Isolation of human foetal myoblasts and its application for microencapsulation.

Foetal cells secrete more growth factors, generate less immune response, grow and proliferate better than adult cells. These characteristics make them desirable for recombinant modification and use in microencapsulated cellular gene therapeutics. We have established a system in vitro to obtain a pure population of primary human foetal myoblasts under several rounds of selection with non-collagen coated plates and identified by desmin staining. These primary myoblasts presented good proliferation ability and better differentiation characteristics in monolayer and after microencapsulation compared to murine myoblast C2C12 cells based on creatine phosphokinase (CPK), major histocompatibility complex (MHC) and multi-nucleated myotubule determination. The lifespan of primary myoblasts was 70 population doublings before entering into senescent state, with a population time of 18-24 hrs. Hence, we have developed a protocol for isolating human foetal primary myoblasts with excellent differentiation potential and robust growth and longevity. They should be useful for cell-based therapy in human clinical applications with microencapsulation technology.

Enhancement of myoblast microencapsulation for gene therapy.

One method of nonviral-based gene therapy is to implant microencapsulated nonautologous cells genetically engineered to secrete the desired gene products. Encapsulating the cells within a biocompatible permselective hydrogel, such as alginate-poly-L-lysine-alginate (APA), protects the foreign cells from the host immune system while allowing diffusion of nutrients and the therapeutic gene products. An important consideration is which kind of cells is the best candidate for long-term implantation. Our previous work has shown that proliferation and differentiation of encapsulated C2C12 myoblasts in vitro are significantly improved by inclusion of basic fibroblast growth factor (bFGF), insulin growth factor II (IGF-II), and collagen within the microcapsules ("enhanced" capsules). However, the effects of such inclusions on the functional status of the microcapsules in vivo are unknown. Here we found that comparing the standard with the enhanced APA microcapsules; there was no difference in the rates of diffusion of recombinant products of different sizes, that is, human factor IX (FIX, 65 kDa), murine IgG (150 kDa), and a lysosomal enzyme, beta-glucuronidase (300 kDa), thus providing a key requirement of such an immunoprotective device. Furthermore, the creatine phosphokinase activity and myosin heavy chain staining (markers for differentiation of the myoblasts) and the cell number per capsule in the enhanced microcapsules indicated a higher degree of differentiation and proliferation when compared to the standard microcapsules, thus demonstrating an improved microenvironment for the encapsulated cells. Efficacy was tested in a melanoma cancer tumor model by treating tumor induced by B16-F0/neu tumor cells in mice with myoblasts secreting angiostatin from either the standard or enhanced APA microcapsules. Mice treated with enhanced APA-microcapsules had an 80% reduction in tumor volume at day 21 compared to a 70% reduction in those treated with standard APA-microcapsules. In conclusion, enhancement of APA microcapsules with growth factors and collagen did not adversely affect their permeability property and therapeutic efficacy. However, the enhanced differentiation and viability of the encapsulated myoblasts in vivo should be advantageous for long-term delivery with this method of gene therapy.

Encapsulation of recombinant cells with a novel magnetized alginate for magnetic resonance imaging.

Implanting recombinant cells encapsulated in alginate microcapsules to express therapeutic proteins has been proven effective in treating several mouse models of human diseases (neurological disorders, dwarfism, hemophilia, lysosomal storage disease, and cancer). In anticipation of clinical application, we have reported the synthesis and characterization of a magnetized ferrofluid alginate that potentially allows tracking of these microcapsules in vivo by magnetic resonance imaging (MRI). We now report the properties of these ferrofluid microcapsules important for applications in gene therapy. When a mouse myoblast cell line was encapsulated in these microcapsules, it showed similar viability as in regular unmodified alginate capsules, both in vitro and in vivo, in mice. The permeability of these magnetized microcapsules, a critical parameter for immunoisolation devices, was comparable to that of classic alginate in the transit of various recombinant molecules of various molecular masses (human factor IX, 65 kDa; murine IgG, 150 kDa; and beta-glucuronidase, 300 kDa). When followed by MRI in vitro and in vivo, the ferrofluid microcapsules remained intact and visible for extended periods, allowing quantitative monitoring of microcapsules. At autopsy, the ferrofluid microcapsules were mostly free within the intraperitoneal cavities, with no overt inflammatory response. Serological analyses demonstrated a high level of biocompatibility comparable to that of unmodified alginate. In conclusion, ferrofluid-enhanced alginate microcapsules are comparable to classic alginate microcapsules in permeability and biocompatibility. Their visibility and stability to MRI monitoring permitted qualitative and quantitative tracking of the implanted microcapsules without invasive surgery. These properties are important advantages for the application of immunoisolation devices in human gene therapy.

