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Development of intracytoplasmic lumens in a colon cancer cell line cultured on a non-adhesive surface.

Cell-matrix interactions have important effects on phenotypic features, such as morphology, differentiation and cell growth. Several papers have suggested that when cell-matrix interactions are interrupted, cells grow as multicellular spheroids and eventually undergo apoptosis. We found that when ET(-), a laminin non-adherent colon cancer cell line, was cultured on poly-2-hydroxyethyl methacrylate (HEMA) coated plastic, the cells floated as cellular aggregates of spheroids or as single cells. Some of the single cells contained a very large intracytoplasmic lumen (ICL) and appeared similar to signet ring cells. These ICL were lined by a layer of short microvilli. The number of the cell did not increased cells when cultured on poly-HEMA. Another type of single cells, usually without ICL, demonstrated the characteristics of apoptotic cells by histologic examination. Acridine orange staining, flow cytometry and electron microscopy confirmed the apoptotic nature of those cells. In immunohistochemical staining for proliferating cell nuclear antigen, spheroids of cells and single cells with ICL were immunoreactive, while most of the single cells without ICL were negative. These results suggest that multicellular aggregation and formation of ICL were induced by the adaptation of ET(-) colon cancer cells in a harmful environment caused by reduced adhesiveness, and these changes might be related to cell survival.

Acridine Orange↗

Plasma-induced graft copolymerization of HEMA onto silicone rubber and TPX film improving rabbit corneal epithelial cell attachment and growth.

A poly(2-hydroxyethyl methacrylate) (pHEMA)-grafted polymer film was prepared by plasma-induced graft copolymerization onto an elastic material, silicone rubber, and a plastic material, poly(4-methyl-1-pentene) (TPX). The control, Ar plasma-treated and pHEMA-grafted silicone rubber and TPX surfaces were characterized by ESCA, FTIR-ATR, SEM and contact angle techniques. ESCA verified the respective chemical shift of control and Ar plasma-treated films. The presence of the grafted pHEMA was also verified by ESCA. The introduction of pHEMA onto a hydrophobic support provided an adequate surface for rabbit corneal epithelium cell attachment and growth. Cell attachment and growth onto these surfaces were examined by light microscopy. Cell attachment onto the control and Ar plasma-treated surfaces was negligible, while improved attachment and growth of rabbit corneal epithelium cells was demonstrated on the pHEMA-grafted polymeric surface. At 72 h, the pHEMA-grafted silicone rubber surface attached and grew more cells as compared with those on a pHEMA-grafted TPX surface. The pHEMA-grafted silicone rubber surface demonstrated a confluent cell layer after 72 h.

Animals↗

Characterization of an in vitro model for evaluating the interface between skin and percutaneous biomaterials.

Percutaneous devices play an essential role in medicine; however, they are often associated with a significant risk of infection. One approach to circumvent infection would be to heal the wound around the devices by promoting skin cell attachment. We used two in vitro assay models to evaluate cutaneous response to poly(2-hydoxyethyl methacrylate) (poly(HEMA)). One approach was to use a cell adhesion assay to test the effects of surface modification of poly(HEMA), and the second used an organ culture system of newborn foreskin biopsies implanted with porous poly(HEMA) rods (20 microm pores) to evaluate the skin/poly(HEMA) interface. Surface modification of poly(HEMA) using 1,1'-carbonyldiimidazole (CDI) enhanced keratinocyte, fibroblast, and endothelial cell adhesion. Keratinocytes in the organ culture model not only remained functionally and structurally viable as observed by immunohistochemistry and electron microscopy, but migrated into the pores of CDI-modified poly(HEMA) rods. No biointegration was seen in the non-CDI-modified poly(HEMA). Laminin 5 immunostaining was seen along the poly(HEMA)/skin interface in a pattern resembling the junctional epithelium of the tooth, the unique natural interface between the skin and tooth that serves as a barrier to bacteria. In vitro systematic evaluation of biomaterials for use in animal implant studies is both cost effective and time efficient.

Aged↗

Microbial adhesion to ionogenic PHEMA, PU and PP implants.

