[Study on relationship of dose-effect and time-effect of APA microencapsulated bovine chromaffin cells on pain treatment].

This study was to investigate the relationship of dose-effect and time-effect of Alginate-Polylysine-Alginate (APA) microencapsulated bovine chromaffin cells on the treatment of pain model rats. Using a rat model of painful peripheral neuropathy, the antinociceptive effects of APA microencapsulated bovine cells transplanted into the subarachnoid space was evaluated by cold allodynia test and hot hyperalgesia test. Compared with control group, the withdrawal difference with cell number 50 thousands groups, 100 thousands groups and 200 thousands groups was reduced (P < 0.05), and the difference decreased with the cells increases, indicating a significant analgesic effect. There was no significant difference between 400 thousands groups and 200 thousands groups. This analgesic effect maintained longer than 12 weeks. There was a positive correlation between the analgesic effect and the quantity of APA microencapsulated bovine chromaffin cells which were transplanted to treat pain model rats, and the effective antinociception remained longer than 12 weeks.

Past, present and future of human chromaffin cells: role in physiology and therapeutics.

Chromaffin cells are neuroendocrine cells mainly found in the medulla of the adrenal gland. Most existing knowledge of these cells has been the outcome of extensive research performed in animals, mainly in the cow, cat, mouse and rat. However, some insight into the physiology of this neuroendocrine cell in humans has been gained. This review summarizes the main findings reported in human chromaffin cells under physiological or disease conditions and discusses the clinical implications of these results.

Reduction of allodynia by intrathecal transplantation of microencapsulated porcine chromaffin cells.

Bovine chromaffin cells (BCCs) are well known to have analgesic effect to reduce acute or chronic pain when transplanted in the subarachnoid space and have been considered as an alternative therapy for pain management. However, due to recent concerns over risks associated with prion transmission, porcine tissue is considered to be an alternate xenogeneic source for clinical use. In the present study, we investigated whether microencapsulated porcine adrenal medullary chromaffin cells (PCCs) also have analgesic effect to reduce allodynia caused by neuropathic pain in chronic constriction injury model of rat. PCCs were isolated from a porcine adrenal medulla and then microencapsulated with alginate and poly. In in vitro tests, the microencapsulated PCCs were investigated whether they have an ability to release catecholamines responding to nicotine stimulation. The levels of catecholamines released from the microencapsulated PCCs were significantly higher than from microencapsulated BCCs. In addition, the microencapsulated PCCs released catecholamines and met-enkephalin responding to cerebral spinal fluid (CSF) retrieved from a neuropathic pain model. In in vivo tests, implantation of microencapsulated PCCs reduced both mechanical and cold allodynia in chronic constriction injury model of a rat whereas the microencapsulated BCCs reduced only cold allodynia under the same conditions. The injection of antagonist of opioid peptides reversed the reduction of cold allodynia in microencapsulated PCC-received animal. The levels of catecholamines in the CSF of rats after implantation of microencapsulated PCCs were significantly higher than in the control group. These data suggest that microencapsulated PCCs may be another effective source for the treatment of neuropathic pain.

Grafts of extra-adrenal chromaffin cells as aggregates show better survival rate and regenerative effects on parkinsonian rats than dispersed cell grafts.

The objective was to discern the neuroregenerative effect of grafts of extra-adrenal cells of the Zuckerkandl's paraganglion (ZP) in the nigrostriatal circuit, by using the retrograde model of parkinsonism in rats. The antiparkinsonian efficacy of two types of grafting procedures was studied (cell aggregates vs. dispersed cells), and GDNF and TGFbeta(1) (dopaminotrophic factors) as well as dopamine presence in extra-adrenal tissue was analyzed. Extra-adrenal chromaffin cells are noradrenergics, tissue dopamine is low, and they express both GDNF and TGFbeta(1). Grafts of cell aggregates, not of dispersed cells, exerted a trophic regeneration of the host striatum, leading to amelioration of motor deficits. Sprouting of spared dopaminergic fibers within the striatum, reduction of dopamine axon degeneration, and/or enhanced phenotypic expression of TH would explain striatal regeneration. Grafted cells as aggregates showed a better survival rate than dispersed cells, and they express higher levels of GDNF. Higher survivability and GDNF content together with the neurorestorative and dopaminotrophic action of both GDNF and TGFbeta(1) could account for striatal recovery and functional amelioration after grafting ZP cell aggregates. Finally, nigral degeneration and partial degeneration of ventral tegmental area were not precluded after transplantation, indicating that the trophic effect of grafts was local within the host striatum.