Biological properties of photocrosslinked alginate microcapsules.

An alternative form of gene therapy using recombinant cell lines delivering therapeutic products encapsulated in alginate hydrogel has proven effective in treating many murine models. The lack of long-term capsule stability has led to a new strategy to reinforce the microcapsules with a photopolymerized interpenetrating covalent network of N-vinylpyrrolidone (NVP) and sodium acrylate. Here the properties for potential application in gene therapy are reported. In assessing potential toxicity of the unpolymerized residues, HPLC showed that even after 1 week of washing, no toxic monomers could be detected. Their ability to sustain cell growth was monitored with growth of the encapsulated cells in vitro and in vivo. Although the initial photopolymerization caused significant cell damage, the cells were able to recover normal growth rates thereafter. After implanting into mice, the NVP-modified capsules showed a high level of biocompatibility as measured by hematological and biochemical functional tests. There was also no difference in the amount and type of plasma proteins adsorbing to the NVP-modified and the classical alginate capsules, thus indicating their similar biological compatibility. Both in vitro and in vivo tests confirmed that the NVP-modified capsules were more resistant to osmotic stress than the alginate microcapsules. Furthermore, when applied to the treatment of a murine model of human cancer by delivering encapsulated cells secreting angiostatin, the NVP-modified microcapsules suppressed tumor growth as successfully as the regular alginate microcapsules. In conclusion, the covalently modified microcapsules have shown a high level of biocompatibility, safety, increase in stability, and clinical efficacy for use as immunoisolation devices in gene therapy.

Quantitative chemical mapping of sodium acrylate- and N-vinylpyrrolidone-enhanced alginate microcapsules.

Alginate microcapsules enclosing recombinant cells secreting therapeutic products have been used successfully to treat several murine models of human diseases. The mechanical and chemical properties of these alginate capsules can be improved by the addition and in situ photo-polymerization of sodium acrylate and N-vinylpyrrolidone in the alginate capsule. The purpose of this modification was to form additional covalent cross-links. In this work we have used scanning transmission X-ray microscopy (STXM) to probe the nature and location of the chemical modifications in the modified capsules by comparison with unmodified capsules. Analysis of X-ray image sequences and selected area spectra has been used to map the calcium gradient in capsules, to identify the presence of polyacrylate throughout the capsules and the localization of poly-N-vinylpyrrolidone in the outer regions of the alginate capsules. The differences in the spatial distributions of these species have led to better understanding of the chemical modifications that provide a mechanically more stable capsule structure.

A novel method to enhance the stability of alginate-poly-L-lysine-alginate microcapsules.

Implantation of microencapsulated recombinant cells is an alternative approach to gene therapy. These genetically-engineered cells enclosed in microcapsules to deliver therapeutic recombinant products have been effective in treating several murine models of human diseases. However, the most commonly used microcapsules fabricated from alginate ionically cross-linked with calcium suffer from loss of long-term mechanical stability. We now report on a method to improve their stability by introducing additional polymers to provide covalent linkages via photopolymerization. Vinyl monomers and a photoinitiator were allowed to diffuse into the initially formed calcium-alginate microcapsules. In situ photopolymerization in the presence of sodium acrylate and N-vinylpyrrolidone substantially enhanced their mechanical strength. After four months of storage in saline, > 70% of these capsules remained intact in the osmotic pressure test, while the un-modified alginate microcapsules totally disintegrated. Tests of their permeability to polyethylene glycol of different molecular weight and their ability to support cell survival showed that these properties remained unaffected by the photopolymerization. Hence, these microcapsules modified by adding a network of vinyl polymers are promising candidates to use for long-term delivery of recombinant gene products in this cell-based method of gene therapy.

