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.

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
<ol>
	<li>Isolation of pancreatic acini
	<ol>
		<li>Prepare 300 ml of fresh Krebs-Ringer bicarbonate HEPES (KRBH) medium before starting. Mix 140 mM NaCl, 4.7 mM KCl, 1.16 mM MgCl2, 1 mM CaCl2, 11 mM Glucose, 10 mM HEPES. Add bovine serum albumin (BSA) and 0.1 mg/ml soybean trypsin inhibitor (STI) (resuspension medium). Prepare 5 - 10 ml of resuspension medium supplemented with Collagenase (~0.75 mg/ml) (digestion medium).</li>
		<li>Oxygenate the KRB medium for ~30 min. Add BSA after oxygenation to prevent bubbling. Bring resuspension and digestion mediums to 37 &#176;C. NOTE: Oxygenating the medium reduces cell mortality and improves responsiveness to secretagogues. The effect of oxygenation is apparent at least for 6 hr.</li>
		<li>Sacrifice the mouse by cervical dislocation or CO2 asphyxiation, according to institutional animal care guidelines. Place the sacrificed animal on its back, attach it to a dissection pad and clean the abdominal surface with ethanol. 
		NOTE: In order to avoid contamination, autoclave the dissection tools and the water used for KRBH medium before use. Filter KRBH medium through a 0.2 &#181;m filter. Prior to excision of the pancreas rinse the dissection tools with 70% ethanol and carry out dissection on a clean bench. Carry out viral infections in a level 3 bio-safety cabinet. Add antibiotics to the O/N culturing medium (See section 4).</li>
	</ol>
	</li>
	<li>Excision of the murine pancreas
	<ol>
		<li>Incision widely the abdominal skin and subcutaneous layer. Identify the liver, stomach, intestine, pancreas and spleen (Figure&#160;1). &#160; NOTE: In the following steps, handle the pancreatic tissue gently and avoid applying excessive mechanical force to prevent auto-digestion of the tissue and cell death.</li>
		<li>By using two blunt forceps, pull out the small intestine. Start at the small intestine proximal end, near the exit from the stomach. Separate the pancreas from the intestine and retain it within the body cavity. Once reaching the large intestine, notice a pink/white connective tissue. Do not confuse it with the pancreas.</li>
		<li>Clear as much pancreatic tissue as possible without collecting fat or connective tissue. Remove the pancreas by detaching it from the spleen and stomach and cutting the abdominal blood vessels beneath it (Figure 1A-C). 
		NOTE: Since the yield of pancreatic acini from a single animal is high, one can refrain from collecting all the pancreatic tissue in order to prevent collecting other tissues.</li>
	</ol>
	</li>
	<li>Collagenase digestion
	<ol>
		<li>Rinse the pancreas a few times in resuspension medium heated to 37 &#176;C and remove remaining parts of non-pancreatic tissues. Immerse the pancreas in ~5 ml of pre-heated digestion medium in a 20 ml scintillation vial. Mince the pancreas to pieces of 1-3mm. Place it in a heated water bath (optimally under agitation of ~100 rpm). To accelerate digestion, pipette the digested tissue up and down once every few minutes. 
		NOTE: The duration of collagenase digestion is dependent mainly on the collagenase concentration and on the amount of mechanical force applied on the tissue. Use of 0.75 mg/ml collagenase for approximately 15 min&#160;and pipetting the tissue with a 5 ml&#160;polystyrene pipette with a 2 mm orifice every 3 - 5 min, resulting in&#160;acini of 10 - 30 cells. Following digestion, the suspension appears turbid containing visible pieces of tissue. Since collagenases efficiencies varies between different commercial types and lots, determine the timing of digestion empirically.</li>
		<li>Washing and filtering
		<ol>
			<li>Transfer the digested tissue into a 50 ml tube and terminate digestion by adding 45 ml of resuspension medium. Filter the suspension through a coarse (~1 mm) metal mesh into a fresh tube. Centrifuge the flow-through at 100 &#215; g for 3 min.</li>
			<li>Discard the supernatant and resuspended the digested pancreas in 50 ml of resuspension medium. Filter the suspension through a 70 or 100 &#181;m nylon mesh. Centrifuge as before.</li>
			<li>In order to remove floating, damaged acini, discard the supernatant and resuspend the pancreatic tissue in 3 - 5 ml of resuspension medium. Apply 1 ml portions of the pancreatic acini into 15 ml conical tubes filled with 7 ml of resuspension medium. Allow the acini to settle for 2 - 3 min. Collect the acini that have settled at the bottom of the tube with a 1&#160;ml pipette, and repeat this step 2 - 3 times.</li>
