SD rats, weighing 50-200 g were used in this study. Pancreas and its amiculas and vessels were washed twice in Hank’s solution and then minced. The fragments were digested with collagenase V (1 g/L, Sigma) at 37°C for 15- 20 min, ground and filtrated through an 80-mesh steal net to remove connective tissue, washed in cold Hank’s solution to stop the activity of collagenase. Then the filtrates were filtrated with a 400-mesh steal net to remove the small exocrine cells, red and white blood cells with the bigger ICCs kept on the net. The cell clusters on the net were collected and put into the Ficoll fluid (Euro-Collin) at the concentrations of 25 g/L, 23 g/L, 20 g/L and 11 g/L. The mixed liquors were centrifuged at 3 500 r/min at 4°C for 15 min. ICCs were washed three times. The purity of ICCs obtained in this way was (88.2 ± 7.6) %. These ICCs were cultured in non-insulin DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10 mmol/L nicotinamide, 5 mmol/L hydroxyethyl methylmethane sulfonic acid, 100 000 U/L penicillin and 1 g/L streptomycin, and maintained at 37°C in a high-humidity incubator with 50 mL/L CO₂ . A total of 400-1 000 purified ICCs were obtained from each rat. These ICCs were stained with trypan blue and the activity was 90%.

Five semipermeable membrane encysts were filled with 1 mL of fresh blood plasma, put into 200 mL of blood and maintained at 37°C in a high-humidity incubator with 50 mL/L CO₂ and kept agitated with magnetic force. Fluid in encysts was suctioned respectively 5 h later, then smeared and observed under a light microscope by Wright’s staining.

Eight semipermeable membrane encysts were filled with 1 mL of isotonic sodium chloride, put into 400 mL of isotonic sodium chloride containing 29.1 mmol/L glucose and kept agitated with magnetic force. The encysts were taken out at 1, 4, 8, 12, 16, 20, 24 and 28 min, respectively, and washed with isotonic sodium chloride. The fluid in the encysts was obtained and the concentration of glucose was detected.

About twelve thousands separated ICCs were divided into ten parts, five were encapsulated by BSM, five were routinely cultured. The culture medium was changed every three days. The concentration of insulin in the medium was detected on d 3, 6, and 15 respectively when the medium was changed.

After ICCs were cultured for 72 h, they were encapsulated by aseptic BSM. The volume of encysts was 1.5 cm × 1.5 cm × 1.5 cm. Each of them contained 6 000 to 10 000 ICCs.

The recipients were normal male local hybrid dogs weighing 7-14 kg. The dogs were intravenously anesthetized and their hair was shaved. The straight nick was made at frontal-occipital middle line perpendicular to the zygomatic arch. The temporalis muscles were pulled back to expose the skull, a hole was burred to enlarge the skull window to 3 cm × 3 cm, and the dura mater was cut open. The pallium was exposed to make a nick under the cerebral lateral fissure, and a fistula was made into ventricle between the rectal fissure and rectal lateral fissure to form a 2 cm × 2 cm × 1.5 cm tunnel. The brain tissue was wet compressed with 0.5 g/L orthophosphoric acid dexamethasone for 15 min[7] , then encysts were transplanted into the tunnel. The dura mater was relocated and compressed with gelatin sponges from the outside. Scalp and muscles were then sutured. Dogs were allowed to take food when consciousness was regained after anesthesia. Penicillin and streptomycin were used 30 min before and 1-3 d after operation.

The dogs were divided into control group ( n = 2) and experimental group( n = 4). In control group, a fistula was made, the stoma at the ventricle side was covered with gelatin sponges. Then the encysts were cut open to let the contents efflux into the brain tissue and the stoma at cortex side was covered with gelatin sponges. In experimental group, brain tissue was obtained 1 mo and 2 mo after encapsulated ICCs were transplanted.

According to the schedule, the brain was perfused with 4 g/L paraformaldehyde phosphate buffer through the ambi-common carotid artery. The grafts’ shape, color, location and changes of surrounding brain tissue were observed. Then part of the grafts and surrounding brain tissue were paraffin embedded, sectioned and stained with HE. Morphological changes of grafts and brain tissue were observed under a light microscope. Some grafts were divided into 1 mm × 1 mm × 1 mm piecers. These pieces were fixed with 25 g/L glutaraldehyde, dehydrated, embedded and then cut into ultrathin sections. The ultramicro-changes of the grafts were observed under a transmission electron microscope (JEM2000).

The first antibody was replced by normal mouse serum in the negative control group. Paraffin-embedded sections were used. The first antibody was mouse anti-human insulin used at 1:100 dilution and stained according to the avidin-biotin compound method (ABC), and then counterstained with hematoxylin to identify B cells in the grafts. The DNA fragmentation of transplanted B cells was identified by insulin-TUNEL double staining .

The BSM was not damaged during the whole process of the experiment in vitro. In lymphocyte-diffusion experiment, lymphocytes and any other blood cells were not immersed into the semipermeable membrane capsule, but glucose could rapidly diffuse into the capsule. The concentration of glucose in the capsule was 14.3 mmol/L after 8 min, which was 50% of the concentration out of the capsule. After 28 min. The concentration of glucose in the capsule reached the same environmental level. The concentration of insulin on d 3, 6 and 15 was (139.0 ± 3.4) mU/L, (78.7 ± 3.9) mU/L and (66.2 ± 2.9) mU/L, respectively in the experimental group , and (145.4 ± 4.2) mU/L, (79.4 ± 2.1) mU/L and (48.2 ± 4.1) mU/L, respectively in the control group. There was no conspicuous variability (P > 0.05) between the two groups at various time points. Insulin secreted from pancreatic islet cells could diffuse rapidly out of the capsule.

The dogs of both groups had no conspicuous behavior disorder. No dog in the experimental group was infected. The graft was deep gray and homogenous in the experimental group. It was connected with the ventricle at the inner side and adhered to the thickening dura mater at the lateral side as well as in contact with the cerebral parenchyma. The boundary was clear. Brain tissue around the graft had no obvious hemorrhage, necrosis and inflammatory abscess. Only residual semipermeable membrane was observed in the control dogs.

Under light microscope, the graft was consisted of epithelial cells and loose connective tissue, including epithelial cells, fibroblasts, collagen fiber, and small vessels. In the middle of them, there were lymphocytes and other kinds of white blood cells. Epithelial cells mostly adhered to the fibroblasts, collagen fiber or small vessels, and were arranged loosely in the connective tissue along the collagen fibers. The structure of small vessels was normal, while that of some parts of the semipermeable membrane was different, but there was no clump-like infiltration of lymphocytes.

Slight hyperplasia and hydropsia of glial cells were observed in the brain tissue around the graft, but severe infiltration of inflammatory cells was not observed.

Under electron microscope, secreting granulae were observed in the endochylema. The granulae were not only different in size but also diferent from those of pancreatic islet cells. Intact and contracted caryons, aggregated chromosome and typical apoptotic bodies were found in some cells (Figure 1).

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Figure 1 Typical morphology of apoptotic cells 1 mo after transplantation (× 6000). ↑collagen fiber, ↑WBC, ▲ apoptosis body.

No xenotransplanted cell disappeared in the control group.

Cytoplasms of many epithelial cells in the grafts of EG were stained yellow, indicating that they were islet B cells (Figure 2). The implanted B cells showed typical apoptosis by insulin-TUNEL double staining.

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Figure 2 Yellow staining of cytoplasma of some graft cells 1 mo after transplantation (× 6000).