Male Sprague-Dawley (SD) rats, weighing 230-250 g, were obtained from Central South University Laboratory Animal Department. For each isolation, one rat was anesthetized with pentobarbital sodium (30 mg/kg) intraperitoneally. Isolation of pancreatic islets was performed as previously described[9]. Briefly, after the output site of the duct from pancreas to duodenum was clamped, the pancreas was distended maximally with 20 mL collagenase (type IV, 1.5 mg/mL, Invitrogen) using a pulsed infusion technique via the portal duct of pancreas. The whole pancreas was removed from male SD rats and placed in a stationary water bath at 37 °C for 25 min to allow it to digest enzymatically. Digestate was agitated gently for 1 min every 5 min during incubation. The digestion process was stopped by adding 30 mL of cold Hank’s balanced salt solution (HBSS) supplemented with 5% fetal bovine serum (FBS). After having vortexed for 1 min to dissociate the islets from adherent acinar elements, the mixture was passed through a steel mesh filter with an aperture of 0.5 mm. The filtrate was subjected to the conventional method of histopaque density gradient centrifugation to separate islets from the digestion mixture.

DTZ stock solution preparation Ten milligrams of DTZ were dissolved entirely in 5 mL of dimethyl sulfoxide (DMSO), then filtered through a 0.2-µm nylon filter and stored briefly at -20 °C.

In vitro DTZ staining Ten microliters of the stock solution was added to 1 mL of culture medium. The culture dishes were incubated at 37 °C for 15 min in the DTZ solution. After the dishes were rinsed thrice with HBSS, clusters stained crimson red were identified as islets. These islets were hand-picked under a stereomicroscope. After hand-picking, the dishes were refilled with DMEM containing 10% FBS. The stain completely disappeared from the cells after 2 h.

Collagen preparation Collagen was extracted from rat tail tendons as previously described[10]. Tendons were excised from rat tails and sheared. Some connective tissues were removed carefully, washed twice with PBS and then soaked in 4 mmol/L acetic acid. After having stirred for two d at 4 °C, collagen was extracted. The extracts were centrifuged for 30 min at 10000 g and supernatant was collected into another distilled vessel for use. Protein concentration in the collagen was 1.0-1.5 mg/mL measured with an ultraviolet spectrophotometer.

Preparation of collagen gels Following sterile components (0.7 mL 10×DMEM/F12, 0.1 mL heat-inactivated low-endotoxin FCS, 0.1 mL 0.1 mol/L NaOH, 0.1 mL islet suspension) were carefully mixed to avoid bubbles. All components required to make collagen gels were placed on ice except for the islet suspension. Thus, the mixture resulted in a physiologic ionic strength and 1×DMEM/F12 in the final gel. The mixture was incubated at 37 °C in 50 mL/L CO₂ and then gelation was completed after 60 min.

Islet culture Islets embedded in collagen gel were divided into three groups and incubated under the following conditions.

3D culture alone (control group) DMEM/F12 (penicillin 100 U/mL, streptomycin 100 μg/mL, 10 mmol/L HEPES)+10% FCS+nicotinamide (10 mmol/L).

3D culture+EGF: DMEM/F12 (penicillin 100 U/ml, streptomycin 100 μg/mL, 10 mmol/L HEPES)+10% FCS +EGF (100 ng/mL)+nicotinamide (10 mmol/L).

3D culture + EGF + CT: DMEM/F12 (penicillin 100 U/mL, streptomycin 100 μg/mL, 10 mmol/L HEPES)+10% FCS+EGF (100 ng/mL)+CT (100 ng/mL)+nicotinamide (10 mmol/L).

Samples were taken at the 1^st, 3^rd, 5^th, 16^th, 28^th, 40^th, 52^nd, 76^th, 88^th,120^th, 168^th h. After old culture medium was piped out thoroughly, islets were rinsed twice and fresh medium was added. Culture media were sampled after 1 h and frozen at -20 °C. Insulin content in samples was detected by radioimmunoassay (RIA).