			<li>View the pancreatic acini under a light or dissecting microscope to test their condition. In Figure 2, observe the purity of isolation and damages inflicted upon the cells during isolation. Use these acini immediately or culture for up to 20 hr.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>Determine the number of experimental conditions to be examined (e.g. non-stimulated vs. stimulated). Use at least three replicates for each condition. In addition, add a set of replicates to determine the total cellular amylase content (&#8216;total&#8217; sample) and a set for the amylase activity of the medium (&#8216;zero&#8217; sample). Label a multi-well plate in which the assay will be conducted, and two sets of microtubes for each of the replicates to be used. 
	NOTE: 12- or 24-well plates can be used to monitor 1 and 0.5 ml of acinar suspensions, respectively.</li>
	<li>Wash the acini twice in 30 ml of KRBH resuspension medium and incubate them in resuspension medium for 30 min&#160;at 37 &#176;C. To the multi-well plate add a 10&#181;l drop of secretagogue to reach the appropriate final concentration.</li>
	<li>Pancreatic acini respond to a gradient of secretagogue in a bi-phasic manner. The concentrations with optimal secretion values are 50 - 100 pM of Cholecystokinin (CCK) and 1&#181;M of Carbachol (Figure&#160;3A)5.</li>
	<li>Wash and resuspend the acini in resuspension medium. Ensure that the acini are distributed homogenously in the suspension and aliquot equal amounts of suspension into the multi-well plate and into the &#8216;total&#8217;-labeled microtubes. Incubate the plate for 30 min&#160;at 37 &#176;C. (Preferably in a water bath under mild agitation (~50 rpm)). 
	NOTE: The exact number of acini may vary between experiments but should be similar in replicates within each experiment. Thus, precise aliquoting of the acini suspension is critical for comparing between different replicates and conditions.</li>
	<li>During the incubation, produce the &#8216;total&#8217; and &#8216;zero&#8217; samples. Centrifuge the &#8216;total&#8217; microtubes for few seconds at 5,000 &#215; g and remove 50 &#181;l from the supernatant into &#8216;zero&#8217;-labeled microtubes and place them at 4&#176;C. Lyse the acini in the &#8216;total&#8217; samples by adding Triton X-100 to the microtubes to reach a final concentration of 1%. Vortex the microtubes vigorously. 
	NOTE: Aliquote the &#8216;total&#8217; replicates similar to the replicates of the experiment. Lysing the acini, leads to the release of the total amylase content. Alternatively, collect the acini at each condition and lyse at the end of the experiment, to determine the cellular content of each replicate.</li>
	<li>Collect most of the acinar suspension into the first set of the pre-labeled microtubes. Centrifuge the microtubes for a few seconds at 5,000 &#215; g. Transfer the supernatant into the second set of the pre-labeled microtubes.</li>
	<li>Assay the supernatant for amylase activity with a commercial kit. Calculate the percentage of amylase secreted. 
	NOTE: It is crucial to read the amylase activity at its linear range. Present the percentage of amylase secreted as the percent of amylase released from initial total content. Subtract the background amylase level of the medium from each triplicate and divide it by the similarly normalized initial total content.</li>
</ol>
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.
<ol>
	<li>Prepare freshly oxygenated resuspension medium as in step 1.1 and bring it to 37 &#186;C. Set the microscope, and if equipped with a temperature controller, adjust it to 37 &#186;C.</li>
	<li>Wash the acinar cells in 30 ml of resuspension medium and place them in a glass or plastic slide. Note: If lipophilic dyes are to be used to label For plasma membrane labeled with lipophilic dyes, stain the acini for not more than 5 min&#160;to prevent labeling of internal membranes by endocytosis. Throughout the work, handle the acini gently and avoid excessive pipetting.</li>
	<li>Once at the microscope, image acini whose morphology is clear, and avoid imaging of acini which display basolateral blebs or intracellular vacuoles. Add secretagogue drop wise or by using a perfusion chamber, either during or immediately prior to the initiation of imaging. 
	NOTE: A high magnification lenses with high numeric apertures (e.g. &#215;63/1.4 or &#215;100/1.4) is recommended to observe subcellular structures.</li>
</ol>
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.
<ol>
	<li>Culture pancreatic acini for up to 20 hr. Resuspended acini obtained from a single pancreas in 25 ml culture medium (DMEM supplemented with fetal calf serum (FCS) (5%), sodium-pyruvate (1%), antibiotics (1%), L-glutamine (0.5%), BSA (10 mg/ml) and STI (0.2 mg/ml), and divided to five 10cm plates. Infect the acini with virus in a level 3 bio-safety cabinet.</li>
	<li>Incubated pancreatic acini at 37&#186;C in a 5% CO2 humidified air. Allow acini to recover in oxygenated resuspension medium for 30 min&#160;before subsequent examination3,8.</li>
</ol>