Spinal subarachnoid adrenal medullary transplants reduce hind paw swelling and peripheral nerve transport following formalin injection in rats.

Previous studies have demonstrated that adrenal medullary chromaffin cells transplanted into the spinal subarachnoid space significantly reduced pain-related behavior following hind paw plantar formalin injection in rats. The data suggests a centrally mediated antinociceptive mechanism. The spinal transplants may have effects on sciatic nerve function as well. To address this, the current study examined the effects of spinal adrenal transplants on hind paw edema and the anterograde transport of substance P (SP) that occur following formalin injection. Robust formalin-evoked edema, as well as hind paw flinching, was observed in striated muscle control-transplanted rats, which were not observed in adrenal-transplanted rats. To visualize transport of SP, the sciatic nerve was ligated ipsilateral to formalin injection and the nerve was processed 48 h later for immunocytochemistry. A significant formalin-induced accumulation of SP immunoreactivity (IR) was observed proximal to the ligation in control-transplanted rats. In contrast, there was significantly less SP IR observed from nerve of adrenal-transplanted rats, suggesting a diminution of anterograde axoplasmic transport by adrenal transplants. The change in SP IR may have been due to an alteration of transport due to formalin injection, thus, transport was visualized by the accumulation of growth-associated protein 43 (GAP43) at the ligation site. Formalin injection did not significantly increase proximal accumulation of GAP43 IR, indicating that formalin does not increase anterograde transport. Surprisingly, however, adrenal transplants significantly diminished GAP43 IR accumulation compared to control-transplanted rats. These data demonstrate that spinal adrenal transplants can attenuate the formalin-evoked response by modulating primary afferent responses.

Alginate bead fabrication and encapsulation of living cells under centrifugally induced artificial gravity conditions.

This study presents a novel method for the direct, centrifugally induced fabrication of small, Ca2+-hardened alginate beads at polymer-tube micronozzles. The bead diameter can arbitrarily be adjusted between 180-800 microm by the nozzle geometry and spinning frequencies between 5-28 Hz. The size distribution of the main peak features a CV of 7-16%, only. Up to 600 beads per second and channel are issued from the micronozzle through an air gap towards the curing agent contained in a standard lab tube ('Eppi'). Several tubes can be mounted on a 'flying bucket' rotor where they align horizontally under rotation and return to a vertical position as soon as the rotor is at rest. The centrifugally induced, ultra-high artificial gravity conditions (up to 180 g) even allow the micro-encapsulation of alginate solutions displaying viscosities up to 50 Pa s, i.e. approximately 50,000 times the viscosity of water! With this low cost technology for microencapsulation, HN25 and PC12 cells have successfully been encapsulated while maintaining vitality.

Testis-derived Sertoli cells survive and provide localized immunoprotection for xenografts in rat brain.

Transplantation of neural tissue into the mammalian central nervous system has become an alternative treatment for neurodegenerative disorders such as Parkinson's disease. Logistical and ethical problems in the clinical use of human fetal neural grafts as a source of dopamine for Parkinson's disease patients has hastened a search for successful ways to use animal dopaminergic cells for human transplantation. The present study demonstrates that transplanted testis-derived Sertoli cells into adult rat brains survive. Furthermore, when cotransplanted with bovine adrenal chromaffin cells (xenograft), Sertoli cells produce localized immunoprotection, suppress microglial response and allow the bovine cells to survive in the rat brain without continuous systemic immunosuppressive drugs. These novel features support Sertoli cells as a viable graft source for facilitating the use of xenotransplantation for Parkinson's disease and suggest their use as facilitators, (i.e., localized immunosuppression) for cell transplantation in general.

Viability and functionality of bovine chromaffin cells encapsulated into alginate-PLL microcapsules with a liquefied inner core.

Implantation of adrenal medullary bovine chromaffin cells (BCC), which synthesize and secrete a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides, has been proposed for the treatment of intractable cancer pain. Macro- or microencapsulation of such cells within semipermeable membranes is expected to protect the transplant from the host's immune system. In the present study, we report the viability and functionality of BCC encapsulated into microcapsules of alginate-poly-L-lysine (PLL) with a liquefied inner core. The experiment was carried out during 44 days. Empty microcapsules were characterized in terms of morphology, permeability, and mechanical resistance. At the same time, the viability and functionality of both encapsulated and nonencapsulated BCC were evaluated in vitro. We obtained viable BCC with excellent functionality: immunocytochemical analysis revealed robust survival of chromaffin cells 30 days after isolation and microencapsulation. HPLC assay showed that encapsulated BCC released catecholamines basally during the time course study. Taken together, these results demonstrate that viable BCC can be successfully encapsulated into alginate-PLL microcapsules with a liquefied inner core.

