Separation of yeast cells from aqueous solutions using magnetically stabilized fluidized beds

AIMS: To separate Saccharomyces cerevisiae cells from aqueous solutions using magnetically stabilized fluidized beds (MSFB) that utilize a horizontal magnetic field, and to study the effect of some parameters, such as bed porosity and height, liquid flow rate and inlet concentration on cell removal efficiency and breakthrough curves. METHODS AND RESULTS: The separation process was conducted in an MSFB under the effect of horizontal magnetic field. The magnetic particles used consist of a ferromagnetic core of magnetite (Fe3O4) covered by a stable layer of activated carbon to adsorb the yeast cells from the suspension. The yeast cell concentration in the effluent was determined periodically by measuring the absorbance at 610 nm. The effect of the magnetic field intensity on the bed porosity and consequently the exit-normalized cell concentration from the bed was studied. It was found that bed porosity increased by 75%, and the normalized cell concentration in the bed effluent decreased by 30%, when the magnetic field intensity was increased from 0 to 110 mT. In addition, increasing the magnetic field intensity and bed height delayed the breakthrough point, and allowed efficient cell removal. These results demonstrate an improved method to separate cells of low concentration from cell suspension. CONCLUSIONS: This study allows the continuous separation of yeast cells from aqueous solutions in an MSFB. The removal efficiency is affected by different parameters including the bed height, flow rate and initial concentration. The removal efficiency reaches 82%, and could be improved by varying the operational parameters. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this investigation show that the MSFB using horizontal fields represents a potential tool for the continuous separation of cell suspension from aqueous solution. This study will contribute to a better understanding of the hydrodynamic parameters on the separation efficiencies of the cell.     

Continuous cell suspension processing using magnetically stabilized fluidized beds

The behavior of a cell suspension in a continuous magnetically stabilized fluidized bed (MSFB) was investigated both experimentally and theoretically. The low, constant pressure drop and fluidity of the solids phase in the MSFB allowed a continuous countercurrent separator to be constructed. The magnetic field eliminated all motion of the solids phase (nickel spheres) and produced a device similar to a packed-bed depth filter. Yeast cells were used as the suspended solids and the performance of the MSFB filter was assessed as a function of the bed height, solids velocity, cell concentration, and liquid composition. Removal rates could be adjusted by controlling the cell/support interaction and were found to be as high as 99%. A mathematical model was used to aid in understanding this filtration and was found to agree qualitatively with all experimental observations. Comparison of the model with the data suggests that both cell/cell binding and cell shadowing are occurring.     

Application of magnetic agarose support in liquid magnetically stabilized fluidized bed for protein adsorption

A novel magnetic agarose support (MAS) was fabricated for application in a liquid magnetically stabilized fluidized bed (MSFB). It was produced by water-in-oil emulsification method using a mixture of agarose solution and nanometer-sized superparamagnetic Fe(3)O(4) particles as the aqueous phase. The MAS showed good superparamagnetic responsiveness in a magnetic field. A reactive triazine dye, Cibacron blue 3GA (CB), was coupled to the gel to prepare a CB-modified magnetic agarose support (CB-MAS) for protein adsorption. Lysozyme was used as a model protein to test the adsorption equilibrium and kinetic behavior of the CB-MAS. The dependence of bed expansion in the MSFB with a transverse magnetic field on liquid velocity and magnetic field intensity was investigated. Liquid-phase dispersion behavior in the MSFB was examined by measurements of residence time distributions and compared with that obtained in packed and expanded beds. Dynamic lysozyme adsorption in the MSFB was also compared with those in packed and expanded beds. The dynamic binding capacity at 10% breakthrough was estimated at 55.8 mg/mL in the MSFB, higher than that in the expanded bed (31.1 mg/mL) at a liquid velocity of 45 cm/h. The results indicate that the CB-MAS is promising for use in liquid MSFB for protein adsorption.     

