This work was supported by NIDDK grants DK098143 and DK101530 from the NIH (JPA), Stanford's Spectrum Child Health Research Institute (CHRI) and SPARK (UL1 TR001085, JPA).

This protocol was carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) of Stanford University School of Medicine. The described protocol is scaled for islet isolation from six 250 - 300 g (8 - 9 weeks old) male Sprague Dawley rats, which is sufficient to generate 228 wells of a 384-well plate for islet-cell replication assessment.
1. Material Preparation
<ol>
	<li>Prepare coating media prior to initiating islet isolation by collecting the conditioned media of 804G rat bladder carcinoma cells maintained at confluence for 3 days (20 ml of RPMI1640) in a 15 cm tissue culture dish 28. Sterile filter and store (-20 &#176;C) the conditioned media for later use.</li>
	<li>Sterilize surgical tools (one 12 cm toothed tissue forceps, one 11.5 cm fine scissors, one 14.5 cm surgical scissors, two 16 cm curved forceps, one 12 cm curved hemostat, one 12 cm scalpel handle) and one 30 mesh tissue sieve prior to initiating islet isolation.</li>
	<li>Prepare pancreatic digestion solution by dissolving a 60%:40% mixture of purified class I:class II collagenases (total collagenase activity of 300,000 - 400,000 units) in 60 ml of 1x Hanks&#39; Balanced Salt Solution (HBSS) supplemented with calcium and magnesium. Load 10 ml of pancreatic digestion buffer into 10 ml syringes (six) with a 1&#34; 22 G needle and place on ice. 
	Note: 10 ml of digestion solution is injected per rat. Scale accordingly.</li>
	<li>Prepare 4.5 ml of anesthetic cocktail in a ventilated hood by mixing 1.3 ml of ketamine (100 mg/ml), 0.2 ml of xylazine (100 mg/ml) and 3 ml of PBS. Prepare the anesthetic cocktail within 1 hr of initiating islet isolation.</li>
	<li>Prepare wash buffer by adding 25 ml of newborn calf serum to 500 ml of HBSS. Place wash buffer on ice for later use.</li>
	<li>Prepare 1 bottle of islet medium which is 500 ml of low glucose Dulbecco&#39;s Modified Eagle Medium (DMEM), 50 ml fetal bovine serum (FBS), 5,000 units/ml of penicillin and 5,000 &#181;g/ml of streptomycin.</li>
	<li>Prepare islet function medium from functionality/viability solution (500 ml) with 2% FBS, 2 mM glutamine, 5 mM glucose, 5,000 units/ml of penicillin and 5,000 &#181;g/ml of streptomycin. Store the islet function media (4 &#176;C) for later use.</li>
	<li>Prepare 40 ml blocking buffer by adding 2.5 ml donkey serum and 0.12 ml triton X-100 to 37.38 ml of 1x phosphate buffered saline (PBS). Store blocking buffer (4 &#176;C) for later use.</li>
	<li>Obtain the required antibodies prior to initiating the protocol. 
	Note: PDX-1 (TRITC) and Ki-67 (FITC) co-staining is used for primary &#946;-cell replication screening. For lineage-specific replication analysis, perform immunofluorescence staining for additional cell identity markers (insulin, glucagon, vimentin or somatostatin; TRITC) along with nuclear (DAPI), PDX (Cy5) and Ki-67 (FITC) staining. Alternative replication markers, e.g., PCNA, may also be used.</li>
	<li>Prepare a 228-well treatment plan with each treatment condition performed in quadruplicate. Include positive- (5-Iodotubercidin [1 &#181;M] or dipyridamole [15 &#181;M]) and vehicle-control (DMSO 1:666 dilution) wells.</li>
</ol>
2. Perfusion of the Pancreas
<ol>
	<li>Anesthetize rats by i.p. injection of diluted anesthetic cocktail solution (0.30 ml/100 g body weight). Once the rat is fully anesthetized and unresponsive to noxious stimuli, perform cervical dislocation.</li>
	<li>Place the euthanized rat in supine position (cranial-caudal orientation) on a stack of paper towel and spray the abdominal area with 70% ethanol.</li>
	<li>Open the abdominal cavity with a U-incision (base at pubic region) and retract skin in the rostral direction over chest to expose the abdominal cavity.</li>
	<li>Carefully grab the duodenum using two pairs of curved forceps, locate the duodenal entry of the common bile duct (sphincter of Oddi) and clamp the duodenal opening at the papilla with a curved hemostat.</li>
	<li>Lift the curved hemostat used to clamp the common bile duct papilla and grab the bile duct close to liver using a curved forceps. Use blunt dissection with the curved forceps to isolate and expose the bile duct.</li>
