Primary acinar cell isolation, protein expression or activity modulation, culture, and down-stream applications are abundantly useful in the ex vivo study of acinar-to-ductal metaplasia (ADM), an early event in the development of pancreatic cancer.
The differentiation of acinar cells to ductal cells during pancreatitis and in the early development of pancreatic cancer is a key process that requires further study. To understand the mechanisms regulating acinar-to-ductal metaplasia (ADM), ex vivo 3D culture and differentiation of primary acinar cells to ductal cells offers many advantages over other systems. With the technique herein, modulation of protein expression is simple and quick, requiring only one day to isolate, stimulate or virally infect, and begin culturing primary acinar cells to investigate the ADM process. In contrast to using basement membrane matrix, the seeding of acinar cell clusters in collagen I extracellular matrix, allows acinar cells to retain their acinar identity before manipulation. This is vital when testing the contribution of various components to the induction of ADM. Not only are the effects of cytokines or other ectopically administered factors testable through this technique, but the contribution of common mutations, increased protein expression, or knockdown of protein expression is testable via viral infection of primary acinar cells, using adenoviral or lentiviral vectors. Moreover, cells can be re-isolated from collagen or basement membrane matrix at the endpoint and analyzed for protein expression.
Acinar-to-ductal metaplasia (ADM) is a protective mechanism during pancreatitis and a key process driving pancreatic cancer development1 that requires further mechanistic insight. While inflammation-induced ADM is reversible2, oncogenic KRAS mutations, which are present in 90% of pancreatic cancer cases3, prevent differentiation back to an acinar phenotype4,5,6. Culturing and differentiating primary acinar cells into ductal cells in 3D culture allows for study of the molecular mechanisms regulating the ADM process, which is difficult to study in vivo. Such ex vivo studies enable real-time visualization of ADM, its drivers and its regulators. Several drivers of the ADM process, as well as mechanistic insight on their downstream signaling pathways, have been identified or verified using the here described method. These include ADM induction by TGF-α (transforming growth factor alpha)-mediated expression of MMP-7 (matrix metalloproteinase-7) and activation of Notch7, as well as RANTES (regulated on activation, normal T cell expressed and secreted; also known as chemokine ligand 5 or CCL5) and TNFα (tumor necrosis factor alpha)-induced ADM through activation of NF-κB (nuclear factor-κB)8. An additional mediator of ADM is oncogenic KRas9,10, which causes a rise in oxidative stress that enhances the ADM process through increased expression of EGFR (epidermal growth factor receptor) and its ligands, EGF (epidermal growth factor) and TGF-α11.
While use of pancreatic cancer cell lines is common for in vitro studies, primary cell cultures offer many advantages. For instance, primary acinar cells from non-transgenic mice or mice harboring initiating mutations, such as KrasG12D, are the most appropriate model for studying the early event of ADM because the cells have few and controlled mutations, which are known to be present early in pancreatic cancer development. This is in contrast to many pancreatic cancer cell lines which have multiple mutations and varied expression based on passage number12. Additionally, the signaling pathways identified by such means have been verified by animal studies. One caveat of using primary acinar cells is that they cannot be transfected as typical cancer cell lines can.
The method herein details the techniques of acinar cell isolation, 3D culture that mimics ADM by adding stimulants or modulating protein expression via adenoviral or lentiviral infection, as well as re-isolation of cells at the endpoint for further analyses.
All animal work was approved by the Mayo Clinic IACUC.
1. Preparation of Materials, Solutions, and 3-dimensional Matrix Bases
2. Acinar Cell Isolation
3. Viral Infection
4. Embedment of cells in Collagen or Basement Membrane Matrix
5. Harvesting Cells from Collagen or Basement Membrane Matrix
Completion of the protocol herein occurs within one day and upon stimulation, ADM is seen in 3-5 days. Figure 1 depicts the sequence of the method, whereby steps 1 through 4 are completed on the first day. This includes preparation, acinar isolation, viral infection and embedment in collagen or basement membrane matrix. While the basement membrane matrix induces ADM, acinar cells within collagen I require a stimulus, such as TGF- α, to undergo differentiation to ductal cells. On day 1, cells in collagen or basement membrane matrix have a grape-like round appearance and if utilizing viral infection with a vector expressing a fluorescent protein, infection efficiency can be checked. By day 5, cells in collagen will form ducts if given a stimulus at the time of embedment and as a supplement in the media, which is changed on days 1, 3, and 5. Cells embedded in basement membrane matrix will form ducts in the absence of a stimulus, but upon stimulation larger ducts will form, as seen in Figure 2.