Preparation of magnetically susceptible polyacrylamide/magnetite beads for use in magnetically stabilized fluidized bed chromatography

Spherical polyacrylamide/magnetite (PAM) composite beads, suitable for use in a magnetically stabilized fluidized bed (MSFB), were manufactured by a suspension polymerization method. Yield of beads depended on the type and concentration of buffer used during polymerization as well as the pH. More stabilizer was needed to prevent bead agglomeration as magnetite concentration increased. Bead diameter ranged from less than 60 to 600 mum, depending on reaction conditions, and the bead mean diameter and size distribution decreased with increasing impeller speed. The density and roundness factor of the beads were 1.19 +/- 0.02 g cm(-3) and 1.08 +/- 0.03, respectively. The beads had high magnetization at a low applied magnetic field strength (60 mT at 75 kA m(-1)) and retained little residual magnetization (<2 mT) after the field was removed. Incorporation of magnetite did not significantly affect the physical strength of the beads: the beads' average elastic modulus was 14 +/- 4 kPa, similar to reported values for polyacrylamide gels (15.8 kPa). The beads were stable in a range of buffers from pH 1 to 10 and were resistant to microbial degradation. The fluidization and stabilization behavior of the beads was examined in a bench-scale MSFB. The minimum fluidization velocity (U(mf)) of the beads (0.035 mm s(-1)) allowed the MSFB to be operated at superficial velocities close to those used in HPLC systems. Against expectations, at high superficial velocities, the stabilized bed of the MSFB had a greater expansion than the unstabilized bed. The PAM beads could be derivatized and activated for soybean trypsin inhibitor immobilization by a standard carbodiimide method, and the affinity separation of trypsin from chymotrypsin was demonstrated. The PAM beads show excellent potential for use in MSFB chromatography. (c) 1997 John Wiley & Sons, Inc.     

Development of the magnetic beads for dye ligand affinity chromatography and application to magnetically stabilized fluidized bed system

Magnetic poly(2-hydroxyethylmethacrylate) [mPHEMA] beads were prepared by suspension polymerization of HEMA in the presence of Fe₃O₄ nano-powder. Cibacron Blue F3GA was covalently immobilized to the mPHEMA beads via nucleophilic substitution reaction between chloride of its triazine ring and hydroxyl groups of HEMA under alkaline conditions. The mPHEMA/Cibacron Blue F3GA beads (100–140 μm in diameter) carrying 68.3 μmol Cibacron Blue F3GA per gram polymer were used for β-casein adsorption studies. Adsorption studies were performed under different conditions in a batch system (i.e., pH, β-casein initial concentration, temperature, and ionic strength) and then in a magnetically stabilized fluidized bed (MSFB) system. The swelling ratio of the mPHEMA was 62.1%. The maximum adsorption capacity for batch system was 20.2% lower as compared to the value obtained in MSFB. The mPHEMA/Cibacron Blue F3GA beads could be repeatedly applied for β-casein adsorption without significant losses in the adsorption capacity.     

Performance of dye-affinity beads for aluminium removal in magnetically stabilized fluidized bed

BACKGROUND: Aluminum has recently been recognized as a causative agent in dialysis encephalopathy, osteodystrophy, and microcytic anemia occurring in patients with chronic renal failure who undergo long-term hemodialysis. Only a small amount of Al(III) in dialysis solutions may give rise to these disorders. METHODS: Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads in the size range of 80-120 microm were produced by free radical co-polymerization of HEMA and ethylene dimethacrylate (EDMA) in the presence of magnetite particles (Fe3O4). Then, metal complexing ligand alizarin yellow was covalently attached onto mPHEMA beads. Alizarin yellow loading was 208 micromol/g. These beads were used for the removal of Al(III) ions from tap and dialysis water in a magnetically stabilized fluidized bed. RESULTS: Al(III) adsorption capacity of the beads decreased with an increase in the flow-rate. The maximum Al(III) adsorption was observed at pH 5.0. Comparison of batch and magnetically stabilized fluidized bed (MSFB) maximum capacities determined using Langmuir isotherms showed that dynamic capacity (17.5 mg/g) was somewhat higher than the batch capacity (11.8 mg/g). The dissociation constants for Al(III) were determined using the Langmuir isotherm equation to be 27.3 mM (MSFB) and 6.7 mM (batch system), indicating medium affinity, which was typical for pseudospecific affinity ligands. Al(III) ions could be repeatedly adsorbed and desorbed with these beads without noticeable loss in their Al(III) adsorption capacity. CONCLUSIONS: Adsorption of Al(III) demonstrate the affinity of magnetic dye-affinity beads. The MSFB experiments allowed us to conclude that this inexpensive sorbent system may be an important alternative to the existing adsorbents in the removal of aluminium.     