	<li>Reposition the rat with the head proximal and the feet distal.</li>
	<li>Cannulate the common bile duct (CBD) at or just distal to the junction of the cystic and common hepatic ducts with a pancreatic digestion solution-loaded 10 ml syringe and slowly inject 10 ml of the pancreatic digestion solution. While injecting, support the CBD with a scalpel handle. Reposition the needle if the duct and pancreas fail to inflate.</li>
	<li>Remove the inflated pancreas using the curved forceps and fine scissors to separate it from the descending colon, intestines, stomach and spleen. Place the inflated pancreas on ice in a 50 ml tube containing 5 ml of pancreatic digestion solution. 
	Note: For maximum islet yield, collect all pancreata within 30 min and place only 1 or 2 pancreata in each 50 ml digestion tube.</li>
</ol>
3. Dissociation of the Pancreas
<ol>
	<li>Once all pancreata are collected, place the digestion tubes into a 37 &#176;C water bath for 15 min. Gently swirl the tubes every 3 min.</li>
	<li>After 15 min, vigorously shake (vertically) the tubes 10 times and add 10 ml of cold wash buffer. Vigorously shake the tubes an additional 5 times and place on ice.</li>
	<li>Fill the digestion tubes to 50 ml with cold wash buffer and mix by inverting tubes 5 times.</li>
	<li>Centrifuge the tubes at 97 x g at 4 &#176;C for 1 min and pour off the supernatant. Be careful not to dislodge the pelleted tissue.</li>
	<li>Add 25 ml wash buffer and re-suspend the tissue by gently vortexing. Repeat steps 3.4 and 3.5 twice more.</li>
	<li>Place a 30 mesh tissue sieve over a sterile 250 ml beaker and pour the tissue suspension onto the sieve. Rinse the digestion tube with an additional 20 ml of wash buffer and pour onto the mesh. The undigested pancreatic and fat tissues are removed at this step.</li>
	<li>Pour the filtered material into two fresh 50 ml tubes and pellet the tissue by spinning at 97 x g for 1 min at 4 &#176;C. Decant supernatant and invert tubes to drain excess buffer.</li>
</ol>
4. Purification of Islets
<ol>
	<li>Add 20 ml of cold polysucrose/sodium diatrizoate solution (1.119 g/ml density) to the pelleted pancreatic tissue. Homogenously re-suspend the contents of each tube by gently pipetting up and down five times.</li>
	<li>Gently overlay the polysucrose/sodium diatrizoate solution with 10 ml HBSS. Maintain a sharp liquid interface by slowly adding the HBSS along the tube wall.</li>
	<li>Centrifuge the digested pancreata at 560 x g for 15 min (4 &#176;C) with slow acceleration and no brake.</li>
	<li>Collect the islet layer from the interface using a 10 ml pipette and place islets in fresh 50 ml tubes. Do not pool islets at this stage.</li>
	<li>Add 40 ml of wash buffer to each 50 ml tube and spin for 1 min at 140 x g at 4 &#176;C.</li>
	<li>Pour off the supernatant and re-suspend pooled islets in 50 ml of wash buffer by inverting the tubes several times. Centrifuge the tubes for 1 min at 97 x g at 4 &#176;C to collect islets in a pellet.</li>
	<li>Pour off the wash buffer and repeat the wash. Bring islets into the tissue culture hood and aspirate the supernatant.</li>
	<li>Re-suspend each tube of islets in 6 ml of islet medium.</li>
	<li>Transfer the islets from each 50 ml tube into a well of six-well tissue culture dish. Swirl the 6-well dish to collect islets into the middle of the well and observe islet quality and purity under a microscope.</li>
	<li>Manually collect islets using a 1 ml pipette and transfer the picked islets into the adjacent well containing 6 ml of islet media. Repeat islet swirling and picking until &#62; 90% purity is achieved.</li>
	<li>Transfer the purified islets to a 10 cm tissue culture dish containing 20 ml of islet medium.</li>
	<li>Place islets in a tissue culture incubator (37 &#176;C, 5% CO2) overnight (Figure 1A).</li>
	<li>In preparation of plating islet cells, coat the wells of a 384-well plate with 40 &#181;l of 804G conditioned media per well. Incubate overnight in a tissue culture incubator.</li>
</ol>
5. Dispersion and Plating of Islet Cells
<ol>
	<li>The following day, gently detach islets from the 10 cm dish with a cell scraper and transfer the islets into a 50 ml tube.</li>
	<li>Pellet the islets by centrifugation for 1 min at 22 x g at room temperature. Aspirate the supernatant.</li>
