A novel technique for measuring solute diffusivities in entrapment matrices used in immobilization

A novel technique has been developed for measuring effective solute diffusivities in entrapment matrices used for cell immobilization. In this technique radiotracers were used to measure effective diffusivities and equilibrium partition coefficients of the solute between the liquid and solid matrix. Ca-alginate was used in this study, because it is one of the most commonly employed matrices for the immobilization of microbial, plant and mammalian cells. The experimental apparatus consisted of a single spherical Ca-alginate bead which was attached to a rotating rod and immersed in water containing C(14)-glucose. The rotational speed of the spherical bead was controlled and resulted in excellent mixing, and negligible external film mass transfer resistance, which allowed the measurement of true effective solute diffusivity within the solid matrix. The rates of C(14)-glucose diffusion within the Ca-alginate sphere were measured using a scintillation spectrometer. A mathematical model of unsteady-state diffusion in a sphere was used with appropriate boundary conditions, and the effective diffusivity of glucose was found from the best fit of the experimental data using a computer regression analysis method. Using 2% (w/v) Ca-alginate beads in this new radiotracer technique the effective diffusivity and partition coefficient of glucose were found to be 6.62 x 10(-10) m(2)/s and 0.98, respectively. The accuracy, advantages, and simplicity of this new method for diffusivity measurements are also compared to other existing methods.     

Spatiotemporal dynamics of glycolytic waves provides new insights into the interactions between immobilized yeast cells and gels

The immobilization of cells or enzymes is a promising tool for the development of biosensors, yet the interactions between the fixative materials and the cells are not fully understood, especially with respect to their impact on both cell metabolism and cell-to-cell signaling. We show that the spatiotemporal dynamics of waves of metabolic synchronization of yeast cells provides a new criterion to distinguish the effect of different gels on the cellular metabolism, which otherwise could not be detected. Cells from the yeast Saccharomyces carlsbergensis were immobilized into agarose gel, silica gel (TMOS), or a mixture of TMOS and alginate. We compared these immobilized cells with respect to their ability to generate temporal, intracellular oscillations in glycolysis as well as propagating, extracellular synchronization waves. While the temporal dynamics, as measured by the period and the number of oscillatory cycles, was similar for all three immobilized cell populations, significant differences have been observed with respect to the shape of the waves, wave propagation direction and velocity in the three gel matrices used.     

Production of molecular hydrogen with immobilized cells ofRhodospirillum rubrum

Summary Cells ofRhodospirillum rubrum have been immobilized in various gels and tested for photobiological hydrogen production. Agar proved to be the best immobilizing agent with respect to production rates as well as stability. Agar immobilized cells were also superior compared to liquid suspension cultures. Growth conditions of the cells prior to immobilization, e.g. cell age, light intensity or nutrient composition, were of primary importance for the activity in the later immobilized state. A reactor with agar immobilized cells has been operated successfully over 3000 h with a loss of the activity of about 60%. Mean rates for hydrogen production for immobilized cells in this work during the first 60 to 70 hours after immobilization were in the range of 18 to 34 μl H₂ mg⁻¹ d.w. h⁻¹ and thus by a factor of up to 2 higher than liquid cultures under the same conditions. Maximal rates of hydrogen production (57 μl H₂ ml⁻¹ immobilized cell suspension) were reached in agar gel beads with cells immobilized after 70 h growth in liquid culture in the light and a cell density of 1.0 mg ml⁻¹, 70 h after immobilization.     

Polymeric cryogels as promising materials of biotechnological interest

Cryogels are gel matrices that are formed in moderately frozen solutions of monomeric or polymeric precursors. Cryogels typically have interconnected macropores (or supermacropores), allowing unhindered diffusion of solutes of practically any size, as well as mass transport of nano- and even microparticles. The unique structure of cryogels, in combination with their osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of biological nanoparticles (plasmids, viruses, cell organelles) and even whole cells. Polymeric cryogels are efficient carriers for the immobilization of biomolecules and cells.     