A multiprong approach to cancer gene therapy by coencapsulated cells.

Immune-isolation of nonautologous cells with microencapsulation protects these cells from graft rejection, thus allowing the same recombinant therapeutic cell line to be implanted in different recipients. This approach was successful in treating HER2/neu-expressing tumors in mice by delivering an interleukin-2 fusion protein (sFvIL-2), or angiostatin. However, treatment with interleukin-2 led to profuse inflammation, while angiostatin delivery did not result in long-term tumor suppression, in part due to endothelial cell-independent neovascularization (vascular mimicry). We hypothesize that coencapsulating the two producer cells in the same microcapsules may enhance the efficacy and ameliorate the above side effects. Hence, B16-F0/neu tumor-bearing mice were implanted with sFvIL-2- and angiostatin-secreting cells coencapsulated in the same alginate-poly-L-lysine-alginate microcapsules. However, this protocol only produced an incremental but not synergistic improvement, as measured with greater tumor suppression and improved survival. Compared to the single sFvIL-2 treatment, the coencapsulation protocol showed improved efficacy associated with: mobilization of sFvIL-2 from the spleen; a higher level of cytokine delivery systemically and to the tumors; increased tumor and tumor-associated endothelial cell apoptosis; and a reduced host inflammatory response. However, compared to the single angiostatin treatment, the efficacy was reduced, primarily due to a "bystander" effect in which the angiostatin-secreting cells suffered similar transgene silencing as the coencapsulated cytokine-secreting cells. Nevertheless, the level of "vascular mimicry" of the single angiostatin treatment was significantly reduced. Hence, while there was no synergy in efficacy, an incremental improvement and some reduction in undesirable side effects of inflammation and vascular mimicry were achieved over the single treatments.

Combined immunotherapy and antiangiogenic therapy of cancer with microencapsulated cells.

An alternative form of gene therapy involves immunoisolation of a nonautologous cell line engineered to secrete a therapeutic product. Encapsulation of these cells in a biocompatible polymer serves to protect these allogeneic cells from host-versus-graft rejection while recombinant products and nutrients are able to pass by diffusion. This strategy was applied to the treatment of cancer with some success by delivering either interleukin 2 or angiostatin. However, as cancer is a complex, multifactorial disease, a multipronged approach is now being developed to attack tumorigenesis via multiple pathways in order to improve treatment efficacy. A combination of immunotherapy with angiostatic therapy was investigated by treating B16-F0/neu melanoma-bearing mice with intraperitoneally implanted, microencapsulated mouse myoblasts (C2C12) genetically modified to deliver angiostatin and an interleukin 2 fusion protein (sFvIL-2). The combination treatment resulted in improved survival, delayed tumor growth, and increased histological indices of antitumor activity (apoptosis and necrosis). In addition to improved efficacy, the combination treatment also ameliorated some of the undesirable side effects from the individual treatments that have led to the previous failure of the single treatments, for example, inflammatory response to IL-2 or vascular mimicry due to angiostatin. In conclusion, the combination of immuno- and antiangiogenic therapies delivered by immunoisolated cells was superior to individual treatments for antitumorigenesis activity, not only because of their known mechanisms of action but also because of unexpected protection against the adverse side effects of the single treatments. Thus, the concept of a "cocktail" strategy, with microencapsulation delivering multiple antitumor recombinant molecules to improve efficacy, is validated.

Effect of growth factors and extracellular matrix materials on the proliferation and differentiation of microencapsulated myoblasts.