	<li>Wash the islets with 20 ml of warm PBS and decant as above.</li>
	<li>Re-suspend islets in 0.25 % trypsin (150 &#181;l per rat pancreas) and incubate at 37 &#176;C for 10 min. Pipette islets up and down 10 times using 1 ml pipette after 5 and 10 min of incubation.</li>
	<li>After 10 min, evaluate a 20 &#181;l sample of the trypsinized islets under the microscope to ensure complete digestion and to count the islet cells using a hemocytometer. If undigested islets remain, continue incubation for an additional 3 - 5 min and pipette up and down 10 more times. Repeat as necessary. 
	Note: The typical yield is ~ 125,000 - 150,000 islet-cells per rat.</li>
	<li>Remove 804G conditioned media-coated 384-well plate from the incubator and remove media using a 12-well manifold aspirator.</li>
	<li>Suspend islet cells at a density of 45,000 cells per ml in Islet function medium and plate 70 &#181;l (3,150 cells) per well with a multichannel pipette (Figure 1B). 
	Note: Because &#946;-cells located in the outer wells tend to migrate towards the perimeter of the plate use rows C to N and columns 4 to 21 to plate 228 wells with the islet-cells isolated from 6 rats.</li>
	<li>Place the plate in the tissue culture incubator for 48 hr to allow cell adhesion.</li>
</ol>
6. Islet-cell Culture Treatment, Fixation and Staining
<ol>
	<li>Prepare 200 &#181;l of vehicle and compounds (treatment conditions are performed in quadruplicate) at a 2x concentration in Islet function medium. Arrange compounds in a sterile 96-well plate to facilitate multi-well pipetting.</li>
	<li>Carefully remove islet medium with a 12-well manifold aspirator and replace with islet function medium (35 &#181;l/well) using a 12-well pipette. Remove and replace media from one row at a time to limit risk of drying (Figure 1C).</li>
	<li>Initiate treatment by transferring 35 &#181;l/well of the 2x compound solutions. Return plate to the incubator for the 48 hr treatment duration.</li>
	<li>After 48 hr of treatment, decant the treatment media by inverting the plate.</li>
	<li>Gently wash the islet cells with 50 &#181;l per well of 1x PBS (1 min). Add the PBS using a multi-channel pipette.</li>
	<li>Decant the PBS and fix the cells by adding 50 &#181;l per well of cold 4% paraformaldehyde (PFA) diluted in 1x PBS. Incubate cells for 15 min at 4 &#176;C.</li>
	<li>Wash the cells three times (5 min each) by inverting the plate to remove the PFA and gently adding 60 &#181;l of PBS per well as above.</li>
	<li>Prepare 15 ml antigen retrieval solution by mixing 14.25 ml formamide and 0.75 ml 0.15 M sodium citrate (pH 6). Make the antigen retrieval solution immediately before use.</li>
	<li>Remove PBS from wells by inverting the plate and add 60 &#181;l antigen retrieval solution to each well. Heat the plate in 70 &#176;C water bath for 45 min. Place a heating block on top of the plate during this incubation to prevent warping of the multi-well plate. 
	Note: The water should be half-way up the plate skirt; the plate should not be submerged.</li>
	<li>Remove the plate from water bath and allow it to cool for 20 min at room temperature. Ensure that the plate is not warped by visual inspection.</li>
	<li>Wash the cells with 60 &#181;l per well of 1x PBS three times (5 min each). Add PBS with a multi-channel pipette and decant PBS by inverting the plate.</li>
	<li>Use a multi-channel pipette to add 40 &#181;l per well of blocking buffer and incubate for 30 min at room temperature. Remove the blocking buffer by inverting the plate and decanting.</li>
	<li>Add 40 &#181;l of mouse anti-human Ki-67 (1:200) and goat anti-human PDX-1 (1:100) antibodies diluted in blocking buffer to each well and incubate at 4 &#176;C overnight.</li>
	<li>The next day, decant the anti-body solution and wash the cells with 1x PBS three times (5 min each) as described above. Decant the PBS.</li>
	<li>Add 40 &#181;l per well of diluted (1:200) biotinylated donkey anti-mouse IgG and rhodamine-conjugated donkey anti-goat IgG in blocking buffer. Incubate for 1 hr at room temperature.</li>
	<li>Wash the cells with 1x PBS three times, 5 min each. Add 40 &#181;l of diluted (1:400 in blocking buffer) fluorescein-conjugated streptavidin to each well. Incubate 30 min at room temperature.</li>
