We would like to thank Dr. Avital Sarusi-Portuguez for help in data analysis and Dr. Dror Kolodkin-Gal for assistance in establishing the protocol in a previous study. We thank all past and present members of the Parnas lab. We thank Dr. Gillian Kay and Dr. Michael Kanovsky for their help in editing. This project has received funding from the Israel Science Foundation grant (No. 526/18 O.P.), the Alex U. Soyka Program, and a grant from the Israel Cancer Research Fund (Research Career Development Award).

The joint ethics committee (Institutional Animal Care and Use Committee) of the Hebrew University (Jerusalem, Israel) and Hadassah Medical Center (Jerusalem, Israel) approved the study protocol for animal welfare (MD-18-15417-5 &#34;Tissue dynamics in pancreatic cancer in mice&#34;), and the protocol presented here complied with all relevant ethical regulations for animal testing and research. The Hebrew University is an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited institute.
NOTE: The mouse strain stock #007908, stock #019378, and stock #008179 were obtained from Jackson&#39;s laboratory. PRT (Kras+/LSL-G12D; Ptf1a-CreER; Rosa26LSL-tdTomato) mice were created by crossing the above strains. Mice from both genders, between 6 weeks and 15 months of age, were used for the study. Tamoxifen was prepared by dissolving the powder in corn oil. Adult mice (6-8 weeks of age, females and males), were injected with tamoxifen subcutaneously on days 0 and 2 at a dose of 400 mg/kg and examined twice a week following the injection. It was not possible to measure tumors as they were internally located; therefore, euthanasia was performed if abnormal clinical signs were observed according to the ethical protocol. Mice were euthanized at different time points post-tamoxifen induction, using isoflurane and cervical dislocation.
1. Pancreatic dissection
NOTE: For optimal yield during extraction and to ensure good cell viability, rapid dissection is critical. To shorten the time required for pancreas isolation, all instruments and equipment must be ready on ice before euthanizing the mouse.
<ol>
	<li>Euthanize the mouse by CO2&#160;asphyxiation and verify using cervical dislocation. From this step on, all procedures must be performed with sterile dissecting instruments.</li>
	<li>Fix the mouse and spray the abdomen with 70% ethanol. Make a V-shaped incision of 2.5 cm in the genital area with scissors and forceps and proceed upward to fully open the abdominal cavity.</li>
	<li>Locate the stomach on the left side of the mouse. Locate the pancreas, which is near the spleen. Separate the pancreas from the stomach and duodenum using two forceps (without tearing). Continue and separate the pancreas from the small intestine, jejunum, and ileum.</li>
	<li>Move the pancreas to the right side of the mouse. Separate the remaining connections between the pancreas and the thoracic cavity with forceps to completely detach the pancreas and attached spleen.</li>
	<li>Remove the pancreas and spread it out for examination in a Petri dish on ice. 
	NOTE: Care must be taken during this step to remove only the pancreas, and not remove mesenteric fat tissue or other adjacent tissue along with the pancreas, to avoid cellular contamination.</li>
</ol>
2. Enzymatic and mechanical dissociation of the pancreas
<ol>
	<li>Prepare the following buffers in advance.
	<ol>
		<li>Dissociation buffer 1: 4 mL of trypsin C + 6 mL of phosphate buffered saline (PBS) (see Table 1) for each sample.</li>
		<li>Dissociation buffer 2: 9 mL of Hanks&#8242; balanced salt solution (HBSS) 1x; 4% bovine serum albumin (BSA); 1 mL of collagenase P (10 mg/mL); 200 &#181;L of trypsin inhibitor (10 mg/mL); and 200 &#181;L of DNase I (10 mg/mL) (see Table 1).</li>
		<li>Wash buffer: 50 mL of HBSS 1x; 2 g of BSA; 1 mL of trypsin inhibitor (10 mg/mL); and 1 mL of DNase 1 (10 mg/mL).</li>
		<li>Enzyme activity stop solution: HBSS 1x containing 5% fetal bovine serum (FBS) and 150 g of DNase I (0.2 mg/mL).</li>
	</ol>
	</li>