This study concentrates on adhesion of microorganisms to polymeric biomaterials implanted in the urinary tract. A number of extensively used polymeric biomaterials (i.e., poly(hydroxyethyl-methacrylate) and its copolymers with a series of acrylic monomers--acrylic acid and dimethylaminoethyl methacrylate--polyetherurethane and polypropylene (PP)) was prepared by using suitable polymerization methods. Their surface structures were characterized by wettability and surface free energy measurements, scanning electron microscopy, electron spectroscopy for chemical analysis, and attenuated total reflection Fourier transform infrared spectroscopy techniques. It was found that these surfaces have different surface free energies in the range of 26.9-63.0 erg cm-2 with different charge and charge densities. The adherence of two strains of Escherichia coli (non-pathogen and pathogen) isolated from indigenous flora of trachea and pathogen flora of urethral surfaces, and uropathogenic Candida albicans to the polymeric surfaces was studied in vitro by means of microscopic techniques. The hydrophobicity of cell surfaces was determined by using two methods including adhesion to hydrocarbons and contact angle measurements. The uropathogenic E. coli and C. albicans were found to be relatively hydrophobic with a similar surface free energy, approximately 40 erg cm-2, when compared with normal E. coli strain which has 61 erg cm-2 free energy. The uropathogenic strains were poorly adherent to hydrophilic polymer surfaces while showing excellent adherence on hydrophobic PP surfaces. However, a relatively hydrophilic, nonpathogenic E. coli strain showed the opposite adhesion behaviour to the same surfaces.

Bacterial Adhesion↗

Performance of a hydrogel composite pericardial substitute after long-term implant studies.

A novel composite patch has been tested as a pericardial substitute to reduce adhesion formation after cardiac surgery. The patch consists of poly 2-hydroxyethyl methacrylate (pHEMA) hydrogel reinforced with a polyethylene terephthalate (PET) mesh. The hydrogel-PET composite pericardial patches were implanted in canines for 6, 9, and 12 months. Upon termination, adhesion formation and epicardial reaction to the implant were rated. No adhesions formed between the patch and the native pericardium or epicardium. A thin fibrous layer on the epicardium progressively developed where the patch contacted the heart. The coronary anatomy remained visible. Histologically, the response to the implant was fibrous in nature. No significant signs of cellular inflammation were found. The gross appearance of the retrieved patches was nearly identical to that of preimplant patches. Mechanical tests showed no significant changes (alpha = 0.05) in patch strength or stiffness. Hydrogel water content initially increased during implantation. The thickness of the patch did not change significantly (alpha = 0.05) throughout the study. Scanning electron microscopy (SEM) revealed unequal layers of hydrogel on either side of the PET mesh and cracks in the hydrogel surfaces of retrieved patches. Both SEM and light microscopic observation of the patches showed traces of calcification in patches in the 9 and 12 month studies.

Animals↗

Complement activation by hydroxyethylmethacrylate-ethylmethacrylate copolymers.

Certain biomaterials, including nylon oxygenator and cellulosic dialysis membranes, are potent activators of human complement. In this study, the effect of polymers containing 2-hydroxyethyl methacrylate (HEMA) on the human complement system was investigated. Copolymers of HEMA with ethylmethacrylate (EMA) varying from 100 to 40% HEMA in the monomer made by radiation initiation were used to coat glass discs. These were equilibrated with human plasma in vitro and the degree of complement activation was quantitated by C3a radio immunoassay. Significant activation was caused by copolymers made from monomers containing 60% or greater HEMA. A direct relationship between the amount of activation and the percentage of HEMA was found. The degree of activation by poly HEMA, when corrected for surface area, was quite similar to that observed for dialysis and oxygenator membranes. Similar observations were made when solid casts of crosslinked HEMA/N-vinylpyrrolidone (NVP) copolymer gels were tested, but the magnitude of activation was much greater. The results are significant because complement activation may play an important role in the response to foreign surfaces, in both extravascular and intravascular settings. A new concept of molecular biocompatibility is proposed in which surfaces eliciting molecular transformations in any of the biological defense systems are deemed nonbiocompatible. By this criterion, the hydrogel poly-HEMA, which has so frequently been thought of as biocompatible, is considered to be a molecularly non-biocompatible material.

Biocompatible Materials↗

Creating porous tubes by centrifugal forces for soft tissue application.