	<li>Wash the cells with 1x PBS three times, 5 min each. Add 40 &#181;l per well of DAPI (300 nM in blocking buffer) and incubate for 15 min at room temperature.</li>
	<li>Wash cells with 1x PBS two times and leave cells in 40 &#181;l 1x PBS. The plate is now ready to be analyzed.</li>
</ol>
7. &#946;-Cell Replication Analysis
Note: An assay protocol must be established for the high content screening microscope used to measure &#946;-cell replication. In its simplest composition, this protocol is a two-color assay where an identity marker is used to define &#946;-cells (PDX-1+-cells) and a replication marker (Ki-67) is used to define cell division events.
<ol>
	<li>Identify objects (&#946;-cells) based upon the average fluorescence intensity of PDX-1 staining (549 nm filter) within an approximate circle (length:width &#60; 1.6) within the expected pixel area of a &#946;-cell nucleus (Figure 2A).</li>
	<li>Next, establish settings to identify replication events (Ki-67-positivity) based upon the average fluorescence intensity (485 nm filter) within the PDX-1+ area (Figure 2B). 
	Note: Exposure times must be fixed to allow pixel intensity comparisons across images. Assay protocol parameters are adjusted such that machine calls consistently exclude nonspecific staining, debris and cell aggregates that could adversely affect data quality.</li>
	<li>Analyze a minimum of 500 &#946;-cells per well to maximize accuracy and limit variability. 
	Note: The basal &#946;-cell replication index in rat islet cultures is expected to be 0.5 - 3%. Typically, 40 images per well is more than sufficient to identify 500 &#946;-cells.</li>
	<li>Prior to analyzing the entire plate, analyze selected negative- (DMSO-treated) and positive-control (5-iodotubercidin [1 &#181;M] or dipyridamole [15 &#181;M]) -treated wells to assess the validity of the assay protocol. When the assay is established correctly, positive control compounds induce at least a two-fold increase in the percentage of replicating &#946;-cells.</li>
	<li>Read the entire plate once the assay protocol is validated.</li>
</ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+screening+for+secreted+proteins+that+modulate+glucose+handling+identifies+interleukin-6+family+members+as+potent+hypoglycemic+agents.">In vivo screening for secreted proteins that modulate glucose handling identifies interleukin-6 family members as potent hypoglycemic agents.</a> PLoS One. 7 (9), e44600 (2012).</li><li>Izumi, K., Hirao, Y., Hopp, L., Oyasu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+induction+of+ornithine+decarboxylase+in+urinary+bladder+carcinoma+cells.">In vitro induction of ornithine decarboxylase in urinary bladder carcinoma cells.</a> Cancer Res. 41 (2), 405-409 (1981).</li><li>Serup, P., 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+homeodomain+protein+IPF-1/STF-1+is+expressed+in+a+subset+of+islet+cells+and+promotes+rat+insulin+1+gene+expression+dependent+on+an+intact+E1+helix-loop-helix+factor+binding+site.">The homeodomain protein IPF-1/STF-1 is expressed in a subset of islet cells and promotes rat insulin 1 gene expression dependent on an intact E1 helix-loop-helix factor binding site.</a> Biochem J. 310, 997-1003 (1995).</li><li>Szabat, M., Luciani, D. S., Piret, J. M., Johnson, 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=Maturation+of+adult+beta-cells+revealed+using+a+Pdx1/insulin+dual-reporter+lentivirus.">Maturation of adult beta-cells revealed using a Pdx1/insulin dual-reporter lentivirus.</a> Endocrinology. 150 (4), 1627-1635 (2009).</li><li>Rieck, 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=Overexpression+of+hepatocyte+nuclear+factor-4alpha+initiates+cell+cycle+entry,+but+is+not+sufficient+to+promote+beta-cell+expansion+in+human+islets.">Overexpression of hepatocyte nuclear factor-4alpha initiates cell cycle entry, but is not sufficient to promote beta-cell expansion in human islets.</a> Mol Endocrinol. 26 (9), 1590-1602 (2012).</li><li>Wang, P., 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=Diabetes+mellitus--advances+and+challenges+in+human+beta-cell+proliferation.">Diabetes mellitus--advances and challenges in human beta-cell proliferation.</a> Nat Rev Endocrinol. 11 (4), 201-212 (2015).</li></ol>

