Isolation and culture of pancreatic stellate cells      

 Pancreatic stellate cells (PaSCs or PSCs) are myofibroblast-like cells found in the areas of the pancreas that have exocrine function.  PaSCs are regulated by autocrine and paracrine stimuli and share many features with their hepatic counterparts, studies of which have helped further our understanding of PaSC biology.  Activation of PaSCs induces them to proliferate, to migrate to sites of tissue damage, to contract and possibly phagocytose, and to synthesize ECM components to promote tissue repair.  Sustained activation of PaSCs has an increasingly appreciated role in the fibrosis that is associated with chronic pancreatitis and with pancreatic cancer. Therefore, understanding the biology of PaSCs offers potential therapeutic targets for the treatment and prevention of these diseases.

1. Cells were isolated either by density gradient centrifugation after collagenase digestion of rat pancreas or by the outgrowth method using pancreas tissue of rats with cerulein pancreatitis.
2. To obtain cells with the inactivated fat-storing phenotype, cells were isolated from the pancreas of untreated male Wistar rats (200–300 g).
3. After the animals were anesthetized with pentobarbital sodium, the abdomen was opened, the common bile duct was ligated, and a cannula was inserted in the biliopancreatic duct.
4. The rats were exsanguinated, and 1 ml collagenase P (6 mg/5 ml) containing McCoy's 5A medium (6 mg/5 ml) was instilled intraductally.
5. The distended pancreas was removed, minced, and shaken in 4 ml collagenase P solution in an Erlenmeyer flask (37°C, 20 min).
6. Thereafter, minced pancreas was incubated three times for 3 min with EDTA containing McCoy's 5A medium followed by three washing steps with McCoy's 5A medium.
7. Thereafter, a second digestion was performed using 5 ml McCoy's 5A medium with 6 mg collagenase P, 5 mg hyaluronidase, 2 mg chymotrypsin, and 250 μl DNase (37°C, 20 min).
8. Dispersion was accomplished by up-and-down suction through cannulas with decreasing diameters.
9. After dissociation, the suspension was filtered through a 100-μm Nylon filter, and the filtrate was placed on top of 7.5 ml McCoy's 5A medium with 15 mg BSA and centrifuged for 5 min at 350 g.
10. Thereafter, supernatant was removed, and the cell pellet was resuspended in Ham's F-12-DMEM (1:1, vol/vol) with 10% FCS.
11. This cell suspension was layered on top of a Percoll-McCoy's 5A medium (7.5:2.5, vol/vol) density gradient and centrifuged for another 5 min at 180 g.
12. Once centrifuged, cells were collected from the top of the gradient, washed, suspended in primary cell system with 20% FCS, antibiotics, amphotericin, and L-glutamine, counted, and seeded in a density of 0.5 × 104 cells/cm2.
13. The purity of the isolated cells (as assessed by light, phase-contrast, fluorescence, and electronmicroscopy) was ∼80%.
14. With the first (24 h after seeding) medium change, most of the contaminating cells were removed, and the cell cultures were almost (>95%) free of impurities.
15. To demonstrate PSC activation (SMA, collagens I and III and fibronectin immunofluorescence, and real-time quantitative RT-PCR) and to study the expression of fibronectin splice variants, cells were cultured in the presence of 10% FCS beginning 24 h after seeding.
16. To demonstrate the effect of growth factors on PSC activation, cells were cultured in the presence of 0.1% FCS.
17. To obtain higher numbers of cells, male Wistar rats weighing between 200 and 450 g were injected intraperitoneally with supramaximal doses (10 μg • kg−1 • h−1) of cerulein.
18. The animals had free access to pelleted food and tap water. The rats were killed 2 days later by exsanguination under ether anesthesia.
19. The pancreas was removed, placed directly in primary cell supplemented with 10% FCS, 4 mmol/l L-glutamine, 100 U/ml penicillin, and 100 U/ml amphotericin, and then cut into small pieces of ∼1 mm3 under sterile conditions.
20. The pieces were placed in six-well plates and allowed to settle and adhere to the bottom of the plates in 4 ml primary cell supplemented with 10% FCS.
21. The plates were placed in a humidified atmosphere with 5% CO^₂ at 37°C.
22. Medium was changed after 12 and 24 h.
23. During the next 3–5 days, cells grew out from the tissue blocks and formed a confluent layer. After reaching confluence, monolayers were trypsinized and passaged 1:3.
24. Cell populations between passages 2 and 6 were used to study the effects of growth factors on cell proliferation and matrix synthesis.

Schematic of the cellular components of the exocrine pancreas.
 

Reference
Eric Schneider, Alexandra Schmid-Kotsas, Jinshun Zhao, Hans Weidenbach, Roland M. Schmid, Andre Menke, Guido Adler, Johannes Waltenberger, Adolf Grünert, and Max G. Bachem. Identification of mediators stimulating proliferation and matrix synthesis of rat pancreatic stellate cells. Am J Physiol Cell Physiol. 2001; 281: C532-C543.