Modeling of encapsulated cell systems

Tissue engineered substitutes consisting of cells in biocompatible materials undergo remodeling with time as a result of cell growth and death processes. With inert matrices that do not directly influence cell growth, remodeling is driven mainly by the concentration of dissolved oxygen (DO). Insulin-secreting cell lines encapsulated in alginate-based beads and used as a pancreatic substitute represent such a case. Beads undergo remodeling with time so that an initially homogeneous distribution of cells is eventually replaced by a dense peripheral ring of primarily viable cells, whereas inner cells are mostly necrotic. This paper develops and analyzes a mathematical model of an encapsulated cell system of spherical geometry that tracks the viable and dead cell densities and the concentration of DO within the construct as functions of radial position and time. Model simulations are compared with experimental histology data on cell distribution. Correlations are then developed between the average intrabead DO concentration (AIDO) and the total viable cell number, as well as between AIDO and the radial cell and DO distributions in beads. As AIDO can be measured experimentally by incorporating a perfluorocarbon emulsion in the beads and acquiring (19)F nuclear magnetic resonance (NMR) spectroscopic data, these correlations can be used to track the remodeling that occurs in the construct in vitro and potentially in vivo. The usefulness of mathematical models in describing the dynamic changes that occur in tissue constructs with time, and the value of these models at obtaining additional information on the system when used interactively with experimental measurements, are discussed.     

Dual perfluorocarbon method to noninvasively monitor dissolved oxygen concentration in tissue engineered constructs in vitro and in vivo

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 ¹⁹F 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     

Bacteriophage T4-Directed DNA Synthesis in Toluene-Treated Cells

DNA synthesis has been studied in T4-infected Escherichia coli cells made permeable to nucleotides by treatment with toluene. The rate of incorporation of labeled deoxyribonucleoside triphosphates into DNA at various times after infection is proportional to the in vivo rate. This in vitro incorporation is dependent on all four deoxyribonucleoside triphosphates (5-hydroxymethyldeoxy-cytidine triphosphate can substitute for dCTP) and Mg(2+). It is stimulated by rATP, partially inhibited by pancreatic DNase, and abolished by N-ethylmalei-mide and 1-beta-d-arabinofuranosylcytosine triphosphate. T4 amber DO (DNA negative) and temperature-sensitive DO mutants under nonpermissive conditions of infection fail to induce DNA synthesis in vitro. The synthesizing activity is intracellular and the DNA product is exclusively T4 DNA. The in vitro synthesis proceeds in a discontinuous manner involving synthesis and subsequent joining of small DNA fragments (about 10S in alkaline sucrose gradients) into larger molecules predominantly one-half the length of mature T4 DNA. No restriction of C-containing or nonglucosylated HMC-containing T4 DNA product is observed in this system.     

^19FNMR在生物医学研究中的应用

核磁共振（ＮＭＲ）是一种无创伤的物理测试方法,它可以直接用于体内和体外的生物样品测定,提供分子水平的信息。正常体内含氟成分很少,测定进无本底信号干扰,因此在体内研究中引进氟代指示剂进行＾１９ＦＮＭＲ研究是目前普遍采用的方法。＾１９ＦＮＭＲ可可以用来测定药物在体内代谢过程、胸内游离的离子如Ｃａ＾２＋和Ｍｇ＾２＋、胞内ｐＨ、氧浓度或氧压力（ｐＯ２）、膜电位、组织温度、血液容积和细胞容积等多项生理生化指     

Development of a bioartificial pancreas: II. Effects of oxygen on long-term entrapped betaTC3 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.     

Allosteric modifiers of fish hemoglobins: in vitro and in vivo studies of the effect of ambient oxygen and pH on erythrocyte ATP concentrations.

