Primary Acinar Cell Isolation: A Method to Isolate Acinar Cells from Mouse Pancreas

Overview

This video describes the technique used for isolating pancreatic acinar cells--exocrine cells--containing a variety of digestive enzymes. Primary acinar cells are useful in the ex vivo study of acinar-to-ductal metaplasia (ADM)--an early event in developing pancreatic cancer.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the jove veterinary review board.

1. Acinar Cell Isolation

1. Sacrifice the mouse via CO₂ induction, and perform cervical dislocation. Immediately dissect the pancreas. To dissect the pancreas, first pin the paws of the mouse to the polystyrene lid, orient the mouse such that the tail is facing the researcher, and spray the abdomen with 70% ethanol.
   NOTE: The mouse used in the representative results was a 12-week old non-transgenic female with C57BL/6 background. Mouse selection is further discussed in the discussion section.
   1. Using a set of autoclaved scissors and forceps, lift the fur/skin with the forceps at the midline and use the scissors to make an incision through the fur and skin from the urethral opening to the diaphragm/ribcage area.
   2. Make additional incisions to the left and right such that the fur/skin is cut away to create a clear view of the abdominal cavity. Then, cut into the peritoneal lining (down the middle and to the right and left) and pull it away from the organs, as was done with the fur/skin.
   3. Lift the intestines with the forceps and put it to the left side of the mouse creating space to see the pancreas, which is light pink in color, and attached to the spleen, which is a dark red oval. The pancreatic tissue is distinguished by its soft, spongy texture. Cut out the pancreas which will run along the stomach and intertwine with the intestines.
   4. Separate the spleen from the pancreas. Then, put the pancreas in a 50 mL tube containing 10 mL of HBSS with 1x penicillin-streptomycin and bring this to the laminar flow hood.
      NOTE: The remaining steps of the protocol should be done utilizing sterile technique in a laminar flow hood.
2. Pour the pancreas and HBSS (with penicillin-streptomycin) into an empty weigh boat and, using forceps, wash the pancreas by swirling it. Then, move the pancreas with the forceps to a second weigh boat containing HBSS (with penicillin-streptomycin). Again, wash the pancreas by swirling.
3. Move the pancreas to the third HBSS (with penicillin-streptomycin)-containing weigh boat and begin cutting the pancreas into small pieces of 5 mm or less. Next, pour the pancreas pieces and HBSS into an empty 50 mL tube.
   1. To do this, use the forceps to move the pancreas pieces into the liquid as the weigh boat is being tipped. Pour once all the pieces are no longer attached to the weigh boat. Pick up any remaining pieces with the forceps and wash the forceps in the 50 mL tube containing the pancreas in HBSS with penicillin-streptomycin.
4. Centrifuge at 931 x g for 2 min at 4 °C and then remove the HBSS (and any fat that is floating) by pipetting it off with a 5 mL pipette.
5. Add 5 mL of collagenase (Make 40 mL of HBSS + 5% FBS, 20 mL of HBSS + 30% FBS, and 5 mL of 2 mg/mL collagenase in HBSS. Sterilize the collagenase by filtration (0.22 µm pore) and keep at room temperature. Keep each of the HBSS solutions on ice) to the pancreas. Ensure a sealed lid by wrapping the 50 mL tube in plastic paraffin film and then place it in an incubator shaking at 220 rpm for 20 min at 37 °C.
   NOTE: The collagenase digestion time may vary, as noted in the discussion. At the end of incubation, no large pieces of tissue should remain.
6. To stop the dissociation, place the pancreas-containing tube on ice and add 5 mL of cold HBSS + 5% FBS. Centrifuge at 931 x g for 2 min at 4 °C and then pipet off the supernatant using a 5 mL pipet.
7. Resuspend in 10 mL of HBSS + 5% FBS and centrifuge at 931 x g for 2 min at 4 °C. Pipet off the supernatant using a 5 mL pipet and repeat this step with another 10 mL of HBSS + 5% FBS.
8. Resuspend in 5 mL of HBSS + 5% FBS and, using a P1000, transfer 1 mL at a time through a 500 µm mesh into a 50 mL tube. Add an additional 5 mL of HBSS + 5% FBS through the mesh with a P1000 to wash any remaining pancreatic cells through the mesh.
9. Put the cell suspension from the 500 µm mesh through the 105 µm mesh by pipetting 1 mL at a time using a P1000. Next, gently pipet the cell suspension into a tube containing HBSS + 30% FBS. A layer of cell suspension will form at the top; upon centrifugation, the acinar cells will sink to form a pellet.
10. Centrifuge at 233 x g for 2 min at 4 °C. Remove the supernatant and resuspend the pellet in 1x Waymouth's complete media.
    1. If proceeding directly to embedment of cells in collagen or basement membrane matrix, resuspend in a volume of 7 mL per pancreas. If proceeding to adenoviral or lentiviral infection, resuspend in 4 mL per pancreas.