	<li>Place the pancreas in a 50 mL tube on ice. Rinse the pancreas in 10% FBS in HBSS 1x. The fatty tissue will float and the pancreas will sink. This is an easy way to visualize and quickly remove the contaminating white adipose tissue still attached to the pancreas.</li>
	<li>Transfer the mouse pancreatic tissue to a sterile Petri dish containing 5 mL of HBSS 1x on ice. Cut the pancreas into small pieces of 1 to 3 mm3 using Noyes scissors and a scalpel (Figure 2A). In case of more than one sample, the samples should be kept on ice in 10% FBS/HBSS 1x.</li>
	<li>Transfer the tissues to a centrifuge tube. Centrifuge at 350 x g at 4 &#176;C for 5 min. Aspirate and discard the supernatant to remove cell fragments and blood cells.</li>
	<li>Resuspend the pieces in dissociation buffer 1 containing 0.02% trypsin C-0.05% EDTA for 10 min at 37 &#176;C with agitation (180 rpm). Immediately wash with 10% FBS/Dulbecco&#39;s modified Eagle&#39;s medium (DMEM). Centrifuge for 5 min at 350 g at 4 &#176;C.</li>
	<li>Wash again by resuspending the pellet in 10 mL of wash buffer and centrifuging at 350 x g for 5 min at 4 &#176;C before the next dissociation step.</li>
	<li>Incubate the pancreas in dissociation buffer 2 for 15 min at 37 &#176;C with agitation (180 rpm).</li>
	<li>After 15 min, perform mechanical dissociation by vigorously pipetting the pancreatic fragments up and down in sterile pipettes of decreasing size (25, 10, and 5 mL serological pipettes) 10 times and bring back to 37 &#176;C.
	<ol>
		<li>After an additional 5 min, repeat the mechanical dissociation and use light microscopy to monitor the dissociation according to the amount of single-cell suspension. Usually if at this stage, less than 90% of the suspension consists of isolated single cells, a longer incubation time is needed. Use trypan blue to monitor cell viability.</li>
		<li>Continue with the incubation and take samples to detect the dissociation every 5 min. 
		NOTE: The total incubation time depends on the tissue and can vary between samples. The incubation and tissue dissociation should end when 90% of the cells are separated into single cells or when a reduction in viability is detected.</li>
	</ol>
	</li>
	<li>After the pancreatic tissue is well dissociated (corresponding to the disappearance of pancreatic fragments and the increasing turbidity of the solution) (Figure 2), stop the enzymatic reaction by washing twice with enzyme activity stop solution for 5 min at 4 &#176;C. From this step, keep the cell suspension on ice.</li>
	<li>Pass the cell suspension through a 70&#181;m nylon mesh and check the cell viability under the microscope. Smaller sizes of nylon mesh may reduce cell viability.</li>
	<li>Resuspend and wash the pellet with 5-10 mL of ice-cold buffered wash solution. Count the cells.</li>
	<li>If several red blood cells are observed, treat the cells with a red blood cell lysis buffer for 2 min at room temperature. In cases where clumps are observed, the sample should be treated again with trypsin, as described in step 2.5. Viability is detected with trypan blue under the microscope, and if viability is less than 80%, live cells should be isolated using the magnetic-activated cell sorting (MACS) dead cells removal kit with MACS MS columns (see Table 1).</li>
</ol>

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D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2019.">Cancer statistics, 2019.</a> CA: a Cancer Journal for Clinicians. 69 (1), 7-34 (2019).</li><li>Yachida, S., 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=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467 (7319), 1114-1117 (2010).</li><li>Peng, 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=Author+correction:+single-cell+RNA-seq+highlights+intra-tumoral+heterogeneity+and+malignant+progression+in+pancreatic+ductal+adenocarcinoma.">Author correction: single-cell RNA-seq highlights intra-tumoral heterogeneity and malignant progression in pancreatic ductal adenocarcinoma.</a> Cell Research. 29 (9), 777 (2019).</li><li>Hruban, R. H., Wilentz, R. E., Kern, S. 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The authors declare no competing interests.

