Hydroxyethyl Methacrylate-methyl Methacrylate Research Articles

Regulation of the outgrowth position of neurites in cell body and the direction of their elongation for PC12 cells in three dimensional hydrogel culture system with feeble flow of the solvent

A rat pheochromocytoma cell line (PC12) was encapsulated in a water-insoluble hydroxyethyl methacrylate-methyl methacrylate copolymer by interfacial precipitation from a polyethylene glycol 200 solution into phosphate-buffered saline. The resulting capsules (660 ± 44 μm in diameter; 84 ± 27 μm wall thickness) contained viable PC12 cells in a spheroidal arrangement, much like tumour spheroids, the latter grown on surfaces unsuitable for cell attachment. In these spheroids, the viable cells formed a band approximately 100 μm thick, surrounding an inner core of necrotic cells. A similar arrangement was seen 14, 28 and 42 days after encapsulation, with capsules maintained in an in vitro tissue culture environment; the annular ring was roughly constant in size, although the packing density appeared to increase over the 6 week observation period. During the first 4 weeks, when measurements were made the encapsulated cells converted a tetrazolium dye (MTT) into an insoluble formazan product, in a time-after-encapsulation-dependent manner. This indicated that PC12 cells retained viability despite encapsulation and an ability to increase (at least in part) their metabolic capacity, presumably by a combination of proliferation and altered cellular activity. The encapsulated PC12 cells also secreted dopamine when incubated in a high potassium release medium but not in a low potassium, conventional tissue culture medium (RPMI 1640). Consistent with the MTT results, the amount of dopamine released was also dependent on the time after encapsulation, as well as the cell density at the time of encapsulation.

Vascularization and Improved In Vivo Survival of VEGF-Secreting Cells Microencapsulated in HEMA-MMA

Vascularization caused by encapsulated cells engineered to secrete vascular endothelial growth factor (VEGF) improved the in vivo survival of the encapsulated cells in a syngeneic mouse Matrigel plug model. Murine fibroblast cells (L929) were engineered to secrete recombinant human vascular endothelial growth factor (rhVEGF(165)). Transfected and nontransfected L929 cells were microencapsulated in a 75:25 hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer. Capsules containing transfected cells induced vascularization in vivo at 1 and 3 weeks postimplantation. In histological sections, a significant positive correlation was seen between the number of capsules and blood vessel density for VEGF-secreting cell capsule implants. New vessels, many positively stained for smooth muscle cells and pericytes, were seen surrounding these VEGF-secreting cell capsule explants. Few vessels were seen in nontransfected L929 capsule implants. The viability of transfected and nontransfected encapsulated cells was assessed on explantation. Although the viability of all encapsulated cells decreased at both 1 and 3 weeks, encapsulated VEGF-secreting cells retained more of the viability than did encapsulated nontransfected control cells. Genetically modified cells promoted vascularization in this context and appeared to enhance the viability of the encapsulated cells, although the extent of the functional benefit was less than expected. Additional effort is required to enhance the benefit, to quantify it, and to understand further the host response to HEMA-MMA microencapsulated cells and tissue constructs, more generally.

Characterization and in vitro application of nano-crystalline calcia stabilized zirconia (CSZ)/copolymer composites

The ideal implant should have long stable life in the physiological environment and induce natural bone growth around implants. In this study, nano-sized particles of calcia stabilized zirconia (CSZ) was used as a filler in poly(hydroxyethylmethacrylate–methylmethacrylate) p(HEMA–MMA) grafted onto chitosan copolymer to produce a bioactive composites analogous to bone. Results coming from this study confirmed that the grafting percentage of CSZ–copolymer composite was enhanced compared to the copolymer as a result of nano-sized filler. Thermo-gravimetric analysis (TGA) proved the presence of attached copolymer layer onto the filler particles for CSZ–copolymer composite. Swelling properties was reduced for CSZ–copolymer composite proving the stability and lower affinity of this composite to water molecules. In vitro tests indicate that the adsorption of calcium ions (Ca 2+) and phosphate ions (PO 4 3−) on the surface of the composites are enhanced. That was confirmed by the formation of a bone-like apatite layer. Fourier transformer infrared spectrophotometer (FT-IR) post-immersion confirmed the formation of carbonate-apatite layer onto the surface of the copolymer and CSZ–copolymer composite at 3 and 21 days, respectively. SEM post-immersion showed enhanced bone-like apatite layer (calcium-phosphate layer) onto the CSZ–polymer composite compared to the copolymer. Subsequently, the formation of a bone-like apatite layer is found to be controlled by the change in content of CSZ filler.