Figure 1: Schematic of the procedure to isolate murine primary acinar cells, modulate protein expression and/or stimulate acinar-to-ductal metaplasia, and embed cells in collagen or basement membrane matrix. The workflow includes isolation of acinar cells, which includes dissection of the pancreas, digestion, and filtration of the cells. Please click here to view a larger version of this figure.
Figure 2: Representative results of primary acinar cells plated within collagen or basement membrane matrix after adenoviral infection and stimulation with TGF-α. Primary acinar cells from a non-transgenic mouse were infected with GFP-adenovirus, embedded in collagen I or basement membrane matrix, and stimulated with or without TGF-α (50 ng/mL). Twenty-four hours post-infection with GFP adenovirus, images were captured to show infection efficiency. At five days post-infection, the images denote the differences in cells stimulated with or without TGF-α in collagen or basement membrane matrix. Duct-like structures are indicated by white arrowheads and scale bars represent 50 µm. Images were obtained using a confocal microscope with the EC Plan-Neofluar 10x/0.3 M27 objective. Please click here to view a larger version of this figure.
The relatively short amount of time for isolation, infection, and plating primary acinar cells is an advantage of this method. In contrast, culturing acinar cells from an explant outgrowth requires little hands-on time, but it takes seven days for the outgrowth of acinar cells13. An alternative protocol for acinar isolation14 notes a very short method for obtaining acinar cells; however, EGF is an essential component in keeping acinar cells alive when isolated using that protocol. Since EGF stimulates differentiation of acinar cells to ductal cells, the method herein, which does not require ADM-stimulating components for culture, is better for studying mechanisms of induction or regulation of ADM. In this method, acinar cell identity is maintained during culturing in collagen unless stimulated to differentiate into ductal cells. Moreover, difficulties in transfecting primary cells are overcome via manipulating protein expression by viral infection.
This procedure offers direct study of ADM, by which visualization of ductal structure induction can occur via brightfield and/or immunofluorescence microscopy. For imaging applications, chamber slides (noted in the Table of Materials) are best. Fixation for 15 min at 37 °C with 4% paraformaldehyde will fix cells embedded in basement membrane matrix without disturbing ductal structures. During this incubation, the basement membrane matrix will liquify and can be gently pipetted off with the paraformaldehyde. Immunofluorescence on collagen embedded cells is described in detail within additional protocols15,16. Further applications for analyses of involved signaling mechanisms at the endpoint of the experiment include Western blot, qPCR, and fluorescence-activated cell sorting (FACS). One-sixth of a pancreas is sufficient material for one sample analysis via Western blot or qPCR.
Limitations of this protocol arise from the use of collagen or basement membrane matrix, where each has their advantages and disadvantages. While embedment of cells in collagen will only produce ducts if stimuli are given, basement membrane matrix embedment will produce ducts without additional stimuli. However, ductal size in basement membrane matrix is a measurable output. An additional limitation is the time involved in the harvesting procedures in step 5, which could affect gene expression. This limitation can be mitigated by confirming results obtained from Western blotting with immunofluorescence, in which the fixation time is considerably less than that of the harvesting time for qPCR or Western blot analysis.
In addition to adenoviral infection, lentiviral infection can be used in primary cells. For lentiviral infection, add 6 µg/mL viral infection enhancer reagent (see table of materials) to the cells and gently swirl the plates. Subsequently, add the lentivirus (with a titer of at least 107 TU/mL) at a 1:1,000 dilution and again, gently swirl. Incubate for 3-5 h (37 °C, 5% CO2) and then continue to embedment of cells in collagen or basement membrane matrix. To generate lentivirus with a plasmid of interest, use the lentiviral packaging mix noted in the table of materials.