Intrathecal implants of microencapsulated xenogenic chromaffin cells provide a long-term source of analgesic substances.

Adrenal medullary chromaffin cells secrete several neuroactive substances including catecholamines and opioid peptides that produce analgesic effects in the central nervous system. This study was designed to investigate whether intrathecal microencapsulated chromaffin cells could release analgesic materials producing antiallodynic effects on the chronic neuropathic pain in rats induced by chronic constriction injury (CCI) of the sciatic nerve. Prior to intrathecal implantation, chromaffin cells were encapsulated with alginate and poly-L-lysine to protect them from the host immune system. Behavior tests were performed before CCI, 1 week later, and at 4, 7, 14, 21, 28 days postimplantation. At the end of study, we performed cerebrospinal fluid (CSF) collection and implant retrieval. We observed that intrathecal implantation of encapsulated xenogenic chromaffin cells reduced the mechanical and cold allodynia in a model of neuropathic pain. CSF levels of catecholamines and metenkephalin in the rats that received implants were higher than the controls. In addition, we observed chronic survival of implants. These results suggested that intrathecal microencapsulated chromaffin cells may represent a new approach to chronic neuropathic pain management.

Intrastriatal Grafting of Chromospheres: Survival and Functional Effects in the 6-OHDA Rat Model of Parkinson's Disease.

Cell replacement therapy in Parkinson's disease (PD) aims at re-establishing dopamine neurotransmission in the striatum by grafting dopamine-releasing cells. Chromaffin cell (CC) grafts produce some transitory improvements of functional motor deficits in PD animal models, and have the advantage of allowing autologous transplantation. However, CC grafts have exhibited low survival, poor functional effects and dopamine release compared to other cell types. Recently, chromaffin progenitor-like cells were isolated from bovine and human adult adrenal medulla. Under low-attachment conditions, these cells aggregate and grow as spheres, named chromospheres. Here, we found that bovine-derived chromosphere-cell cultures exhibit a greater fraction of cells with a dopaminergic phenotype and higher dopamine release than CC. Chromospheres grafted in a rat model of PD survived in 57% of the total grafted animals. Behavioral tests showed that surviving chromosphere cells induce a reduction in motor alterations for at least 3 months after grafting. Finally, we found that compared with CC, chromosphere grafts survive more and produce more robust and consistent motor improvements. However, further experiments would be necessary to determine whether the functional benefits induced by chromosphere grafts can be improved, and also to elucidate the mechanisms underlying the functional effects of the grafts.

Functional regeneration in a rat Parkinson's model after intrastriatal grafts of glial cell line-derived neurotrophic factor and transforming growth factor beta1-expressing extra-adrenal chromaffin cells of the Zuckerkandl's organ.

Intrabrain transplantation of chromaffin cell aggregates of the Zuckerkandl's organ, an extra-adrenal paraganglion that has never been tested for antiparkinsonian treatment, induced gradual improvement of functional deficits in parkinsonian rats. These beneficial effects were related to long survival of grafted cells, striatal reinnervation, and enhancement of dopamine levels in grafted striatum. Grafted cells were not dopaminergics, but they expressed glial cell line-derived neurotrophic factor (GDNF) and transforming growth factor-beta(1). These factors were detected in the host striatal tissue, indicating that chromaffin cells secreted them after grafting. Because glial cell line-derived neurotrophic factor possesses neurorestorative properties over dopaminergic neurons, and transforming growth factor-beta(1) is a cofactor that potentiates the neurotrophic actions of GDNF, functional regeneration was likely caused by the chronic trophic action of neurotrophic factors delivered by long-surviving grafted cells. This work should stimulate research on the clinical applicability of transplants of the Zuckerkandl's organ in Parkinson's disease.

NMDA antagonist peptide supplementation enhances pain alleviation by adrenal medullary transplants.