Fundulus heteroclitus decreases erythrocyte adenosine triphosphate and increases blood hematocrit when acclimated to hypoxic conditions. A defined medium has been developed which allows isolated F. heteroclitus erythrocytes to be maintained for several hours without an appreciable loss of cellular ATP. The effect of oxygen tension, pH and metabolic inhibitors on the cellular concentration of ATP of fish red cells has been investigated as an in vitro model to explain in vivo responses to environmental changes. The isolated red cells significantly decrease their ATP/Hb molar ratio when exposed either to anaerobiosis or metabolic inhibitors. It is concluded that the in vivo response is mediated at the red cell level via decreased oxidative phosphorylation in the presence of low environmental oxygen. The length of time necessary to elicit the responce both in vivo and in vitro is also discussed.     

Plantlet regeneration from encapsulated somatic embryos of hybrid Solanum melongena L

High frequency somatic embryogenesis was induced from leaf expiants of F1 hybrid Solanum melongena L. on Murashige and Skoog's medium supplemented with 8.0 mg/1 NAA and 0.1 mg/1 Kn. The somatic embryos were encapsulated in various concentrations (2–6%) of sodium alginate and complexed with calcium chloride (25–100mM): 3% sodium alginate and 75 mM calcium chloride were found to be optimal for encapsulation. The encapsulated somatic embryos were transferred to various conversion media in vitro and in vivo. The frequency of plantlet regeneration varied from 27.0–49.7% in vitro and 2.0–4.5% in vivo.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid     

A kinetic model describing Shewanella oneidensis MR-1 growth, substrate consumption, and product secretion

Aerobic growth of Shewanella oneidensis MR-1 in minimal lactate medium was studied in batch cultivation. Acetate production was observed in the middle of the exponential growth phase and was enhanced when the dissolved oxygen (DO) concentration was low. Once the lactate was nearly exhausted, S. oneidensis MR-1 used the acetate produced during growth on lactate with a similar biomass yield as lactate. A two-substrate Monod model, with competitive and uncompetitive substrate inhibition, was devised to describe the dependence of biomass growth on lactate, acetate, and oxygen and the acetate growth inhibition across a broad range of concentrations. The parameters estimated for this model indicate interesting growth kinetics: lactate is converted to acetate stoichiometrically regardless of the DO concentration; cells grow well even at low DO levels, presumably due to a very low K(m) for oxygen; cells metabolize acetate (maximum specific growth rate, micro(max,A) of 0.28 h(-1)) as a single carbon source slower than they metabolize lactate (micro(max,L) of 0.47 h(-1)); and growth on acetate is self-inhibiting at a concentration greater than 10 mM. After estimating model parameters to describe growth and metabolism under six different nutrient conditions, the model was able to successfully estimate growth, oxygen and lactate consumption, and acetate production and consumption under entirely different growth conditions.     

Determination of external and internal mass transfer limitation in nitrifying microbial aggregates

In this article we present a study of the effects of external and internal mass transfer limitation of oxygen in a nitrifying system. The oxygen uptake rates (OUR) were measured on both a macro-scale with a respirometric reactor using off-gas analysis (Titrimetric and Off-Gas Analysis (TOGA) sensor) and on a micro-scale with microsensors. These two methods provide independent, accurate measurements of the reaction rates and concentration profiles around and in the granules. The TOGA sensor and microsensor measurements showed a significant external mass transfer effect at low dissolved oxygen (DO) concentrations in the bulk liquid while it was insignificant at higher DO concentrations. The oxygen distribution with anaerobic or anoxic conditions in the center clearly shows major mass transfer limitation in the aggregate interior. The large drop in DO concentration of 22-80% between the bulk liquid and aggregate surface demonstrates that the external mass transfer resistance is also highly important. The maximum OUR even for floccular biomass was only attained at much higher DO concentrations (approximately 8 mg/L) than typically used in such systems. For granules, the DO required for maximal activity was estimated to be >20 mg/L, clearly indicating the effects of the major external and internal mass transfer limitations on the overall biomass activity. Smaller aggregates had a larger volumetric OUR indicating that the granules may have a lower activity in the interior part of the aggregate.     

Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic β-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic β-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated β-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.     

Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis

Summary The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations. At high DO concentration, e.g. at 1.25 mM DO, the growth rate was decreased up to 36 % compared with that of 0.063 mM DO. The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O₂ evolution. Under higher DO concentrations, superoxide dismutase and ascorbate peroxidase activities tended to increase, while the contents of photosynthetic pigment, such phycocyanin, carotenoid and chlorophyll-a decreased distinctly.     

In vivo imaging of transplanted islets with 64Cu-DO3A-VS-Cys40-Exendin-4 by targeting GLP-1 receptor

Glucagon-like peptide 1 receptor (GLP-1R) is highly expressed in pancreatic islets, especially on β-cells. Therefore, a properly labeled ligand that binds to GLP-1R could be used for in vivo pancreatic islet imaging. Because native GLP-1 is degraded rapidly by dipeptidyl peptidase-IV (DPP-IV), a more stable agonist of GLP-1 such as Exendin-4 is a preferred imaging agent. In this study, DO3A-VS-Cys(40)-Exendin-4 was prepared through the conjugation of DO3A-VS with Cys(40)-Exendin-4. The in vitro binding affinity of DO3A-VS-Cys(40)-Exendin-4 was evaluated in INS-1 cells, which overexpress GLP-1R. After (64)Cu labeling, biodistribution studies and microPET imaging of (64)Cu-DO3A-VS-Cys(40)-Exendin-4 were performed on both subcutaneous INS-1 tumors and islet transplantation models. The subcutaneous INS-1 tumor was clearly visualized with microPET imaging after the injection of (64)Cu-DO3A-VS-Cys(40)-Exendin-4. GLP-1R positive organs, such as pancreas and lung, showed high uptake. Tumor uptake was saturable, reduced dramatically by a 20-fold excess of unlabeled Exendin-4. In the intraportal islet transplantation models, (64)Cu-DO3A-VS-Cys(40)-Exendin-4 demonstrated almost two times higher uptake compared with normal mice. (64)Cu-DO3A-VS-Cys(40)-Exendin-4 demonstrated persistent and specific uptake in the mouse pancreas, the subcutaneous insulinoma mouse model, and the intraportal human islet transplantation mouse model. This novel PET probe may be suitable for in vivo pancreatic islets imaging in the human.     

脂质体包埋猪血红蛋白的制备及其注入小鼠体内的初步试验

 为探索一条研制猪血红蛋白 (pHb)为基础的血液代用品新途径 ,开发了干膜超声法将猪血红蛋白和别构效应剂、超氧化物歧化酶等联合包埋于脂质体的技术 .考察了氢化大豆卵磷脂、二肉豆蔻酰磷脂酰胆碱、胆固醇、二硬脂酰磷脂酰乙醇胺 甲氧基聚乙二醇和维生素E等在制备脂质体包埋血红蛋白 (LEH)中的作用 .通过控制磷脂与其他成分的配比 ,制得包埋率为 10 3%,pHb浓度达 16 %的稳定的LEH ;进一步将pHb微囊通过小鼠尾静脉多次注入其体内 ,检测受试小鼠血液中红细胞和白细胞数、抗体滴度、血小板聚集率及肾脏组织学等方面的变化 .小鼠体内试验表明了所制备的LEH具有低免疫原性、对肾脏无明显损害等特点 .脂质体包埋pHb是一种极具开发前景的稳定的人工载氧系统     

Stimulation of 6-keto-prostaglandin F1 alpha release from isolated pancreatic islets by an insulinotropic concentration of glucose

In a glucose-free medium, the release of 6-keto-prostaglandin F1 alpha from isolated pancreatic islets was 1.67 pg/islet/60 min. The release was not altered by increasing the glucose concentration to 3.3 mM, whereas the release was significantly increased to 3.79 pg/islet/60 min by 16.7 mM glucose. Indomethacin (10 microM) and mepacrine (100 microM) markedly suppressed the 6-keto-prostaglandin F1 alpha release. The results indicate that the insulinotropic concentration of glucose enhances the enzymatic formation of 6-keto-prostaglandin F1 alpha in pancreatic islets. It seems highly possible that glucose enhances 6-keto-prostaglandin F1 alpha formation by stimulating phospholipase A2 in pancreatic islets.     