Countercurrent gradient chromatography: A continuous focusing technique

The continuous separation of proteins was performed in a countercurrent gradient chromatography (CGC) system. A magnetically stabilized fluidized bed (MSFB) was used to establish true countercurrent contact of a solid resin with a liquid buffer. STable pH gradients were formed in the system in less than 10 min and remained stable throughout the course of the separation experiment (>2 h). The shape of the pH gradient, which ultimately controls the resolution and purity of the separation, can be controlled by making simple adjustments in the interstitial velocities of the liquid and solid phases. We have performed the separation of myoglobin and human serum albumin (HSA) using this device and achieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been developed that predicts the focusing behaviour and capabilities of the CGC system. Using the model, we have estimated the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations. (c) 1995 John Wiley & Sons, Inc.     

Continuous protein separations in a magnetically stabilized fluidized bed using nonmagnetic supports

Continuous protein separations were performed using a magnetically stabilized fluidized bed (MSFB) and a commercially available affinity adsorption resin that contained no magnetically susceptible material. These nonmagnetic materials can be stabilized at relatively low fields (<75 G requiring <30 W) if sufficient magnetically susceptible particles are also present in the stabilized bed. The minimum amount of magnetic particles necessary to stabilize the bed is as low as 20% by volume and is a function of various parameters including the size and density of both particles, the magnetic field strength, and the fluidization velocity. Advantages of these beds for performing separations include true continuous, countercurrent liquid-solids contact, mass-transfer efficiencies nearly equal to that of packed beds, and the ability of handle suspended cells or cell debris. A variety of commercially available affinity, ion-exchange, and adsorptive supports can be used in the bed for continuous separations; results are presented for the adsorption and recovery of lysozyme from an aqueous mixture of lysozyme and myoglobin using an affinity resin.     

A novel magnetic silica support for use in chromatographic and enzymatic bioprocessing

A limited number of support matrices have so far been developed for use in magnetically stabilized fluidized bed (MSFB) applications. We have developed a versatile magnetic silica support which can be derivatized readily for both adsorption chromatography and enzyme immobilization by well-known techniques. A magnetic pellicular bead is prepared by electrostatically depositing alternating layers of colloidal silica and cationic polymer onto macroscopic nickel core particles. The polymer is then burned out and the silica partially sintered to yield a porous shell with 5-80 m(2)/g of surface area. This magnetic composite was tested as a support for immobilizing invertase. Sucrose was continuously converted to its component monosaccharides with nearly constant activity over the first 8 days and retention of 50% of initial activity after 25 days.     

The immune system as a regulator of thyroid hormone activity

It has been known for decades that the neuroendocrine system can both directly and indirectly influence the developmental and functional activity of the immune system. In contrast, far less is known about the extent to which the immune system collaborates in the regulation of endocrine activity. This is particularly true for immune-endocrine interactions of the hypothalamus-pituitary-thyroid axis. Although thyroid-stimulating hormone (TSH) can be produced by many types of extra-pituitary cells--including T cells, B cells, splenic dendritic cells, bone marrow hematopoietic cells, intestinal epithelial cells, and lymphocytes--the functional significance of those TSH pathways remains elusive and historically has been largely ignored from a research perspective. There is now, however, evidence linking cells of the immune system to the regulation of thyroid hormone activity in normal physiological conditions as well as during times of immunological stress. Although the mechanisms behind this are poorly understood, they appear to reflect a process of local intrathyroidal synthesis of TSH mediated by a population of bone marrow cells that traffic to the thyroid. This hitherto undescribed cell population has the potential to microregulate thyroid hormone secretion leading to critical alterations in metabolic activity independent of pituitary TSH output, and it has expansive implications for understanding mechanisms by which the immune system may act to modulate neuroendocrine function during times of host stress. In this article, the basic underpinnings of the hematopoietic-thyroid connection are described, and a model is presented in which the immune system participates in the regulation of thyroid hormone activity during acute infection.     

