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Isolated pancreatic acini retain their in vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.
Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex vivo culture will represent the in vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.
The exocrine pancreas constitutes most of the mass of the mammalian pancreas and is dedicated to production and secretion of digestive enzymes1. The functional unit of the exocrine pancreas, the acinus, is a cluster of epithelial cells which forms and shares a joint lumen. Upon hormonal or neuronal stimulation, vesicles filled with enzymes are transported to the apical cell surface, fuse with it and expel their content into the lumen1-3. The secreted enzymes are then drained by a coalescing set of ducts into the small intestine, where they catalyze the breakdown of food into nutrients.
This video demonstrates how intact acini are isolated from the whole pancreas and how their secretory capacity can be assessed. Using this setup, pancreatic secretion is quantified by measuring the relative amount of amylase that was released following stimulation. Alternatively, secretion can be imaged live by the use of different sensors and dyes.
The ex vivo setup of pancreatic acini is advantageous since isolated acini, which retain many characteristics of the intact pancreas, can be manipulated and monitored more readily than in the whole animal4-6. This setup was originally developed in the late 1970’s and was since extended for the study of several exocrine tissues such as the salivary and mammary glands3-7. Notably, it allows the study of secretion with minimal interference to the complex cellular organizations of these polarized epithelia.
NOTE: Procedures involving animal subjects have been approved by the Institutional Animal Care and Use Committee (IACUC) at the Weizmann Institute of Science.
1. Sample Preparation
2. Amylase Secretion Assay
NOTE: The amylase secretion assay serves as a global measure for the secretory capacity of pancreatic acini. This is achieved by collecting the medium in which the pancreatic acini were incubated, and determining the amylase activity of the medium, usually by using a commercial kit.
3. Live Imaging of Pancreatic Secretion
Secretion from pancreatic acini can be monitored directly by live imaging. Using various fluorescent dyes and sensors, live imaging allows the characterization of secretion at a sub-cellular resolution.
4. O/N Culture of Pancreatic Acini (Alternate Cell Culture Technique)
NOTE: O/N culturing is often used for Adeno virus infection. Infection is carried out at 106-107 pfu/ml for 9 - 16 hr before examination.
Pancreatic acini that were isolated properly display a stereotypic morphology when viewed by transmitted light. Their basolateral domains should appear round and devoid of blebs. The apical domain is surrounded by hundreds of secretory vesicles and appears darker in color (Figure 2A, B). The nuclei are located basal to the vesicular area. Cell debris and components of the pancreatic ductal system and of the endocrine pancreas, which can be detected at early stages of acini isolation (Figure 2C, D...
Besides the ex vivo culture, alternative setups for the study of exocrine secretion include intravital microscopy and measurement of fluids from the pancreatic duct. Intravital microscopy was shown to operate well in the mouse salivary glands10. This method can record secretion in real time in the intact animal, but is limited in its resolution and by the means for manipulating the exocrine tissue. Assessing secretion by collecting fluids from the pancreatic duct was achieved in anesthetized rats...
The authors declare they have no competing financial interests.
This work was funded by a grant from the US-Israel BSF to B.S. and E.D.S. B.S. is an incumbent of the Hilda and Cecil Lewis professorial chair in Molecular Genetics.
Name | Company | Catalog Number | Comments |
ICR mice | Harlan | Any strain will do | |
HBSS | Sigma Aldrich | H8264 | Can substitude for KRB |
BSA | Sigma Aldrich | A7906 | Fraction V |
Trypsin inhibitor | Sigma Aldrich | T9003 | |
Collagenase XI | Sigma Aldrich | C0130 | |
Nylon mesh | BD Falcon | 352360 | 70-100µm |
Amylase infinity reagent | Thermo | TR25421 | |
CCK | Sigma Aldrich | C2175 | |
FM4-64 | Lifetechnologies | F34653 | Diluted to 16µM |
20mL scintillation vial | Sigma Aldrich | Z190527 | |
DeltaVision system | Applied Precision | Consisting of an inverted microscope IX71 equipped with 60x/1.4 or 100x/1.3 objectives (Olympus) | |
Zeiss 510 or 710 confocal microscope | Zeiss | 60x/1.4 or 100x/1.4 objectives | |
Ultra Microplate reader | BioTek | ELx808 |
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