An alternative approach to gene therapy via non-autologous somatic gene therapy is to implant genetically-engineered cells protected from immune rejection with microcapsules to deliver a therapeutic gene product. This delivery system may be optimized by using myoblast cell lines which can undergo terminal differentiation into myotubes, thus removing the potential problems of tumorigenesis and space restriction. However, once encapsulated, myoblasts do not proliferate or differentiate well. We now report the use of extracellular matrix components and growth factors to improve these properties. Addition of matrix material collagen, merosin or laminin all stimulated myoblast proliferation, particularly when merosin and laminin were combined; however, none, except collagen, stimulated differentiation. Inclusion of basic fibroblast growth factor (bFGF) within the microcapsules in the presence of collagen stimulated proliferation of C2C12 myoblasts, as well as differentiation into myotubes. Inclusion of insulin growth factor (IGF-II) in the microcapsules had no effect on proliferation but accelerated myoblasts differentiation. When the above matrix material and growth factors were provided in combination, the use of merosin and laminin together with bFGF and IGF-II stimulated myoblast proliferation but had no effect on differentiation. In contrast, the cocktail containing bFGF, IGF-II and collagen induced increased myoblasts proliferation and subsequent differentiation. Hence, the combination of bFGF, IGF-II and collagen appears optimal in improving proliferation and differentiation in encapsulated myoblasts.

Antiangiogenic cancer therapy with microencapsulated cells.

Inhibition of angiogenesis has led to tumor suppression in several cancer models. Although administering purified recombinant antiangiogenic product is effective, alternative approaches through genetic manipulation may be more cost-effective. We propose to implant nonautologous recombinant cells secreting angiostatin for systemic delivery of angiostatin in cancer treatment. These cells are protected from graft rejection in alginate microcapsules to function as "micro-organs" to deliver angiostatin in vivo. This approach was tested by implanting encapsulated mouse myoblast C2C12 cells genetically modified to secrete angiostatin into mice bearing solid tumor. Angiostatin was detected in sera of the treated mice. Efficacy was demonstrated by suppression of palpable tumor growth and improved survival. At autopsy, angiostatin localized to residual tumors and high levels of angiostatic activity were detected in tumor extracts. Tumor tissues showed increased apoptosis and necrosis compared with those from untreated or mock-treated mice. Immunohistochemical staining against von Willebrand factor, an endothelial cell marker, showed that within tumors from the treated mice, the neovasculature was poorly defined by endothelial cells, many of which were undergoing apoptosis. However, the tumors eventually developed neovasculature independent of endothelial cells. Such vascular mimicry would account for the lack of long-term efficacy despite persistent angiostatin delivery. In conclusion, implantation with nonautologous microencapsulated cells is feasible for systemic delivery of angiostatin, resulting in localization of angiostatin to tumors and targeted apoptosis of the endothelial cells. Clinical efficacy was demonstrated by suppression of tumor growth and extension of life span. Although the potential of this cell-based approach for angiostatin-mediated cancer therapy is confirmed, long-term efficacy must take into account the possible escape by some tumors from angiogenesis inhibition.

Properties of a novel magnetized alginate for magnetic resonance imaging.

Implanting recombinant cells encapsulated in alginate microcapsules to secrete therapeutic proteins has been proven clinically effective in treating several murine models of human diseases. However, once implanted, these microcapsules cannot be assessed without invasive surgery. We now report the preparation and characterization of a novel ferrofluid to render these microcapsules visible with magnetic resonance imaging (MRI). The ferrofluid was prepared as a colloidal iron oxide stabilized in water by alginate. The presence of iron particles in the ferrofluid was verified with chemical titration, dynamic light scattering, and magnetization measurement. The microcapsules fabricated with various concentrations of the ferrofluid in the core, or on the surface of alginate microcapsules, or both, all produced microcapsules with smooth surfaces as shown with light and scanning electron microscopy. However, at the nanoscale level, as revealed with atomic force microscopy, the ferrofluid-fabricated microcapsules demonstrated increased granularity, particularly when the ferrofluid was used to laminate the surface. From the force spectroscopy measurements, these modified microcapsules showed increasing surface rigidity in the following order: traditional alginate < ferrofluid in the core < ferrofluid on the surface. Although the mechanical stability of low-concentration ferrofluid (0.1%) microcapsules was reduced, increasing concentrations, up to 20%, were able to improve stability. When these ferrofluid microcapsules were examined with MRI, their T(2) relaxation time was reduced, thereby producing increased contrast readily detectable with MRI, whereas the traditional alginate microcapsules showed no difference when compared with water. In conclusion, such ferrofluid-enhanced alginate is suitable for fabricating microcapsules that offer the potential for in vivo tracking of implanted microcapsules without invasive surgery.