Stem cells of the respiratory epithelium and their in vitro cultivation

Summary Parenchymal (epithelial or mesenchymal) stem cells are rapidly drawing both scientific and clinical attention in solid organs like the liver, skin, intestine and abdominal mesothelium, just as has been the case in the hematopoietic system. For the stem cells of these organs various definitions, markers for identification, methods of isolation and in vitro cultivation, and lineage mechanisms have been proposed and some of them are now proven to be valid and useful. In this article attempts will be made to explore whether there are stem cells in the lower respiratory system (from the trachea to the lung periphery) and what they look like. Because of its anatomical and functional complexity the stem cell concept for the respiratory system has been developing rather slowly. Nevertheless, the data available seem to indicate that in analogy to the above mentioned organs there is only one type of epithelial stem cells throughout all sections of the lower respiratory system during fetal through adult stages. They are multipotent for cell differentiation and able to yield lineage progenitors for ciliated, goblet, basal, Clara, neuroendocrine, alveolar type 1 and alveolar type 2 cells.     

Diffusion chamber colony-forming unit (CFU-d): a primitive stem cell

Assay of hematopoietic precursor cells in diffusion chambers (DCs) implanted intraperitoneally in experimental animals provides a powerful tool for studying stem cell kinetics in vivo. In this system, the effect of cell migration (which complicates whole animal studies) is eliminated because the membranes utilized in the construction of the chambers are impermeable for cells, while permitting free passage of molecules present in the humoral phase of the host. As judged by light microscopy, conditions in the DC cultures primarily favor macrophage and granulocyte growth. However, the use of in vitro and in vivo subculture to further analyze chamber contents has demonstrated that the system supports proliferation of early hematopoietic progenitors. Additionally, cells capable of rescuing lethally irradiated mice proliferate in DC cultures. Development of the plasma clot DC technique has revealed that most of the growth occurs in colonies which are derived from single cells (CFU-d). Characterization of these cells indicates that they are at least as primitive as other colony-forming cells and, also based on subculture studies, can differentiate along several hematopoietic lineages. In addition to normal CFU-d, both embryonal and leukemic cells can give rise to granulocytes, macrophages, megakaryocytes and erythroid cells in the DC cultures. Evaluation of the effects of humoral factors on hematopoietic cell proliferation and differentiation in the system has led to the identification of both stimulators and inhibitors that may be different from the well-characterized cytokines. Thus, the system seems to be useful for detecting molecules controlling the most primitive stages of hematopoiesis. We believe that the DC culture technique holds enormous potential in the study of stem cell proliferation and differentiation in vivo.     

NK细胞与HIV/SIV感染相关性的研究进展

NK细胞作为天然免疫系统的重要组成部分,其在HIV/SIV感染后的免疫机制及如何发挥抗病毒作用成为近几年艾滋病研究的热点之一。研究中发现,伴随HIV/SIV的感染,NK细胞亚群比例发生改变同时伴有功能缺陷,这种变化与HIV/SIV慢性感染阶段病毒复制水平有显著相关性。并且由于归巢受体表达的改变引起NK细胞在HIV/SIV感染者体内不同组织间的重新分布。NK细胞表面的受体KIR3DL1和KIR3DS也表现出对HIV感染的抵抗作用。这些发现为我们进一步研究NK细胞的抗HIV/SIV病毒感染的免疫机制提供了新的思路和方向。     

Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

An early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished in lepidopteran insect cells following baculovirus infection

The expression of recombinant proteins in their native state has become a prerequisite for a variety of functional and structural studies, as well as vaccine development. Many biochemical properties and functions of proteins are dependent on or reside in posttranslational modifications, such as glycosylation. The baculovirus system has increasingly become the system of choice due to it capabilities of performing posttranslational modifications and usually high yields of recombinant proteins. The Toxoplasma gondii surface antigen SAG1 was used as a model for a glycosylphosphatidyl-inositol (GPI)-anchored protein and expressed in insect cells using the baculovirus system. We show that the T. gondii SAG1 surface antigen expressed in this system was not modified by a GPI-anchor. In vitro and in vivo studies demonstrate that uninfected insect cells are able to produce GPI-precursors and to transfer a mature GPI-anchor to nascent proteins. These cells however are not capable to produce GPI-precursors following infection. We also show that the biosynthesis of the early GPI intermediate GlcNH(2)-PI is blocked in baculovirus-infected H5 cells, thus preventing the subsequent mannosylation steps for the synthesis of the conserved GPI-core-glycan. We therefore conclude that the baculovirus system is not appropriate for the expression of GPI-anchored proteins.     