Diffusion in immobilized-cell agar layers: influence of bacterial growth on the diffusivity of potassium chloride

Summary The diffusitivity of potassium chloride in composite agar slab/microporous membrane structures loaded with various amounts of Escherichia coli whole cells was determined using both time-lag and steady-state methods. The diffusion coefficient of KCl decreased linearly with the logarithm of the immobilized-cells content. The effect exerted by bacterial growth inside the immobilization matrices on KCl diffusivity was then investigated. The diffusion coefficient of KCl obtained by time-lag analysis decreased during incubation of the immobilized-cell structures, whereas less consitent results arose from the steady-state method. An apparent doubling time for immobilized E. coli, increasing with the initial cell content of the gel, was obtained from the calibration relationship between KCl diffusivity and the number of organisms in agar. Offprint requests to: G.-A. Junter     

无载体固定化培养模式下HEK293细胞的生长和代谢特征

利用HEK293细胞在悬浮培养体系中下具有聚集成团的体外培养特性,在250ml的spinner flask搅拌式细胞培养瓶中以悬浮细胞团的形式实施HEK293细胞的无载体固定化培养,以细胞密度、细胞活力、细胞团粒径分布和葡萄糖比消耗率 (qglc)、乳酸比产率 (qlac)、乳酸转化率 (Ylac/glc)、氨基酸消耗为观察指标,同时设置静止培养体系作为参照,考察无载体固定化培养模式下的HEK293细胞生长和代谢特征。观察结果表明,HEK293细胞在搅拌式细胞培养瓶中无载体固定化培养和在组织培养瓶中静止贴壁培养表现为基本相同的细胞生长和代谢特征,平均粒径小于300μm的细胞团中的物质传递能够满足HEK293细胞维持正常生长和代谢的基本需要。HEK293细胞的无载体固定化培养便于实施灌注操作、提高生物反应器单位体积的生产效率。     

Physical Studies on Cell Immobilization Using Calcium Alginate Gels

Columns of calcium alginate gel pellets have excellent physical properties when used as a cell immobilization support. Columns of pellets were very resistant to compression and abrasion during passage of high concentrations of sucrose at high flow rates, but if the pellets were formed using low alginate and Ca²⁺ concentrations, compression occurred and flow out of the column was reduced and pressure built up. Transfer of sucrose into the pellets was controlled by internal diffusion, the rate of diffusion being increased by reductions in the alginate and Ca²⁺ concentrations used for immobilization and by the presence of entrapped active cells. Some leakage of cells occurred during use especially when cell division of the entrapped cells took place, but leakage could be minimized by using more highly polymerized pellets. Therefore, immobilization conditions can be chosen so as to form strong pellets, possessing high substrate transfer rates and low rates of cell leakage.     

Evaluation of some gelling agents for immobilization of aerobic microbial cells in alginate and carrageenan gel beads

Summary Different gelling agents were used to immobilized viable cells in either alginate or -carrageenan gel beads. Based on cell leakage from the gel beads, oxygen and glucose diffusion coefficients and toxicity of the gelling agents, SrCl₂ was found to be the best for immobilization of aerobic microbial cells in, not only alginate but also carrageenan gel beads.     

Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices

The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 x 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 +/- 0.8 x 10(8) cells/cm3 after 5 weeks, compared to 2.0 +/- 1.1 x 10(8) cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 +/- 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development.     

Ethanol fermentation by ionically adsorbed Zymomomas mobilis: Environmental effects on cell immobilization

Batch ethanol fermentation by cells of Zymomomas mobilis ATCC 29191, ionically adsorbed on a DEAE-cellulose ion exchanger, was investigated in a stirred fermentor. Adsorption isotherms in different media were determined and used to interpret the effects of the environment on cell immobilization. Other factors affecting cell immobilization during an actual fermentation were studied. Mechanical agitation was found to cause detachment of cells from the ion exchange particles. The results suggest that the amount of cells adsorbed during a fermentation process is different from that found from adsorption isotherm data. Consequently, application of equilibrium adsorption data to actual fermentations should be done with caution.     