<ol>
	<li>Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+pancreatic+acinar+cell+function.">Regulation of pancreatic acinar cell function.</a> Current Opinion in Gastroenterology. 22, 498-504 (2006).</li><li>Schnekenburger, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+kinesin,+dynein+and+microtubules+in+pancreatic+secretion.">The role of kinesin, dynein and microtubules in pancreatic secretion.</a> Cellular and Molecular Life Sciences : CMLS. 66, 2525-2537 (2009).</li><li>Geron, E., Schejter, E. D., Shilo, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+exocrine+secretory+vesicles+to+the+apical+membrane+by+actin+cables+generated+by+the+formin+mDia1.">Directing exocrine secretory vesicles to the apical membrane by actin cables generated by the formin mDia1.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 10652-10657 (2013).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+isolated+pancreatic+exocrine+cells.">Structural and functional characterization of isolated pancreatic exocrine cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 69, 3028-3032 (1972).</li><li>Williams, J. A., Korc, M., Dormer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+secretagogues+on+a+new+preparation+of+functionally+intact,+isolated+pancreatic+acini.">Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.</a> The American journal of physiology. 235, 517-524 (1978).</li><li>Jerdeva, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+and+non-muscle+myosin+II+facilitate+apical+exocytosis+of+tear+proteins+in+rabbit+lacrimal+acinar+epithelial+cells.">Actin and non-muscle myosin II facilitate apical exocytosis of tear proteins in rabbit lacrimal acinar epithelial cells.</a> Journal of Cell Science. 118, 4797-4812 (2005).</li><li>Menozzi, D., Jensen, R. T., Gardner, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersed+pancreatic+acinar+cells+and+pancreatic+acini.">Dispersed pancreatic acinar cells and pancreatic acini.</a> Methods in Enzymology. , 192-271 (1990).</li><li>Bi, Y., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Rho+and+Rac+in+secretagogue-induced+amylase+release+by+pancreatic+acini.">A role for Rho and Rac in secretagogue-induced amylase release by pancreatic acini.</a> Am J Physiol Cell Physiol. 289, C22-C32 (2005).</li><li>Palade, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+aspects+of+the+process+of+protein+synthesis.">Intracellular aspects of the process of protein synthesis.</a> Science. 189, 867 (1975).</li><li>Masedunskas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+for+the+actomyosin+complex+in+regulated+exocytosis+revealed+by+intravital+microscopy.">Role for the actomyosin complex in regulated exocytosis revealed by intravital microscopy.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 13552-13557 (2011).</li><li>Petersen, H., Grossman, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+exocrine+secretion+in+anesthetized+and+conscious+rats.">Pancreatic exocrine secretion in anesthetized and conscious rats.</a> The American Journal of Physiology. 233, E530-E536 (1977).</li><li>Riedl, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nat Methods. 5, 605-607 (2008).</li><li>Porat-Shliom, N., Milberg, O., Masedunskas, A., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+for+the+actin+cytoskeleton+during+regulated+exocytosis.">Multiple roles for the actin cytoskeleton during regulated exocytosis.</a> Cellular and Molecular Life Sciences : CMLS. 70, 2099-2121 (2013).</li></ol>

The authors declare they have no competing financial interests.

Besides the ex&#160;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 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&#160;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&#8217;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.