Metabolomic, proteomic and biophysical analyses of Arabidopsis thaliana cells exposed to a caesium stress. Influence of potassium supply

The incorporation and localisation of 133Cs in a plant cellular model and the metabolic response induced were analysed as a function of external K concentration using a multidisciplinary approach. Sucrose-fed photosynthetic Arabidopsis thaliana suspension cells, grown in a K-containing or K-depleted medium, were submitted to a 1 mM Cs stress. Cell growth, strongly diminished in absence of K, was not influenced by Cs. In contrast, the chlorophyll content, affected by a Cs stress superposed to K depletion, did not vary under the sole K depletion. The uptake of Cs was monitored in vivo using 133Cs NMR spectroscopy while the final K and Cs concentrations were determined using atomic absorption spectrometry. Cs absorption rate and final concentration increased in a K-depleted external medium; in vivo NMR revealed that intracellular Cs was distributed in two kinds of compartment. Synchrotron X-ray fluorescence microscopy indicated that one could be the chloroplasts. In parallel, the cellular response to the Cs stress was analysed using proteomic and metabolic profiling. Proteins up- and down-regulated in response to Cs, in presence of K+ or not, were analysed by 2D gel electrophoresis and identified by mass spectrometry. No salient feature was detected excepting the overexpression of antioxidant enzymes, a common response of Arabidopsis cells stressed whether by Cs or by K-depletion. 13C and 31P NMR analysis of acid extracts showed that the metabolome impact of the Cs stress was also a function of the K nutrition. These analyses suggested that sugar metabolism and glycolytic fluxes were affected in a way depending upon the medium content in K+. Metabolic flux measurements using 13C labelling would be an elegant way to pursue on this line. Using our experimental system, a progressively stronger Cs stress might point out other specific responses elicited by Cs.     

Effect of sodium ascorbate on the chemiluminescent response of murine peritoneal exudate cells.

Vitamin C is an effective antioxidant that neutralizes reactive oxygen radicals. The purpose of this study was to determine if sodium ascorbate would neutralize the reactive oxygen products generated during the respiratory burst of thioglycollate-elicited murine peritoneal exudate cells (PEC). In vitro and in vivo studies were done. Cells treated in vitro showed a significant, dose-dependent reduction in chemiluminescence (CL) after activation with opsonized zymosan. Higher concentrations of sodium ascorbate (24.2 mM) produced a significantly greater reduction in CL than did lower concentrations (0.242 mM). This range of sodium ascorbate concentrations overlaps those found in normal leukocytes (1-4 mM). Sodium ascorbate at physiological plasma concentrations (0.09 mM) did not reduce CL. Cells incubated with 500 mM sodium ascorbate in vitro and then washed once prior to zymosan activation also showed a significant reduction in CL. In contrast, PEC harvested from mice treated in vivo with sodium ascorbate (one or five daily doses of 1.0 M sodium ascorbate, 0.01 ml/g body weight) did not show a reduction in CL. This concentration of sodium ascorbate represents a dose that is 2310 times greater than the Recommended Dietary Allowance (RDA). These studies show that physiological doses of sodium ascorbate can quench CL in vitro, but even large doses of sodium ascorbate administered in vivo do not affect the CL of harvested murine PEC.     