Suppressed Splenocyte Proliferation Following a Xenogeneic Skin Graft due to Implanted Biomaterials

Immune system responses to antigens in the context of biomaterials are poorly understood. Biomaterial implantation results in an inflammatory reaction, which is anticipated to alter the adaptive immune response, in the case presented here, to a skin xenograft. Our earlier work showed unexpectedly low splenocyte proliferation following a xenogeneic cell implant in tandem with a biomaterial in the form of a microcapsule. Here we explore whether that effect was due to the cells or the biomaterial, and attempt to dissect the mechanism of immune deviation. We assessed the immune response of Balb/c mice to hamster skin grafts accompanied by one of three implants: encapsulated xenogeneic cells; free cells accompanied by the same encapsulation biomaterials; and the encapsulation biomaterials without cells. Cells were encapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer then embedded in an agarose gel. Splenocyte proliferation upon re-challenge in vitro, antibody titer in serum, and Th1/2 polarization (by cytokines in splenocyte challenge supernatants and antibody isotypes in serum) were measured. All skin grafts with encapsulation materials (even without cells) suppressed subsequent splenocyte proliferation at 10 days postimplant, although this effect disappeared by two months. In contrast, the antibody response was equal to or greater than that for a skin graft alone. Th1/2 polarization could not explain these observations because it did not correlate with the suppression of splenocyte proliferation. Implanted biomaterials caused nonspecific, transient suppression of splenocyte responses to hamster cells following hamster skin grafts, which is potentially important in the context of tissue engineering.

Biological Response to a SupraDescemetic Synthetic Cornea in Rabbits

To evaluate the biocompatibility of a novel nonpenetrating keratoprosthesis (supraDescemetic synthetic cornea) in a rabbit model. Seven rabbits received a supraDescemetic synthetic cornea (7-mm diameter, 350-microm-thick optical zone, 100-microm-thick peripheral flange) in their healthy right eyes. A surgical technique was developed that allowed implantation of the device on top of the bare Descemet membrane. Three rabbits received a supraDescemetic synthetic cornea made of hydroxyethyl methacrylate-methyl methacrylate(26), 1 received a hydroxyethyl methacrylate-N-vinyl pyrrolidone(75) mesoplant, and 3 were implanted with devices made of polymethyl methacrylate. All rabbits were euthanized after 8 weeks; the eyes were enucleated and examined by conventional histological and immunohistochemical evaluations. All eyes became quiet within several days. The Descemet membrane remained transparent during the observation period. Indirect ophthalmoscopy performed through the prosthesis allowed accurate examination of the posterior pole. Histological evaluation of the implanted corneas displayed no signs of an acute or chronic inflammatory reaction to the supraDescemetic synthetic cornea in 5 eyes; a few inflammatory cells were detected in the corneas of 2 rabbits. The interface between the Descemet membrane and the mesoplant displayed ingrowth of very thin (<10-microm) tissues colonized by keratocytes in 3 of the 7 corneas. This study validates the biocompatibility of this new type of nonpenetrating keratoprosthesis. Because opening of the anterior chamber is not required with the supraDescemetic synthetic cornea, the risk for intraocular infection is minimal, and the implantation procedure is less traumatic compared with a penetrating device.

In Vivo Recognition by the Host Adaptive Immune System of Microencapsulated Xenogeneic Cells

