Frank T. Gentile

Implantation of encapsulated catecholamine and GDNF-producing cells in rats with unilateral dopamine depletions and parkinsonian symptoms

Studies in rodents suggest that PC12 cells, encapsulated in semipermeable ultrafiltration membranes and implanted in the striatum, have some potential efficacy for the treatment of age- and 6-OHD-induced sensorimotor impairments (22, 70, 71, 74). The objectives of this study were to: (1) determine if baby hamster kidney cells engineered to secrete glial cell line-derived neurotrophic factor (BHK-GDNF) would survive encapsulation and implantation in a dopamine-depleted rodent striatum, (2) compare polymer-encapsulated PC12 and PC12A cells in terms of their ability to survive and produce catecholamines in vivo in a dopamine-depleted striatum, and (3) determine if BHK-GDNF, PC12, or PC12A cells reduce parkinsonian symptoms in a rodent model of Parkinsons disease. Capsules with BHK-GDNF or PC12 cells contained viable cells after 90 days in vivo, with little evidence of host tissue damage/gliosis. In rats with tyrosine hydroxylase (TH)-positive fibers remaining in the lesioned striatum, there was TH-positive fiber ingrowth into the membranes of the BHK-GDNF capsules. PC12-containing capsules had higher basal release of both dopamine and L-DOPA after 90 days in vivo than before implantation, while basal release of both dopamine and L-DOPA decreased in the PC12A-containing capsules. Both encapsulated PC12 and PC12A cells, but not encapsulated BHK-GDNF cells, decreased apomorphine-induced rotations. Parkinsonian symptoms (akinesia, freezing/bracing, sensorimotor neglect) related to the extent of dopamine depletion were evident even in rats with dopamine depletions of only 25%. Evidence that encapsulated cells may attenuate these parkinsonian symptoms was not detected but most of the rats were more severely depleted of dopamine than Parkinsons patients (less than 2% dopamine remaining in the entire striatum), and these tests were not sensitive to differences between rats with less than 10% dopamine remaining. These results suggest that cell encapsulation technology can safely provide site-specific delivery of dopaminergic agonists or growth factors within the CNS, without requiring suppression of the immune system, and without using fetal tissue. Of the three types of encapsulated cells examined in the present study, PC12 cells seem to offer the most therapeutic potential in rats with severe dopamine depletions.

Transport characterization of hydrogel matrices for cell encapsulation

Current membrane‐based bioartificial organs consist of three basic components: (1) a synthetic membrane, (2) cells that secrete the product of interest, and (3) an encapsulated matrix material. Alginate and agarose have been widely used to encapsulate cells for artificial organ applications. It is important to understand the degree of transport resistance imparted by these matrices in cell encapsulation to determine if adequate nutrient and product fluxes can be obtained. For artificial organs in xenogeneic applications, it may also be important to determine the extent of immunoprotection offered by the matrix material. In this study, diffusion coefficients were measured for relevant solutes [ranging in size from oxygen to immunoglobulin G (IgG)] into and out of agarose and alginate gels. Alginate gels were produced by an extrusion/ionic crosslinking process using calcium while agarose gels were thermally gelled. The effect of varying crosslinking condition, polymer concentration, and direction of diffusion on transport was investigated. In general, 2–4% agarose gels offered little transport resistance for solutes up to 150 kD, while 1.5–3% alginate gels offered significant transport resistance for solutes in the molecular weight range 44–155 kD—lowering their diffusion rates from 10‐ to 100‐fold as compared to their diffusion in water. Doubling the alginate concentration had a more significant effect on hindering diffusion of larger molecular weight species than did doubling the agarose concentration. Average pore diameters of approximately 170 and 147 Å for 1.5 and 3% alginate gels, respectively, and 480 and 360 Å for 2 and 4% agarose gels, respectively, were estimated using a semiempirical correlation based on diffusional transport of different‐size solutes. The method developed for measuring diffusion in these gels is highly reproducible and useful for gels crosslinked in the cylindrical geometry, relevant for studying transport through matrices used in cell immobilization in the hollow fiber configuration.