Chemically crosslinked poly(2-hydroxyethyl methacrylate) (PHEMA) tubes were synthesized by applying centrifugal forces to propagating polymer chains in solution. Initiated monomer solutions, with a composition typical for PHEMA sponges, were placed into a cylindrical mold that was rotated about its long axis. As polymerization proceeded, phase separated PHEMA formed a sediment at the periphery under centrifugal action. The solvent remained in the center of the mold while the PHEMA phase gelled, resulting in a tube. By controlling the rotational speed and the formulation chemistry (i.e., monomer, initiator and crosslinking agent concentrations), the tube dimensions and wall morphology were manipulated. Tube manufacture was limited by a critical casting concentration [M]c, above which only rods formed. All tubes had an outer diameter of 2.4 mm, reflecting the internal diameter of the mold and a wall thickness of approximately 40-400 microm. Wall morphologies varied from interconnecting polymer and water phases to a closed cell, gel-like, structure. Concentric tubes were successfully prepared by using formulations that enhanced phase separation over gelation/network formation. This was achieved by using formulations with lower concentrations of monomer and crosslinking agent and higher concentrations of initiator. This technique offers a new approach to the synthesis of polymeric tubes for use in soft tissue applications, such as nerve guidance channels.

Biocompatible Materials↗

Viability of HEMA-MMA microencapsulated model hepatoma cells in rats and the host response.

Small diameter hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA; 75% HEMA) microcapsules containing an aggregate of viable rat hepatoma H4IIEC3 cells, after implantation into an omental pouch in Wistar rats, contained viable cells at 7 days but not 14 days. A similar transplantation of microencapsulated aggregates of human hepatoma HepG2 cells did not result in viable cells even at 7 days. The loss of viability was attributed to the tissue reaction, because both encapsulated cell types remained viable in vitro. However, it is not clear if the cells lost their viability in vivo, leading to the aggressive tissue reaction or if the latter caused the cells to starve or otherwise die. The tissue reactions to microcapsules containing rat or human hepatoma cells at day 1 was one cell layer thick and avascular. At later times, tissue reactions were comprised of three regions: macrophages, fibroblasts, and some foreign body giant cells apposed to the polymer membrane, a dense region of fibroblasts and collagen, and a region of vascularized granulation tissue. Prompt vascularization of the tissue reactions occurred after 4 days and was maintained for up to 14 days. Even at 14 days, immune cells were observed, suggesting a continued immune response toward antigens shed from the encapsulated cells.

Animals↗

Platelet adherence and detachment with adsorbed fibrinogen: a flow study with a series of hydroxyethyl methacrylate-ethyl methacrylate copolymers using video microscopy.

The adhesion and detachment of platelets were studied on glass coatings of a series of copolymers of hydroxyethyl methacrylate (HEMA) and ethyl methacrylate (EMA) with preadsorbed fibrinogen. Observations of the interactions of acridine-orange-labeled washed platelets with these surfaces from a flowing (500 s-1 wall shear rate) suspension in Tyrode's solution containing albumin and red cells were made with epifluorescent video microscopy (EVM). In some cases preadsorbed materials were incubated for 24 h, during which little or no loss of protein occurred. Protein surface concentration, by itself, was a poor indicator of expected cell adhesion and morphology. Surface chemistry was a second important factor which must be considered. A third observation is that for the 100% EMA copolymer, 24 h of incubation led to a large reduction in platelet adhesion when compared to the 100% EMA material without incubation. For the 0% and 100% EMA polymers, the percentage of contacting platelets which adhere and detach is greater for the 24-h incubation cases than for those not incubated. These results led to the conclusion that our most hydrophilic surface favors adhesion with detachment, transient cell contact, over long-term adhesion, as does incubation of adsorbed protein. A brief discussion is presented of a possible connection between this behavior and platelet consumption in vivo for hydrogels.

Adsorption↗

Microencapsulated human hepatoma (HepG2) cells: in vitro growth and protein release.