Delivery of recombinant gene product to canine brain with the use of microencapsulation.

An alternative approach to somatic gene therapy is to deliver a therapeutic protein by implanting "universal" recombinant cells that are immunologically protected from graft rejection with alginate microcapsules. This strategy has proved successful in reversing pathologic conditions in several rodent models of human disease (dwarfism, lysosomal storage disease, hemophilia, cancer). In particular, neurologic disease and behavioral deficit in the mouse model of a neurodegenerative disease (mucopolysaccharidosis [MPS] VII) were significantly improved through the intraventricular implantation of the recombinant encapsulated cells. Here we report the feasibility of delivering recombinant gene products to the central nervous systems (CNSs) of dogs, first using human growth hormone as a marker for delivery in normal dogs and then using alpha-iduronidase as a therapeutic product for delivery in the MPS I dog that is genetically deficient in this lysosomal enzyme. Madin-Darby canine kidney cells were genetically modified to express either human growth hormone or canine alpha-iduronidase, then enclosed in alginate-poly-l-lysine-alginate microcapsules of about 500 microm in diameter. The encapsulated cells were implanted into the brain under steoreotaxic guidance. The brains were monitored with computed tomographic scans before and after surgery and examined biochemically and histologically. Delivery of gene products, as measured in the plasma and cerebrospinal fluid sampled periodically through 21 days or in various regions of the brain after death showed that the delivery of both gene products was extremely low but detectable. However, we noted extensive inflammatory reactions, both at the sites of implantation and in the immediate vicinity of the implanted microcapsules. Hence for this technology to be applicable to the CNSs of larger animals and human beings, a more accurate and less invasive neurosurgical procedure, more biocompatible microcapsule-recombinant cell combinations, and higher output of recombinant products must be developed.

A novel approach to tumor suppression with microencapsulated recombinant cells.

A novel approach to cancer gene therapy is to implant microcapsules containing nonautologous cells engineered to secrete molecules with antineoplastic properties. The efficacy of this treatment is now tested in a mouse model bearing HER-2/neu-positive tumors. Nonautologous mouse myoblasts (C(2)C(12)) were genetically modified to secrete interleukin-2 linked to the Fv region of a humanized antibody with affinity to HER-2/neu. The resulting fusion protein, sFvIL-2, would encompass immune-stimulatory cytokine activity now targeted to the HER-2/neu-expressing tumor. These recombinant cells were then immunoprotected with alginate-poly-L-lysine-alginate microcapsules before implantation into tumor-bearing mice. Treatment with these encapsulated cells led to a delay in tumor progression and prolonged survival of the animals. The long-term efficacy was limited by an inflammatory reaction against the implanted microcapsules probably because of the secreted cytokine and antigenic response against the xenogeneic fusion protein itself. However, over the short term (initial 2 weeks), efficacy was confirmed when a significant amount of biologically active interleukin-2 was detected systemically, and targeting of the fusion protein to the HER-2/neu-expressing tumor was shown immunohistochemically. The tumor suppression in the treated animals was associated with increased apoptosis and necrosis in the tumor tissue, thus demonstrating successful targeting of the antiproliferative effect to the tumors by this delivery paradigm. In conclusion, this new approach to systemic cancer gene therapy needs to be modified to provide long-term delivery, but has demonstrated short-term efficacy and potential to become a cost-effective, benign, and non-viral-based adjunct to the current armory of anticancer strategies.