Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Podsumowanie

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Streszczenie

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10⁶ acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

Wprowadzenie

A frequently encountered problem for research laboratories working on exocrine pancreatic tissue is the difficulty of cultivating acinar cells in vitro for a period of time long enough to allow a long-term experiment.

One factor impeding development of such culture systems is the intrinsic sensitivity of pancreatic tissue to experimental manipulation due to the high content in glycolytic, proteolytic, and lipolytic enzymes, which literally digest the pancreatic tissue when they are released during the isolation of pancreatic cells.

A second factor is the remarkable in vitro plasticity of acinar cells, which tend to lose their secretory characteristics and transdifferentiate to other mature cells, such as pancreatic ductal cells or hepatocyte-like cells ¹. In vitro, this cell plasticity varies with the experimental conditions (such as culture medium composition) ² and introduces a degree of complexity into the design of appropriate culture conditions for exocrine pancreatic cells ¹.

Several methods have been developed for the isolation and culture of acinar cells, first from the guinea pig pancreas ^3-5. Initially, those protocols involved digestion of pancreatic tissue with collagenase, chymotrypsin, and a protease cocktail, with ultimate isolation by vigorous mechanical dissociation. The pancreatic cells isolated in this way displayed abnormal structural and functional characteristics, notably a loss of apical structures and significant damage to their membrane receptors. Isolated cells remained viable for only 1 or 2 days.

Preparation of dispersed acini maintains their intra- and intercellular architecture, preserving cell membranes, limiting damage to surface receptors, and thus improving exocrine secretion in response to secretagogues ^6-8. As a result, this method offers the major advantage of extending acinar cell viability to 7-10 days in vitro. Furthermore, this method is currently preferred to acinar cell isolation ^9-12 because maintenance of intercellular contacts, including cell coupling by gap junctions, is an essential determinant of the exocrine pancreatic acinar cell phenotype ¹³.

As the dedifferentiation of acinar cells and their transdifferentiation to ductal cells is one of the proposed mechanisms for the genesis of aggressive exocrine pancreatic cancers ¹⁴, the dispersed acini model is also an adequate system to study pancreatic plasticity and its subsequent molecular mechanisms. Furthermore, in combination with the use of genetically modified animals ^15,16 and the development of gene transfer techniques (adenoviral ² or lentiviral transduction, use of nanoparticles, etc), this in vitro primary acinar cell model can be very useful in determining how various genetic dysfunctions affect the regulation of acinar cell differentiation or dedifferentiation and should provide better understanding of the molecular events responsible for the onset of pancreatitis, precancerous lesions, and changes in cell plasticity.

Isolation of dispersed acini is the approach we use in our laboratory to culture pancreatic acinar cells. We here describe and discuss the method used. It involves enzymatic dissociation of pancreatic tissue (with a bacterial collagenase) coupled to mechanical disruption without dissociation of acinar cells. While most protocols involve culturing the acini, either in suspension or on specially treated plastic substrates, we grow them in suspension only briefly (for 24 hr), seeding them afterwards onto matrix scaffolds if prolonged cell culture is required.

This protocol allows rapid isolation (in less than 1 hr) of dispersed pancreatic acini, sustainable for more than one week in culture. It allows isolation of more than 20 x 10⁶ acinar cells per mouse pancreas. Its simplicity makes it possible to process independently as many as 10 pancreases in parallel. By maintaining the intra- and intercellular architecture of acini and thus the acinar phenotype of isolated primary cells, this model constitutes a system of choice for the study of transdifferentiation mechanisms, as all other exocrine pancreatic models currently available are derived from pancreatic tumors displaying many genetic alterations leading to cellular transformation.

Protokół

All procedures were approved by an ethic committee under regulatory of governmental authority ("Comité d'Evaluation Commun au Centre Léon Bérard, à l'Animalerie de transit de l'ENS, au PBES et au laboratoire P4" (CECCAPP)). Mice were maintained in a specific pathogen-free animal facility at the "Plateforme AniCan, Centre Léon Bérard" (Lyon, France) and handled in compliance with the institutional guidelines.

A schematic representation of the procedure is shown in Figure 1.

1. Pancreas Dissection and Dilaceration (Day 0)

A very rapid dissection is critical for an optimal yield of extraction and to insure a good viability of cells in culture. In order to reduce the time needed for pancreas isolation, all instruments and equipment must be ready before the mouse euthanasia.

1. Euthanize mouse by CO₂ asphyxiation or cervical dislocation.

From this step, all procedures have to be performed under a sterile atmosphere (microbiological safety cabinet, level II) with sterile dissection equipment.