The feasibility of a microencapsulation process ultimately for cell transplantation was investigated by encapsulating human hepatoma (HepG2) cells in hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) membranes through an interfacial precipitation process. Changes in viability and metabolic activity as well as protein secretion by the encapsulated cells were studied in vitro. When encapsulated at either low or high density (1 or 5 x 10(6) cells/mL, respectively), HepG2 cells retained their active metabolic state and/or proliferated during the initial 1-week period, after which a significant drop in cell viability was obtained. Encapsulation of a biological attachment substrate, Matrigel, along with the cells, however, resulted in rapid proliferation in both low and high density capsules with prolonged maintenance of an active metabolic state. The secretion of four model proteins (alpha 1-acid glycoprotein, alpha 1-antitrypsin, haptaglobin and fibrinogen) was demonstrated during the 2-week study period for the Matrigel encapsulated cells. Furthermore, the encapsulated cells remained responsive to interleukin 6 (IL6), a physiological stimulator of plasma protein secretion, as determined by the elevated secretion of haptaglobin in response to IL6 treatment. We conclude that HEMA-MMA capsules, in the presence of an attachment substrate, provide a suitable environment for the growth and expression of differentiated functions of encapsulated hepatoma cells.

Biocompatible Materials↗

Study on antithrombogenicity of poly[beta-(acetylsalicylyloxy)ethyl methacrylate] relative to poly(hydroxyethyl methacrylate).

The antithrombogenicity of a polymer made of aspirin bound to hydroxyethyl methacrylate (HEMA), abbreviated as ASA-polymer, was compared with that of poly(hydroxyethyl methacrylate) (PHEMA). Platelet from platelet rich plasma (PRP) incubated with ASA-polymer surface exhibited noticeable decreases in adhesion and aggregation as compared to platelets incubated with PHEMA. Low molecular weight components other than aspirin, which may be released from ASA-polymer during the incubation with PRP, or contact with ASA-polymer causing denaturation of platelets without morphological changes could be responsible for the decrease of adhesion and aggregation. Both PRP and PPP exposed to ASA-polymer-coated surfaces exhibited a much smaller partial thromboplastin time (PTT) than if exposed to PHEMA-coated surfaces; the PTT of ASA-polymer was similar to that of glass exposed plasma. With respect to the in vivo antithrombogenicity, the ASA-polymer surface led to thrombus formation. This may be due to the partial hydrolysis of the acetyl groups resulting in the formation of a negatively charged surface which in turn accelerates the coagulation cascade despite its inhibitory effects on platelet adhesion and aggregation. On the other hand, neointima formed around a thrombus layer on PHEMA-coated sutures after 14 days.

Animals↗

Poly(2-hydroxyethyl methacrylate) biomimetic coating to improve osseointegration of a PMMA/HA/glass composite implant: in vivo mechanical and histomorphometric assessments.

Bone implants must simultaneously satisfy many requirements, even though the surface properties remain a crucial aspect in osseointegration success. Since a single material with a uniform structure cannot satisfy all of these requirements, composite materials specifically designed for orthopedic or dental implant application should be envisaged. Two poly(methylmethacrylate)/hydroxyapatite composites reinforced by E-glass fibres, uncoated (PMMA/HA/Glass) and poly(2-hydroxyethyl methacrylate) (PMMA/HA/Glass+pHEMA) coated by the biomimetic method, were mechanically (push-out test) and histomorphometrically (Affinity Index, AI) investigated in an in vivo rabbit model. Cylindrical implants (diameter 2 mm x 5 mm length) were inserted into rabbit femoral cortical (mid-diaphysis) and cancellous (distal epiphysis) bone, under general anesthesia. The highest values of push-out force and ultimate shear strength were observed for the PMMA/HA/Glass at 12 weeks, which significantly (p < 0.001) differed from those of PMMA/HA/Glass+pHEMA at the same experimental time and from those of PMMA/HA/Glass at 4 weeks. At both experimental times, significantly (p < 0.0005) lower values of AI were observed in the PMMA/HA/Glass+pHEMA versus PMMA/HA/Glass (distal femoral epiphysis: 4 weeks = 33%; 12 weeks = 19%; femoral diaphysis: 4 weeks = 15%; 12 weeks = 11%). The good mechanical and histomorphometric results obtained with PMMA/HA/Glass should be followed by further evaluation of bone remodeling processes and mechanical strength around loaded PMMA/HA/Glass implants at longer experimental times. Finally, the biomimetic method applied to pHEMA needs to be further investigated in order to improve the positive effect of SBF on pHEMA and to enhance the coating adhesion.

Animals↗

Covalent immobilization of alpha-amylase onto pHEMA microspheres: preparation and application to fixed bed reactor.

