Metabolic activity of CHO fibroblasts in HEMA-MMA microcapsules

Chinese hamster ovary (CHO) fibroblast cells were microencapsulated in polyacrylate membranes (HEMA-MMA: 75% HEMA) via an interfacial precipitation process. The CHO cells were observed to grow in large aggregates, attached to each other instead of to the capsule wall. When CHO cells were encapsulated at high density (4 x 10(6) cells/mL), the initial metabolic activity in microcapsules, as determined by the MTT assay, correlated with the polymer-cell extrusion ratio, presumably because of the dependence of encapsulation efficiency on the relative flow rates. However, there was a large variation in the metabolic activity among individual microcapsules throughout the present study. Capsules with low encapsulation efficiency (at a "seeding" density of 4 x 10(6) cells/mL) exhibited a rapid increase in the metabolic activity during the following week. When CHO cells were encapsulated at low density (4 x 10(5) cells/mL), there was only a small increase in the metabolic activity. Only a small fraction ( approximately 5%) of the capsules exhibited a high level of metabolic activity and 40% of the capsules exhibited undetectable metabolic activity even after 2 weeks. We conclude that CHO cells, which served as model cells, survive the encapsulation process and retain an active metabolic state once enclosed by the HEMA-MMA membranes. However, the resultant microcapsules are extremely heterogeneous in the amount of retained metabolic activity.     

Microencapsulation of human fibroblasts in a water-insoluble polyacrylate

Viable human diploid fibroblasts have been micro-encapsulated in EUDRAGIT RL, a commercially available water-insoluble polyacrylate, by an interfacial precipitation technique. Cells in medium and polymer solution (in diethyl phthalate) were coextruded and formed into droplets by a coaxial air stream. The droplets fell into a corn-oil/mineral-oil mixture to extract the solvent to precipitate the polymer around the cells. Capsules were ca. 500 mum in diameter depending on the air flowrate with a ca. 10-mum thick wall. When collagen (1 mg/mL) was added to the cell suspension prior to encapsulation and base-washed corn oil was used, cell growth occurred with one doubling achieved after five to six days as the collagen gel contracted inside the capsule. In the absence of collagen, cells spread on the inner wall of the capsule but did not grow, presumably because the surface charge on the capsule was inadequate. In similar fashion fibroblasts spread but did not grow on films of EUDRAGIT RL but did grow on blends of EUDRAGIT RL and EUDRAGIT E containing 10-30% of the latter more highly aminated polyacrylate. Although not suitable for anchorage-dependent cell growth by itself, EUDRAGIT RL has been suitable as a model polymer to demonstrate the feasibility of using water insoluble polyacrylates and organic solvents and nonsolvents for the micro-encapsulation of fibroblasts. Such microcapsules are of potential interest as a mode of large scale tissue culture for the production of novel therapeutic agents.     

Viability and protein secretion from human Hepatoma (HepG2) cells encapsulated in 400-mum polyacrylate microcapsules by submerged nozzle-liquid jet extrusion

An interfacial precipitation process to encapsulate mammalian cells in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) microcapsules of approximately 750 in approximately m diameter was previously described. It was not possible to produce smaller capsules due to low shearing force. A new droplet generation scheme was developed by suspending the cell and polymer co-extrusion nozzle in a uniform co-axial fluid jet which enabled the production of 300 to 600-microm diameter capsules. HepG2 hepatoma cells in 400-microm-diameter HEMA-MMA capsules were able to retain their metabolic activity during and after the encapsulation process. The in vitro secretion of plasma proteins alpha(1)-acid glycoprotein, alpha(1)-antitrypsin, and fibrinogen by the encapsulated cells was retained. The encapsulated cells secreted less fibrinogen (340 kD) relative to alpha(1)-acid glycoprotein (42kD), indicating the sieving effect (but not absolute cut-off) of the HEMA-MMA membrane. (c) 1994 John Wiley & Sons, Inc.     