2. Fix the mouse and spray the mouse abdomen with 70% ethanol. With any dissecting scissors and forceps, make a V-shaped incision at the genital area and continue it up to the diaphragm to open completely the abdominal cavity.
3. Position the liver lobes against the diaphragm; they should remain there if the body cavity is open far enough. Pull the gut and the colon outside the abdominal cavity to your left, and find the rectum. With a pair of curved forceps and dissecting scissors, grab and section the rectum.
4. With the same pair of forceps, carefully unroll entirely the bowel from the rectum to the stomach by pulling the intestine on your left.

At this step, the pancreas can be distinguished as a small strip between the stomach and the beginning of the bowel. Its ligations with the spleen remain intact.

5. Using Noyes scissors and a pair of forceps, carefully cut the pancreas along the bowel and liberate it with the spleen from the rest of the digestive tract.
6. Grab the spleen and section the pancreas attached to it (Figure 2).

At this step, be sure that no mesenteric fat tissue and/or other adjacent tissue (spleen, bowel, etc) could be collected with the pancreas, to avoid cellular contamination.

For the rest of the procedure, all buffers must be prepared without calcium ion Ca²⁺ chelators to avoid the complete dissociation of the exocrine pancreatic tissue in single acinar cells.

7. Rinse the pancreas twice in Hank's Balanced Salt Solution (HBSS) 1x.

At this step, as fat tissue will float contrary to pancreas that will sink, it is easily possible to visualize and rapidly remove the contaminant white adipose tissue still attached to pancreas.

If the pancreas needs to be transported to the cell culture facility, it must be kept on ice in HBSS 1x.

8. Transfer the pancreas in a sterile Petri dish containing 5 ml of HBSS 1x. Using Noyes scissors and a scalpel, slice the pancreas in small pieces of 1 to 3 mm³ (Figure 3A).

2. Enzymatic and Mechanical Dissociations of Pancreas (Day 0)

1. Transfer them into a sterile 50 ml polypropylene tube.
2. Centrifuge for 2 min at 450 x g and 4 °C. Aspirate and discard the supernatant to remove cell fragments and blood cells.
3. Add 10 ml of collagenase IA solution (HBSS 1x containing 10 mM HEPES, 200 U/ml of collagenase IA, and 0.25 mg/ml of trypsin inhibitor) to pancreas sections. Using a 25 ml serological pipette, transfer them to a 25 cm² flask. Incubate it for 20-30 min at 37 °C. During this time (every 5 min), perform a mechanical dissociation by energetically moving back-and-forth the pancreas fragments about ten times, in sterile pipettes of decreasing size (25, 10, and 5 ml serological pipettes).

At this step, it is essential to frequently monitor the extent of the enzymatic dissociation of pancreatic sections.

4. When the pancreatic tissue seems to be well-dissociated (according to the disappearance of pancreatic fragments and to the increased turbidity of the solution) (Figure 3B), stop the enzymatic reaction by adding 10 ml of cold buffered washing solution (HBSS 1x containing 5 % Fetal Bovine Serum (FBS) and 10 mM HEPES).
5. Transfer it into a sterile 50 ml polypropylene tube and centrifuge for 2 min at 450 x g and 4 °C. Carefully aspirate and discard the supernatant to remove the collagenase IA solution.
6. Resuspend and wash the pellet with 10 ml of buffered washing solution. Centrifuge for 3 min at 450 x g and 4 °C. Carefully aspirate and discard the supernatant. Repeat this step two more times.

3. Filtration and Seeding of Dispersed Acini (Day 0)

1. Resuspend the cell pellet in 7 ml of Waymouth's medium containing 2.5 % FBS, 1 % Penicillin-Streptomycin mixture (PS), 0.25 mg/ml of trypsin inhibitor, and 25 ng/ml of recombinant human Epidermal Growth Factor (EGF).
2. Filtrate the cell mixture by allowing it to pass through a 100 μm filter to retain the non-digested fragments (ducts, blood vessels, and Langerhans islets). Pancreatic acinar structures (acinus of 10-15 cells) pass through.
3. Rinse the filter with 6 ml of Waymouth's medium containing FBS, PS, trypsin inhibitor, and EGF.

After this step, the cells have to be treated very carefully, to avoid any acini dissociation.

4. Seed the isolated acini in a 6-well culture dish (2 ml per well) (Figure 3C). Culture them at 37 °C under 5% (v/v) CO₂ atmosphere.

After this step, the acinar cells are cultured in suspension.

4. Acinar Cell Culture (Day 1 to 10)

1. Twenty-four hours after, transfer the acini (in suspension) into a new 6-well culture dish, to eliminate the contaminant cells and cellular remnants that have adhered overnight (Figure 4).

If the cell culture needs to be extended for several days or if the experimental conditions require cells grown in monolayer, it is recommended to transfer and seed acini on matrix scaffolds.