<table><tbody><tr><td>ICR mice</td><td>Harlan</td><td></td><td>Any strain will do</td></tr><tr><td>HBSS</td><td>Sigma Aldrich</td><td>H8264</td><td>Can substitude for KRB</td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>A7906</td><td>Fraction V</td></tr><tr><td>Trypsin inhibitor</td><td>Sigma Aldrich</td><td>T9003</td><td></td></tr><tr><td>Collagenase XI&amp;nbsp;</td><td>Sigma Aldrich</td><td>C0130</td><td></td></tr><tr><td>Nylon mesh</td><td>BD Falcon</td><td>352360</td><td>70-100&amp;micro;m</td></tr><tr><td>Amylase infinity reagent&amp;nbsp;</td><td>Thermo</td><td>TR25421</td><td></td></tr><tr><td>CCK</td><td>Sigma Aldrich</td><td>C2175</td><td></td></tr><tr><td>FM4-64</td><td>Lifetechnologies</td><td>F34653</td><td>Diluted to 16&amp;micro;M</td></tr><tr><td>20mL scintillation vial</td><td>Sigma Aldrich</td><td>Z190527</td><td></td></tr><tr><td>DeltaVision system&amp;nbsp;</td><td>Applied Precision</td><td></td><td>Consisting of an inverted microscope IX71 equipped with 60x/1.4 or 100x/1.3 objectives (Olympus)</td></tr><tr><td>Zeiss 510 or 710 confocal microscope</td><td>Zeiss</td><td></td><td>60x/1.4 or 100x/1.4 objectives&amp;nbsp;</td></tr><tr><td>Ultra Microplate reader</td><td>BioTek</td><td>ELx808</td><td></td></tr></tbody></table>

assessing the secretory capacity of pancreatic acinar cells

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&#160;vivo culture will represent the in&#160;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.

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...

Watch this Scientific Journal Video about Assessing the Secretory Capacity of Pancreatic Acinar Cells at JoVE.com

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Isolated pancreatic acini retain their in&#160;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 ...

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Research

JoVE Journal

Biology

16.6K Views.  Weizmann Institute of Science. 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.

Video: Assessing the Secretory Capacity of Pancreatic Acinar Cells

Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture

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.

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 ...

Cancer Research

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within pancreatic islets and act in a coordinated fashion to maintain glucose homeostasis. It is becoming increasingly clear that interactions between transmembrane proteins on the surfaces of adjacent beta cells are important determinants of beta-cell function.
Elucidation of the roles of particular transcellular interactions by knockdown, knockout or overexpression studies in cultured beta cells or in vivo necessitates direct perturbation of mRNA and protein expression, potentially affecting beta-cell health and/or function in ways that could confound analyses of the effects of specific interactions. These approaches also alter levels of the intracellular domains of the targeted proteins and may prevent effects due to interactions between proteins within the same cell membrane to be distinguished from the effects of transcellular interactions.
Here a method for determining the effect of specific transcellular interactions on the insulin secreting capacity and responsiveness of beta cells is presented. This method is applicable to beta-cell lines, such as INS-1 cells, and to dissociated primary beta cells. It is based on coculture models developed by neurobiologists, who found that exposure of cultured neurons to specific neuronal proteins expressed on HEK293 (or COS) cell layers identified proteins important for driving synapse formation. Given the parallels between the secretory machinery of neuronal synapses and of beta cells, we reasoned that beta-cell functional maturation might be driven by similar transcellular interactions. We developed a system where beta cells are cultured on a layer of HEK293 cells expressing a protein of interest. In this model, the beta-cell cytoplasm is untouched while extracellular protein-protein interactions are manipulated. Although we focus here primarily on studies of glucose-stimulated insulin secretion, other processes can be analyzed; for example, changes in gene expression as determined by immunoblotting or qPCR.

Transcellular protein interactions are important determinants of pancreatic beta-cell function. Detailed here is a method&#x2014;adapted from a coculture model of synaptogenesis&#x2014;for investigating how specific transmembrane proteins influence insulin secretion. Transfected HEK293 cells express proteins of interest; beta cells do not need to be transfected or otherwise directly perturbed.

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

Medicine

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix ECM Production

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

Bioengineering

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

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.

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.

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 ...