Calculations on O2 transfer in capsules with animal cells for the determination of maximum capsule size without O2 limitation

The size of the capsules without O2 limitation and maximum allowable capsule size for different cell densities were calculated by the observable modulus (modified Thiele modulus). When hybridoma (ATCC HB-8852) at 2.5 3 × 105cells/ ml was encapsulated and grown, the cell density reached to 2.6 × 107cells/ml in the 7th day of incubation. The average diameter of the capsule was 2.1mm. The maximum cell density obtained from experiment agreed with the calculated cell density for the given size of the capsules. © Rapid Science Ltd. 1998     

Microencapsulation of mammalian cells in a water-insoluble polyacrylate by coextrustion and interfacial precipitation

A process for the microencapsulation of mammalian cells in a commercially available water-insoluble polyacrylate (EUDRAGIT RL) is described, and the effects of process parameters are outlined The polymer dissolved in diethyl phthalate was pumped along the annulus formed from two concentric needles, while the cell suspension was pumped inside the inner needle Droplets of polymer solution containing cells were blown off the end of the needles by a coaxial air stream. The droplets fell into a corn oil-mineral oil curing bath, in which the solvent was removed from the nascent capsule causing the polymer to precipitate around the cell suspension core. Capsules were washed free of oils and solvent in a fractionated plasma that allowed for quantitative transfer of capsules from the oil phase to an aqueous medium. By appropriate adjustment of the coaxial air flow rate, capsule size could be varied from 250-1000 mum, although the most convenient size was found to be 400-700 mum. Adding Ficoll 400 to the cell suspension to match the density of the suspension to the polymer solution resulted in capsules with a well-centered core but did not affect capsule strength. It appeared that increasing the polymer solution concentration or the polymer to the cell flow rate ratio resulted in an increased capsule strength, although differences in capsule size made unequivocal conclusions difficult. These capsules are of potential use as an artificial pancreas for the treatment of diabetes (with pancreatic islets) or for large-scale tissue culture and the production of bioactive products (e.g., with fibroblasts).     

Ethanol production from glucose and dilute-acid hydrolyzates by encapsulated S. cerevisiae

The performance of encapsulated Saccharomyces cerevisiae CBS 8066 in anaerobic cultivation of glucose, in the presence and absence of furfural as well as in dilute-acid hydrolyzates, was investigated. The cultivation of encapsulated cells in 10 sequential batches in synthetic media resulted in linear increase of biomass up to 106 g/L of capsule volume, while the ethanol productivity remained constant at 5.15 (+/-0.17) g/L x h (for batches 6-10). The cells had average ethanol and glycerol yields of 0.464 and 0.056 g/g in these 10 batches. Addition of 5 g/L furfural decreased the ethanol productivity to a value of 1.31 (+/-0.10) g/L x h with the encapsulated cells, but it was stable in this range for five consecutive batches. On the other hand, the furfural decreased the ethanol yield to 0.41-0.42 g/g and increased the yield of acetic acid drastically up to 0.068 g/g. No significant lag phase was observed in any of these experiments. The encapsulated cells were also used to cultivate two different types of dilute-acid hydrolyzates. While the free cells were not able to ferment the hydrolyzates within at least 24 h, the encapsulated yeast successfully converted glucose and mannose in both of the hydrolyzates in less than 10 h with no significant lag phase. However, since the hydrolyzates were too toxic, the encapsulated cells lost their activity gradually in sequential batches.     

Capsule walls as barriers to oxygen availability: Implications for the development of brooded embryos by the estuarine gastropod Crepipatella dilatata (Calyptraeidae)

Encapsulation of developing embryos imposes potential restrictions, because the capsule wall must allow for adequate inward diffusion of oxygen and for increased diffusion of oxygen as metabolic demand increases with continued development. Samples of egg capsules from the gastropod Crepipatella dilatata were used to document surface characteristics, composition of the different capsule wall layers, and alterations in wall thickness during development. The diffusion coefficient and capsule wall permeability were determined experimentally for capsules containing embryos at different developmental stages. We also determined oxygen consumption rates for various embryonic stages and for nurse eggs, which provide food for embryos during development. The capsule wall of C. dilatata possesses 2 differentiated layers: the external capsular wall (ECW) and the internal capsular wall (ICW). The ECW is compact and fibrous, features that remain invariable during development, and lacks surface features that might make some portions of the capsule wall more permeable to oxygen than others. On the other hand, the ICW is initially spongy and thick, but significantly decreases in thickness over time, particularly before the embryos begin feeding on nurse eggs. Although the capsule wall is a serious barrier to diffusion, permeability to oxygen increases over time by 112% due to the dramatic thinning of the inner capsule wall layer. Nurse eggs consume oxygen but at very low rates, supporting the idea that they correspond to living embryonic cells that have stopped their development. Respiration measurements indicated that embryos are initially supplied with enough oxygen within the egg capsules to carry out the activities characteristic of embryogenesis, even though the capsular walls show their maximum thickness and lowest permeability at this time. However, as the embryo develops its velum and becomes more active, capsule wall thickness decreases and capsule permeability to oxygen increases. Correspondingly, the oxygen demands of metamorphosed but still encapsulated specimens are approximately 135% higher than those of pre-metamorphosed sibling embryos.     