In this article, we present a protocol for pancreatic tissue dissociation. The protocol is simple, easy to use, and provides a tool to isolate viable single cells from pancreatic tissue at different stages during the malignancy process, including solid tumors. In previous studies, different types of collagenases were used to digest the pancreas8,9. Using a very potent collagenase, such as collagenase D, results in a large population of immune cells and a lower pe...

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types1. Clinical evidence supports the notion that PDAC develops from exocrine-system cells, including acinar cells, over many years, driven by mutations in the KRAS proto-oncogene2.
Pancreatic tumors include many different cell types, and it has been demonstrated that malignant cells count for only 20%-50% of the tumor mass3. Different cell types interact with the epithelial cells, support their transformation, and enhance tumor formation and growth. Early events cause acinar metaplasia, which gives rise to microscopic lesions called pancreatic intraepithelial neoplasia (PanINs), which can in some cases develop into PDAC4.
There is a critical need to investigate these interactions and target pivotal signals. Single-cell RNA-sequencing (scRNA-seq) is a powerful method that reveals gene expression at a single-cell resolution, thereby tracking the changes that epithelial cells undergo, thus enabling the exploration of pancreatic cancer development. 
Tissue dissection and digestion to single cells is the first stage in a scRNA-seq experiment. Several factors make pancreatic tissue digestion especially challenging: i) acinar cells account for more than 90% of the pancreas and acinar cells contain large amounts of digestive enzymes, including proteases and RNases that reduce the quality of RNA-based libraries; (ii) acinar cells are very sensitive and may lyse if standard protocols are used; (iii) acinar cells express a small number of genes at very high levels. Therefore, if these cells are lysed during the experiment, this can contaminate the observed gene expression profile of other cells; (iv) pancreatic tissue recovered from tumors is desmoplastic, making it hard to dissect without damaging the cells. Thus, even though maintaining high viability of all the cell types is required, the large number and sensitivity of acinar cells add additional complexity. These factors impose difficulties in achieving a single-cell suspension that is more than 80% viable and has no clumps, as is required for scRNA-seq experiments. 
Here, we developed a protocol using trypsin C and collagenase P, along with frequent tissue monitoring. This supports dissociation to single cells while retaining high viability to support the success of scRNA-seq experiments5,6.

<table><tbody><tr><td>Reagent or Resource</td><td></td><td></td><td></td></tr><tr><td>70 &amp;micro;m nylon mesh&amp;nbsp;</td><td>Corning</td><td>cat##431751</td><td></td></tr><tr><td>BSA</td><td>Sigma Aldrich</td><td>cat# A7906</td><td></td></tr><tr><td>Collagenase P</td><td>Roche</td><td>cat# 11213857001</td><td></td></tr><tr><td>Critical Commercial Assay</td><td></td><td></td><td></td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>cat#MBD0015</td><td></td></tr><tr><td>Dnase I</td><td>Roche</td><td>cat# 10104159001</td><td></td></tr><tr><td>Experimental Models: Organisms/Strains</td><td></td><td></td><td></td></tr><tr><td>Fetal Bovine Serum South American</td><td>ThermoFisher</td><td>Cat#10270106</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution</td><td>Biological industries</td><td>cat#02-018-1A&amp;nbsp;</td><td></td></tr><tr><td>&lt;em&gt;KRAS&lt;sup&gt;LSL-G12D&lt;/sup&gt;&lt;/em&gt; mice</td><td>Jackson Laboratory</td><td>JAX008179</td><td></td></tr><tr><td>MACS dead cells removal kit</td><td>Milteny Biotec</td><td>cat#130-090-101</td><td></td></tr><tr><td>PBS</td><td>Biological industries</td><td>cat#02-023-1A&amp;nbsp;</td><td></td></tr><tr><td>&lt;em&gt;Ptf1a-Cre&lt;sup&gt;ER &lt;/sup&gt;&lt;/em&gt;mice</td><td>Jackson Laboratory</td><td>JAX019378</td><td></td></tr><tr><td>&lt;em&gt;Ptf1a-Cre&lt;sup&gt;ER&lt;/sup&gt;&lt;/em&gt;; &lt;em&gt;Rosa26&lt;sup&gt;LSL-tdTomato &lt;/sup&gt;&lt;/em&gt;mice</td><td>Jackson Laboratory</td><td>JAX007908</td><td></td></tr><tr><td>Trypsin C-EDTA 0.05%</td><td>Biological industries</td><td>cat# 03-053-1A</td><td></td></tr><tr><td>Trypsin inhibitor</td><td>Roche</td><td>cat#T6522</td><td></td></tr></tbody></table>

