Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine)

In general, microcapsules prepared from alginate and polycations lack mechanical strength because the interaction between alginate and polycations is ionic instead of covalent, which represents a much stronger bond. To increase the mechanical strength of the capsule, we prepared photosensitive microcapsules that could form covalent bonds between polymers in the capsular membrane by light irradiation. Two types of photosensitive poly(allylamine), with 5% and 10% of amino groups modified by alpha-phenoxycinnamylidene acetylchloride, were synthesized. Both photopolymers exhibited an absorption maximum at 325 nm and were capable of crosslinking upon light exposure. These photosensitive polymers were used for the preparation of microcapsules. The capsules formed from this photosensitive poly(allylamine) and alginate were strengthened significantly by light irradiation. Only 28% of the microcapsules prepared from the 5%-modified photopolymer fractured after 48 h of shaking at 150 rpm. This fracture percentage is much lower when compared with the 60% of capsules fractured when prepared from the untreated poly(allylamine). By using poly(allylamine) at 10% modification, the mechanical strength was improved only slightly, with 26% of capsules fractured. Analysis of the permeability test indicated that the photo-crosslinked capsular membrane was freely permeable to cytochrome c and myoglobin, but less permeable to serum albumin. The encapsulation method was used to entrap and culture IW32 mouse leukemia cells. The cells proliferated to a density of about 1.1 x 10(7) cells/mL in the capsules after 7 days of cultivation. Concurrently, the concentration of erythropoietin in the microcapsules increased to 800 mU/mL. This new encapsulation technique has great potential in the application of a bioindustrial cell-culturing process.     

Proliferation,morphology, and pluripotency of mouse induced pluripotent stem cells in three different types of alginate beads for mass production

Induced pluripotent stem cells (iPSCs) are expected to be an ideal cell source for biomedical applications, but such applications usually require a large number of cells. Suspension culture of iPSC aggregates can offer high cell yields but sometimes results in excess aggregation or cell death by shear stress. Hydrogel‐based microencapsulation can solve such problems observed in Suspension culture, but there is no systematic evaluation of the possible capsule formulations. In addition, their biological effects on entrapped cells are still poorly studied so far. We, therefore, immobilized mouse iPSCs in three different types of calcium–alginate (Alg–Ca) hydrogel‐based microcapsules; (i) Alg–Ca capsules without further treatment (Naked), (ii) Alg–Ca capsules with poly‐l ‐lysine (PLL) coating (Coated), and (iii) Alg–PLL membrane capsules with liquid cores (Hollow). After 10 days of culture within the medium containing serum and leukemia inhibitory factor, we obtained good cellular expansions (10–13‐fold) in Coated and Hollow capsules that were similar to Suspension culture. However, 32 ± 9% of cellular leakage and lower cell yield (about threefold) were observed in Naked capsules. This was not observed in Coated and Hollow capsules. In addition, immunostaining and quantitative RT‐PCR showed that the formation of primitive endodermal layers was suppressed in Coated capsules contrary to all other formulations. This agenesis of primitive endoderm layers in Coated capsules is likely to be the main cause of the significantly better pluripotency maintenance in hydrogel‐based encapsulation culture. These results are helpful in further optimizing hydrogel‐based iPSC culture, which can maintain better local cellular environments and be compatible with mass culture. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:896–904, 2014     

Production and characterization of liquid-core capsules made from cross-linked acrylamide copolymers for biotechnological applications

A novel chemistry has been developed for the production of capsules composed of a hydrophobic liquid core surrounded by a cross-linked polyacrylamide/alginate membrane. These liquid-core capsules may be used in capsular perstraction for the removal of inhibitory products from bioprocesses and bioconversions. They have the advantage of having a high surface area to promote rapid mass transfer, while separation of the organic core phase from the aqueous environment by the capsule membrane prevents the formation of stable emulsions and potential problems associated with toxicity of the organic phase for microbial cells or enzymes. Monodisperse spherical liquid-core capsules of between 800 microm and 1.6 mm diameter, with high mechanical resistance, have been prepared by co-extrusion, using the jet break-up technique. Capsules produced from a solution of MBA/total monomer (5%) were found to be more elastic and have a higher burst force when exposed to chelating agents such as phosphate or citrate. The mechanical resistance was unaffected by buffer solutions in the pH range 4-9 and after sterilization at 121 degrees C for 20 min. Capsules having membranes composed of a copolymer of acrylamide and N-hydroxymethylacrylamide exhibited even higher mechanical stability toward chelating agents.     