The cellular basis for the Ia restriction in murine experimental autoimmune thyroiditis

Susceptibility to experimental autoimmune thyroiditis (EAT) in the mouse is linked to the I-A subregion of the major histocompatibility complex. EAT can be induced in susceptible strains of mice by immunization with mouse thyroglobulin (MTg) and adjuvant. We have described a cell transfer system wherein spleen cells from EAT-susceptible CBA/J mice primed in vivo with MTg and lipopolysaccharide (LPS) can be activated in vitro with MTg to transfer EAT to naive syngeneic recipients. This cell transfer system was used to elucidate the cellular basis for the I-A restriction in EAT. While the cell active in transferring EAT was Thy 1+ I-A-, depletion of I-A+ cells from the in vitro culture prevented the activation of EAT effector T cells. MTg-pulsed mitomycin C-treated naive syngeneic spleen cells as antigen-presenting cells (APCs) could replace the I-A+ cells in vitro. Allogeneic (Balb/c) APCs were ineffective. Using APCs from several recombinant inbred strains of mice, it was shown that C3H/HEN and B10.A(4R) APCs were effective in activating MTg/LPS-primed CBA/J spleen cells to transfer EAT while B10.A(5R) APCs were ineffective. This maps the H-2 restriction to the K or I-A subregions. Addition of polyclonal anti-Iak or monoclonal anti-I-Ak or anti-L3T4 during in vitro activation inhibited both the generation of EAT effector cells and the proliferative response to MTg. Irrelevant anti-Ia reagents, monoclonal anti-I-Ek, and monoclonal anti-I-Jk were ineffective. Thus the I-A restriction in murine EAT appears to result from an I-A restricted interaction between Ia+ APCs and Ia- EAT effector T cells.     

Macroporous gel particles as robust macroporous matrices for cell immobilization

A new design of robust matrices for cell immobilization is described. Macroporous gels (MGs) with immobilized microbial cells were prepared at subzero temperatures and were formed inside a plastic core (so-called, protective housing). Due to the protective housing the macroporous gel particles with immobilized cells can be used in well-stirred bioreactors. High retained activity of yeast (77-92%) and Escherichia coli (50-91%) cells immobilized in MGs after drying and storage in the dried state was due to the high structural stability and heterogeneous porous structure of the MGs.     

Effects of the adjuvants SGP and Quil A on the induction of experimental autoimmune thyroiditis in mice

In the present study, two adjuvants, SGP and Quil A, were assessed for their ability to induce experimental autoimmune thyroiditis (EAT) in mice. SGP (a synthetic copolymer of starch, acrylamide, and sodium acrylate) and Quil A (a plant saponin) were compared with lipopolysaccharide (LPS) and complete Freund's adjuvant (CFA) given together with mouse thyroglobulin (MTg) for their ability to induce EAT in CBA/J mice. Immunization with MTg and LPS, MTg and CFA, or MTg with SGP was effective in inducing anti-MTg antibodies and histologic EAT, while MTg with Quil A was ineffective in inducing either anti-MTg antibodies or EAT. MTg with LPS was able to prime mice for the development of an in vitro spleen cell proliferative response to MTg while MTg with SGP or with Quil A was unable to prime spleen cells to proliferate detectably in response to MTg. MTg with LPS given in vivo primes CBA/J spleen cells for further activation by in vitro culture with MTg to transfer EAT to naive CBA/J recipients. MTg with SGP was also effective in priming CBA/J spleen cells for in vitro activation and transfer of EAT while MTg with Quil A was ineffective. The effective adjuvant activity of SGP and its lack of toxicity relative to LPS should make it a useful agent for further studies in murine models of EAT.     

Diffusion in lyotropic mesophases used as matrix for biocatalysts in organic solvents

Summary Lyotropic liquid crystals are appropriate matrices for the immobilization of biocatalysts used for biocatalysis in organic solvents. For the reactants of an microbial epoxidation reaction, oxygen, propene, and propene oxide, the diffusion constants in lyotropic mesophases were determined by a special diffusion cell. The diffusion rate strongly depends on the water content of the liquid crystals and the hydrophobicity of the reactant.     