Release studies of Lactobacillus casei strain Shirota from chitosan-coated alginate-starch microcapsules in ex vivo porcine gastrointestinal contents

AIMS: To investigate the release characteristics of Lactobacillus casei strain Shirota (LCS) from chitosan-coated alginate-starch (CCAS) capsule in different regions of ex vivo porcine gastrointestinal (GI) contents. METHODS AND RESULTS: A 0.1 g of CCAS encapsulated bacteria (containing c. 10(8) CFU of LCS) were incubated in different sections of ex vivo porcine GI contents (10 ml) anaerobically at 37 degrees C. Samples were taken from different GI contents at different time intervals (up to 24 h) and estimated for the release of LCS from capsules. There was almost a complete release (1.1 x 10(8) CFU) of LCS from capsules within 8 h of incubation in ileal contents, while it took nearly 12 h to release the completely encapsulated bacteria in colon content under similar conditions. There was only a partial release of encapsulated LCS, incubated in duodenal and jejunal contents, while there was no significant (P > 0.05) release of encapsulated bacteria in gastric contents even after 24 h of incubation. CONCLUSIONS: The capsules were able to release viable probiotic cells completely in ex vivo porcine ileal and colon contents. SIGNIFICANCE AND IMPACT OF THE STUDY: CCAS capsule can be an effective way of protecting probiotic bacteria from adverse gastric conditions and delivering viable bacteria to the host intestinal systems.     

Encapsulation of mammalian cell with chitosan-CMC capsule

Viable hybridoma cells were encapsulated. The capsules were formed in one step by placing a drop of cell suspension mixed with negatively charged carboxymethylcellulose (CMC) into a positively charged chitosan solution through the interpolymeric ionic interaction between two oppositely charged polymers. These capsules were found to have a mean diameter of about 1. 5 mm and wall thickness of 3 mum. The cells grew in the capsules using supplemented DMEM/F12 (four kinds of growth factor). The maximum cell density in encapsulating cell culture reached 1 x 10(7) cells/ml, 10 times higher than that obtained in the free cell culture. The maximum monoclonal antibody concentration in the free cell culture was 15mug/mL, but that in the capsule was 45mug/mL The antibody produced by the cell was concentrated about four times higher inside than outside of the capsules.     

Gelation conditions and transport properties of hollow calcium alginate capsules

The diameter, membrane thickness, and compression intensity of hollow Ca-alginate capsules were measured at different gelation conditions, such as the reactant concentration, dropping velocity, and gelation time. The optimum operation conditions for preparing capsules were determined at 100 g/L CaCl(2), 10 g/L sodium alginate (Na-alginate), a dropping velocity of 150 droplets/min, and a gelation time of 10 min. Diffusion of some saccharide and amino acid from bulk solution into capsules was investigated, and the diffusion coefficients were calculated by the developed mathematical model. All the tested substances can diffuse easily into the capsules. The combined diffusion coefficients of the capsule D(m) are 92-99% as large as their diffusion coefficients in pure water, while the diffusion coefficients in the capsule membrane D(1) are 60-95% as large as those. By employing polyethylene glycol (PEG) and bovine serum albumin (fraction V) (BSA(V)), the molecular weight cut-off of the capsule was determined. For linear macromolecules, hollow Ca-alginate capsules have a molecular weight cut-off of 4000. No diffusion of BSA(V) into the capsules was observed.     

Metabolic response to carbohydrate ingestion during exercise in males and females

The present study investigated potential sex-related differences in the metabolic response to carbohydrate (CHO) ingestion during exercise. Moderately endurance-trained men and women (n = 8 for each sex) performed 2 h of cycling at approximately 67% Vo(2 max) with water (WAT) or CHO ingestion (1.5 g of glucose/min). Substrate oxidation and kinetics were quantified during exercise using indirect calorimetry and stable isotope techniques ([(13)C]glucose ingestion, [6,6-(2)H(2)]glucose, and [(2)H(5)]glycerol infusion). In both sexes, CHO ingestion significantly increased the rates of appearance (R(a)) and disappearance (R(d)) of glucose during exercise compared with WAT ingestion [males: WAT, approximately 28-29 micromol x kg lean body mass (LBM)(-1) x min(-1); CHO, approximately 53 micromol x kg LBM(-1) x min(-1); females: WAT, approximately 28-29 micromol x kg LBM(-1) x min(-1); CHO, approximately 61 micromol x kg LBM(-1) x min(-1); main effect of trial, P < 0.05]. The contribution of plasma glucose oxidation to the energy yield was significantly increased with CHO ingestion in both sexes (from approximately 10% to approximately 20% of energy expenditure; main effect of trial, P < 0.05). Liver-derived glucose oxidation was reduced, although the rate of muscle glycogen oxidation was unaffected with CHO ingestion (males: WAT, 108 +/- 12 micromol x kg LBM(-1) x min(-1); CHO, 108 +/- 11 micromol x kg LBM(-1) x min(-1); females: WAT, 89 +/- 10 micromol x kg LBM(-1) x min(-1); CHO, 93 +/- 11 micromol x kg LBM(-1) x min(-1)). CHO ingestion reduced fat oxidation and lipolytic rate (R(a) glycerol) to a similar extent in both sexes. Finally, ingested CHO was oxidized at similar rates in men and women during exercise (peak rates of 0.70 +/- 0.08 and 0.65 +/- 0.06 g/min, respectively). The present investigation suggests that the metabolic response to CHO ingestion during exercise is largely similar in men and women.     

