentiated cells were present. The cells were subcultured. After three passages, the doubling time had stabilized so that subcultures could be made at 7 to 10 day intervals. When subcultured, the cells attached and grew on the surface of the flasks and formed dense cell sheets and clumps. The derived cell line has been maintained for over 2 years, and the nutritional requirements have been studied. Cytogenic studies of the line at passages 14, 27, and 48 showed that the line has a chromosome count of 11 (2n = 12) and the missing chromosome is probably the Y chromosome. Thus we believe that the line is fundamentally diploid.     

Effects of millimeter-wave radiation on monolayer cell cultures. II. Scanning and transmission electron microscopy

Both thermal and athermal effects of millimeter-wave radiation on BHK-21/C13 cells were sought using scanning and transmission electron microscopy in conjunction with an in vitro technique that allows direct exposure of monolayer cultures to high average power densities. Culture dishes were irradiated by placing them on the open end of an E- or U-band wave guide. This technique exposes different regions of the cell monolayer lying along the longer axis of the wave guide aperture to varying power densities ranging from zero at each edge to twice the average power density at the center. Cell ultrastructure was unaffected by microwave radiation for 1 hour (41.8 or 74.0 GHz, average power densitites = 320 or 450 mW/cm², respectively) with or without cooling by rapid recirculation of the culture medium. Temperature in recirculated cultures was held at 37.2 °C, and that in noncooled cultures never exceeded 42 °C during irradiation at either power density. In contrast, cell morphology was affected by microwave exposure whenever irradiation conditions were altered so that the temperature of the monolayer reached or exceeded 44.5 °C. Ultrastructural alterations included breakage of cell processes, progressive detachment of cells from the substrate, increased clumping of heterochromatin in the nuclei, and the appearance of large empty vesicles in the cytoplasm. Such morphological changes resulted from either application of higher average power densities or irradiation at the power densities described above at a higher ambient temperature (>38.5°C).     

Development and utilization of crustacean long-term primary cell cultures: Ecdysteroid effects in vitro

Summary

Long-term maintenance of lobster, Homarus americanus and crayfish, Pacifasticus leniusculus primary cell cultures of testicular and hematopoietic tissues, for 11 and 3 months, respectively, succeeded in a modified Medium 199 supplemented with 10% fetal bovine serum (pH 7.5, 200°C). In addition, NaC1 was used to adjust the lobster culture medium to 1000 mOsm and the crayfish medium to 400 mOsm. Proline concentration was also elevated. Testes were dissociated with 200 U/ml type II collagenase 2–3 days prior to culture.

Lobster hemocytes reacted to 10^?7 M 20-hydroxyecdysone (20-HE) by reducing contact inhibition and increasing invasive behavior one week after hormonal exposure. The presence of 10^?7 M 20-HE caused mesodermal cell death and spermatogonial proliferation in lobster testicular cell cultures within one week. Crayfish testicular mesodermal cells formed vacuoles 5 days after exposure to 10^?8 M 20-HE. These results are discussed in relation to the cellular events that occur in vivo during premolt.     

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.     

Development of a bioartificial nerve graft. I. Design based on a reaction-diffusion model

A simple reaction-diffusion model has been developed to describe the mass transport of nutrients and nerve growth factor within a bioartificial nerve graft (BNG). The BNG consists of a porous polymer conduit that is preseeded with Schwann cells in its lumen. The Schwann cells produce growth factors to stimulate nerve regeneration within the lumen of the conduit. The model can predict the wall thickness, porosity, and Schwann cell seeding density needed to maximize the axon extension rate while ensuring that sufficient nutrients, especially oxygen, are made available to the neurons until the formation of the neovasculature. The model predicts a sixteen-fold increase in the levels of nerve growth factor by dropping the porosity from 95 to 55% but only at the expense of reducing the oxygen concentration. At higher porosities, increasing the wall thickness and increasing the Schwann cell seeding density both have the same effect of increasing the concentration of nerve growth factor within the lumen of the conduit. This model provides a simple tool for evaluating various conduit designs before continuing with experimental studies in vivo.     

Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

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 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 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.     

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.     

Effects of long-term preservation on growth and productivity of Panax ginseng and Catharanthus roseus cell cultures

Cell cultures of Panax ginseng and Catharanthus roseus producing secondary metabolites were preserved in liquid nitrogen or under mineral oil for six months. The growth behaviour and the ability of the cultures to produce ginsenosides or indole alkaloids were measured after a recovery period and compared with cultures maintained by frequent subcultivation during the same period. Neither growth kinetics nor the degree of vacuolization during growth were affected by the long term preservation. Some changes in secondary metabolism were however found, indicating that preservation under mineral oil does not preserve the productivity of cell cultures whereas the cryogenic method does.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulphoxide - HPLC high performance liquid chromatography