Encapsulated PC12 Cells Research Articles

Harnessing nerve-muscle cell interactions for biomaterials-based skeletal muscle regeneration.

Nerve cells secrete neurotrophic factors that play a critical role in neuronal survival, proliferation, and regeneration. However, their role in regulating myoblast behavior and skeletal muscle repair remains largely unexplored. In the present study, we investigated the effects of PC12 secreted signaling factors in modulating C2C12 myoblast behavior under physiologically relevant conditions. We showed that PC12 conditioned media modulated myoblast proliferation and differentiation in both 2D culture and 3D aligned electrospun fiber scaffold system in a dose-dependent manner. We further developed a biomimetic, tunable hydrogel consisting of hyaluronic acid, chondroitin sulfate, and polyethylene glycol as a 3D matrix encapsulating PC12 cells. The hydrogel-encapsulated PC12 cells promoted survival and proliferation of myoblasts in co-culture. Further proteomics analysis identified a total of 2,088 proteins from the secretome of the encapsulated PC12 cells and revealed the biological role and overlapping functions of nerve-secreted proteins for skeletal muscle regeneration, potentially through regulating myoblast behavior, nerve function, and angiogenesis. These experiments provide insights into the nerve-muscle interactions and pave the way for developing advanced biomaterials strategies incorporating nerve cell secretome for accelerated skeletal muscle regeneration.

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.

Grafting of encapsulated dopamine-secreting cells in Parkinson's disease: long-term primate study.

The transplantation of encapsulated dopamine-secreting cells into the striatum represents one potential means of treating Parkinson's disease. The present study investigated the ability of encapsulated PC12 cells, which are derived from rat pheochromocytoma, to supply L-dopa and dopamine into the primate brain in the long term and to effect functional improvement in the animals. Following polymer encapsulation, PC12 cells were transplanted into the striatum of hemiparkinsonian monkeys. The secretion of L-dopa and dopamine from the encapsulated cells, the morphology of these cells, the histology of the host striatum surrounding the capsule, and functional changes in the host animals were examined 1, 6, and 12 months after transplantation. Analysis of retrieved capsules revealed that the PC12 cells survived and continued to release L-dopa and dopamine even 12 months after transplantation. The histological response of the host brain surrounding the capsules was minimal and there were no signs of immunological rejection or tumor formation. The physical condition of the host animals was good for 12 months, and hematologic and cerebrospinal fluid analysis revealed that no animals suffered from infection or immunological reaction. These PC12 cell-grafted monkeys showed improvements in hand movements after transplantation, effects that lasted for at least 12 months. These results further support the potential use of this approach for the treatment of Parkinson's disease.

Dose control with cell lines used for encapsulated cell therapy.

Cell therapy-use of cells to deliver active factors-is an emerging technique in treatment of neurodegenerative disease. Successful devices maintain cell viability and functionality over extended implant periods. Use of dividing cell lines to deliver therapeutic factors has been studied extensively. One emerging issue is the tendency of cells to continue proliferation within the intracapsular environment-potentially outstripping nutrient supply. This work presents a method of controlling proliferation and delivering therapeutic molecules within a dose range. The method entails encapsulation into a hollow fiber device of discrete numbers of cell-containing microcarriers. Proliferation control is attained by embedding cell-containing microcarriers in nonmitogenic hydrogels. PC-12 cells secreting L-dopa and dopamine was the model cell line tested. Feasibility of the method in controlling growth of normally rapidly dividing cells in the intracapsular environment was demonstrated in vitro and in vivo. Control nonmicrocarrier PC-12 cell devices had approximately fourfold greater expansion in cell number compared to experimental microcarrier-containing devices over 4 weeks in vitro and in vivo after implant into rat striatum. Ability to control dose released over a several-fold range was demonstrated with encapsulated PC-12 cells delivering neurotransmitters and C2C12 mouse myoblast cells delivering neurotrophic factors (CNTF).