Technology and applications for encapsulated spermatozoa

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus, a potential use in artificial insemination (AI). Membranes made of the following polymers have proven best: poly-l-lysine, polyarginine, polyvinylamine, and protamine sulfate. Successful encapsulation has been achieved for capsules ranging in size from 0.75 to 1.5 mm, and sperm concentrations from 45 to 180 x 10(6) cells/ml. Successful buffers include Cornell University Extender and egg yolk citrate - glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under aging and physical stress) was negatively related to membrane thickness which ranges from 1.92 microm to 5.32 microm (controlled by polymer concentration, molecular weight, and exposure time) and positively to concentration of sperm encapsulated. On delivery to the porcine reproductive tract, the capsule constructed of poly-l-lysine membranes ruptured between 12 and 24 hr after insemination. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when AI is at conventional times following detection of estrus.     

乳杆菌微胶囊化培养的研究

采用MaCSPDMDAAC微胶囊对两种乳杆菌进行了固定化发酵研究。结果表明,两种乳杆菌能够在微胶囊内很好地生长和繁殖,菌浓度可分别达到18×1011/mL 胶囊和269×1011/mL胶囊,比游离培养高出十倍以上,并且能与游离发酵一样产生乳酸,糖耗时间可缩短1/3～2/3。进行的多批次发酵显示了巨大的产酸能力。     

Encapsulated transgene cells attenuate hypertension, cardiac hypertrophy and enhance renal function in Goldblatt hypertensive rats

BACKGROUND: The success of any gene-therapy approach depends on the survival of the genetically engineered cells that are implanted in the patient to deliver the therapeutic product. Immunoisolation of nonautologous cells within a microcapsule is a unique approach for gene therapy. METHODS: We employed an immunoisolation device that protects nonautologous cells from destruction, to implant human atrial natriuretic peptide (hANP)-producing Chinese hamster ovary (CHO) cells in two-kidney, one-clip (2K1C) hypertensive rats. CHO cells transfected with the plasmid CMV-cANP were encapsulated in biocompatible polycaprolactone (PCL) capsules, and then the PCL capsules were implanted into 2K1C hypertensive rats intraperitoneally. RESULTS: The implantation of encapsulated hANP-producing cells caused a significant delay of blood pressure (BP) increase 2 weeks post-implantation and the effect lasted for more than 5 months. The implantation of encapsulated hANP-producing cells also caused significant increases in renal blood flow (RBF), glomerular filtration rate (GFR), sodium output, urine excretion, and urinary cGMP levels. These beneficial effects were reflected morphologically by an attenuation of the glomerular sclerotic lesions, reduction in cardiomyocyte size, tubular injury and renal arterial thickening. Immunoreactive hANP can be detected in the blood of 2K1C rats after implantation of the PCL capsules containing hANP-producing cells. CONCLUSIONS: This study demonstrates the usefulness of encapsulated ANP gene transfected cells as a new tool for ANP gene delivery in studying renovascular hypertension and cardiovascular diseases. Thus, our results may have important implications for clinical use of transgene cells as therapeutic agents in the treatment of cardiovascular diseases.     