Spinal transplantation of adrenal medullary chromaffin cells has been shown to decrease pain responses in several animal models. Improved potency may be possible by engineering cells to produce greater levels of naturally derived analgesics. As an initial screen for potential candidates, adrenal medullary transplants were evaluated in combination with exogenously administered neuropeptides in rodent pain models. Histogranin is a 15-amino acid peptide that exhibits NMDA receptor antagonist activity. The stable derivative [Ser1]histogranin (SHG) can attenuate pain symptoms in some animal models. The formalin model for neurogenic inflammatory pain and the chronic constriction injury (CCI) model for neuropathic pain were used to evaluate the combined effects of chromaffin cell transplantation and intrathecal (IT) SHG injections. Animals were implanted with either adrenal medullary or control striated muscle tissue in the spinal subarachnoid space. For evaluation of formalin responses, animals were pretreated with SHG (0.5, 1.0, 3.0 microg) followed by an intraplantar injection of formalin, and flinching responses were quantified. Pretreatment with SHG had no significant effect on flinching behavior in control animals at lower doses, with incomplete attenuation only at the highest dose. In contrast, 0.5 microg SHG significantly reduced flinching responses in animals with adrenal medullary transplants, and 1.0 microg nearly completely eliminated flinching in these animals in the tonic phase. For evaluation of effects on neuropathic pain, animals received transplants 1 week following CCI, and were tested for thermal and mechanical hyperalgesia and cold allodynia before and following SHG treatment. The addition of low doses of SHG nearly completely eliminated neuropathic pain symptoms in adrenal medullary transplanted animals, while in control transplanted animals only thermal hyperalgesia was attenuated, at the highest dose of SHG. These results suggest that SHG can augment adrenal medullary transplants, and the combination may result in improved effectiveness and range in the treatment of chronic pain syndromes.

Intrathecal grafting of porcine chromaffin cells reduces formalin-evoked c-Fos expression in the rat spinal cord.

Chromaffin cells from the adrenal gland secrete a combination of neuroactive compounds including catecholamines, opioid peptides, and growth factors that have strong analgesic effects, especially when administered intrathecally. Preclinical studies of intrathecal implantation with xenogeneic bovine chromaffin cells in rats have provided conflicting data with regard to analgesic effects, and recent concern over risk of prion transmission has precluded their use in human clinical trials. We previously developed a new, safer source of adult adrenal chromaffin cells of porcine origin and demonstrated an in vivo antinociceptive effect in the formalin test, a rodent model of tonic pain. The goal of the present study was to confirm porcine chromaffin cell analgesic effects at the molecular level by evaluating neural activity as reflected by spinal cord c-Fos protein expression. To this end, the expression of c-Fos in response to intraplantar formalin injection was evaluated in animals following intrathecal grafting of 10(6) porcine or bovine chromaffin cells. For the two species, adrenal chromaffin cells significantly reduced the tonic phases of the formalin response. Similarly, c-Fos-like immunoreactive neurons were markedly reduced in the dorsal horns of animals that had received injections of xenogeneic chromaffin cells. This reduction was observed in both the superficial (I-II) and deep (V-VI) lamina of the dorsal horn. The present study demonstrates that both xenogeneic porcine and bovine chromaffin cells transplanted into the spinal subarachnoid space of the rat can suppress formalin-evoked c-Fos expression equally, in parallel with suppression of nociceptive behaviors in the tonic phase of the test. These findings confirm previous reports that adrenal chromaffin cells may produce antinociception by inhibiting activation of nociceptive neurons in the spinal dorsal horn. Taken together these results support the concept that porcine chromaffin cells may offer an alternative xenogeneic cell source for transplants delivering pain-reducing neuroactive substances.

Human chromaffin cell graft into the CSF for cancer pain management: a prospective phase II clinical study.

A number of pre-clinical studies have demonstrated the value of adrenal medullary allografts in the management of chronic pain. The present longitudinal survey studied 15 patients transplanted for intractable cancer pain after failure of systemic opioids due to the persistence of undesirable side-effects. Before inclusion, all the patients had their pain controlled by daily intrathecal (I-Th) morphine administration. The main evaluation criteria of analgesic activity of the chromaffin cell allograft was the complementary requirement of analgesics and in particular the consumption of I-Th morphine required to maintain effective pain control. Out of the 12 patients who profited from enhanced analgesia with long-term follow-up (average 4.5 months), five no longer required the I-Th morphine (with prolonged interruption of systemic opioids as well), two durably decreased I-Th morphine intake and five were stabilized until the end of their follow-up. Durable decline and stabilization were interpreted as indicative of analgesic activity by comparison with the usual dose escalation observed during disease progression. In most cases, we noted a relationship between analgesic responses and CSF met-enkephalin levels. The results of this phase II open study demonstrate the feasibility and the safety of this approach using chromaffin cell grafts for long-term relief of intractable cancer pain. However, while analgesic efficacy was indicated by the reduction or stabilization in complementary opioid intake, these observations will need to be confirmed in a controlled trial in a larger series of patients.