Swelling of cellulose derivative (HPMC) matrix systems for drug delivery

The water swellable hydrogels are commonly used in the production of solid pharmaceutical dosage systems for oral administration (matrices). Their use allows to obtain the controlled drug release. The key role is played by the transport phenomena which take place: water up-take, gel swelling and erosion, increase in diffusivity due to hydration. Thus, knowledge of these phenomena is fundamental in designing and realizing the pharmaceutical systems.In this work, tablets made of pure hydrogel, HydroxyPropyl-MethylCellulose (HPMC), were produced and immersed in a thermostatic bath filled with stirred distilled water (37 °C). The water up-take was allowed only by radial direction (from the lateral surface) by confining the tablet between two glass slides. Two distinct methods, an optical technique already described in a previous work, and a gravimetric procedure described here, were applied to measure the water concentration profiles along the radial direction in the tablets. The data obtained were used both to clarify the nature of the transport phenomena involved, and to perform a better tuning of a mathematical model previously proposed.     

Diffusion of biologically relevant molecules through gel-like tissue scaffolds

Encapsulation of living cells into gel-like matrices that are capable of maintaining their viability over an extended time period is starting to play a major role in medicine in applications such as, cell-based sensors, cellular therapy, and tissue engineering. The permeability of nutrients and waste products through these matrices is critical to their performance. In this article, we report a methodology for selecting scaffolds with different permeabilities and surface area/volume ratios that can be used to house a 3D cell aggregate. Such a system can be modeled if the consumption or production rates for metabolites and waste products, respectively and the diffusion coefficients of these solutes in culture medium and the encapsulating gel matrix are known. A transient finite volume mass diffusion model, based on Fick's law, is derived where the consumption of a solute by the cells is modeled through a source term. The results show that the "performance" of cell-doped gel is critically dependent on the rate at which cells consume key molecules e.g., glucose. Pragmatically, the model also provides insight as to how many cells a given gel geometry and structure can support. The approach used applies to any porous structure where mass transport occurs through diffusion.     

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane-based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2-4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5-3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44-155 kD-lowering their diffusion rates from 10- to 100-fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 A for 1.5 and 3% alginate gels, respectively, and 480 and 360 A for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different-size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration. (c) 1996 John Wiley & Sons, Inc.     

Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.     

In situ analysis of T cell subset composition in experimental autoimmune thyroiditis after adoptive transfer of activated spleen cells

T cells from genetically susceptible mice developing experimental autoimmune thyroiditis (EAT) proliferate in response to restimulation with mouse thyroglobulin (MTg) in vitro. The in vitro-activated cells adoptively transfer EAT as well as differentiate into cells cytotoxic for syngeneic thyroid monolayers. To examine the kinetics of T cell subset infiltration and distribution in situ after adoptive transfer, we applied the avidin-biotin-peroxidase labeling technique to thyroid sections, utilizing rat monoclonal antibodies followed by a biotinylated rabbit anti-rat antibody. Female CBA donor mice were immunized with MTg and lipopolysaccharide. Their spleen cells were obtained 7 days later, cultured with MTg, and transferred into recipient mice. The thyroids were removed on Days 7, 10, and 14 after transfer and serially sectioned. The early phase of transferred EAT showed a higher percentage of L3T4+ cells compared to Lyt-2+ cells, yielding a ratio of 2.3 and total T cells of about 35%. By Day 10, both T cell subsets had increased to a total of about 56%. However, the relative increase was greater in the Lyt-2+ subset; the nearly doubled percentage was statistically significant, resulting in a downward shift in the subset ratio to 1.7. Little change in the in situ distribution was seen on Day 14. The percentages of F4/80+ (macrophage) population in lesions examined on Days 10 and 14 were fairly constant and B cell involvement was minimal. These findings illustrate the pathogenic role of both T cell subsets in adoptively transferred EAT and the time-dependent changes in their relative proportions leading to thyroid gland destruction.     

Oxygen uptake rate of immobilized growing hybridoma cells

Summary The specific oxygen uptake rate of hybridoma cells immobilized in calcium alginate gel particles was measured, and the observed data was compared with those of non-immobilized cells. The uptake rate of the immobilized cells coincided with that of the non-immobilized hybridoma cells just after immobilization, but increased with cell growth. On the other hand, the cellular glucose consumption rate decreased slightly during the experiments. The increased oxygen uptake rate by immobilized cells was closely related to the formation of cell colonies in the gel particles.