Microencapsulation is under consideration as a means of enabling pancreatic islet transplantation. To understand better the ongoing destructive host response, we examined whether the adaptive immune system of the recipient recognized polymer-encapsulated xenogeneic cells implanted intraperitoneally. Balb/c mice were implanted with xenogeneic Chinese hamster ovary cells, inside and outside poly(hydroxyethyl methacrylate-methyl methacrylate) microcapsules, and responses were compared with xenografted Chinese hamster skin (positive control). Capsules were localized within an agarose rod. Splenocyte proliferation upon rechallenge in vitro, antibody titer in serum, and Th1/2 polarization (assessed by interleukin-4 and interferon-gamma in supernatants of antigen-challenged splenocytes and immunoglobulin [Ig]G1 and IgG2a antibody isotypes in serum) were measured. Encapsulation did not prevent a strong recipient antibody response. Splenocyte proliferation in vitro did not differ after priming by implanted cells, inside or outside capsules. Thus, the capsule membrane did not prevent indirect recognition of shed antigens. However, after 10 days of implantation, proliferation was lower than that induced by skin grafts, although this difference disappeared by 2 months. This transient T-cell suppression was unexpected because encapsulated cell viability was already compromised by 10 days. The influence of Th1/2 bias did not explain the observed suppression. Cells inside capsules elicited a consistent Th2 response, whereas cells outside capsules elicited a mixed response, and skin xenografts showed an initial Th2 response that became mixed by 2 months. Encapsulation does not prevent host immune responses, but the inflammatory response to the implanted biomaterials or xenogeneic cells may be responsible both for encapsulated cell death and transient T-cell suppression.

Influence of cataract surgery with implantation of different intraocular lenses on scanning laser tomography and polarimetry

Purpose To describe the influence of cataract surgery with implantation of different intraocular lenses on scanning laser tomography (SLT) and scanning laser polarimetry (SLP) and to measure the birefringent properties of intraocular lenses in a laboratory setting. Design Prospective cohort study. Methods The influence of phacoemulsification cataract surgery with intraocular lens implantation (56 polymethylmethacrylate [PMMA; 31 Pharmacia/Upjohn 811 B and 25 Domilens Flex 60]; 25 hydroxyethylmethacrylate/methylmethacrylate (HEMA/MMA) [Technomed, Memory Lens]; 32 silicone [Allergan Si40]; 25 acrylic [Storz Hydroview]) on SLP and SLT measurements was evaluated preoperatively and 3 to 4 weeks postoperatively in 138 eyes of otherwise healthy patients. Lens opacities were classified according to the Lens Opacities Classification System III (LOCS III). Imaging was performed with TopSS and GDx (Laser Diagnostic Technologies, San Diego, California, USA) for SLT and SLP, respectively. Additionally, birefringent properties of intraocular lenses were measured in a laboratory setting. Results Our results show that imaging with SLT and SLP is possible with lens opacity–induced visual acuity reductions down to 0.2 and 0.16, respectively. There were no significant differences in the topographic parameters assessed before and after cataract surgery with SLT. However, standard deviations between three within-session single measurements were smaller postoperatively. Nerve fiber layer patterns were similar before and after cataract surgery with SLP, whereas certain total nerve fiber layer thickness values were significantly higher postoperatively. As a consequence, “the number” is significantly lower in the Domilens Flex 60 group ( P = .01) and in the Storz Hydroview group ( P = .02), and with a tendency also in the other groups as well. Measurements with a high-resolution optical bench revealed no birefringent properties of the implanted lenses. Conclusions Our results indicate that after cataract surgery with intraocular lens implantation some SLP values are altered significantly, whereas SLT parameters are not influenced. These findings are of clinical interest, especially in the follow-up of glaucoma patients.

Viability of hydroxyethyl methacrylate-methyl methacrylate-microencapsulated PC12 cells after omental pouch implantation within agarose gels.

Hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA, 75 mol% HEMA). Microcapsules containing viable PC12 cells (as an allogeneic transplant model) were implanted into omental pouches in Wistar rats. Two different capsule preparations were tested, based on differences in polymer solutions during extrusion: 10% HEMA-MMA in TEG, and 9% HEMA-MMA in TEG with 30% poly(vinyl pyrrolidone) (PVP). The omental pouch proved to be an ideal transplant site in terms of implantation, recovery, and blood vessel proximity (nutrient supply). To minimize the fibrous overgrowth and damaged capsules previously seen on implantation of individual capsules, agarose gels were used to embed the capsules before implantation. Cells proliferated within the microcapsule-agarose device during the first 7 days of implantation, but overall cell viability declined over the 3-week period, when compared with similar capsules maintained in vitro. Nonetheless, approximately 50% of the initial encapsulated cells were still viable after 3 weeks in vivo. This approach to HEMA-MMA microcapsule implantation improved cell viability and capsule integrity after 3 weeks in vivo, compared with capsules implanted without agarose.