Therapeutic Potential of a Polymer-Encapsulated l-DOPA and Dopamine-Producing Cell Line in Rodent and Primate Models of Parkinson’s Disease

Encapsulation of cells within polymer membranes prior to transplantation provides a novel means of achieving continuous, site-specific delivery of therapeutic molecules to the CNS. The use of encapsulated dopamine-secreting cells that can be transplanted directly into the striatum has particular appeal for the treatment of Parkinson’s disease. This article provides a brief and timely review of the progress that has been made over the past decade using encapsulated PC12 cells as a means of delivering dopamine and l-DOPA to the striatum in rodent and primate models of Parkinson’s disease. The polymer membranes are well tolerated and biocompatible. Encapsulated PC12 cells survive in vivo for up to 6 mo, they release dopamine into the surrounding host striatum, and they clearly improve behavioral function in both dopamine-depleted rodents and primates. Although these results are promising, fundamental issues remain concerning the extent of dopamine diffusion from the polymer membranes and the number of devices needed for behavioral improvement, and the duration and consistency of cell viability and device output. Nevertheless, this technology appears to be a promising means of avoiding many of the practical, societal, and ethical issues that have been associated with other transplantation approaches.

Therapeutic potential of a polymer-encapsulated L-DOPA and dopamine-producing cell line in rodent and primate models of Parkinson's disease.

Encapsulation of cells within polymer membranes prior to transplantation provides a novel means of achieving continuous, site-specific delivery of therapeutic molecules to the CNS. The use of encapsulated dopamine-secreting cells that can be transplanted directly into the striatum has particular appeal for the treatment of Parkinson's disease. This article provides a brief and timely review of the progress that has been made over the past decade using encapsulated PC12 cells as a means of delivering dopamine and L-DOPA to the striatum in rodent and primate models of Parkinson's disease. The polymer membranes are well tolerated and biocompatible. Encapsulated PC12 cells survive in vivo for up to 6 mo, they release dopamine into the surrounding host striatum, and they clearly improve behavioral function in both dopamine-depleted rodents and primates. Although these results are promising, fundamental issues remain concerning the extent of dopamine diffusion from the polymer membranes and the number of devices needed for behavioral improvement, and the duration and consistency of cell viability and device output. Nevertheless, this technology appears to be a promising means of avoiding many of the practical, societal, and ethical issues that have been associated with other transplantation approaches.

Polymer-encapsulated PC-12 cells demonstrate high-affinity uptake of dopamine in vitro and 18F-DOPA uptake and metabolism after intracerebral implantation in nonhuman primates

Intracranial implantation of polymer-encapsulated PC-12 cells has been shown to improve motor behavioral performance in animal models of Parkinson's disease. The purpose of this blinded study was to examine whether such improvement is associated with the active uptake and metabolism of dopamine precursors by intracerebrally implanted polymer-encapsulated PC-12 cells. In an in vitro experiment we demonstrate that 3H-dopamine uptake by PC-12 cells was 10 8 fmol/min x 10 6 cells, and that this uptake can be specifically blocked 88% by the addition of 10 nM of nomifensine. In the in vivo experiments, polymer-encapsulated PC-12 cells were implanted in four MPTP-treated monkeys into the left deep parietal white matter (R1) or left striatum (R2–4). A fifth MPTP-treated monkey (R5) served as a control and received left striatal implants of empty capsules. 18F-Dopa Positron Emission Tomography (PET) imaging was performed on each monkey before and after implantation surgery by blinded investigators. PET images obtained 5–13 wk after implantation demonstrated well delineated focal areas of high 18F-dopa uptake in R1, R2, and R4. The focal area of high 18F-Dopa uptake in R1 precisely coregistered on a brain magnetic resonance image to the site of implantation. R3 (in whom the polymer-encapsulated PC-12 cells demonstrated poor cell survival upon explantation) and R5 (empty capsules) failed to demonstrate any area of increased 18F-dopa uptake in their PET images. Histological examination of the host brain revealed no sprouting of dopaminergic nerve terminals around the implantation sites of the polymer-encapsulated PC-12 cells. These results indicate that the previously noted behavioral improvement after intrastriatal implantation of polymer encapsulated PC-12 cells is at least in part due to their highly specific uptake and metabolism of dopamine precursors. Furthermore, these data suggest that polymer-encapsulated PC-12 cells can store, reuptake, and functionally replenish dopamine and therefore, may be an effective treatment for Parkinson's disease.