Materials

Name	Company	Catalog Number	Comments
37 °C shaking incubator	Thermo Scientific	SHKE4000-7
5% CO2, 37 °C Incubator	NUAIRE	NU-5500
50 ml tubes	Falcon	352070
Absorbent pad, 20" x 24"	Fisherbrand	1420662
BD Precision Glide Needle 21G x 1/2	Fisher Scientific	305167	Use as pins for dissection
Beaker, 600 mL	Fisherbrand	FB-101-600
Centrifuge	Beckman Coulter	Allegra X-15R Centrifuge
Collagenase Type I, Clostridium histolyticum	Sigma	C0130-1G	Create a 100 mg/ml solution by dissolving powder in sterile molecular biology grade water. When thawing one aliquot, dilute to 10 mg/ml, filter sterilize and place 1 ml aliquots at -20 °C.
Dexamethasone	Sigma	D1756	Create a 4 mg/ml solution by dissolving powder in methanol, aliquoting and storing at -20 °C.
Ethanol, 200 proof	Decon Laboratories	2701
Fetal Bovine Serum	Sigma	F0926-100mL
Forceps	Fine Science Tools	11002-12
Forceps	Fine Science Tools	91127-12
Hank's Balanced Salt Solution (HBSS), No calcium, No magnesium, No phenol red	Fisher Scientific	SH3048801
Ice bucket, rectangular	Fisher Scientific	07-210-103
LAB GUARD specimen bags (for mouse after dissection)	Minigrip	SBL2X69S
Parafilm	Bemis	PM992	Referred to as 'plastic paraffin film' in the manuscript
PBS	Fisher Scientific	SH30028.02
Penicillin-Streptomycin	ThermoFisher Scientific	15140122
Pipet tips, 10 µl	USA Scientific	1110-3700
Pipet tips, 1000 µl	Olympus Plastics	24-165RL
Pipet tips, 200 µl	USA Scientific	1111-1700
Pipet-Aid	Drummond
PIPETMAN Classic P10, 1-10 μl	Gilson	F144802
PIPETMAN Classic P1000, 200-1000 μl	Gilson	F123602
PIPETMAN Classic P20, 2-20 μl	Gilson	F123600
PIPETMAN Classic P200, 20-200 μl	Gilson	F123601
Pipettes, 10 ml	Falcon	357551
Pipettes, 25 ml	Falcon	357525
Pipettes, 5 ml	Falcon	357543
Polypropylene Mesh, 105 µm	Spectrum Labs	146436
Polypropylene Mesh, 500 µm	Spectrum Labs	146418
Scissors	Fine Science Tools	14568-12
Scissors	Fine Science Tools	14568-13
Sodium Bicarbonate (Fine White Powder)	Fisher Scientific	BP328-500
Sodium Hydroxide	Fisher Scientific	S318-500
Soybean Trypsin Inhibitor	Gibco	17075029
Spatula	Fisherbrand	21-401-10
Steriflip 50 ml, 0.22 micron filters	Millipore	SCGP00525
Type I Rat Tail Collagen	Corning	354236
Waymouth MB 752/1 Medium (powder)	Sigma	W1625-10X1L
Weigh boat, hexagonal, medium	Fisherbrand	02-202-101