Dioxygen transmembrane distributions and partitioning thermodynamics in lipid bilayers and micelles

Cellular respiration, mediated by the passive diffusion of oxygen across lipid membranes, is key to many basic cellular processes. In this work, we report the detailed distribution of oxygen across lipid bilayers and examine the thermodynamics of oxygen partitioning via NMR studies of lipids in a small unilamellar vesicle (SUV) morphology. Dissolved oxygen gives rise to paramagnetic chemical shift perturbations and relaxation rate enhancements, both of which report on local oxygen concentration. From SUVs containing the phospholipid sn-2-perdeuterio-1-myristelaidoyl, 2-myristoyl-sn-glycero-3-phosphocholine (MLMPC), an analogue of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), we deduced the complete trans-bilayer oxygen distribution by measuring (13)C paramagnetic chemical shifts perturbations for 18 different sites on MLMPC arising from oxygen at a partial pressure of 30 bar. The overall oxygen solubility at 45 °C spans a factor of 7 between the bulk water (23.7 mM) and the bilayer center (170 mM) and is lowest in the vicinity of the phosphocholine headgroup, suggesting that oxygen diffusion across the glycerol backbone should be the rate-limiting step in diffusion-mediated passive transport of oxygen across the lipid bilayer. Lowering of the temperature from 45 to 25 °C gave rise to a slight decrease of the oxygen solubility within the hydrocarbon interior of the membrane. An analysis of the temperature dependence of the oxygen solubility profile, as measured by (1)H paramagnetic relaxation rate enhancements, reveals that oxygen partitioning into the bilayer is entropically favored (ΔS° = 54 ± 3 J K(-1) mol(-1)) and must overcome an enthalpic barrier (ΔH° = 12.0 ± 0.9 kJ mol(-1)).     

Development and characterization of a tissue engineered pancreatic substitute based on recombinant intestinal endocrine L‐cells

A tissue engineered pancreatic substitute (TEPS) consisting of insulin‐producing cells appropriately designed and encapsulated to support cellular function and prevent interaction with the host may provide physiological blood glucose regulation for the treatment of insulin dependent diabetes (IDD). The performance of agarose‐based constructs which contained either a single cell suspension of GLUTag‐INS cells, a suspension of pre‐aggregated GLUTag‐INS spheroids, or GLUTag‐INS cells on small intestinal submucosa (SIS), was evaluated in vitro for total cell number, weekly glucose consumption and insulin secretion rates (GCR and ISR), and induced insulin secretion function. The three types of TEPS studied displayed similar number of cells, GCR, and ISR throughout 4 weeks of culture. However, the TEPS, which incorporated SIS as a substrate for the GLUTag‐INS cells, was the only type of TEPS tested which was able to retain the induced insulin secretion function of non‐encapsulated GLUTag‐INS cells. Though improvements in the expression level of GLUTag‐INS cells and/or the number of viable cells contained within the TEPS are needed for successful treatment of a murine model of IDD, this study has revealed a potential method for promoting proper cellular function of recombinant L‐cells upon incorporation into an implantable three‐dimensional TEPS. Biotechnol. Bioeng. 2009;103: 828–834. © 2009 Wiley Periodicals, Inc.     

Sodium: a regulator of glucose uptake in virus-transformed and nontransformed cells.

Observations of cells transformed by the Bryan strain of Rous sarcoma virus (RSV-BH) suggested that the intracellular concentrations of sodium ion (Na+) may play a critical role in cellular metabolism. In an attempt to manipulate intracellular Na+, chick embryo cells were exposed to graded concentrations of Na+ in the cellular growth medium, and the effects on capacity for glucose uptake was examined. After incubation for six hours, the incorporation rate of 2-deoxyglucose (used as a substitute for glucose) was proportional to the external Na+ concentration over the range, 100 mM to 200 mM. Cells transformed by RSV-BH were less responsive than nontransformed cells to differences in Na+ at low concentrations. The changes were specifically dependent upon Na+, since K+, Li+, or choline + were ineffective as substitutes, and increasing the ionic strength above that of 120 mM Na+ was effective only when Na+ was the added cation.