Evaluation of intracerebral grafting of dopamine-secreting PC12 cells into allogeneic and xenogeneic brain

The PC12 pheochromocytoma tumor cell line is derived from a rat adrenal medullary tumor and secretes dopamine. We have previously reported that grafted microencapsulated PC12 cells using agarose and poly(styrene sulfonic acid) survived in the xenogeneic brain without immunosuppression. To investigate whether unencapsulated PC12 cells form a tumor and how they provoke immunological reaction, PC12 cell suspension was implanted into the striatum of Sprague-Dawley rat (allogeneic graft) or guinea pig (xenogeneic graft) and histological analysis using Nissl stain and immunocytochemical analysis using antityrosine hydroxylase (TH) antibody were performed 1, 2, and 4 wk after transplantation. Host animals were not immunosuppressed. PC12 cells formed a mass 1 and 2 wk after transplantation both in allogeneic and xenogeneic brain. These grafted PC12 cells were immunoreactive to anti-TH antibody. Four weeks after transplantation, however, grafted PC12 cells in the allogeneic brain were only found within the restricted area near the site of implantation. In the xenogeneic brain, only the trace of grafted PC12 cells were found around the site of implantation 4 wk after transplantation. In both allogeneic and xenogeneic animals, a number of lymphocytes were found in and around the grafts at all period investigated. These findings indicate that PC12 cells could survive in the allogeneic or xenogeneic brain for 2 wk and were ultimately rejected by immunological reaction by 4 wk after transplantation. Implantation of encapsulated PC12 cells in the allogeneic or xenogeneic brain is considered a safe and effective method for delivering dopamine into the brain because PC12 cells will not form a tumor in the long-term even if capsules are damaged in some reason.

Evaluation of intracerebral grafting of dopamine-secreting PC12 cells into allogeneic and xenogeneic brain.

The PC12 phenochromocytoma tumor cell line is derived from a rat adrenal medullary tumor and secretes dopamine. We have previously reported that grafted microencapsulated PC12 cells using agarose and poly (styrene sulfonic acid) survived in the xenogeneic brain without immunosuppression. To investigate whether unencapsulated PC12 cells form a tumor and how they provoke immunological reaction, PC12 cell suspension was implanted into the striatum of Sprague-Dawley rat (allogeneic graft) or guinea pig (xenogeneic graft) and histological analysis using Nissl stain and immunocytochemical analysis using antityrosine hydroxylase (TH) antibody were performed 1, 2, and 4 wk after transplantation. Host animals were not immunosuppressed. PC12 cells formed a mass 1 and 2 wk after transplantation both in allogeneic and xenogeneic brain. These grafted PC12 cells were immunoreactive to anti-TH antibody. Four weeks after transplantation, however, grafted PC12 cells in the allogeneic brain were only found within the restricted area near the site of implantation. In the xenogeneic brain, only the trace of grafted PC12 cells were found around the site of implantation 4 wk after transplantation. In both allogeneic and xenogeneic animals, a number of lymphocytes were found in and around the grafts at all period investigated. These findings indicate that PC12 cells could survive in the allogeneic or xenogeneic brain for 2 wk and were ultimately rejected by immunological reaction by 4 wk after transplantation. Implantation of encapsulated PC12 cells in the allogeneic or xenogeneic brain is considered a safe and effective method for delivering dopamine into the brain because PC12 cells will not form a tumor in the long-term even if capsules are damaged in some reason.