2. The day before seeding on matrix support (Day 0), coat a 6-well culture dish with type I collagen (5 μg/cm²). Add 1 ml of type I collagen solution (50 μg/ml in 0.02 M acetic acid, 0.2 μm-filtered) to each well and allow it to passively adsorb on plastic, during 1 hr at 37 °C (or overnight at 4 °C).
3. Aspirate the type I collagen solution and rinse the coated well twice with Phosphate-Buffered Saline 1x.
4. Allow the coated well to dry (under a microbiological safety cabinet) at least 12 hr before use.
5. Transfer the isolated primary acini (obtained at Step 4.1) into the type I collagen-coated 6-well culture dish and culture them in the same conditions as previously described (at 37 °C under 5% (v/v) CO₂ atmosphere). The cells adhere to the type I collagen substrate for 2 days.
6. On Day 3, change the culture medium to eliminate non-viable cells that have not adhered. With time in culture, the cells will progressively spread on the collagen-containing support. Change the culture medium every 3 days (Figure 5).

The isolated acinar cells obtained can be counted, after a complete mechanical dissociation by using a Thoma cell counting chamber. Note that isolated acinar cells cannot be maintained in culture afterwards.

The quality of the acinar culture obtained can be controlled by checking the expression of acinar specific markers such as Trypsinogen, Pancreas Transcription Factor 1 subunit Alpha, or Carboxypeptidase A1 (by immunocytochemistry or immunofluorescence experiments).

Wyniki

Figure 1 schematizes the "dispersed" acini method for primary acinar cells isolation. The critical steps, which have to be strictly respected during the protocol, are described in the discussion part.

To facilitate its removal, the pancreas has to be collected from the abdomen along with the attached spleen (Figure 2). Both organs need to be cut apart, and the residual fat tissue that could be still attached to the pancreas must be removed (Step 1.6).

...

Dyskusje

In this protocol, we describe a procedure for isolating pancreatic acinar cells. This method makes possible to isolate more than 20 x 10⁶ acinar cells per animal in less than 1 hr. Thanks to its rapid and simple implementation (as many as 10 pancreases can be independently processed per experiment in parallel), this protocol appears as a good compromise between existing isolation methods ^3-5,9-12,17 .

Critical steps/Trouble-shooting

Materiały

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
0.2 μm filter	Dutscher	146560
10 ml serological pipettes	Beckton Dickinson	357551
100 μm filter	Beckton Dickinson	352360
100 mm Petri dish	Beckton Dickinson	353003
1000 μl filter tips	Starlab	S1122-1830
20 μl filter tips	Starlab	S1120-1810
200 μl filter tips	Starlab	S1120-8810
25 ml serological pipettes	Beckton Dickinson	357535
5 ml serological pipettes	Beckton Dickinson	357543
50 ml polypropylene tube	Beckton Dickinson	352070
6-well plate	Beckton Dickinson	353046
Acetic acid 100%	VWR BDH Prolabo	20104.298
Collagenase IA	Sigma-Aldrich	C2676
Curved forceps, Dumont #7	World Precision Instruments	14188	To sterilize before use
Dissecting scissors, straight	World Precision Instruments	14393	To sterilize before use
Epidermal Growth Factor, human	Promokine	C-60180
Ethanol absolute (AnalaR Normapur)	VWR BDH Prolabo	20821.310
Fetal Bovine Serum	Lonza	14-801F
Forceps, Dumont #5	World Precision Instruments	14098	To sterilize before use
Hank's Balanced Salt Solution 1x	Gibco	14025050
HEPES 1 M (pH 6.98-7.30)	Lonza	17-737F
Incubator O2/CO2	Sanyo	MCO-19M
Inverted microscope	Nikon	Eclipse TS100
Matrigel	Beckton Dickinson	356234
Microbiological Safety Cabinet, level II	Faster	SafeFast Elite 212 S
Noyes scissors, sharp/sharp tips, German	World Precision Instruments	500228-G	To sterilize before use
Penicillin-Streptomycin mixture	Gibco	15140122
Phosphate Buffer Saline 10x	Gibco	14200067
Pipet-Aid	Drummond Scientific Company	Pipet-Aid XP
Pipetman P1000	Gilson	F123602
Pipetman P20	Gilson	F123600
Pipetman P200	Gilson	F123601
Refrigerated centrifuge	Eppendorf	5810R
Scalpel	Paramount Surgimed Ltd.	Disposable Scalpel Size 23
T25 flask, 25 cm2	Sigma-Aldrich	Z707481
Trypsin inhibitor, from Glycine Max	Sigma-Aldrich	T6522
Type I collagen	Beckton Dickinson	354236
Waymouth's medium	Gibco	31220-023