Modeling regulation mechanisms in the immune system

We develop a mathematical framework for modeling regulatory mechanisms in the immune system. The model describes dynamics of key components of the immune network within two compartments: lymph node and tissue. We demonstrate using numerical simulations that our system can eliminate virus-infected cells, which are characterized by a tendency to increase without control (in absence of an immune response), while tolerating normal cells, which are characterized by a tendency to approach a stable equilibrium population. We experiment with different combinations of T cell reactivities that lead to effective systems and conclude that slightly self-reactive T cells can exist within the immune system and are controlled by regulatory cells. We observe that CD8+ T cell dynamics has two phases. In the first phase, CD8+ cells remain sequestered within the lymph node during a period of proliferation. In the second phase, the CD8+ population emigrates to the tissue and destroys its target population. We also conclude that a self-tolerant system must have a mechanism of central tolerance to ensure that self-reactive T cells are not too self-reactive. Furthermore, the effectiveness of a system depends on a balance between the reactivities of the effector and regulatory T cell populations, where the effectors are slightly more reactive than the regulatory cells.     

Introduction and expression of the bacterial PaeR7 restriction endonuclease gene in mouse cells containing the PaeR7 methylase.

To study the factors essential for a functional restriction system, the PaeR7 restriction-modification system has been introduced and expressed in murine cells. Transfer of this system was accomplished in two steps. First, cells containing sufficient PaeR7 methylase to completely methylate the mouse genome were constructed. In the second step, the mouse metallothionein promoter-regulated, endonuclease expression vector linked to the hygromycin B resistance selection marker was used to transfect the high methylase-expressing cells. Sixty percent of the clones isolated contained PaeR7 endonuclease enzymatic activity. Transfected cells expressing both methylase and endonuclease were incapable of blocking infection by DNA viruses, and possible explanations are discussed.     

Isolation of gametes and central cells from Oryza sativa L.

In vitro fertilization system of higher plants has been well established using maize gametes and central cells, which can produce embryos and endosperms. In the present study, procedures for isolating gametes and central cells from rice (Oryza sativa L. cv. Nipponbare), a model plant, are reported with the goal of establishing rice in vitro fertilization system. Egg cells and central cells were isolated by manual manipulation of enzyme-treated unpollinated ovules, and an alternative direct isolation method for egg cells that does not use enzymatic treatment was also established. Fluorescent visualization of the granular structures in the cytoplasm of isolated egg cells and the nucleoli in two polar nuclei of isolated central cells suggest that these cells are reliable gametes and central cells. For sperm cell isolation, the contents of rice pollen grains were released by osmotic pressure-induced bursting of the grains. In addition, electrofusion with isolated gametes was successfully conducted.     

Mean-field coupling of calcium oscillations in a multicellular system of rat hepatocytes

In a multicellular system of rat hepatocytes and even in an intact liver, cytoplasmic calcium oscillations are synchronized and highly coordinated. In this paper, the mean-field coupling term has been introduced to describe the coupling flux, which is more efficient than gap junctional coupling terms. An optimal coupling strength and an optimal stimulation level for the synchronization of the coupled system have been observed in this paper. Moreover, it has been proved that these results are independent of the cells number. Interestingly, it has been observed that the intracellular noise and the extracellular noise have different effects on the synchronization of the coupled system.     

Establishment of fetal gonad/mesonephros coculture system using EGFP transgenic mice

In developing mouse embryos, the Sertoli cells, Leydig cells, and seminiferous cords are differentiated in the XY gonads. The migration of mesonephric cells into the gonads is required during the developmental stage for seminiferous cord formation in the male gonads. In previous experiments, an organ coculture system has been used to examine morphologically developing gonads. However, by the process used in this system for fixing and staining the gonad/mesonephros complexes for examination, the kinetics of cell migration and the character of migrating cells cannot be observed. In the present study, we established an improved organ coculture system, using transgenic mice ubiquitously expressing Enhanced Green Fluorescent Protein (EGFP). In this system, time-dependent morphological changes in male-specific migration were observable in the gonad/mesonephros complex. The cell migration occurred at around 20 hr of coculture and began to spread at 25 hr with increases in the number of migrating cells occurring at 45 hr of coculture. No degenerative changes were detected at the end of coculture. Our results indicate that the present coculture system is very useful for investigating the mechanism of cell migration, as well as the characteristics of the migrating cells, in developing gonads. J. Exp. Zool. 286:320-327, 2000.