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.

Conformal coating of small particles and cell aggregates at a liquid-liquid interface.

Polymer encapsulation of allogeneic or xenogeneic tissue is under active investigation as a means of isolating transplanted cells, such as pancreatic islets, from the immune system. We report here a method for coating small particles and cell aggregates with a very thin water insoluble hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) membrane that conforms to the shape of the aggregate, and minimizes the polymer's contribution to the total transplant volume. Cell aggregates were coated at a liquid-liquid interface of a discontinuous density gradient composed of both aqueous and organic liquids. By increasing the viscosity difference and decreasing the density difference between the two liquids of the coating interface, coatings from approximately 1 to 15 microns thick were formed. Aggregates of HepG2 cells and pancreatic islets were coated and remained viable.

Immunoblot analysis of proteins associated with HEMA-MMA microcapsules: Human serum proteins in vitro and rat proteins following implantation

Human serum proteins and their fragments, associated with hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) copolymer microcapsules, were characterized using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Capsules were incubated with serum for 1 week in vitro and then dissolved in ethanol to also precipitate the adsorbed protein. The precipitate was dissolved in 2% (w/v) SDS (the ‘capsule eluate’) to be assayed by electrophoresis. The majority of proteins probed for in the immunoblots were detected in the capsule eluates. These included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H, Factor I, C3, but not β-lipoprotein, fibrinogen, HMWK, or IgM. Complement activation fragments were detected in both the immunoblots of the capsule eluates and the medium containing serum without capsules. Thus, the adsorption of these fragments, formed independent of capsule presence, may be partially or completely responsible for the complement fragments associated with capsules. The prevention of complement activation by the addition of 5.8 mM EDTA, at the begining of the week-long incubation, resulted in fewer low-molecular-weight C3 fragments associated with capsules. Rat proteins were also detected in immunoblots of the eluate of ‘free-floating’ capsules from the rat peritoneal cavity following implantation for 1 day using anti-human antibodies. Detected proteins included HMWK, fibrinogen, antithrombin III, transferrin, α 1-antitrypsin, fibronectin, albumin, α 2-macroglobulin, vitronectin, β 2-microglobulin, Factor B and Factor I. Rat fibrinogen, IgG, and complement C3 fragments were also detected in these immunoblots, but with monoclonal antibodies against the rat proteins.

Microcapsules as bio-organs for somatic gene therapy.

Current human gene therapy relies on genetic modification of the patient's own cells. An alternate non-autologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immuno-isolation devices would allow the same recombinant cell line to be used for different patients, thus potentially lowering the cost of treatment. The feasibility of this idea has now been demonstrated in vitro and in vivo. Recombinant gene products with potential therapeutic applications (human growth hormone, factor IX, lysosomal enzymes, adenosine deaminase) have been expressed from genetically modified cells after encapsulation with alginate-poly-L-lysine-alginate or hydroxyethyl methacrylate-methyl methacrylate. We have also demonstrated the feasibility of this idea in vivo. After intraperitoneal implantation, genetically modified mouse Ltk- fibroblasts or C2C12 myoblasts encapsulated in alginate-poly-L-lysine-alginate could deliver recombinant gene products (human growth hormone, human factor IX) to the systemic circulation of mice. The clinical efficacy of this novel approach to gene therapy has now been shown in murine models of human diseases. In the Snell dwarf mice deficient in growth hormone production, implantation of encapsulated mouse myoblasts engineered to secrete mouse growth hormone resulted in increases in body weight, length and organ sizes, some to > 25% above those of the controls. In the Gus/Gus mice suffering from the lysosomal storage disease mucopolysaccharidosis type VII due to deficient beta-glucuronidase, implantation of encapsulated mouse fibroblasts engineered to secrete mouse beta-glucuronidase resulted in delivery of normal levels of the enzyme in the plasma and significant correction of the organ histopathology. Hence, delivery of recombinant gene products through bioartificial devices appears to be a promising strategy for the treatment of genetic diseases.

Selected aspects of the microencapsulation of mammalian cells in HEMA-MMA.

