Adrenal chromaffin glands from the adrenal mass of patients with renal cell cancer were isolated and transported at 4℃ in Locke's solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO₃, 5.6 mM glucose, and 5 mM HEPES) to the laboratory within 1 h. After removing connective tissue and blood vessels using an aseptic technique, the exterior surfaces of the exposed adrenal glands were rinsed and perfused with Locke's solution including 1% penicillin/streptomycin (10,000 unit/ml; Gibco, Carlsbad, CA, USA). The glands were then incubated in digestion buffer (0.2% [wt/vol] collagenase, Worthington, Lakewood, NJ, USA in Locke's solution) at 37℃ for 20 min. The adrenal medullary tissue was dissected from the surrounding cortical tissue, triturated, and incubated in digestion buffer at 37℃ for 1 h. The digested medullary tissue was then passed through a polypropylene screen (100 µm mesh). The cell suspension was centrifuged for 5 min at 70 × g in 40 ml Locke's solution. The supernatant was discarded and the cell pellet was resuspended and centrifuged for 5 min. The cell pellet was then resuspended in Dulbecco's Modified Eagle's Medium (DMEM)/F12 (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen) and inoculated into non-tissue culture dishes at a density of ~5 × 10⁶ cells/dish. The cells were incubated at 37℃ (5% CO₂) in a humidified, enriched environment overnight to allow for differential adherence of unwanted cell types such as fibroblasts and endothelial cells. The next day, the less adherent chromaffin cells were separated by gentle agitation and were plated into non-tissue culture-treated Petri dishes at the same density after centrifugation at 80 × g at 25℃ for 10 min.

The procedure was repeated once again to obtain highly purified chromaffin cells and replated into tissue culture-treated dishes at the same density in DMEM/F12 including 10% FBS, antibiotics (100 µM/ml penicillin/streptomycin, Gibco), and antimytotic agents (10^-5 M uridine and 10^-5 M 5-fluoro5-deoxyurdine, Sigma-Aldrich, St. Louis, MO, USA). A 10 µl aliquot of cells was stained with trypan blue solution (0.2%) and counted in a hemocytometer to estimate both yield and cell viability. To confirm the purification of HCCs, a 10 µl aliquot was stained with neutral red (0.9% in phosphate buffered solution), a dye that selectively reacts with monoamine-containing cells. In all, 95% HCCs were obtained through the multiple purification process.

Function of HCCs inside the capsules was evaluated by measuring the release of catecholamine by nicotine stimulation in vitro for 30 days. At various times during cell culture, microencapsulated HCCs were incubated in 12-well plates at a density of 1 × 10⁶ cells/well and washed three times with HBSS (Hanks' balanced salt solution, Gibco) containing 50 µM ascorbic acid. They were incubated in 1 ml HBSS for 60 min at 37℃ to obtain basal levels of catecholamines. These samples were collected, filtered with 0.2 µm filter and immediately frozen in at -70℃. After basal sampling, HCCs were incubated in 1 ml HBSS with 50 µM nicotine for 60 min under the same condition as the basal samples. Catecholamine levels were quantified using reverse phase high-performance liquid chromatography (HPLC, BAS-480; Bioanalytical Systems, West Lafayette, IN, USA) with electrochemical detection. The mobile phase was 0.07 M NaH₂PO₄, 0.2 mM octylsodium sulfate, 0.1 mM EDTA, and 8% methanol. The flow rate through a Waters Resolve C18µ Bondapak column was 1.0 ml/min. The electrochemical detector was set at +0.6 mV versus an Ag-AgCl reference electrode.