大孔型NaCS-PDMDAAC微胶囊固定化细胞在摇床和鼓泡塔中的培养研究

NaCS-PDMDAAC生物微胶囊囊膜较为致密,影响胶囊内外物质的交换,从而影响胶囊内细胞的生长。利用淀粉酶对致孔剂淀粉的降解作用制备了一种大孔型的纤维素硫酸钠_聚二甲基二烯丙基氯化铵（NaCS-PDMDAAC）生物微胶囊,实验表明胶囊的孔径和通透性能都有了很大的提高。将酵母和大肠杆菌作为模型细胞包埋于胶囊中分别通过摇瓶和鼓泡塔半连续培养,在鼓泡塔中胶囊内细胞的密度要高于摇床,表明氧气的传递是胶囊内好氧细胞生长的限制因素,大孔胶囊由于囊膜孔径变大,氧气的传递更为快速,在鼓泡塔中大孔型胶囊内的最大细胞密度比常规胶囊要高出20%～110%。由于对氧气的需求量的不同,大肠杆菌菌浓提高的程度要高于酵母。     

The permeability of EUDRAGIT RL and HEMA-MMA microcapsules to glucose and inulin

Measurement of the rate of glucose diffusion from EUDGRAGIT RL and HEMA-MMA microcapsules coupled with a Thiele modulus/Biot number analysis of the glucose utilization rate suggests that pancreatic islets and CHO (Chinese hamster ovary) cells (at moderate to high cell densities) should not be adversely affected by the diffusion restrictions associated with these capsule membranes. The mass transfer coefficients for glucose at 20 degrees C were of the same order of magnitude for both capsules, based on release measurements: approximately 5 x 10(-6) cm/s for EUDRAGIT RL and approximately 2 x 10(-6) for HEMA-MMA. Inulin release from EUDRAGIT RL was slower than for glucose (mass transfer coefficient 14 +/- 4 x 10(-8) cm/s). The Thiele moduli were much less than 1, either for a single islet at the center of a capsule or CHO cells uniformly distributed throughout a capsule at 10(-6) cells/ mL, so that diffusion restrictions within the cells in EUDRAGIT RL or 800 mum HEMA-MMA capsules should be negligible. The ratio of external to internal diffusion resistance (Biot number) was less than 1, so that at most, only a small diffusion effect on glucose utilization should be expected (i.e., the overall effectiveness factors were greater than 0.8). These calculations were consistent with experimental observation of encapsulated islet behavior but not fully with CHO cell behavior. Permeability restricted cell viability and growth is potentially a major limitation of encapsulated cells; further analysis is warranted.     

Novel reactive perstraction system applied to the hydrolysis of penicillin G

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range of organic solvents were tested for use in in situ product recovery. Of these solvents dibutyl sebacate (DBS) was chosen due to the rapid extraction rate, the high logP and to facilitate capsule production. The extraction efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coefficients of 8 and 0.7 for PAA and penicillin G (PenG), respectively, thereby showing that PAA could be selectively extracted at pH 3.5 and 25 degrees C. Liquid-core capsules containing DBS were shown to efficiently remove PAA selectively and the PAA could be effectively back-extracted and the capsules re-used in a three-stage process resulting in high product separation. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concentration over sevenfold higher than in the aqueous phase. Higher extraction efficiencies could be obtained by varying the substrate concentration and number of capsules. The enzyme immobilized on capsules could be stored for over 4 months at pH 8 and 4 degrees C with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated.     