The authors declare that they have no competing financial interests.

Experimental methods for studying the molecular pathways that control &#946;-cell growth and regeneration are important tools for diabetes researchers. Herein, a rat-islet-based screening platform to identify and characterize small-molecule stimulators of &#946;-cell replication is presented.
While most aspects of this protocol are easily performed by experienced researchers, a few steps require particular technique. First, during islet isolation, cannulation of the bile-duct without disruptin...

Diabetes encompasses a collection of disorders sharing the common end-point of disrupted glucose homeostasis. Although the pathogenic mechanisms of diabetes subtypes are distinct, they share the consequence of decreased &#946;-cell mass, i.e., loss of insulin production capacity1,2. Presently, diabetes treatment strategies rely upon chronic administration of exogenous insulin, pharmacologic stimulation of insulin production or enhancement of insulin sensitivity, and rarely, the transplantation of pancreatic islets or whole pancreas3,4. Regrettably, the success of these strategies is short-lived and/or fails to sufficiently recapitulate the function of endogenous insulin production. Despite the utility of developing a method to stimulate &#946;-cell regeneration, no such approach exists. Consequently, a major diabetes research goal is to develop methods to generate new &#946;-cells or to expand endogenous &#946;-cell mass5. Although &#946;-cell regeneration from renewable sources such as embryonic stem cells is advancing, safety and efficiency concerns make the pursuit of alternative strategies, including expansion of mature &#946;-cells, a priority6,7. Importantly, the predominant source of new &#946;-cells in vivo is pre-existing &#946;-cells rather than specialized progenitor cells8,9. Although &#946;-cells appear to have limited replication capacity, a small increase in &#946;-cell mass (~ 30%) may be sufficient to restore glucose homeostasis in many diabetics. Furthermore, in situ pharmacologic stimulation of &#946;-cell mass is a potentially inexpensive and scalable treatment strategy. Herein a high-content screening method for identifying and characterizing small molecules that stimulate &#946;-cell growth is presented.
A variety of in vitro experimental methods may be used to identify gene products and/or molecules that promote primary &#946;-cell replication. Early efforts for measuring &#946;-cell replication induction used fetal rodent pancreata culture or intact isolated islet cultures to measure [3H] thymidine incorporation, BrdU incorporation or mitotic bodies within the aldehyde-thionine or insulin stained population in response to specific treatment conditions10,11. These in vitro approaches and close variants thereof have several limitations. Prominent deficiencies include (1) the use of fetal cells which, unlike mature &#946;-cells, display a high basal &#946;-cell replication rate and are growth regulated in a distinct manner12; (2) the subjective nature of experimenter-dependent adjudication of &#946;-cell replication events; (3) the labor and time intensive nature of experimenter-dependent counting of &#946;-cell replication events retards experimental throughput; (4) the use of nuclear incorporation/stain/appearance to identify replication events and a non-overlapping cytoplasmic stain to identify &#946;-cells leads to misattribution of proximate non-&#946;-cell replication events to &#946;-cells.
More recently mature primary &#946;-cells have been used to assess the impact of transgene over-expression as well as gene product or compound treatment on &#946;-cell replication13-16. However, these studies have also relied upon subjective counting of replication events, cytoplasmic staining- or non-specific methods for &#946;-cell identification and/or labor-intensive steps that limit throughput, e.g., individual slide-well plating of cells or intact islet paraffin embedding and processing17. Notably, an image-based human &#946;-cell replication screening methodology, similar to the one presented herein, has been published18; however, successful use of this assay has not been demonstrated and the use of human islets for primary screening may not be broadly feasible.
An alternative strategy for identifying replication-promoting substances is to assess growth induction of &#946;-cell lines. Initial efforts used transformed &#946;-cell-lines such as min6 cells or INS 832/13-cells14,19-21. However, these cell-lines demonstrate unrestrained growth and bear little resemblance to well-differentiated &#946;-cells22. Consequently, growth-induction capacity is minimal, of unclear relevance and sometimes difficult to recapitulate. An improved strategy for cell-line based screening utilizes &#34;reversibly transformed&#34; cells that are growth arrested in the absence of tetracycline (doxycycline)-dependent SV40 T antigen expression23,24. However, it is unclear whether these cells revert to a &#34;normal&#34; &#946;-cell-like state upon doxycycline removal. Unfortunately, use of these cells has yielded generalized growth-promoting compounds that don&#39;t appear to have immediate utility24. Overall, the use of cell-lines to study growth regulation of a cell-type displaying minimal spontaneous replication activity may have limited applicability.
The &#946;-cell replication screening platform presented herein utilizes mature primary rat &#946;-cells to retain in vivo growth regulation to the extent possible, islet-cell cultures of mixed cell-type composition to enable identification of lineage-restricted growth-promoting activities, multi-well formatting to maximize throughput and automated analysis to eliminate bias and facilitate throughput. Successful use of this platform has enabled identification of several compounds that promote &#946;-cell replication25,26. Additionally, the assay has been used for structure-activity relationship studies and chemical epistasis experiments to provide mechanistic insights into the molecular regulation of &#946;-cell replication. The presented platform was successfully adapted for lentiviral RNAi-based investigation of &#946;-cell replication pathways25. Limitations of the assay include restricted scalability (use of primary cells), use of rodent rather than human islet-cells (though the assay may be adapted for human islet studies), expenses associated with antibody-based imaging and primary islet use, the use of dispersed islets (disrupted islet architecture) to facilitate automated image acquisition and dependence upon the availability of an automated microscope with image acquisition and analysis capability. Although a facile in vivo screening methodology for identifying gene products or compounds that stimulate &#946;-cell regeneration in situ would be ideal, such a platform is not yet available27. Consequently, the described platform is appropriate for researcher interested in investigating most aspects of &#946;-cell replication.