Microencapsulation of live mammalian cells is one means of creating hybrid artificial organs, like an artificial pancreas or an artificial liver. In addition to creating and developing the methodologies for enclosing cells within the appropriate semipermeable and biocompatible membranes, novel techniques are needed to assess the various features of the resulting capsules. The small size of a capsule or its heterogeneity can lead to additional complexities that go beyond the problem of examining cell behavior in the presence of biomaterials. These problems are illustrated here by comparison of protein release by microencapsulated HepG2 cells within large and small HEMA-MMA (hydroxyethyl methacrylate-methyl methacrylate) capsules, by assessment of the effect of processing conditions on HEMA-MMA microcapsule permeability to horseradish peroxidase at the individual capsule level, and by a confocal microscopy technique for assessing intracapsule cell viability.

X-Ray photoelectron spectroscopy (XPS) surface analysis of HEMA-MMA microcapsules

High resolution carbon, C 1s, X-ray photoelectron spectroscopy (XPS) of the surface of hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA; 75 mol% HEMA) capsules maintained in PBS for 1 week showed that the surface was not pure HEMA-MMA. In these spectra, more carbon was bonded in the C-O form than in the C-C form indicating the presence of the Pluronic surfactant, L101, adsorbed from the precipitation bath to the surface during microcapsule preparation. Capsules maintained in medium containing fetal bovine serum for 1 week showed a nitrogen signal consistent with the presence of adsorbed serum proteins. There was a decrease in the amount of nitrogen on the surface after phosphate buffered saline (PBS) washing, however this did not decrease to zero. These preadsorbed proteins, present on the surface of capsules incubated in serum-containing medium before their implantation, may affect the tissue response to these capsules. Calcium was not detected on freshly-made capsules or capsules maintained in PBS for 1 week but was detected on capsules maintained in medium containing serum. Calcium deposits, if formed in vitro, could act as nucleation sites for calcification of the polymer in vivo.

Secretion of recombinant proteins from hydroxyethyl methacrylate-methyl methacrylate capsules

Current human gene therapy relies on genetic modification of the patient's own cells. An alternate nonautologous approach is to use universal cell lines engineered to secrete therapeutic products. Protection with immunoisolation devices before implantation would allow the use of the same recombinant cell line for treating different patients, thus potentially lowering the cost of treatment. To study the properties of a mechanically stable synthetic biomaterial, hydroxyethyl methyacrylate-methyl methacrylate (HEMA-MMA) as the immuno-isolation device, we encapsulated recombinant mouse fibroblast cells engineered to secrete products ranging from 27 to 300 kDa in size (human growth hormone, mouse beta-hexosaminidase and beta-glucuronidase) in the presence or absence of the extracellular matrix Matrigel. Both viability and cell number in the microcapsules increased with time after encapsulation and cell morphology indicated viable cell growth, thus showing that the capsule membrane barrier was compatible with nutrient/waste exchange necessary for normal metabolic activity.The intracellular levels of these recombinant gene products were constant throughout the experimental period of 22 days in the presence or absence of Matrigel, thus demonstrating that the microenvironment did not lead to downregulation of the transgenes. However, the extracellular levels of the gene products secreted from the cells and trapped within the microcapsules were dependent on the molecular size of the product and presence of Matrigel. With the 27-kDa human growth hormone, the presence of Matrigel caused its retention within this intracapsular space, but its release from the microcapsules to the culture medium was not impeded. With the 120-kDa beta-hexosaminidase or the 300-kDa beta-glucuronidase, they were retained within the microcapsule space regardless of the presence or absence of Matrigel, and their passage from the microcapsules to the media was totally blocked. In conclusion, the HEMA-MMA microcapsules are supportive of recombinant cell growth and maintained their molecular cutoff at approximately 100 kDa. Inclusion of extracellular matrix was unable to improve cell growth and may impede the exit of some gene products.

A thyroid-stimulating hormone immuno-electrode

A non-labelled thyroid-stimulating hormone (TSH) immuno-electrode is described in this paper. The chloride electrodes are used as the basic electrode and the reference electrode. The hydroxyethyl methacrylate-methyl methacrylate copolymer was used as the membrane matrix material covering the basic electrode. The TSH immuno-electrode can be used for TSH determination up to 17.5 μIU/ml with a determination limit of 1.4 μIU/ml. The excellent selectivity of the electrode was guaranteed by the highly selective immunoreaction. The analytical results obtained corresponded satisfactorily with those obtained using the radio immunoassay procedure.