After 3-4 days of primary culture, HCCs were suspended in 1.4% (wt/vol) sodium alginate (Junsei, Tokyo, Japan) and 0.85% NaCl at a density of 5,106 cells/ml. Spherical droplets of this suspension were formed by extrusion through a syringe pump and gelled in 1.1% CaCl₂. Cell capsules were microcapsules having a diameter ranging from 100-300 mm. After washing twice in 0.85% NaCl, the capsules were coated with 0.05% poly-l-lysine (Sigma, St. Louis, MO, USA). The coated capsules were washed with 0.85% NaCl and then suspended for 7 min in 0.12% sodium alginate, which formed the outer layer of the membrane. After another wash with 0.85% NaCl, the capsules were then treated with 1 mM sodium citrate (pH 7.4) for 2 min. The sodium citrate was removed by washing twice with 0.85% NaCl and the encapsulated cell suspension was then distributed equally into 6-well culture dishes with DMEM/F12 supplemented with 10% FBS. The encapsulated cells were maintained at 37℃ (95% relative humidity, 5% CO₂).

Animals: Male Sprague-Dawley rats (200 to 300 g, Taconic) were group-housed with two per polycarbonate cage, in a temperature controlled room with a 12 : 12-hour light/dark cycle. Food and water were available ad libitum. All surgical procedures were performed by trained surgeons using aseptic techniques and anesthesia (pentobarbital, 50 mg/kg intraperitoneally, supplemented as necessary).

Neuropathic pain model: For the CCI surgery, the left common sciatic nerve was exposed at the mid-thigh level under 25x magnification. Four ligatures of size 40 chromic gut were tied loosely around the nerve with 1 mm spacing between knots so that the epineural circulation was preserved. At the time of tying, the ligatures just barely reduced the nerve diameter [7]. Over time, the ligatures evoked intraneural edema, resulting in constriction of the nerve. After surgery, the wound was washed with saline and the layers (fascia and skin) were closed with size 30 silk thread.

Implantation: One week after CCI surgery, rats were randomly allocated into the two groups. The cell loaded group received encapsulated chromaffin cells (n = 10), while control group received empty capsules (n = 8). The microcapsules were implanted into the lumbar subarachnoid space. A posterior L5 laminectomy was performed and then a small incision was made in the dura. Approximately 500 microcapsules (5 × 10² cells/capsule) were implanted into the subarachnoid space via a 24-gauge teflon catheter. After surgery, the wound was washed with saline and the layers (fascia and skin) were closed with size 30 silk thread.

Cold allodynia: A drop of acetone solution (97.7%) was applied under the hind paw:acetone evaporates quickly producing a sensation of cold. The duration of the withdrawal response (cold allodynia) was recorded with an arbitrary minimum value of 0.5 s and a maximum value of 40 s.

Opioid antagonist (Naloxone): To assess the potential contribution to pain reduction by opioid peptides released from the implanted microcapsules, all animals were injected with the opioid antagonist, naloxone, during the peak period of analgesia exerted by the chromaffin cells. Following behavioral testing 3 weeks after nerve ligation (2 weeks after implantation), the animals were injected with naloxone (2.0 mg/kg, S.C.). Fifteen minutes later, the degree of cold allodynia was assessed.

Data analysis: Results are reported as means ± SD. Statistical analysis of the cold allodynia data was performed using SPSS for Windows version 10.0 (SPSS Inc., Chicago, IL). We applied a student t test to compare the values from two independent samples and a paired t test to compare the values of means from two related samples. Statistical significance was considered when P < 0.05.

Quantities of norepinephrine are shown in Fig. 1. The concentration of norepinephrine after nicotine stimulation was significantly increased compared to basal levels (P < 0.05).

As shown in Fig. 2, animals exhibited little or no response to a cold stimulus (acetone) prior to ligation of the sciatic nerve. One week after nerve ligation, there was an enhanced withdrawal response to acetone evaporation. After the implantation of microencapsulated HCCs, the response duration was significantly reduced as compared to the controls which received empty microcapsules (P < 0.05, Fig. 2).

As shown in Fig. 3, fifteen minutes after the injection of naloxone, the degree of cold allodynia in control animals was unaltered. However, cold allodynia significantly increased after naloxone injection in rats with HCCs (P < 0.05). The severity of cold allodynia in rats with HCCs was less than in the control animals (P < 0.05).