William J. Bell

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.

Numerous adrenal chromaffin cell preparations fail to produce analgesic effects in the formalin test or in tests of acute pain even with nicotine stimulation

&NA; Previous studies have reported that intrathecal implants of a variety of adrenal chromaffin cell preparations all produce analgesic effects in rodents. The major objective of the present study was to determine if any adrenal chromaffin cell preparations produce more robust analgesic effects than other cell preparations. The present study included adult rat adrenal chromaffin tissue allografts, purified adult bovine chromaffin cells, and polymer‐encapsulated calf adrenal chromaffin cells, all prepared according to previously published procedures, as well as purified calf adrenal chromaffin cells. Previous studies have also suggested that immunosuppression may play a role in graft survival, and potentially increase the magnitude of analgesic effects, so the present study included both immunosuppressed and non‐immunosuppressed groups (cyclosporin A, 10 mg/kg per day). Behavioral tests included the formalin test; and a dorsal tail‐flick, hot‐plate, and paw‐pinch test, conducted sequentially 2 min after systemic nicotine (0.1 mg/kg) to evoke release from the chromaffin cells, as previously reported. Analgesic effects related to morphine and nicotine were detected, and consistent differences in performance could be detected between individual animals. Surprisingly, no analgesic effects were detectable with any of the four chromaffin cell preparations, with or without immunosuppression, in the formalin test or with nicotine stimulation in tests of acute pain.

Intraventricular encapsulated calf adrenal chromaffin cells: viable for at least 500 days in vivo without detectable adverse effects on behavioral/cognitive function or host immune sensitization in rats

Numerous studies have reported that adrenal chromaffin cell transplants, including encapsulated xenogeneic adrenal chromaffin cells, have analgesic effects. However, in addition to efficacy, the clinical utility of encapsulated xenogeneic adrenal chromaffin cells for treatment of chronic pain is dependent on the duration of cell viability in vivo, and their relative safety. The objectives of the present study in rats were to: (1) examine encapsulated calf adrenal chromaffin (CAC) cells for evidence of viable cells and continued release of analgesic agents after an extended period in vivo; (2) determine if intraventricular encapsulated CAC cells produce detectable adverse effects on behavioral/cognitive function; and (3) test for evidence of host immune sensitization after an extended period of exposure to encapsulated xenogeneic adrenal chromaffin cells. Results of the present study suggest that some encapsulated CAC cells remain viable for nearly 1.5 years in vivo and continue to produce catecholamines and met-enkephalin. Post-explant device norepinephrine output was equivalent to amounts previously shown to produce analgesic effects with intrathecal implants. Encapsulated adrenal chromaffin cells also appeared relatively safe, even when implanted in the cerebral ventricals, with a lower side-effect profile than systemic morphine (4 mg/kg). There was no evidence that encapsulated CAC-cells implanted in the ventricles affected body weight, spontaneous activity levels, or performance in the delayed matching to position operant task which is sensitive to deficits in learning, memory, attention, motivation, and motor function. Finally, encapsulated CAC cells produced no detectable evidence of host immune sensitization after 16.7 months in vivo, although unencapsulated CAC cells produced a robust immune response even in aged rats. The results of the present study suggest that adrenal chromaffin cells remain viable in vivo for long periods of time, and that long-term exposure to encapsulated xenogeneic adrenal chromaffin cell implants appears relatively safe.

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.

The efficacy of intracranial PLG-based vaccines is dependent on direct implantation into brain tissue.

We previously engineered a macroporous, polymer-based vaccine that initially produces GM-CSF gradients to recruit local dendritic cells and subsequently presents CpG oligonucleotides, and tumor lysate to cell infiltrates to induce immune cell activation and immunity against tumor cells in peripheral tumor models. Here, we demonstrate that this system eradicates established intracranial glioma following implantation into brain tissue, whereas implantation in resection cavities obviates vaccine efficacy. Rats bearing seven-day old, intracranial glioma tumors were treated with PLG vaccines implanted into the tumor bed, resulting in retention of contralateral forelimb function (day 17) that is compromised by tumor formation in control animals, and 90% long-term survival (>100 days). Similar benefits were observed in animals receiving tumor resection plus vaccine implants into the adjacent parenchyma, but direct implantation of PLG vaccines into the resection cavity conferred no benefit. This dissociation of efficacy was likely related to GM-CSF distribution, as implantation of PLG vaccines within brain tissue produced significant GM-CSF gradients for prolonged periods, which was not detected after implantation in resection cavities. These studies demonstrate that PLG vaccine efficacy is correlated to GM-CSF gradient formation, which requires direct implantation into brain tissue, and justify further exploration of this approach for glioma treatment.

Seizure-Suppressant and Neuroprotective Effects of Encapsulated BDNF-Producing Cells in a Rat Model of Temporal Lobe Epilepsy

Brain-derived neurotrophic factor (BDNF) may represent a therapeutic for chronic epilepsy, but evaluating its potential is complicated by difficulties in its delivery to the brain. Here, we describe the effects on epileptic seizures of encapsulated cell biodelivery (ECB) devices filled with genetically modified human cells engineered to release BDNF. These devices, implanted into the hippocampus of pilocarpine-treated rats, highly decreased the frequency of spontaneous seizures by more than 80%. These benefits were associated with improved cognitive performance, as epileptic rats treated with BDNF performed significantly better on a novel object recognition test. Importantly, long-term BDNF delivery did not alter normal behaviors such as general activity or sleep/wake patterns. Detailed immunohistochemical analyses revealed that the neurological benefits of BDNF were associated with several anatomical changes, including reduction in degenerating cells and normalization of hippocampal volume, neuronal counts (including parvalbumin-positive interneurons), and neurogenesis. In conclusion, the present data suggest that BDNF, when continuously released in the epileptic hippocampus, reduces the frequency of generalized seizures, improves cognitive performance, and reverts many histological alterations associated with chronic epilepsy. Thus, ECB device-mediated long-term supplementation of BDNF in the epileptic tissue may represent a valid therapeutic strategy against epilepsy and some of its co-morbidities.