No detectable analgesic effects in the formalin test even with one million bovine adrenal chromaffin cells.

The present experiments were conducted to identify analgesic agents for transfection into immortalized adrenal chromaffin cell lines to maximize their analgesic potential. Analgesic agents known to be produced by adrenal chromaffin cells were infused intrathecally at a low dose (0.2 microg) which might conceivably be attained by adrenal chromaffin cell transplants. Numerous agents, administered individually and in two-factor combinations, produced significant analgesic effects in the formalin test. Before assessing the potential additive or synergistic effects of these analgesic agents with adrenal chromaffin cells, studies were conducted to demonstrate analgesic effects with adrenal chromaffin cells alone. Analgesic effects were previously reported in the literature with 80-100k intrathecal bovine adrenal chromaffin (BAC) cells; but in the present study 500k purified BAC cells failed to produce detectable analgesic effects. One million purified BAC cells also failed to produce analgesic effects in the formalin test. In a final study, even nicotine-stimulated release from one million purified BAC cells failed to produce analgesic effects in the formalin test. The fact that even one million nicotine-stimulated BAC cells failed to demonstrate therapeutic potential in these blinded experiments under conditions which were clearly sensitive to the analgesic agents produced by BAC cells, raises serious questions about the clinical utility of this experimental treatment.

Long-term alleviation of allodynia-like behaviors by intrathecal implantation of bovine chromaffin cells in rats with spinal cord injury.

Adrenal chromaffin cells produce analgesic substances, such as catecholamines and enkephalins, and intrathecal (i.t.) implantation of either allografted adrenal tissue or xenogenic chromaffin cells produce antinociception in animals. We evaluated the analgesic effect of bovine chromaffin cells in a model of central pain in which rats exhibit chronic allodynia-like behavior after photochemically induced ischemic spinal cord injury. Bovine chromaffin cells or endothelial cells were injected i.t. onto the lumbar spinal cord and their effects on mechanical and cold allodynia-like behaviors were studied for up to 8 weeks. The chronic allodynia-like behavior was stable for months without signs of remission and i.t. implantation of human endothelial cells did not alleviate the chronic allodynia-like behavior for the entire observation period. In contrast, 2 weeks after i.t. implantation of bovine chromaffin cells, the mechanical allodynia was abolished in the spinally injured rats, and the enhanced response to cold stimuli was significantly reduced. The overall effects were significant up to 8 weeks after i.t. implantation, although the anti-allodynic effect decreased towards the end of the observation period. No signs of side-effects were noted after i.t. implantation. The allodynia-like state was temporarily restored by naloxone (0.5 mg/kg) or phentolamine (0.3 mg/kg) injected intraperitoneally. Immunohistochemical examination revealed that tyrosine hydroxylase (TH)-positive chromaffin cells could be identified adjacent to the spinal cord up to 4 weeks after i.t. implantation, whereas at 8 weeks the TH-positive cells were sparse. It is concluded that bovine chromaffin cells stay viable in rat spinal cord for a considerable period of time after i.t. administration and alleviate chronic allodynia-like behavior in spinally injured rats, possibly through activation of opioid and alpha-adrenoceptors. The present results further document a new therapeutic approach for the treatment of chronic neuropathic pain.

Adrenal chromaffin cells on microcarriers exhibit enhanced long-term functional effects when implanted into the mammalian brain.

Rat adrenal chromaffin cells attached to either collagen-coated dextran (Cytodex 3) or glass bead microcarriers, both of 90-200 microns diameter, were used as dopamine-secreting implants in the caudate-putamen of rats with 6-hydroxydopamine-induced unilateral lesions of the substantia nigra. As controls, beads without cells and cells in suspension alone were implanted. Chromaffin cells adhered to microcarriers reduced apomorphine-induced rotation by 75% in lesioned animals. Animals that were lesioned but not receiving cell implants or receiving beads alone showed no reduction. Animals implanted with cells not attached to beads also showed a reduction in rotation but this effect lasted less than three months. Microcarrier-attached cells, however, maintained their effect in reducing rotation for at least eight months (rotations were reduced from a control mean of 10.9 +/- 1.4 to 3.6 +/- 1.1 turns/min) without any "drop-off" of the effect. Histological examination showed that eight months post-implant the cells pre-adhered to beads were still present and could be stained by anti-tyrosine hydroxylase antibody. Sections stained with hematoxylin-eosin showed no signs of an inflammatory response. In contrast to beads implanted into the striatum, Cytodex bead implants injected into the lateral ventricle induced a histopathological response appearing to involve the ependyma and choroid plexus. Results suggest that the striatal parenchyma but not the ventricle is amenable to studies using the microcarrier approach to transplantation.