Polymer-encapsulated PC-12 cells demonstrate high-affinity uptake of dopamine in vitro and 18F-Dopa uptake and metabolism after intracerebral implantation in nonhuman primates.

Intracranial implantation of polymer-encapsulated PC-12 cells has been shown to improve motor behavioral performance in animal models of Parkinson's disease. The purpose of this blinded study was to examine whether such improvement is associated with the active uptake and metabolism of dopamine precursors by intracerebrally implanted polymer-encapsulated PC-12 cells. In an in vitro experiment we demonstrate that 3H-dopamine uptake by PC-12 cells was 10(8) fmol/min x 10(6) cells, and that this uptake can be specifically blocked 88% by the addition of 10nM of nomifensine. In the in vivo experiments, polymer-encapsulated PC-12 cells were implanted in four MPTP-treated monkeys into the left deep parietal white matter (R1) or left striatum (R2-4). A fifth MPTP-treated monkey (R5) served as a control and received left striatal implants of empty capsules. 18-F-Dopa Positron Emission Tomography (PET) imaging was performed on each monkey before and after implantation surgery by blinded investigators. PET images obtained 5-13 wk after implantation demonstrated well delineated focal areas of high 18F-dopa uptake in R1, R2, and R4. The focal area of high 18F-dopa uptake in R1 precisely coregistered on a brain magnetic resonance image to the site of implantation. R3 (in whom the polymer-encapsulated PC-12 cells demonstrated poor cell survival upon explantation) and R5 (empty capsules) failed to demonstrate any area of increased 18F-dopa uptake in their PET images. Histological examination of the host brain revealed no sprouting of dopaminergic nerve terminals around the implantation sites of the polymer-encapsulated PC-12 cells. These results indicate that the previously noted behavioral improvement after intrastriatal implantation of polymer encapsulated PC-12 cells is at least in part due to their highly specific uptake and metabolism of dopamine precursors. Furthermore, these data suggest that polymer-encapsulated PC-12 cells can store, reuptake, and functionally replenish dopamine and therefore, may be an effective treatment for Parkinson's disease.

Somatic Delivery of Catecholamines in the Striatum Attenuate Parkinsonian Symptoms and Widen the Therapeutic Window of Oral Sinemet in Rats

Guidelines for clinical transplantation studies for Parkinson's disease emphasize that transplants should be considered as an adjunct to systemicl-DOPA, yet few preclinical studies have specifically assessed the potential of transplants as an adjunct to the clinical gold standard treatment. The objectives of the present study were to determine if encapsulated PC12 cells implanted in rats with severe unilateral dopamine depletions: (i) have a direct therapeutic effect on measures of parkinsonian symptoms; and/or (ii) increase the therapeutic window of oral sinemet in this model. Rats with severe unilateral dopamine depletions received striatal implants of encapsulated PC12 cells producing dopamine andl-DOPA. These rats were tested on a battery of behavioral measures of parkinsonian symptoms, at a range of doses of oral sinemet (0, 12, 24, and 36 mg/kg). Stereotypies/dyskinesias were also quantified after high doses of oral sinemet (36 and 50 mg/kg). The results confirm that parkinsonian symptoms can be quantified in rats with severe dopamine depletions, and the validity and clinical relevance of these measures are supported by the fact that the clinical gold standard treatment, oral sinemet, attenuates these parkinsonian symptoms. Somatic delivery of dopamine andl-DOPA, directly to the dopamine-depleted striatum, also attenuates parkinsonian symptoms. In fact, the magnitude of the therapeutic effect produced by continuous, site-specific, somatic delivery of dopamine andl-DOPA was larger than the effect produced by acute, systemic, oral sinemet. The beneficial effects of oral sinemet and striatal implants of catecholamine-producing devices were additive, but there were no adverse effects related to striatal catecholamine-producing devices, and these devices did not increase the adverse effects related to oral sinemet. Therefore, striatal implants of catecholamine-producing devices have direct therapeutic effects which are fairly robust, and they widen the therapeutic window of oral sinemet.

