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% CO2 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.