Expression and regulation of CNTF receptor-alpha in the in situ and in oculo grafted adult rat adrenal medulla.

Cultured and transplanted adrenal medullary cells respond to ciliary neurotrophic factor (CNTF) with neurite formation and improved cell survival although the presence of the CNTF receptor-alpha (CNTFRalpha) has been unclear. This study show that CNTFRalpha mRNA was expressed in the postnatal day 1 as well as in the adult rat adrenal medulla. The highest CNTFRalpha mRNA signal was found in the ganglion cells of the adrenal medulla. After transplantation of adrenal medullary tissue the CNTFRalpha mRNA levels were down-regulated in the chromaffin cells. CNTF treatment of grafts did not normalize the receptor levels, but treatment with nerve growth factor (NGF) did. Thus, we demonstrate that CNTFRalpha mRNA is expressed in adrenal medulla, the levels becomes down-regulated after transplantation, but normalized after treatment with NGF.

One-year chromaffin cell allograft survival in cancer patients with chronic pain: morphological and functional evidence.

The control of chronic pain through transplantation of chromaffin cells has been reported over the past few years. Analgesic effects are principally due to the production of opioid peptides and catecholamines by chromaffin cells. Clinical trials have been reported with allografts consisting of whole-tissue fragments implanted into the subarachnoid space of the lumbar spinal cord (14,19,36). In the present study, allogeneic grafts were successfully used to control chronic pain in two patients over a period of 1 yr based on patient reported pain scores, morphine intake, and CSF levels of Met-enkephalin. Macroscopic examination at autopsy located the transplanted tissue fragments in the form of multilobulated nodules at the level of the spinal axis and cauda equina. Immunocytochemical microscopy showed neuroendocrine cells are positive for chromagranin A (CGA), and enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DbetaH). The results suggest that there is a relationship between analgesic effect, Met-enkephalin levels in CSF, and the presence of chromaffin cells surviving in spinal subarachnoid space.

Purification of adrenal chromaffin cells increases antinociceptive efficacy of xenotransplants without immunosuppression.

We have found that immunosuppression is necessary for the survival of xenogeneic adrenal medullary transplants. Because chromaffin cells are essentially nonimmunogenic, it is likely that the highly immunogenic "passenger" cells in the transplant preparation bring about rejection. This article describes a procedure that produces an essentially pure preparation of chromaffin cells for transplantation. Bovine adrenal medullary cells were isolated and differentially plated, resulting in a semipurified preparation of chromaffin cells. Ferromagnetic beads were added to the cell suspension, some of which were phagocytized by endothelial cells, which allowed their removal by exposure to a magnet. The remaining cells were then exposed to ferromagnetic beads coated with isolectin B4 from Griffonia simplicifolia and once again to a magnetic field. The "semipurified" preparation contained approximately 90% chromaffin cells, whereas the "highly purified" preparation was > 99.5% chromaffin cells as determined immunohistochemically. The immunogenicity of the two cell preparations was assessed in vitro by determining their capacity to evoke lymphocyte proliferation. Rat spleen lymphocytes were mixed with either a highly purified or semipurified population of bovine chromaffin cells. The results of this assay demonstrated that the highly purified preparation was a much weaker stimulant of lymphocyte proliferation than was the semipurified preparation and may demonstrate better graft survival in vivo. Transplantation via intrathecal catheter of either 80,000 or 250,000 cells from the highly or partially purified preparations onto the lumbar spinal cord of nonimmunosuppressed and non-nicotine-stimulated rats produced a cell number-dependent antinociception for both A(delta) and C fiber-mediated thermonociception at 6 days after transplantation. After 6 days and up to 28 days, only the "highly purified" preparation showed antinociception. These results suggest that nearly complete purification of bovine chromaffin cells minimizes immunorejection of xenogeneic transplants of these cells.