Microspheres of poly(2-hydroxyethyl methacrylate) with and without cross-linker were prepared by suspension polymerization. As the amount of cross-linker increased, the equilibrium water content, enzyme loading, immobilization efficiency and recovered activity were all adversely affected. Enzyme alpha-amylase was immobilized onto the microspheres after activation with epichlorohydrin. The Km value for the immobilized enzyme (0.90% w/v) was much greater than that of the free enzyme (0.53% w/v). It was found that the inactivation constant (ki) increased from 2.23 x 10(-8) min-1 at 20 degrees C to 1.45 x 10(-4) min-1 at 60 degrees C. Since the enzyme activity increased as the temperature increased, the temperature profile yielded a peak at 50 degrees C. For free enzyme this is at 45 degrees C. The residence time was proportional to the percentage hydrolysis until a residence time of 12 min was reached. Beyond this the activity increase could not match the increase in residence time. The pH profile yielded a broadening upon immobilization in addition to a small shift to higher pH (from 5.5 to 6.0). The continuous run at 30 degrees C, 1.0% w/v starch concentration and flow rate of 40 cm3 h-1 led to only 20% loss in activity after a 120 h operation.

Biocompatible Materials↗

Pathogenic antibody removal using magnetically stabilized fluidized bed.

Magnetic poly(2-hydroxyethyl methacrylate) (mPHEMA) beads were used in the removal of anti-dsDNA antibodies from systemic lupus erythematosus (SLE) patient plasma in a magnetically stabilized fluidized bed. mPHEMA beads, in the size range of 80-120 microm, were produced by suspension technique. Then, DNA was immobilized onto mPHEMA beads by carbodiimide activation. Magnetic beads were contacted with blood in in vitro systems. Loss of blood cells and clotting times were followed. mPHEMA beads were characterized by scanning electron microscopy (SEM). Important results obtained in this study are as follows: the mPHEMA beads have a spherical shape and porous structure. Loss of cells in the blood contacting with mPHEMA/DNA was negligible. The anti-dsDNA adsorption capacity decreased significantly with the increase of the flow-rate. With increasing anti-dsDNA antibody concentration, the amount of antibody adsorbed per unit mass increased, then reached saturation. Maximum anti-dsDNA antibody adsorption capacity was found to be 97.8 mg/g. Pathogenic antibody molecules could be repeatedly adsorbed and desorbed with these magnetic beads without noticeable loss in their antibody adsorption capacity. Because of the good blood-compatibility, mPHEMA is hopeful for the treatment of SLE by magnetically stabilized fluidized bed systems in the future.

Antibodies, Antinuclear↗

Tissue interaction with hydrogel sponges implanted in the rabbit cornea.

We proposed poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel sponges as potential materials for the peripheral zone of a composite keratoprosthesis. It was previously shown that such sponges allowed cellular invasion when implanted s.c. in rabbits. To evaluate the reaction elicited by these materials in the corneal tissue, a PHEMA sponge, with pore size diameters of 10-30 microns, was produced, impregnated with collagen, and implanted in the rabbit cornea in both central and limbal regions of separate eyes. The eyes were examined by slit-lamp biomicroscopy up to 6 months postoperatively. Enucleation was performed at 3 weeks, 6 weeks, and 6 months, then excised implants were examined by light and transmission electron microscopy. Both clinical and histopathological examination indicated that sponges were well tolerated by the stromal and limbal tissues. No capsule was formed around any of the implants. Corneal fibroblasts and capillaries readily invaded the internal voids in the sponge to all depths. The invading cells remained viable and active up to the end of the follow-up period. Our findings suggest that hydrophilic PHEMA sponges may be successful as peripheral keratoprosthetic materials able to provide a permanent and tight fusion of the keratoprosthesis with the host tissue.

Animals↗

Immunocytochemical demonstration of rabbit ribonuclease and phospholipase A2 by the peroxidase-antiperoxidase technique in professional phagocytes (pulmonary alveolar macrophages and granulocytic and mononuclear peritoneal exudate cells) and in glycol methacrylate sections of dermal tuberculous (BCG) lesions.