Low‐adhesive ethylene vinyl alcohol–based packaging to xenogeneic islet encapsulation for type 1 diabetes treatment

Transplantation of encapsulated porcine islets is proposed to treat type 1 diabetes. However, the envelopment of fibrous tissue and the infiltration of immune cells impair islet function and eventually cause implant failure. It is known that hemodialysis using an ethylene vinyl alcohol (EVOH) membrane results in minor tissue responses. Therefore, we hypothesized that using a low‐adhesive EVOH membrane for encapsulation may prevent host cell accumulation and fibrous capsule formation. In this study, rat islets suspended in chitosan gel were encapsulated in bags made from highly porous EVOH membranes, and their in vitro insulin secretion function as well as in vivo performance was evaluated. The results showed that the EVOH bag did not affect islet survival or glucose‐stimulated insulin secretion. Whereas naked islets were dysfunctional after 7 days of culture in vitro, islets within the EVOH bag produced insulin continuously for 30 days. Streptozotocin‐induced diabetic mice were given islets–chitosan gel–EVOH implants intraperitoneally (650–800 islets equivalent) and exhibited lower blood glucose levels and regained body weight during a 4‐week observation period. The transplanted mice had higher levels of serum insulin and C‐peptide, with an improved blood glucose disappearance rate. Retrieved implants had minor tissue adhesion, and histology showed a limited number of mononuclear cells and fibroblasts surrounding the implants. No invasion of host cells into the EVOH bags was noticed, and the encapsulated islets were intact and positive for insulin–glucagon immunostaining. In conclusion, an EVOH bag can protect encapsulated islets, limit fibrous capsule formation, and extend graft function.     

Characterisation of small molecules diffusion in hydrogel-membrane liquid-core capsules

The overall diffusion coefficients for several low molecular weight solutes, such as glucose, fructose, sucrose, lactose, and vitamin B(12) have been determined in Ca-alginate membrane liquid-core capsules using the unsteady-state method following the release of solutes from the capsules to a well-stirred solution of limited volume. The diffusion coefficients obtained for saccharides were 5-20% lower than the corresponding diffusivity in water while for vitamin B(12) about 50% that of water. The diffusion coefficients of the investigated capsules were not influenced by the change in alginate concentration in the capsule membrane from 0.5 to 1.0%. Lower diffusivities and higher deviations from the diffusivity in water were obtained for higher molecular weight solutes.     

Locally controlled release of basic fibroblast growth factor from multilayered capsules

Biodegradable multilayered capsules encapsulating basic fibroblast growth factor (bFGF) were developed as a cytokine release carrier for drug delivery systems. The multilayered hollow capsules were fabricated via the layer-by-layer (LbL) assembly of chitosan (CT) and dextran sulfate (Dex). The bFGF was encapsulated into the CT/Dex multilayered capsules by controlling the membrane permeability, and the local and sustained release of bFGF from the capsules was examined. At pH < 8.0, the capsule membrane tightened, and FITC-dextran ( Mw = 4000) could not enter the capsules. However, FITC-dextran ( M w = 250000) easily entered the capsules at pH > 8.0, which can be attributed to the electrostatic repulsion of Dex caused by the deprotonation of the amine group in CT. After treatment with acetic acid buffer (pH 5.6), FITC-dextran or bFGF was successfully encapsulated into the capsules. The amount of encapsulated bFGF was approximately 34 microg/1 mg of capsule. Initially, about 30% of the encapsulated bFGF was released in serum-free medium within a few hours, however, the release was sustained over 70 h. When the bFGF encapsulating capsules were added to cell culture medium (serum-free), the mouse L929 fibroblast cells proliferated well for 2 weeks as compared to cultures, where bFGF was added to the medium or where bFGF and empty hollow capsules were added separately. The proliferation is due to the local and sustained release of bFGF from the adsorbent capsule to the cell surface.     

Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase

Stable polyelectrolyte capsules were produced by the layer-by-layer (LbL) assembling of biodegradable polyelectrolytes, dextran sulfate and protamine, on melamine formaldehyde (MF) microcores followed by the cores decomposition at low pH. The mean diameter of the capsules at pH 3-5 was 8.0 +/- 0.2 microm, which is more than that diameter of the templates (5.12 +/- 0.15 microm). With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 microm. The microcapsules were loaded with horseradish peroxidase. Seemingly, peroxidase is embedded in the gellike structure in the microcapsule interior formed by MF residues in the complex with polymers used for LbL coating as proved by Raman confocal spectroscopy. The amount of finally incorporated peroxidase increased from 0.2 x 10(8) to 2.2 x 10(8) peroxidase molecules per capsule with pH growing from 5 to 8. The pH shifts causing changes in capsule swelling and the replacement of solutions without pH shifts lead to the protein loss. The encapsulated peroxidase showed a high activity (57%), which remained stable for 12 months.     

Controlled aggregation of primary human pancreatic islet cells leads to glucose‐responsive pseudoislets comparable to native islets

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three‐dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose‐responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re‐associated human islet cells showed an a‐typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C‐peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.