Progress technology in microencapsulation methods for cell therapy

Cell encapsulation in microcapsules allows the in situ delivery of secreted proteins to treat different pathological conditions. Spherical microcapsules offer optimal surface‐to‐volume ratio for protein and nutrient diffusion, and thus, cell viability. This technology permits cell survival along with protein secretion activity upon appropriate host stimuli without the deleterious effects of immunosuppressant drugs. Microcapsules can be classified in 3 categories: matrix‐core/shell microcapsules, liquid‐core/shell microcapsules, and cells‐core/shell microcapsules (or conformal coating). Many preparation techniques using natural or synthetic polymers as well as inorganic compounds have been reported. Matrix‐core/shell microcapsules in which cells are hydrogel‐embedded, exemplified by alginates capsule, is by far the most studied method. Numerous refinement of the technique have been proposed over the years such as better material characterization and purification, improvements in microbead generation methods, and new microbeads coating techniques. Other approaches, based on liquid‐core capsules showed improved protein production and increased cell survival. But aside those more traditional techniques, new techniques are emerging in response to shortcomings of existing methods. More recently, direct cell aggregate coating have been proposed to minimize membrane thickness and implants size. Microcapsule performances are largely dictated by the physicochemical properties of the materials and the preparation techniques employed. Despite numerous promising pre‐clinical results, at the present time each methods proposed need further improvements before reaching the clinical phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

CHO immobilization in alginate/poly-l-lysine microcapsules: an understanding of potential and limitations

Microencapsulation offers a unique potential for high cell density, high productivity mammalian cell cultures. However, for successful exploitation there is the need for microcapsules of defined size, properties and mechanical stability. Four types of alginate/poly-l-Lysine microcapsules, containing recombinant CHO cells, have been investigated: (a) 800 μm liquid core microcapsules, (b) 500 μm liquid core microcapsules, (c) 880 μm liquid core microcapsules with a double PLL membrane and (d) 740 μm semi-liquid core microcapsules. With encapsulated cells a reduced growth rate was observed, however this was accompanied by a 2–3 fold higher specific production rate of the recombinant protein. Interestingly, the maximal intracapsular cell concentration was only 8.7 × 10⁷ cell mL^-1, corresponding to a colonization of 20% of the microcapsule volume. The low level of colonization is unlikely to be due to diffusional limitations since reduction of microcapsule size had no effect. Measurement of cell leaching and mechanical properties showed that liquid core microcapsules are not suitable for continuous long-term cultures (>1 month). By contrast semi-liquid core microcapsules were stable over long periods with a constant level of cell colonization (ϕ = 3%). This indicates that the alginate in the core plays a predominant role in determining the level of microcapsule colonization. This was confirmed by experiments showing reduced growth rates of batch suspension cultures of CHO cells in medium containing dissolved alginate. Removal of this alginate would therefore be expected to increase microcapsule colonization.     

Capsular perstraction as a novel methodology for the recovery and purification of geldanamycin

The molecular complex “Heat shock protein 90” has become a novel target for anticancer drugs in recent times on account of its ability to perform as a chaperone toward proteins involved in cancer progression. The geldanamycin binds to this complex with high affinity and prevents it from performing correctly, which results in tumor destruction. The aim of this study was to investigate the feasibility of applying liquid‐core microcapsules as a novel technique (termed “capsular perstraction”), for the recovery and purification of geldanamycin from culture media. Results demonstrated how this procedure was capable of rapidly extracting >70% of geldanamycin from culture media using a liquid‐core volume to medium ratio of only 1%. Optimum conditions for removal, including agitation speed, microcapsule size, and membrane thickness were examined, and it was shown how the stagnant aqueous film around the microcapsules was the main resistance to mass transfer. A volumetric mass transfer coefficient of 5.66 × 10^?6 m/s was obtained for the highest agitation speed (400 rpm), which was considerable greater compared to the value of 0.88 × 10^?6 m/s achieved for the lowest speed of 100 rpm. Removal of geldanamycin from microcapsules was also examined to fully investigate the potential of such particles for in situ product recovery, and it was demonstrated how the methodology can be used as a simple mechanism for purifying the compound (>99%) through solvent extraction and crystallization. The results of this work demonstrate the novel use of capsular perstraction as a methodology for the recovery and purification of geldanamycin from culture environments. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