Further modification can include isolation of cells from mice expressing KrasG12D. In these mice, which already have fibrotic areas with ADM and PanIN lesions, collagenase digestion takes longer. Careful monitoring of collagenase digestion, as described in the critical steps and troubleshooting, is required. The more abnormal tissue a mouse has, the longer the digestion will take (around an hour for a twelve-week-old p48Cre; LSL-KrasG12D mouse). Since there can be immense variation between mice of the same age, we advise to isolate acinar cells from an LSL-KrasG12D mouse and perform adenoviral or lentiviral infection utilizing cre to obtain oncogenic KRas expression. It also should be noted that our protocol is optimized for isolation of acinar cell to investigate the ADM process. The isolation of ADM and PanIN cells from KrasG12D-expressing mice for organoid culture requires altered protocols.
One critical step is the amount of time that the chopped pancreas spends in collagenase. The ideal collagenase digestion time can vary from mouse to mouse and between lots of collagenase from the same company. The time noted in the protocol is appropriate for pancreata weighing about 0.250 g. Too much time can damage and kill cells, while too little time will result in incomplete digestion and therefore fewer cells that will make it through the filtering process onto the final plate. Check the dissociation visually by looking for breakdown of the chopped-up pancreas pieces; the solution should turn cloudy. Additionally, before stopping the collagenase reaction with cold HBSS, the solution can be taken up with a 5 mL pipet, where the ease with which the solution can be taken up will indicate if it is appropriate to stop the reaction. An additional consideration is the number of pancreata harvested. If more than one pancreas is isolated at the same time, the procedure works best when the pancreata are kept separate.
Another important point to ensure healthy culture of primary acinar cells is that care should be taken when pipetting the acinar cell solution. Vigorous pipetting may cause cell damage and result in a lack of duct formation, even with addition of known ADM inducers, such as TGF-α. Further, if there are viability issues, the centrifugation in steps 2.4, 2.6, and 2.7 can be taken down to 300 x g to produce a softer pellet.
The authors have nothing to disclose.
This work was supported by an R01 grant (CA200572) from the NIH to PS. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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 | |
Adenovirus, Ad-GFP | Vector Biolabs | 1060 | |
Aluminum foil | Fisherbrand | 01-213-101 | |
BD Precision Glide Needle 21G x 1/2 | Fisher Scientific | 305167 | Use as pins for dissection |
Beaker, 600 mL | Fisherbrand | FB-101-600 | |
Bleach, 8.25% | Clorox | 31009 | |
Cell Recovery Solution | Corning | 354253 | Referred to as 'basement membrane matrix recovery solution' in the manuscript. |
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 | |
Glass slide, 8-well | Lab-Tek | 177402 | |
Hank's Balanced Salt Solution (HBSS), No calcium, No magnesium, No phenol red | Fisher Scientific | SH3048801 | |
Ice bucket, rectangular | Fisher Scientific | 07-210-103 | |
Instant Sealing Sterilization Pouch | Fisherbrand | 01-812-51 | |
LAB GUARD specimen bags (for mouse after dissection) | Minigrip | SBL2X69S | |
Lentiviral Packaging Mix, Virapower | Invitrogen | 44-2050 | |
Matrigel | Corning | 356234 | Referred to as 'basement membrane matrix' in the manuscript. |
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 | |
Plate, 12-well | Corning Costar | 3513 | |
Plate, 24-well plate | Corning Costar | 3524 | |
Plate, 35 mm | Falcon | 353001 | |
Plate, 6-well | Falcon | 353046 | |
Polybrene | EMD Millipore | TR-1003-G | Create a 40 mg/ml solution by dissolving powder in sterile molecular biology grade water, aliquoting and storing at -20 °C. |
Polypropylene Mesh, 105 µm | Spectrum Labs | 146436 | |
Polypropylene Mesh, 500 µm | Spectrum Labs | 146418 | |
Scissors | Fine Science Tools | 14568-12 | |
Scissors | Fine Science Tools | 91460-11 | |
Sodium Bicarbonate (Fine White Powder) | Fisher Scientific | BP328-500 | |
Sodium Hydroxide | Fisher Scientific | S318-500 | |
Soybean Trypsin Inhibitor 8 | Gibco | 17075029 | |
Spatula | Fisherbrand | 21-401-10 | |
Steriflip 50 ml, 0.22 micron filters | Millipore | SCGP00525 | |
TGF-α | R&D Systems | 239-A-100 | |
Type I Rat Tail Collagen | Corning | 354236 | |
Waymouth MB 752/1 Medium (powder) | Sigma | W1625-10X1L | |
Weigh boat, hexagonal, medium | Fisherbrand | 02-202-101 |