Islets were hand-picked from slides pretreated with poly-L-lysine and fixed in 4% paraformaldehyde (PFA). Then the fixed islets were processed for routine histology and immun-ostained for insulin (mouse anti-insulin, Boster, Wuhan) and cytokeratin 19 (mouse anti-cytokeratin 19, Boster, Wuhan) using the streptavidin-biotin complex (SABC) method following the instructions of kit (Boster, Wuhan). For cytokeratin 19, islet cells were pretreated with 0.1% trypsin. The sections were incubated overnight at 4 °C with appropriate primary antibodies. Negative controls involved omission of the primary antibodies.

The collagen matrix was dissolved in 0.25 mg/mL collagenase and islets were washed thrice. Total RNA was extracted from cultured rat islets using TRIzol (GIBCO BRL) and reverse-transcribed into cDNA with TaKaRa RNA PCR kit (AMV) Ver. 2.1 (TaKaRa, Japan) according to the standard procedure. cDNA samples were subjected to PCR amplification with specific primers. The cycling parameters were pre-denaturation at 94 °C for 2 min, denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and elongation at 72 °C for 1 min (34 cycles). Table 1 summarizes the sequences of the PCR primers used in this study.

All data were expressed as mean±SD with n = 3 at each time point. The difference between time points with respect to insulin content was evaluated by one-way ANOVA. P<0.05 was considered statistically significant.

We used DTZ to identify islets which were stained crimson while acinar and ductal tissue failed to incorporate the stain. Islets were stained with DTZ and hand-picked after density gradient centrifugation. The stain completely disappeared from the cells after 2 h (Figure 1).

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Figure 1 Islets stained crimson with DTZ (A), purified by density gradient separation and hand-picking (B), and loss of color 2 h after islet staining (C) (×100).

Islets embedded in collagen gel could retain their natural shape, while those in monolayer cultures could not (Figure 2).

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Figure 2 Shape of islets culture in collagen gel (A ×100) and in monolayer (B ×200 ).

The islets in groups 1 and 2 were not different in shape on d 1, 3 and 5. But some islets in group 3 continually enlarged in these days (Figure 3). The percentage of islets undergoing cystic transformation was increased over the time-course of the culture period in group 3.

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Figure 3 Islets of group 3 cystic transformed gradually in the process of culture. A: d 1 in culture; B: d 3 in culture; C: d 5 in culture (inverted microscope ×100).

During the culture time, the abilities of islets to secrete insulin in three groups were all decreased. Five hours later, islets lost their secreting ability faster in group 3 than in groups 1 and 2 (P<0.05 or 0.01) (Figure 4).

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Figure 4 Insulin contents in culture media of three groups at different time points were detected using RIA. (^aP<0.05: group 1 vs group 3; ^bP<0.01: group 1 vs group 3; ^cP<0.05: group 2 vs group 3; ^dP<0.01: group 2 vs group 3).

Islets cultured for 1, 3, 5 d were insulin positive and cytokeratin 19 negative in groups 1 and 2.

In group 3, islets were insulin positive and cytokeratin 19 negative on d 1. There was a decrease in expression of insulin mainly in the center of islets and some islet cells around the cystic spaces began to express protein cytokeratin 19 on d 3. Only a few cells in the islets expressed insulin continually on d 5, but most cells expressed cytokeratin 19 (Figures 5 and 6).

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Figure 5 Islets of group 3 stained for insulin by immunocytochemistry. A: d 1 (strongly stain); B: d 3 (decreased stain); C: d 5 (lightly stain). (DAB stain and counterstained with hematine crystal ×100).

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Figure 6 Islets of group 3 stain for cytokeratin 19. A: d 1 (did not stain); B: d 3 (lightly stain); C: d 5 (strongly stain).

Freshly isolated islets expressed islet characteristic genes (pdx-1 and insulin) but did not express duct characteristic gene (cytokeratin 19) (Figure 7). After incubation for 7 d, islets in the three groups could express genes (insulin and pdx-1), but cytokeratin 19 was only expressed in group 3 (Figure 8).

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Figure 7 Pdx-1, insulin genes were expressed in freshly isolated islets, but cytokeratin 19 was not expressed (lane 1: Pdx-1; lane 2: Insulin; lane 3: β-actin; lane 4: cytokeratin 19).

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Figure 8 Gene’s expression of islets of three groups cultured for 7 d; A: all of three groups expressed insulin; B: all of three groups expressed pdx-1; C: only group 3 expressed CK19 which group 1 and group 2 did not express.