基于海藻酸钠羟基改性的MPEG原位共价修饰微胶囊及抗蛋白性能

目的:通过对海藻酸钠链段羟基位点改性制备甲氧基聚乙二醇(MPEG)原位共价修饰的海藻酸钠/壳聚糖(AC)微胶囊,在保证MPEG修饰微胶囊机械强度不受影响的基础上,有效提高表面MPEG修饰密度,实现兼具良好机械稳定性及抗蛋白性能的微胶囊制备方法。方法:利用溴化氰对海藻酸钠羟基进行活化并将末端氨基的点击化学linker(BAT)接枝在主链上进而制备MPEG原位共价修饰微囊A_(B(OH))CP_N,用球磨法表征微囊机械强度,用Ig G和Fgn为模型考察微囊表面抗蛋白吸附性能,以L929细胞在其二维模拟平板膜上的黏附情况作为衡量指标,考察MPEG修饰微胶囊表面细胞粘附情况,并最终通过体内移植考察MPEG修饰微囊的生物相容性。结果:基于海藻酸钠羟基位点的MPEG原位共价修饰微胶囊能够实现与常规条件制备的微胶囊接近的机械强度;同时与对照组相比Ig G吸附量降低87.4%,Fgn吸附量降低75.5%,实现了良好的抗蛋白吸附性能;二维模拟平板膜表面L929细胞粘附情况显著改善,细胞粘附数与对照组相比降低了76.9%;体内移植结果证明MPEG修饰微囊细胞粘附极少,微囊与纤维层分离明显。结论:基于海藻酸钠羟基位点的MPEG原位修饰能够实现兼具良好机械稳定性及抗蛋白吸附性能的微胶囊。     

Spontaneous loading of positively charged macromolecules into alginate-templated polyelectrolyte multilayer microcapsules

A simple and high-efficiency approach to loading macromolecules into microscale carriers is presented. Calcium-cross-linked alginate hydrogel microspheres were fabricated by an emulsification technique and then used as negatively charged templates to form polyelectrolyte multilayer coatings. A calcium ion chelator, EDTA, was used to free the Ca(2+)-cross-linked alginate hydrogel within {poly(allylamine hydrochloride)/poly (styrene sulfonate)}(4) ({PAH/PSS}(4)) coating, allowing partial release of alginate. The retention of alginate in {PAH/PSS}(4) microcapsule was confirmed by FTIR spectroscopy and confocal microscopy. Real-time confocal microscopy was used to investigate the loading process of positively charged macromolecules (dextran-amino, and peroxidase) into alginate-templated microcapsules, which showed the loading occurred in <2 min for dextran-amino and <10 min for peroxidase, respectively. A high loading efficiency of 25 mug peroxidase in approximately 1.0 x 10(7) microcapsules (2.5 pg POx/capsule) was achieved with a low concentration of peroxidase loading solution (10 mug/mL). This spontaneous loading technique for encapsulating positively charged molecules in alginate-templated polyelectrolyte microcapsules shows strong potential for biosensor and drug delivery applications.     

Towards a fully synthetic substitute of alginate: optimization of a thermal gelation/chemical cross-linking scheme ("tandem" gelation) for the production of beads and liquid-core capsules

Fully synthetic polymers were used for the preparation of hydrogel beads and capsules, in a processing scheme that, originally designed for calcium alginate, was adapted to a "tandem" process, that is the combination a physical gelation with a chemical cross-linking.The polymers feature a Tetronic backbone (tetra armed Pluronics), which exhibits a reverse thermal gelation in water solutions within a physiological range of temperatures and pHs. The polymers bear terminal reactive groups that allow for a mild, but effective chemical cross-linking. Given an appropriate temperature jump, the thermal gelation provides a hardening kinetics similar to that of alginate. With slower kinetics, the chemical cross-linking then develops an irreversible and elastic gel structure, and determines its transport properties. In the present article this process has been optimized for the production of monodisperse, high elastic, hydrogel microbeads, and liquid-core microcapsules. We also show the feasibility of the use of liquid-core microcapsules in cell encapsulation. In preliminary experiments, CHO cells have been successfully encapsulated preserving their viability during the process and after incubation. The advantages of this process are mainly in the use of synthetic polymers, which provide great flexibility in the molecular design. This, in principle, allows for a precise tailoring of mechanical and transport properties and of bioactivity of the hydrogels, and also for a precise control in material purification.     

Alginate concentration: a key factor in growth of temperature-sensitive baculovirus-infected insect cells in microcapsules

The desire to increase cell density and product concentration has been the primary driving force for the development of better animal cell culture processes. In the technique used in our laboratory-microencapsulation-insect cells (Spodoptera frugiperda), infected with a temperature-sensitive mutant of the Autographa californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate-polylysine (PLL) microcapsules which had a controlled membrane molecular-weight cutoff and an intracapsular alginate concentration which was ca. 16% lower than that obtained in the commercially available single-membrane system. Cell culture experiments indicated that the intracapsular alginate concentration appears to be a key factor in achieving good cell growth. It was possible to obtain intracapsular cell densities of 8 x 10(7) cells/mL capsules and virus concentrations to 10(9) IFU/mL capsules. The virus litre in the supernatant was ca. 300 times lower, indicating that virtually all of the virus was retained within the capsules.     