<table><tbody><tr><td>250 g male Male Sprague Dawly Rat</td><td>Charles River</td><td>Stain # 400</td><td></td></tr><tr><td>12 cm teeth tisuue forceps</td><td>Fine Science Tools</td><td>11021-12</td><td></td></tr><tr><td>11.5 cm fine scissors</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>14.5 cm surgical scissors</td><td>Fine Science Tools</td><td>14001-14</td><td></td></tr><tr><td>16 cm curved forceps</td><td>Fine Science Tools</td><td>11003-16</td><td></td></tr><tr><td>12 cm curved hepostat</td><td>Fine Science Tools</td><td>13011-12</td><td></td></tr><tr><td>12 cm scalpel handle</td><td>Fine Science Tools</td><td>10003-12</td><td></td></tr><tr><td>Tissue sieve-30 mesh</td><td>Bellco Glass</td><td>1985-85000</td><td></td></tr><tr><td>Cizyme RI, 375,000 CDA units</td><td>VitaCyte</td><td>005-1030</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution (Ca&lt;sup&gt;++&lt;/sup&gt; and Mg&lt;sup&gt;++&lt;/sup&gt;)</td><td>Gibco</td><td>24020-117</td><td></td></tr><tr><td>Ketamine HCl (200 mg/20 ml)</td><td>JHP Pharmaceuticals</td><td>NDC# 42023-113-10</td><td>to make anesthetic cocktail&amp;nbsp;</td></tr><tr><td>Xylazine (5 g/50 ml)</td><td>LLOYD</td><td>NADA# 139-236</td><td>to make anesthetic cocktail&amp;nbsp;</td></tr><tr><td>Histopaque 1077</td><td>Sigma</td><td>H-1077</td><td>to make histopaque 1100</td></tr><tr><td>Histopaque 1119</td><td>Sigma</td><td>H-1119</td><td>to make histopaque 1100</td></tr><tr><td>Newborn Calf Serum 500 ml</td><td>Hyclone</td><td>SH30118.03</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt solution</td><td>Hyclone</td><td>SH30268.01</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/Low Glucose&amp;nbsp;</td><td>Hyclone</td><td>SH30021.01</td><td></td></tr><tr><td>Functionality/Viability Solution&amp;nbsp;</td><td>Mediatech</td><td>99-768-CV</td><td></td></tr><tr><td>RPMI1640 media&amp;nbsp;</td><td>Hyclone</td><td>SH30096.01</td><td>to make conditioned medium</td></tr><tr><td>804G rat bladder carcinoma cell-line</td><td>Available upon request</td><td></td><td>to make conditioned medium</td></tr><tr><td>Fetal Bovine Serum, Qualified</td><td>Gibco</td><td>26160</td><td></td></tr><tr><td>GlutaMax-I</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicillin (5,000 IU/ml/Strptomycin (5 mg/ml)&amp;nbsp;</td><td>MP Biomedicals</td><td>1670049</td><td></td></tr><tr><td>Formamide 500 ml</td><td>Fisher BioReagents</td><td>BP227-500</td><td></td></tr><tr><td>Antigen Unmasking Solution 250 ml (pH 6.0)</td><td>Vector Laboratories</td><td>H-3300</td><td>to make 0.15 M Sodium Sitrate solution</td></tr><tr><td>Dextrose, Anhydrous</td><td>EMD Chemicals</td><td>DX0145-1</td><td>to make 1 M glucose solution</td></tr><tr><td>Nomal Donkey Serum (Powder)</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787-100ML</td><td></td></tr><tr><td>Mouse anti-human Ki67 antibody</td><td>BD Biosciences</td><td>556003</td><td></td></tr><tr><td>Goat anti-human PDX-1 antibody</td><td>R&amp;amp;D Systems</td><td>AF2419</td><td></td></tr><tr><td>Polyclonal Guinea Pig anti-insulin antibody</td><td>Dako</td><td>2016-08</td><td></td></tr><tr><td>Polyclonal Rabbit anti-glucagon antibody</td><td>Dako</td><td>2014-06</td><td></td></tr><tr><td>Polyclonal Rabbit anti-somatostatin antibody</td><td>Dako</td><td>2011-08</td><td></td></tr><tr><td>Polyclonal chicken anti-vimentin antibody</td><td>abcam</td><td>ab24525</td><td></td></tr><tr><td>Biotin-SP-conjugated, Donkey Anti-Mouse IgG</td><td>Jackson ImmunoResearch</td><td>715-065-150</td><td></td></tr><tr><td>StreptAvidin, Alex Flour 488 conjugated&amp;nbsp;</td><td>Invitrogen</td><td>S32354</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Goat IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>705-025-147</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Guinea Pig IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>706-025-148</td><td></td></tr><tr><td>Rhodamine-conjugated Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>711-025-152</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Guinea Pig IgG&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>706-175-148</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Goat IgG</td><td>Jackson ImmunoResearch</td><td>705-175-147</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Rabbit IgG</td><td>Jackson ImmunoResearch</td><td>711-175-152</td><td></td></tr><tr><td>Cy 5-conjugated Donkey Anti-Chicken IgG</td><td>Jackson ImmunoResearch</td><td>703-175-155</td><td></td></tr><tr><td>DAPI</td><td>Millipore</td><td>S7113</td><td></td></tr><tr><td>Disposable Reagent Reservoir 25 ml</td><td>Sorenson BioScience</td><td>39900</td><td></td></tr><tr><td>384 well, black/clear, tissue culture treated plate</td><td>BD Falcon</td><td>353962</td><td></td></tr><tr><td>96 well, black/clear, tissue culture treated plate</td><td>Costar</td><td>3603</td><td></td></tr><tr><td>Multi-channel pipettor</td><td>Costar</td><td>4880</td><td></td></tr><tr><td>12-channel vaccume aspirator</td><td>Drummond</td><td>3-000-096</td><td></td></tr><tr><td>Cell Scraper</td><td>Falcon</td><td>353085</td><td></td></tr><tr><td>Isotemp Water Bath Model 2223&amp;nbsp;</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>High-content screening instrument: ArrayScan VTI</td><td>Thermo Scientific</td><td></td><td></td></tr></tbody></table>