Protection of Encapsulated Human Islets Implanted Without Immunosuppression in Patients With Type I or Type II Diabetes and in Nondiabetic Control Subjects

Human islets were macroencapsulated in permselective hollow fiber membrane devices and successfully allotransplanted subcutaneously with > 90% viability after 2 weeks in situ. Recipients were patients with type I or type II diabetes and normal control subjects; none was immunosuppressed. Between 150 and 200 islet equivalents were implanted in each of the nine patients. No adverse patient complications were observed. Biocompatibility of devices was excellent. Insulin-positive β-cells were confirmed in encapsulated islets recovered from the implanted devices in all patient populations including the type I diabetic patients. Glucose-stimulated insulin release could be demonstrated in vitro from recovered islets. These data demonstrate that macroencapsulated human islets can survive at the subcutaneous site and that permselective membranes can be designed to protect against both allogeneic immune responses as well as the autoimmune component of type I diabetes.

A novel approach to neural transplantation in Parkinson's disease: Use of polymer-encapsulated cell therapy

Transplantation of dopaminergic neurons derived from fetal or adrenal tissue into the striatum is a potentially useful treatment for Parkinsons disease (PD). Although initially promising, recent clinical studies using adrenal autografts have demonstrated limited efficacy. The use of human fetal cells, despite promising preliminary results, is complicated by tissue availability and ethical concerns. An attractive alternative is based on encapsulating dopamine-producing cells into polymer capsules prior to transplantation. Polymer capsules can be fabricated to surround the cells with a semi-permeable and immunoprotective barrier. The semi-permeable membrane allows nutrients to enter the capsule, so the encapsulated cells will survive and function, and dopamine and other low molecular weight constituents to diffuse out into the host tissue. Thus, the technique allows use of unmatched human tissue (allografts), or even animal tissue (xenografts) without immunosuppression of the recipient. Cell-loaded polymer capsules can also be retrieved if necessary or desired. The demonstration that striatal implants of encapsulated dopamine-producing cells promote behavioral recovery in rodent and primate models of PD further suggests that cellular encapsulation may be a useful strategy for ameliorating the behavioral consequences of PD.

Transport characterization of membranes for immunoisolation

This study relates to the diffusive transport characterization of hollow fibre membranes used in implantable bio-hybrid organs and other immunoisolatory devices. Techniques were developed to accurately determine the mass transfer coefficients for diffusing species in the 10(2)-10(5) MW range, validated and then used to study one membrane type known to effectively immunoisolate both allografts and xenografts in vivo. Low-molecular-weight diffusing markers included glucose, vitamin B12 and cytochrome C; higher-molecular-weight molecules were bovine serum albumin, immunoglobulin G, apoferritin and a range of fluorescein-tagged dextrans. Overall and fractional mass transfer coefficients through the hollow fibres were determined using a resistance-in-series model for transport. A flowing dialysis-type apparatus was used for the small-molecular-weight diffusants, whereas a static diffusion chamber was used for large-molecular-weight markers. For diffusion measurements of small-molecular-weight solutes, convective artefacts were minimized and the effect of boundary layers on both sides of the membrane were accounted for in the model. In measuring diffusion coefficients of large-molecular-weight species, boundary layer effects were shown to be negligible. Results showed that for small-molecular-weight species (< 13,000 MW) the diffusion coefficient in the membrane was reduced relative to diffusion in water by two to four times. The diffusion rate of large-molecular-weight species was hindered by several thousand-fold over their rate of diffusion in water.

Polymer science for macroencapsulation of cells for central nervous system transplantation

The goal of encapsulated cell therapy research is to develop implants containing living xenogeneic cells to treat serious and disabling human conditions. The enabling concept is straightforward: cells or small clusters of tissue are surrounded by a selective membrane barrier which admits oxygen and required metabolites, releases bioactive cell secretions but restricts the transport of the larger cytotoxic agents of the bodys immune defense system. Use of a selective membrane both eliminates the need for chronic immunosuppression in the host and allows cells to be obtained from non-human sources, thus avoiding the cell-sourcing constraints which have limited the clinical application of general successful investigative trials of unencapsulated cell transplantation for chronic pain, Parkinsons disease, and type I diabetes. Target applications for encapsulated cell therapy include these same disorders as well as other disabilities caused by loss of secretory cell function which cannot be adequately treated by current organ transplantation or drug therapies and conditions potentially capable of responding to local sustained delivery of growth factors and other biologic response modifiers. Several types of device configurations are possible. Here we focus on easily retrieved, non-vascularized, macrocapsules. Such devices have four basic components: a hollow fiber or flat sheet membrane (usually thermoplastic based), cells (primary or dividing), and extracellular matrix (natural or synthetic) to promote cell viability and function, and other device components such as seals, tethers and radio-opaque markers. Choice of membrane and extracellular matrix polymers as well as issues surrounding implantation and biocompatibility evaluation are complex, inter-related, and ultimately driven by implantation site and delivery requirements. Cross species immunoisolated cell therapy has been validated small and large animal models of chronic pain, Parkinsons disease, and type 1 diabetes and is under active investigation by several groups in animal models of Huntingtons, Hemophilia, Alzheimers, ALS, and other CNS disorders.