Continued presence of intrastriatal but not intraventricular polymer-encapsulated PC12 cells is required for alleviation of behavioral deficits in Parkinsonian rodents

To date, few studies have systematically evaluated the most appropriate location for grafting catecholaminergic cells as a potential treatment for Parkinson's disease (PD). The following study was conducted to determine 1) if placement of catecholamine-secreting encapsulated PC12 cells into the lateral ventricle of 6-OHDAtreated rats is as effective as intrastriatal implants on reducing apomorphine-induced rotational behavior, and 2) to determine if the survival of encapsulated PC12 cells is differentially affected by the implant site. Polymer-encapsulated PC12 cells were implanted into either the striatum or lateral ventricle of unilateral 6-OHDA-lesioned rats. Animals were tested for apomorphine-induced rotations over a 6-wk period. Only those animals that received intrastriatal implants of encapsulated PC12 cells showed a reduction in rotation behavior. Moreover, removal of the devices from the striatum resulted in a return to preimplant rotation levels. Postexplant neurochemical analyses demonstrated that the potassium-evoked L-dopa device output increased in vivo while the potassium-evoked dopamine output from the devices decreased over time in vivo. The location of the implant significantly affected catecholamine output from the PC12 cell-loaded devices. The increase in potassium-evoked L-dopa output was greatest, as was the decrease in potassium-evoked dopamine output, from those devices implanted in the striatum. Basal output of dopamine and DOPAC was also significantly higher from devices explanted from the lateral ventricle. These results demonstrate that the continued presence of intrastriatal implants of encapsulated PC12 cells is required to maintain the behavioral effects in 6-OHDA-lesioned rats. In addition, the site of implantation appears to affect device output. These results provide additional support for intrapa-renchymal delivery of L-dopa and dopamine via polymer encapsulation as a possible treatment for PD.

Continued presence of intrastriatal but not intraventricular polymer-encapsulated PC12 cells is required for alleviation of behavioral deficits in Parkinsonian rodents.

To date, few studies have systematically evaluated the most appropriate location for grafting catecholaminergic cells as a potential treatment for Parkinson's disease (PD). The following study was conducted to determine 1) if placement of catecholamine-secreting encapsulated PC12 cells into the lateral ventricle of 6-OHDA-treated rats is as effective as intrastriatal implants on reducing apomorphine-induced rotational behavior, and 2) to determine if the survival of encapsulated PC12 cells is differentially affected by the implant site. Polymerencapsulated PC12 cells were implanted into either the striatum or lateral ventricle of unilateral 6-OHDA-lesioned rats. Animals were tested for apomorphine-induced rotations over a 6-wk period. Only those animals that received intrastriatal implants of encapsulated PC12 cells showed a reduction in rotation behavior. Moreover, removal of the devices from the striatum resulted in a return to preimplant rotation levels. Postexplant neurochemical analyses demonstrated that the potassium-evoked L-dopa device output increased in vivo while the potassium-evoked dopamine output from the devices decreased over time in vivo. The location of the implant significantly affected catecholamine output from the PC12 cell-loaded devices. The increase in potassium-evoked L-dopa output was greatest, as was the decrease in potassium-evoked dopamine output, from those devices implanted in the striatum. Basal output of dopamine and DOPAC was also significantly higher from devices explanted from the lateral ventricle. These results demonstrate that the continued presence of intrastriatal implants of encapsulated PC12 cells is required to maintain the behavioral effects in 6-OHDA-lesioned rats. In addition, the site of implantation appears to affect device output. These results provide additional support for intraparenchymal delivery of L-dopa and dopamine via polymer encapsulation as a possible treatment for PD.