Extraction of volatile fatty acids from diluted aqueous solution using a supported liquid membrane

Experimental and analytical studies on the extraction of volatile fatty acids (VFAs) from an aqueous solution using a supported liquid membrane (SLM) were carried out. Teflon and 20% (w/w) tri-n-octyl phosphine oxide (TOPO) in kerosene were used as the supporting membrane and liquid, respectively. The extraction rate of VFAs transferred from the source across the SLM to the sink was measured. It was observed that only undissociated forms of VFAs can penetrate into the SLM and that the complex formation between TOPO and VFA (1/1 molar ratio) inside the membrane enhanced the transfer rate of VFA across the membrane. This phenomenon was explained by mathematical models based on mass transfer and the chemical reactions occurring inside the membrane, suggesting that diffusion of the VFA-TOPO complex inside the membrane may be the rate-limiting step in this experiment.     

Characterization of alginate lyase activity on liquid, gelled, and complexed states of alginate

A study of alginate lyase was carried out to determine if this enzyme could be used to remove alginate present in the core of alginate/poly-L-lysine (AG/PLL) microcapsules in order to maximize cell growth and colonization. A complete kinetic study was undertaken, which indicated an optimal activity of the enzyme at pH 7-8, 50 degrees C, in the presence of Ca2+. The buffer, not the ionic strength, influenced the alginate degradation rate. Alginate lyase was also shown to be active on gelled forms of alginate, as well as on the AG/PLL complex constituting the membrane of microcapsules. Batch cultures of CHO cells in the presence of alginate showed a decrease of the growth rate by a factor of 2, although the main metabolic flux rates were not modified. The addition of alginate lyase to cell culture medium increased the doubling time 5-7-fold and decreased the protein production rate, although cell viability was not affected. The addition of enzyme to medium containing alginate did not improve growth conditions. This suggests that alginate lyase is probably not suitable for hydrolysis of microcapsules in the presence of cells, in order to achieve high cell density and high productivity. However, the high activity may be useful for releasing cells from alginate beads or AG/PLL microcapsules.     

Competing two enzymatic reactions realizing one‐step preparation of cell‐enclosing duplex microcapsules

The usefulness of cell‐enclosing microcapsules in biomedical and biopharmaceutical fields is widely recognized. In this study, we developed a method enabling the preparation of microcapsules with a liquid core in one step using two enzymatic reactions, both of which consume H₂O₂ competitively. The microcapsule membrane prepared in this study is composed of the hydrogel obtained from an alginate derivative possessing phenolic hydroxyl moieties (Alg‐Ph). The cell‐enclosing microcapsules with a hollow core were obtained by extruding an aqueous solution of Alg‐Ph containing horseradish peroxidase (HRP), catalase, and cells into a co‐flowing stream of liquid paraffin containing H₂O₂. Formation of the microcapsule membrane progressed from the surface of the droplets through HRP‐catalyzed cross‐linking of Ph moieties by consuming H₂O₂ supplied from the ambient liquid paraffin. A hollow core structure was induced by catalase‐catalyzed decomposition of H₂O₂ resulting in the center region being at an insufficient level of H₂O₂. The viability of HeLa cells was 93.1% immediately after encapsulation in the microcapsules with about 250 µm diameter obtained from an aqueous solution of 2.5% (w/v) Alg‐Ph, 100 units mL^?1 HRP, 9.1 × 10⁴ units mL^?1 catalase. The enclosed cells grew much faster than those in the microparticles with a solid core. In addition, the thickness of microcapsule membrane could be controlled by changing the concentrations of HRP and catalase in the range of 13–48 µm. The proposed method could be versatile for preparing the microcapsules from the other polymer derivatives of carboxymetylcellulose and gelatin. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1528–1534, 2013     