Two PC12 pheochromocytoma lines sealed in hollow fiber-based capsules tonically release L-dopa in vitro.

Two PC12 cell-derived lines have been studied following encapsulation into polymer-based hollow fibers with respect to secreted catecholamines and their metabolites. Cellular encapsulation provides a chronic microperfusion environment within which basally secreted PC12 products can be readily measured. Encapsulated PC12 cells grown and held under the conditions specified in this report basally release amounts exceeding their total cellular stores of the dopamine precursor L-DOPA and the electrochemically active dopamine metabolites DOPAC and HVA during 45-min static incubations. Under these same conditions, these cells release less than 0.1% of their total cellular store of dopamine. Depolarizing incubations enhance dopamine secretion eightyfold and enhance secretion of L-DOPA, HVA, and DOPAC about twofold. The relative composition of products basally secreted differs between PC12-derived cell lines, and an inverse relationship exists between basal release of L-DOPA and total cellular store of dopamine. These results further indicate that selected PC12 cell lines are potentially a source of both dopamine and L-DOPA in therapeutic cellular replacement applications.

DESIGN OF MEMBRANE-BASED BIOARTIFICIAL ORGANS

The goal of encapsulated cell therapy research is to develop implants containing living xenogeneic cells to treat serious and disabling human conditions. The concept is straightforward: cells or small clusters of tissue are surrounded by a selective membrane barrier which admits oxygen and required metabolites, releases bioactive cell secretions but restricts the transport of the larger cytotoxic agents of the body’s immune defense system. Use of a selective membrane both eliminates the need for chronic immuno-suppression in the host and allows cells to be obtained from non-human sources, thus avoiding the cell-sourcing constraints which have limited the clinical application of generally successful investigative trials of unencapsulated cell transplantation for chronic pain1, Parkinson’s disease2, and type I diabetes.3–5 Cross-species immunoisolated cell therapy has been validated in small and large animal models of chronic pain6,7, Parkinson’s disease8,9, and Type 1 diabetes10–12, and is under active investigation by several groups in animal models of Huntington’s13, ALS14 and Alzheimer’s15–19. In addition, the first encapsulated therapy using xenografts in humans has been performed in chronic nain20 and ALS21.

Treatment of Central Nervous System Diseases with Polymer-Encapsulated Xenogeneic Cells

One of the major goals of neuroscience research is to develop effective treatments for clinical disorders. It is generally accepted that the discovery and development of effective, novel treatments is more efficient (i.e., faster and less costly) if those efforts are guided by a rational plan of action, based on careful consideration of the available data. Although tremendous technical and conceptual advances have been made in the neurosciences and considerable information about many neurological disorders has become available, it is still difficult to formulate a plan of action that can assure success because so many fundamental questions remain unanswered. For example, among the most problematic of the neurological disorders are those associated with the loss of brain neurons. Although we continue to learn more and more about the pathology and molecular biology of neurodegenerative diseases, congenital disorders, and strokes, very little is known about the specific mechanisms that mediate cell death. In fact, some research findings in these areas serve more to elucidate how little we truly understand about the etiology of these disorders and to stimulate the articulation of new questions that need to be addressed than to point the way toward a specific solution. As long as questions remain about the primary etiology and pathological mechanisms that mediate cell death, some uncertainty will remain about which avenues of research will produce effective preventative or palliative treatments for these disorders.

Chapter III.11 – Site-specific Treatment of Central Nervous System Disorders Using Encapsulated Cells

Publisher Summary The goal of encapsulated cell therapy research is to develop implants containing living xenogeneic cells to treat serious and disabling human conditions. The concept is straightforward: Cells or small clusters of tissue are surrounded by a selective membrane barrier that admits oxygen and required metabolites, releases bioactive cell secretions, but restricts the transport of the larger cytotoxic agents of the bodys immune defense system. Use of a selective membrane both eliminates the need for chronic immunosuppression in the host and allows cells to be obtained from nonhuman sources, thus avoiding the cell-sourcing constraints that have limited the clinical application of generally successful investigative trials of unencapsulated cell transplantation for chronic pain, Parkinsons disease, and Type I diabetes.