Preliminary report of polymer-encapsulated dopamine-secreting cell grafting into the brain

Polymer-encapsulated dopamine-secreting cell grafting is one of the most promising approaches for the treatment of Parkinson's disease. We microencapsulated dopamine secreting PC12 cells into agarose/poly(styrene sulfonic acid) complex and grafted them into the xenogeneic brain without immunosuppression. Dopamine secretion from the encapsulated cells was confirmed by high-performance liquid chromatography (HPLC) analysis before grafting. A large number of encapsulated PC12 cells survived in the brain 1 mo after transplantation and these cells were immunoreactive to tyrosine hydroxylase (TH) antibody, suggesting that these cells were secreting dopamine into the brain. There was no apparent immunological rejection or tumor formation. We concluded that microencapsulated PC12 cells survive in the xenogeneic brain without immunosuppression, and this grafting procedure is expected to be applied for the treatment of Parkinson's disease in the near future in combination with stereotaxic thalamotomy or pallidotomy.

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.

Functional Recovery in Hemiparkinsonian Primates Transplanted with Polymer-Encapsulated PC12 Cells

Cross-species neural grafting of cell lines immunoisolated by a permselective polymer membrane represents a promising source of neuroactive molecules for the treatment of neurodegenerative diseases. Utilization of a cell line of xenogeneic origin is advantageous since the transplanted cells will be rejected by the host immune system in case of breakage of the immunoisolating envelope. Polymer-encapsulated PC12 cells, a dopaminergic cell line derived from a rat pheochromocytoma, were transplanted in five Macaca fascicularis monkeys which had been previously rendered hemiparkinsonian by a unilateral carotid injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Well-preserved, tyrosine hydroxylase positive encapsulated PC12 cells were observed in the lesioned striatum for up to 5 months after implantation. Four out of five monkeys which received polymer-encapsulated PC12 cells showed significant behavioral improvement, whereas three monkeys implanted with either encapsulated bovine chromaffin cells or empty polymer envelopes showed no amelioration.

Polymer-Encapsulated PC12 Cells Promote Recovery of Motor Function in Aged Rats

The feasibility of using polymer-encapsulated PC12 cells to ameliorate the motor deficits in aged rats was evaluated. Spontaneous locomotion and motor coordination was evaluated in young (5-6 month) and aged (24-25 month) rats. Aged animals tested for spontaneous locomotor activity in Digiscan animal activity monitors were found to be hypoactive relative to young animals. Compared to the young animals the aged animals: (1) remained suspended from a horizontal wire for less time, (2) were unable to descend a wooden pole covered with wire mesh in a coordinated manner, (3) fell more rapidly from a rotating rod, and (4) were unable to maintain their balance on a series of wooden beams with either a square or rounded top of varying widths. Prior to implantation PC12 cell-loaded capsules were chromatographically characterized for catecholamine release. Following baseline testing, aged animals received either no implant, empty capsules, or PC12 cell-loaded capsules implanted bilaterally into the striatum. Three weeks following surgery, animals were retested and a significant improvement in balance on the rotorod and wooden beams was observed in those aged animals receiving PC12 cell-loaded capsules. No recovery was observed in the animals receiving PC12 cell-loaded capsules on any of the other motor tasks. Likewise, no improvement was observed on any behavioral measure in those animals receiving empty capsules. Histological analysis revealed the presence of numerous surviving tyrosine hydroxylase-positive PC12 cells within the capsules. Encapsulated PC12 cells survive following implantation into aged rats and such a technique may be useful for treating some of the behavioral consequences of aging.

TRANSPLANTATION OF POLYMER ENCAPSULATED PC12 CELLS: USE OF CHITOSAN AS AN IMMOBILIZATION MATRIX.