pancreatic tissue dissection to isolate viable single cells

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for approximately 90% of the pancreas and includes cells that produce and secrete digestive enzymes. The digestive enzymes are produced in the pancreatic acinar cells, stored in vesicles called zymogens, and are then released into the duodenum via the pancreatic duct to initiate metabolic processes. The enzymes produced by the acinar cells can kill cells or degrade cell-free RNA. In addition, acinar cells are fragile, and common dissociation protocols result in a large number of dead cells and cell-free proteases and RNases. Therefore, one of the biggest challenges in pancreatic tissue digestion is recovering intact and viable cells, especially acinar cells. The protocol presented in this article shows a two-step method that we developed to meet this need. The protocol can be used to digest normal pancreata, pancreata that include pre-malignant lesions, or pancreatic tumors that include a large number of stromal and immune cells.

In a recently published work5, we applied the protocol described above to explore the early stages of PDAC development using a mouse model. The mouse was genetically engineered to include the cassettes&#160;Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato7, which allow the expression of constitutively active KRAS in acinar cells after tamoxifen injection.
After cervical dislocation (according to the mouse ethi...

Watch this Scientific Journal Video about Pancreatic Tissue Dissection to Isolate Viable Single Cells at JoVE.com

Pancreatic Tissue Dissection to Isolate Viable Single Cells

Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing.

The pancreas includes two major systems: the endocrine system, which produces and secretes hormones, and the exocrine system, which accounts for ...

pancreatic-tissue-dissection-to-isolate-viable-single-cells

Research

JoVE Journal

Cancer Research

2.4K Views.  The Hebrew University of Jerusalem. Pancreatic metaplastic cells are precursors of malignant cells that give rise to pancreatic tumors. However, isolating intact viable pancreatic cells is challenging. Here, we present an efficient method for pancreatic tissue dissociation. The cells can then be used for single-cell RNA sequencing (scRNA-seq) or for two- or three-dimensional co-culturing. 

Video: Pancreatic Tissue Dissection to Isolate Viable Single Cells

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. However, it is still unknown whether stem and progenitor cells exist in the adult pancreas. Research strategies using cre-lox lineage-tracing in adult mice have yielded results that either support or refute the idea that beta cells can be generated from the ducts, the presumed location where adult pancreatic progenitors may reside. These in vivo cre-lox lineage-tracing methods, however, cannot answer the questions of self-renewal and multi-lineage differentiation-two criteria necessary to define a stem cell. To begin addressing this technical gap, we devised 3-dimensional colony assays for pancreatic progenitors. Soon after our initial publication, other laboratories independently developed a similar, but not identical, method called the organoid assay. Compared to the organoid assay, our method employs methylcellulose, which forms viscous solutions that allow the inclusion of extracellular matrix proteins at low concentrations. The methylcellulose-containing assays permit easier detection and analyses of progenitor cells at the single-cell level, which are critical when progenitors constitute a small sub-population, as is the case for many adult organ stem cells. Together, results from several laboratories demonstrate in vitro self-renewal and multi-lineage differentiation of pancreatic progenitor-like cells from mice. The current protocols describe two methylcellulose-based colony assays to characterize mouse pancreatic progenitors; one contains a commercial preparation of murine extracellular matrix proteins and the other an artificial extracellular matrix protein known as a laminin hydrogel. The techniques shown here are 1) dissociation of the pancreas and sorting of CD133+Sox9/EGFP+ ductal cells from adult mice, 2) single cell manipulation of the sorted cells, 3) single colony analyses using microfluidic qRT-PCR and whole-mount immunostaining, and 4) dissociation of primary colonies into single-cell suspensions and re-plating into secondary colony assays to assess self-renewal or differentiation.