Observing Islet Function and Islet-Immune Cell Interactions in Live Pancreatic Tissue Slices

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared from live human and mouse pancreatic tissue embedded in agarose and cut using a vibratome. This method allows for the tissue to maintain viability and function in addition to preserving underlying pathologies such as type 1 (T1D) and type 2 diabetes (T2D). The slice method enables new directions in the study of the pancreas through the maintenance of the complex structures and various intercellular interactions that comprise the endocrine and exocrine tissues of the pancreas. This protocol demonstrates how to perform staining and time-lapse microscopy of live endogenous immune cells within pancreatic slices along with assessments of islet physiology. Further, this approach can be refined to discern immune cell populations specific for islet cell antigens using major histocompatibility complex-multimer reagents.

This study presents the application of live pancreatic tissue slices to the study of islet physiology and islet-immune cell interactions.

Live pancreatic tissue slices allow for the study of islet physiology and function in the context of an intact islet microenvironment. Slices are prepared ...

Immunology and Infection

Inducing Pancreatic Injury in Mice Through Intraperitoneal Drug Injection

Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological changes associated with SAP using animal models can aid in identifying potential therapeutic targets and exploring novel treatment approaches. Previous studies primarily induced pancreatic injury through retrograde bile duct injection of sodium taviaurocholate, but the impact of surgical damage on the quality of the animal model remains unclear. In this study, we employed various frequencies of intraperitoneal Caerulein injections combined with different doses of LPS to induce pancreatic injury in C57BL/6J mice and compared the extent of injury across five intraperitoneal injection protocols. Regarding inducing acute pancreatitis in mice, an intraperitoneal injection protocol is proposed that results in a mortality rate as high as 80% within 5 days. Specifically, mice received ten daily intraperitoneal injections of Caerulein (50 &#181;g/kg), followed by an injection of LPS (15 mg/kg) one hour after the last Caerulein administration. By adjusting the frequency and dosage of injected medications, one can manipulate the severity of pancreatic injury effectively. This model exhibits strong controllability and has a short replication cycle, making it feasible for completion by a single researcher without requiring expensive equipment. It conveniently and accurately simulates key disease characteristics observed in human SAP while demonstrating a high degree of reproducibility.

Intraperitoneal drug administration is a safe and effective non-invasive approach for inducing pancreatic injury. This study compared five distinct intraperitoneal injection protocols on mice to induce varying degrees of pancreatic injury and established a model of severe pancreatic injury to investigate the pathological changes and treatment strategies for severe acute pancreatitis (SAP).

The treatment of severe acute pancreatitis (SAP), with high mortality rates, poses a significant clinical challenge. Investigating the pathological ...

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. 
The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Availability of somatic SCs is crucial for regenerative medicine, disease modeling and to gain insight into SC properties. Here we present experimental strategies to reprogram, in vitro, differentiated adult cells into their corresponding expandable tissue-specific stem/progenitor cells by the transient expression of the single transcriptional co-activator YAP.

Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient ...

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails significantly fewer preparation-induced changes than isolated islets, acini, ducts, or dispersed cells described in typical in vitro studies. By combining the acute pancreatic tissue slice with live-cell calcium imaging in confocal laser scanning microscopy (CLSM), calcium signals can be studied in a large number of endocrine and exocrine cells simultaneously, with a single-cell or even subcellular resolution. The sensitivity permits the detection of changes and enables the study of intercellular waves and functional connectivity as well as the study of the dependence of physiological responses of cells on their localization within the islet and paracrine relationship with other cells. Finally, from the perspective of animal welfare, recording signals from a large number of cells at a time lowers the number of animals required in experiments, contributing to the 3R-replacement, reduction, and refinement-principle.

We present the preparation of acute pancreatic tissue slices and their use in confocal laser scanning microscopy to study calcium dynamics simultaneously in a large number of live cells, over long time periods, and with high spatiotemporal resolution.

The acute mouse pancreatic tissue slice is a unique in situ preparation with preserved intercellular communication and tissue architecture that entails ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Summary

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Preparing a Mice Model of Severe Acute Pancreatitis via a Combination of Caerulein and Lipopolysaccharide Intraperitoneal Injection

De Novo Generation of Somatic Stem Cells by YAP/TAZ

Confocal Laser Scanning Microscopy of Calcium Dynamics in Acute Mouse Pancreatic Tissue Slices