Polymer capsules were fabricated to encapsulate PC12 cells within a semipermeable and immunoprotective barrier. The inclusion of precipitated chitosan as an immobilization matrix within the polymer capsules increased the survival and physiological functioning of the PC12 cells. In an initial study, HPLC analysis revealed that the inclusion of a chitosan matrix resulted in an increased output of catecholamines from the encapsulated PC12 cells under both basal conditions, and following high potassium depolarization at 2 and 4 wk following encapsulation in vitro. Furthermore, implantation of cohort PC12 cell-loaded capsules into guinea pig striata revealed that chitosan enhanced PC 12 cell survival after 6 wk. A second study determined that 12 wk after implantation into guinea pig striatum, abundant tyrosine hydroxylase-positive PC12 cells were evenly distributed within capsules containing chitosan. The long-term biocompatibility of these implants was good as determined by the absence of inflammatory or immune cells, and minimal GFAP reactivity surrounding the implant site. In contrast, implantation of unencapsulated PC12 cells resulted in a marked host tissue reaction, and destruction of the implanted cells within 4 wk. It is concluded that the inclusion of precipitated chitosan as an immobilization matrix enhanced the viability of encapsulated PC12 cells, and that altering the internal milieu of polymeric capsules may represent an effective transplant strategy for ameliorating human diseases characterized by secretory cell dysfunction.

Growth of tumour cell lines in polymer capsules: ultrastructure of encapsulated PC12 cells.

Recent studies indicate that polymer-encapsulated PC12 cells release sufficient amounts of dopamine to significantly alter behavioural paradigms in animals with unilateral lesions of dopaminergic midbrain neurons. Because cell fine structure provides a useful measure for assessment of storage function, exocytosis, metabolism, cell activity and cell viability, we examined the ultrastructure of PC12 cells grown in semi-permeable polymer capsules maintained in vitro or implanted into the forebrain of rats or guinea pigs. Encapsulated PC12 cells remained viable and continued to divide for the entire evaluation period of six months. Overall morphologies of encapsulated PC12 cells were similar in both environments and they resembled PC12 cells grown in monolayer cultures. In short-term cultures, encapsulated PC12 cells typically contained abundant quantities of chromaffin cell-like granules. The encapsulated cells had initially abundant microvilli on their surfaces which decline in frequency over time. After long-term enclosure for ten weeks or more, fewer secretory granules were detected in the cytoplasm of cells in capsules cultured in vitro and in brain-implanted capsules. Some cells in implanted capsules had long slender filipodia that were not present on PC12 cells in cultured capsules. The morphological changes of PC12 cells may correlate with altered growth conditions such as serum and oxygen concentrations, the presence or absence of growth factors in different environments, and with changes of cell interactions related to cell densities and build up of debris within the capsules over time. Since dopaminergic PC12 pheochromocytoma cells remain viable in semi-permeable polymer capsules for at least six months, such 'cell-capsules' could provide an alternative to dopamine-secreting embryonic neural grafts in dopamine replacement therapies.

Polymer-encapsulated PC12 cells: long-term survival and associated reduction in lesion-induced rotational behavior.

Intrastriatal implantation of a dopaminergic cell line surrounded by a permeable, thermoplastic membrane was investigated as a method of long-term dopamine (DA) delivery within the central nervous system (CNS). An increase in DA release from PC12 cell-loaded capsules maintained in vitro was associated with an increase in mitotic activity of the encapsulated cell line. A significant reduction in apomorphine-induced rotational behavior was observed after PC12 cell-containing capsules were implanted into unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats, which was sustained for 24 wk. Four wk after implantation, microdialysis studies revealed the presence of DA near PC12 cell-containing capsules, which was comparable to extracellular striatal levels of unlesioned controls. Extracellular striatal DA was undetectable by microdialysis in lesioned animals near empty polymer capsules. Histological analysis after 24 wk in vivo demonstrated that encapsulated PC12 cells survived, continued to express tyrosine hydroxylase, and that encapsulation prevented tumorigenesis. The data suggested that the release of a diffusible substance, most likely DA, from an implant is sufficient to exert a long-term functional influence upon 6-OHDA unilaterally lesioned rats and that capsules containing DA-secreting cells may be an effective method of long-term DA delivery in the CNS.