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

In vitro colony assays to detect self-renewal and differentiation of progenitor cells isolated from adult murine pancreas are devised. In these assays, pancreatic progenitors give rise to cell colonies in 3-dimensional space in methylcellulose-containing semi-solid medium. Protocols for handling single cells and characterization of individual colonies are described.

Stem and progenitor cells from the adult pancreas could be a potential source of therapeutic beta-like cells for treating patients with type 1 diabetes. ...

Developmental Biology

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 ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and tumorigenesis.

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Medicine

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

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 ...

Biology

Streamlining Mouse Pancreatic Endothelial Cell Extraction for Disease Studies

Isolation of Mouse Pancreatic Endothelial Cells

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and glucagon, as well as digestive enzymes. The pancreas is also a highly vascularized organ, a feature facilitated by the intricate network of pancreatic capillaries. This extensive capillary network is made up of highly fenestrated endothelial cells (ECs) important for pancreas development and function. Accordingly, the dysfunction of ECs can contribute to that of the pancreas in diseases like diabetes and cancer. Thus, researching the function of pancreatic ECs (pECs) is important not only for understanding pancreas biology but also for developing its pathologies. Mouse models are valuable tools to study metabolic and cardiovascular diseases. However, there has not been an established protocol with sufficient details described for the isolation of mouse pECs due to the relatively small population of ECs and the abundant digestive enzymes potentially released from the acinar tissue that can lead to cell damage and, thus, low yield. To address these challenges, we devised a protocol to enrich and recover mouse pECs, combining gentle physical and chemical dissociation and antibody-mediated selection. The protocol presented here provides a robust method to extract intact and viable ECs from the whole mouse pancreas. This protocol is suitable for multiple downstream assays and may be applied to various mouse models.

This protocol describes the isolation of mouse endothelial cells from whole pancreas.

The pancreas is a vital organ for maintaining metabolic balance within the body, in part due to its production of metabolic hormones such as insulin and ...

Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells

Source: Villarreal D. et al.&#160;<a href="https://www.jove.com/v/57048/" target="_blank">A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice</a>. J. Vis. Exp. (2019)
Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions.&#160;This protocol explains a method for isolating pancreatic islets by first perfusing collagenase via Ampulla of Vater to digest the exocrine pancreatic tissue and a simplified density gradient centrifugation to purify the islets from mice.

Source: Villarreal D. et al.&#160;A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice. J. Vis. Exp. (2019)
Pancreatic islets, also ...

JoVE Encyclopedia of Experiments

Pancreatic Cancer

Cell isolation & culture

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

Source: Fleming Martinez, A. K., et al. <a href="https://www.jove.com/v/59096/" target="_blank">Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture</a>. J. Vis. Exp. (2019).
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.

Source: Fleming Martinez, A. K., et al. Mimicking and Manipulating Pancreatic Acinar-to-Ductal Metaplasia in 3-dimensional Cell Culture. J. Vis. Exp. ...

Pancreatic Tissue Dissection to Isolate Viable Single Cells

Summary

Explore More Videos

Chapters in this video

In Vitro Colony Assays for Characterizing Tri-potent Progenitor Cells Isolated from the Adult Murine Pancreas

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

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Isolation of Mouse Pancreatic Endothelial Cells

Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells

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