Dopamine secretion by PC12 cells microencapsulated in a hydroxyethyl methacrylate-methyl methacrylate copolymer

A rat pheochromocytoma cell line (PC12) was encapsulated in a water-insoluble hydroxyethyl methacrylate-methyl methacrylate copolymer by interfacial precipitation from a polyethylene glycol 200 solution into phosphate-buffered saline. The resulting capsules (660 ± 44 μm in diameter; 84 ± 27 μm wall thickness) contained viable PC12 cells in a spheroidal arrangement, much like tumour spheroids, the latter grown on surfaces unsuitable for cell attachment. In these spheroids, the viable cells formed a band approximately 100 μm thick, surrounding an inner core of necrotic cells. A similar arrangement was seen 14, 28 and 42 days after encapsulation, with capsules maintained in an in vitro tissue culture environment; the annular ring was roughly constant in size, although the packing density appeared to increase over the 6 week observation period. During the first 4 weeks, when measurements were made the encapsulated cells converted a tetrazolium dye (MTT) into an insoluble formazan product, in a time-after-encapsulation-dependent manner. This indicated that PC12 cells retained viability despite encapsulation and an ability to increase (at least in part) their metabolic capacity, presumably by a combination of proliferation and altered cellular activity. The encapsulated PC12 cells also secreted dopamine when incubated in a high potassium release medium but not in a low potassium, conventional tissue culture medium (RPMI 1640). Consistent with the MTT results, the amount of dopamine released was also dependent on the time after encapsulation, as well as the cell density at the time of encapsulation.

The effects of polymer concentration and a pore-forming agent (PVP) on HEMA-MMA microcapsule structure and permeability

The permeability to horseradish peroxidase, (HRP, MW 40 kDa) increased approximately sixty-fold by decreasing the concentration of polymer (HEMA-MMA, hydroxyethyl methacrylate-methyl methacrylate) in triethylene glycol (TEG) from 10 or 11% to 9% (w/v), in microcapsules prepared by interfacial precipitation into phosphate buffered saline. Permeability was determined at the individual capsule level by enzyme-chromogenic substrate assay. With the lower polymer concentration, aggregates and the interconnecting structures formed large interstitial spaces and what appeared to be large pores on the external surface. Adding poly(vinyl pyrrolidone) (PVP) to the HEMA-MMA solution increased the capsule permeability, as much as 25-fold, apparently by increasing the external pore density (defined as the number of pores per unit surface area) without significant deviation in pore size and its distribution. Thus PVP is potentially useful for enhancing the permeability of these capsules which are used to immunoisolate cells used for transplantation. Measurements of molecular weight cut-off are needed before demonstrating that PVP/HEMA-MMA microcapsules are capable of immunoisolating the entrapped cells while maintaining high permeability to the materials essential to encapsulated cell survival and the bioactive cellular products such as insulin.

Microencapsulated human hepatoma (HepG2) cells: capsule-to-capsule variations in protein secretion and permeability

Human hepatoma (HepG2) cells were microencapsulated in hydroxyethyl methacrylate-methyl methacrylate by an interfacial precipitation process. The secretion of a,-antitrypsin (AT: 52 kDa) and fibrinogen (Fbg: 340 kDa) from individual capsules was determined using sensitive enzyme-linked immunosorbent assays. A significant capsule-to-capsule variation in the secretion rates of both AT and Fbg was found. After 2 weeks of in vitro culture, ∼ 20 and 70% of the capsules did not secrete any detectable AT and Fbg, respectively. The relative secretion of Fbg with respect to AT (Fbg/AT) for those capsules which secreted AT also varied among the individual capsules: ∼50% of capsules had zero Fbg/AT, presumably indicating the fraction of the capsules exhibiting the expected sieving effect to the larger protein Fbg but not to the smaller protein AT. Using a new procedure for the permeability of individual capsules (horseradish peroxidase, 40 kDa, as the model protein), a significant capsule-to-capsule variation in protein permeability was found consistent with the observed variation in Fbg and AT release.

Viability and protein secretion from human Hepatoma (HepG2) cells encapsulated in 400‐μm 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.