Superior Cell Delivery Features of Genipin Crosslinked Polymeric Microcapsules: Preparation,In Vitro Characterization and Pro-Angiogenic Applications Using Human Adipose Stem Cells

The ability of mesenchymal stem cells to self-renew and differentiate into specialized cell lineages makes them promising tools for regenerative medicine. Local injection and use of scaffolds had been employed earlier to deliver these cells; yet, an optimal delivery system remains to be identified. Here, using genipin, which is a non-toxic natural cross linker for proteins, we prepared alginate–chitosan polymeric microcapsules (GCAC) to develop an efficient stem cell delivery system. We investigated the properties of this membrane along with the encapsulated adipose tissue-derived stem cells (ASCs) and compared that with the widely used alginate poly-lysine (APA) membranes. The GCAC membrane was able to support cell viability, augment cell growth, and showed better results under external rotational and osmotic pressures with about 30% of the ruptured capsules in comparison to 60% ruptured APA capsules. The membrane also provided immune-protection to the entrapped cells as demonstrated by the lymphocyte proliferation assay. The capsule also has potential for long-term storage. The encapsulated four million ASCs also showed steady secretion of approximately 4600 pg vascular endothelial growth factor (VEGF) over 15-day time period comparable to that of free cells. Furthermore, the encapsulated ASCs showed around 3.8-fold increase in VEGF secretion after 72 h hypoxic conditions in comparison to normoxic conditions. This increased VEGF expression resulted in improved angiogenic potential of the bioactive capsules as noted by enhanced endothelial cell growth. GCAC encapsulation also did not show any effect on their differentiation ability. Thus, because of these biocompatible and bioactive attributes, genipin cross-linked polymeric microcapsules can emerge as a potentially important tool for improved stem cell-based therapy and cell delivery applications.     

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.     

Performance of an external loop air-lift bioreactor for the production of monoclonal antibodies by immobilized hybridoma cells

Summary The performance of an external loop air-lift bioreactor was investigated by assessing the inter-relationships between various hydrodynamic properties and mass transfer. The feasibility of using this bioreactor for the production of monoclonal antibodies by mouse hybridoma cells immobilized in calcium alginate gel beads and alginate/poly-l-lysine microcapsules was also examined. When the superficial gas velocity, V _g , in the 300 ml reactor was varied from 2 to 36 cm/min, the average liquid velocity increased from 3 to 14 cm/sec, the gas hold-up rose from 0.2 to 3.0%, and the oxygen mass transfer coefficient, k _L a, increased from 2.5 to 18.1 h^-1. A minimum liquid velocity of 4 cm/s was required to maintain alginate gel beads (1000 m diameter, occupying 3% of reactor volume) in suspension. Batch culture of hybridoma cells immobilized in alginate beads followed logarithmic growth, reaching a concentration of 4×10⁷ cells/ml beads after 11 days. Significant antibody production did not occur until day 9 into the culture, reaching a value of 100 g/ml of medium at day 11. On the other hand, bioreactor studies with encapsulated hybridoma cells gave monoclonal antibody concentrations of up to 800 g/ml capsules (the antibody being retained within the semipermeable capsule) and maximum cell densities of 2×10⁸ cells/ml capsule at day 11. The volumetric productivities of the alginate gel immobilized cell system and the encapsulated cell system were 9 and 3 g antibody per ml of reactor volume per day, respectively. The main advantage of the bioreactor system is its simple design, since no mechanical input is required to vary the hydrodynamic properties.     

A novel cytomedical vehicle capable of protecting cells against complement

We have developed "Cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that APA microcapsules could protect the entrapped cells from injury by cellular immune system. However, microencapsulated cells were not protected from humoral immune system. Here, we developed a novel APA microcapsule, in which APA microbeads (APA(Ba) microbeads) were modified to contain a barium alginate hydrogel within their centers in an attempt to make it more difficult for antibody and complement to permeate the microcapsules. The permeability of APA(Ba) microbeads was clearly less than that of APA microcapsules, presumably due to the presence of barium alginate hydrogel. Cells encapsulated within APA(Ba) microbeads were protected against treatment with xenogeneic anti-serum. Furthermore, murine pancreatic beta-cells encapsulated in APA(Ba) microbeads remained viable and continued to secrete insulin in response to glucose. Therefore, APA(Ba) microbeads may be a useful carrier for developing anti-complement device for cytomedical therapy.