a high-content in vitro pancreatic islet &#946;-cell replication discovery platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion of endogenous insulin-producing pancreatic islet &#946;-cells remains an attractive strategy. &#946;-cells have limited spontaneous regenerative activity; consequently, a crucial research effort is to develop a precise understanding of the molecular pathways that restrain &#946;-cell growth and to identify drugs capable of overcoming these restraints. Herein an automated high-content image-based primary-cell screening method to identify &#946;-cell replication-promoting small molecules is presented. Several, limitations of prior methodologies are surmounted. First, use of primary islet cells rather than an immortalized cell-line maximizes retention of in vivo growth restraints. Second, use of mixed-composition islet-cell cultures rather than a &#946;-cell-line allows identification of both lineage-restricted and general growth stimulators. Third, the technique makes practical the use of primary islets, a limiting resource, through use of a 384-well format. Fourth, detrimental experimental variability associated with erratic islet culture quality is overcome through optimization of isolation, dispersion, plating and culture parameters. Fifth, the difficulties of accurately and consistently measuring the low basal replication rate of islet endocrine-cells are surmounted with optimized immunostaining parameters, automated data acquisition and data analysis; automation simultaneously enhances throughput and limits experimenter bias. Notable limitations of this assay are the use of dispersed islet cultures which disrupts islet architecture, the use of rodent rather than human islets and the inherent limitations of throughput and cost associated with the use of primary cells. Importantly, the strategy is easily adapted for human islet replication studies. This assay is well suited for investigating the mitogenic effect of substances on &#946;-cells and the molecular mechanisms that regulate &#946;-cell growth.

To assess &#946;-cell or &#945;-cell replication, a four-color assay protocol is required. First, objects are identified by DAPI staining (Channel 1, 386 nm). Next, &#946;-cells (event 1) are counted: objects that co-express PDX-1+ (channel 2, 650 nm) and peri-nuclear insulin (channel 3, 549 nm). Subsequently, replicating &#946;-cells (event 2) are counted: &#946;-cells (event 1) that co-express Ki-67 (channel 4, 485 nm) (Figure 3). The percentage of replicatin...

Watch this Scientific Journal Video about A High-content In Vitro Pancreatic Islet Î²-cell Replication Discovery Platform at JoVE.com

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Critical challenges for the diabetes research field are to understand the molecular mechanisms that regulate islet &#946;-cell replication and to develop methods for stimulating &#946;-cell regeneration. Herein a high-content screening method to identify and assess the &#946;-cell replication-promoting activity of small molecules is presented.

A High-content In Vitro Pancreatic Islet &#946;-cell Replication Discovery Platform

Loss of insulin-producing &#946;-cells is a central feature of diabetes. While a variety of potential replacement therapies are being explored, expansion ...

a-high-content-vitro-pancreatic-islet-cell-replication-discovery

Research

JoVE Journal

Medicine

11.1K Views.  Stanford University School of Medicine.  Cruciale uitdagingen voor het onderzoeksgebied diabetes zijn de moleculaire mechanismen die eilandje β-cellen replicatie en op werkwijzen voor het stimuleren van β-celregeneratie ontwikkelen begrijpen. Hierin een high-gehalte screening methode voor het identificeren en evalueren van de β-cel-replicatie bevorderende activiteit van kleine moleculen wordt gepresenteerd. 