Acid-acting (pH 6-7) (presumably lysosomal) ribonuclease and neutral-acting (pH 7-8) calcium-dependent phospholipase A2 (presumably the enzyme releasing arachidonic acid from membrane phospholipids) were demonstrated by the peroxidase-antiperoxidase (PAP) immunocytochemical technique in rabbit professional phagocytes: pulmonary alveolar macrophages (AM), oil-induced peritoneal exudate macrophages (M phi) and glycogen-induced peritoneal exudate polymorphonuclear granulocytes (PMN). All three cell types stained positively with antisera to purified rabbit lung RNase and purified rabbit granulocyte phospholipase A2. The RNase and phospholipase A2 were also demonstrated by the PAP technique in the activated macrophages and granulocytes present in tissue sections of tuberculous (BCG) lesions. The intensity of staining of these two enzymes in individual macrophages did not change appreciably as the BCG lesions developed and regressed, but there were more macrophages rich in both enzymes when the lesions reached their peak size at 21 days. When the anti-RNase serum was fractionated by immunoabsorbent chromatography, the anti-delta RNase serum fraction stained exudate M phi and PMN better than AM; and the anti-beta RNase fraction stained AM better than M phi and PMN. Similar to isolated phagocytes, tissue granulocytes stained best with the anti-delta fraction; and activated tissue macrophages stained best with the anti-beta fraction. Thus, macrophages and granulocytes contain two types of RNase, beta and delta; and the beta RNase is associated with macrophage activation.

Animals↗

Molding mineral within microporous hydrogels by a polymer-induced liquid-precursor (PILP) process.

Natural biominerals often have exquisite morphologies, where the cells exercise a high degree of crystallographic control through secretion of biological macromolecules and regulation of ion transport. One important example is the sea urchin spine. It has recently been shown to be formed through deposition of a transient amorphous calcium carbonate (ACC) precursor phase that later transforms to single-crystalline calcite, ultimately forming an elaborate three-dimensional microporous calcium carbonate structure with interconnected pores. Macromolecules associated with the mineral phase are thought to play a key role in regulating this transformation. The work described here mimics this type of morphological control by "molding" an amorphous calcium carbonate precursor within a porous poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogel that has been prepared as a negative replica from the void space of an urchin spine. Using an acidic biomimetic polymer as a process-directing agent, we show that polyaspartic acid induces amorphous calcium carbonate (ACC) nanoparticles, which have fluidic character and therefore are able to infiltrate the PHEMA hydrogel replica and coalesce into the convoluted morphology that replicates the original microporous structure of the sea urchin spine. By "molding" calcium carbonate into a complex morphology at room temperature, using a precursor process that is induced by a biomimetic acidic macromolecule, the PILP process is a useful in vitro model for examining different aspects of the amorphous-to-crystalline transformation process that is apparently used by a variety of biomineralizing organisms. For example, although we were able to replicate the overall morphology of the spine, it had polycrystalline texture; further studies with this system will focus on controlling the nucleation event, which may help to elucidate how such a convoluted structure can be prepared with single-crystalline texture via an amorphous precursor. Through a better understanding of the mechanisms used by organisms to regulate crystal properties, such biomimetic processes can lead to the synthesis of materials with superior electronic, mechanical, and optical properties.

Animals↗

Covalent immobilization of Aspergillus niger on pHEMA membrane: application to continuous flow reactors.

Poly(2-hydroxyethyl methacrylate) (pHEMA) membrane was prepared via photopolymerization and activated with epichlorohydrin. The conidia of Aspergillus niger strains (wild type 'NRRL-3' and genetically improved strain 'NRRL-3/2-2A') were covalently-immobilized on the membranes. Uniform growth of A. niger cells on membrane surfaces was verified by SEM. The glucose oxidase (GOD) activity of the immobilized cells was determined in a continuous flow membrane reactor (CFMR) by assaying for hydrogen peroxide produced. The activity was also determined in the culture fluids of A. niger strains, freely grown in batch cultures. The CFMR was run with 0.1 mol dm-3 glucose with a fixed flow rate of 20 cm3 h-1 for 60 h during which a 10% loss of the original activity was detected. The loss of the activity with the freely cultivated mycelia was about 50% after 30 h. The GOD activity of the improved strain NRRL-3/2-2A was about 20 times higher whether in immobilized or in free form. The GOD activity of the immobilized A. niger strains in the continuous flow membrane reactor was found to be 2.5 times better than their counterparts freely grown in batch cultures indicating that immobilization increases the activity and the stability of the microorganisms.

Aspergillus niger↗