Video: A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform

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

Assessing Replication and Beta Cell Function in Adenovirally-transduced Isolated Rodent Islets

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, respectively. Functional beta cell mass is determined by the anatomical beta cell mass as well as the ability of the beta cells to respond to a nutrient load. A loss of functional beta cell mass is central to both major forms of diabetes 1-3. Whereas the declining functional beta cell mass results from an autoimmune attack in type 1 diabetes, in type 2 diabetes, this decrement develops from both an inability of beta cells to secrete insulin appropriately and the destruction of beta cells from a cadre of mechanisms. Thus, efforts to restore functional beta cell mass are paramount to the better treatment of and potential cures for diabetes.
Efforts are underway to identify molecular pathways that can be exploited to stimulate the replication and enhance the function of beta cells. Ideally, therapeutic targets would improve both beta cell growth and function. Perhaps more important though is to identify whether a strategy that stimulates beta cell growth comes at the cost of impairing beta cell function (such as with some oncogenes) and vice versa.
By systematically suppressing or overexpressing the expression of target genes in isolated rat islets, one can identify potential therapeutic targets for increasing functional beta cell mass 4-6. Adenoviral vectors can be employed to efficiently overexpress or knockdown proteins in isolated rat islets 4,7-15. Here, we present a method to manipulate gene expression utilizing adenoviral transduction and assess islet replication and beta cell function in isolated rat islets (Figure 1). This method has been used previously to identify novel targets that modulate beta cell replication or function 5,6,8,9,16,17.

This protocol allows one to identify factors that modulate functional beta cell mass to find potential therapeutic targets for the treatment of diabetes. The protocol consists of a streamlined method to assess islet replication and beta cell function in isolated rat islets following manipulation of gene expression with adenoviruses.

Glucose homeostasis is primarily controlled by the endocrine hormones insulin and glucagon, secreted from the pancreatic beta and alpha cells, ...

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 ...

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, ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Bioengineering

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

Differentiation of Human Pluripotent Stem Cells into Insulin-Producing Islet Clusters

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and diabetes treatment. However, challenges remain in obtaining stem cell-derived beta cells that adequately mimic native human beta cells. Building upon previous studies, hPSC-derived islet cells have been generated to create a protocol with improved differentiation outcomes and consistency. The protocol described here utilizes a pancreatic progenitor kit during Stages 1-4, followed by a protocol modified from a paper previously published in 2014 (termed "R-protocol" hereafter) during Stages 5-7. Detailed procedures for using the pancreatic progenitor kit and 400 &#181;m diameter microwell plates to generate pancreatic progenitor clusters, R-protocol for endocrine differentiation in a 96-well static suspension format, and in vitro characterization and functional evaluation of hPSC-derived islets, are included. The complete protocol takes 1 week for initial hPSC expansion followed by ~5 weeks to obtain insulin-producing hPSC islets. Personnel with basic stem cell culture techniques and training in biological assays can reproduce this protocol.

The differentiation of stem cells into islet cells provides an alternative solution to conventional diabetes treatment and disease modeling. We describe a detailed stem cell culture protocol that combines a commercial differentiation kit with a previously validated method to aid in producing insulin-secreting, stem cell-derived islets in a dish.

Differentiation of human pluripotent stem cells (hPSCs) into insulin-secreting beta cells provides material for investigating beta cell function and ...

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

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Intracellular Signaling Events and Hormone Secretion in Human Pseudoislets

Human Pseudoislet System for Synchronous Assessment of Fluorescent Biosensor Dynamics and Hormone Secretory Profiles

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, have a central role in the control of glucose homeostasis through the secretion of insulin by beta cells, which lowers blood glucose, and glucagon by alpha cells, which raises blood glucose. Intracellular signaling pathways, including those mediated by cAMP, are key for regulated alpha and beta cell hormone secretion. The 3D islet structure, while essential for coordinated islet function, presents experimental challenges for mechanistic studies of the intracellular signaling pathways in primary human islet cells. To overcome these challenges and limitations, this protocol describes an integrated live-cell imaging and microfluidic platform using primary human pseudoislets generated from donors without diabetes that resemble native islets in their morphology, composition, and function. These pseudoislets are size-controlled through the dispersion and reaggregation process of primary human islet cells. In the dispersed state, islet cell gene expression can be manipulated; for example, biosensors such as the genetically encoded cAMP biosensor, cADDis, can be introduced. Once formed, pseudoislets expressing a genetically encoded biosensor, in combination with confocal microscopy and a microperifusion platform, allow for the synchronous assessment of fluorescent biosensor dynamics and alpha and beta cell hormone secretory profiles to provide more insight into cellular processes and function.

Author Spotlight: Investigating Islet Abnormalities and Function with a Pseudoislet Protocol 

This protocol describes a method for the synchronous acquisition and co-registration of intracellular signaling events and the secretion of insulin and glucagon by primary human pseudoislets using the adenoviral delivery of a cyclic adenosine monophosphate (cAMP) biosensor, a cAMP difference detector in situ (cADDis), and a microperifusion system.

The pancreatic islets of Langerhans, which are small 3D collections of specialized endocrine and supporting cells interspersed throughout the pancreas, ...

A High-content In Vitro Pancreatic Islet β-cell Replication Discovery Platform

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