The authors would like to thank Bethany Carboneau (Vanderbilt University) for critical reading of this manuscript. We also thank Anastasia Coldren (Vanderbilt University Medical Center Islet Procurement and Analysis Core) for islet isolations, and Dr. Alvin C. Powers (Vanderbilt University Medical Center) and Dr. David Jacobson (Vanderbilt University) for use of their centrifuge and tissue culture facility. This research involved use of the Islet Procurement and Analysis Core of the Vanderbilt Diabetes Research and Training Center supported by NIH grant DK20593. This work was supported by an American Heart Association Postdoctoral Fellowship (14POST20380262) to R.C.P., and grants from the Juvenile Diabetes Research Foundation (1-2011-592), and Department of Veterans Affairs (1BX00090-01A1) to M.A.G.

All procedures were approved and performed in accordance with the Vanderbilt Institutional Animal Care and Use Committee.
1. Labeling COI with Fluorophore (Optional)
<ol>
	<li>Choose a fluorescent dye that will react with a free primary amine (e.g., on a protein), such as succinimidyl esters or fluorescein derivatives, to visualize microsphere cargo. Dissolve 8x molar excess (relative to moles of COI) of fluorophore into 200 &#181;l of dimethyl sulfoxide (DMSO).</li>
	<li>Resuspend 50 mg of COI up to a final volume of 800 &#181;l in a vehicle solution (final concentration of 62.5 ng/ &#181;l). The vehicle solution will vary depending on the source of the COI. 
	NOTE: Typical vehicle solutions include phosphate buffered saline (PBS) or DMSO.</li>
	<li>Add all of the fluorophore/DMSO solution from step 1.1 and resuspend the COI. Vortex overnight at 4 &#176;C. Alternatively, vortex the reaction mixture at room temperature (RT) for 4 hr.</li>
	<li>Following the labeling reaction, remove excess fluorophore using a desalting column.
	<ol>
		<li>Drain storage buffer from desalting column and rinse with 16 ml of deionized water. Discard all flow through.</li>
		<li>Add the fluorescently labeled COI to the desalting column. Elute with 1.2 ml of deionized water and collect the flow through.</li>
		<li>Repeat elution step an additional four times, each time adding 1.2 ml of deionized water and collecting the flow through as a separate sample.</li>
		<li>Freeze all collected flow through samples at -80 &#176;C and lyophilize according to manufacturer&#39;s instructions for 24 hr.</li>
	</ol>
	</li>
</ol>
2. COI-loaded Microsphere Preparation via the Water-in-oil-in-water Emulsion Solvent Evaporation Method
<ol>
	<li>Add 1 mg of fluorescently labeled COI to 100 &#181;l of deionized water to form the first water phase (W1).</li>
	<li>Dissolve 65 mg of poly(lactic-co-glycolic acid) (50:50 Lactide:Glycolide, molecular weight 54,000 - 69,000) in 750 &#181;l of dichloromethane in a microcentrifuge tube. Ultrasonicate for 10 - 30 sec (at 160 W) to completely dissolve the PLGA. This forms the oil phase (O).</li>
	<li>Add all of the W1 phase generated in step 2.1 to the O phase in a drop-wise manner. Emulsify using a hand-held homogenizer at 20,000 rpm for 30 sec to form the W1/O phase.</li>
	<li>Add all of the W1/O phase in a drop-wise manner to 15 ml of a 1% (weight/volume) aqueous poly(vinyl alcohol) (PVA) solution and emulsify using a hand-held homogenizer at 20,000 rpm for 30 sec.</li>
	<li>Transfer all of the emulsion generated in step 2.4 to a 200 ml round-bottom flask and subject to a 635 mm Hg vacuum using a rotary evaporator for 1 hr to remove the solvent and generate the aqueous phase.</li>
	<li>Aliquot 1 ml of the aqueous phase generated in step 2.5 to fourteen microcentrifuge tubes. Centrifuging the aqueous solution in the microcentrifuge tubes at 7,500 x g for 8 min. 
	NOTE: At this step, the microspheres are present in the remaining aqueous solution and are concentrated to the bottom of the microcentrifuge tube.</li>
	<li>Carefully remove 900 &#181;l of aqueous solution from the microcentrifuge tubes with a micropipette, pipetting so as to not disturb the microspheres on the bottom of the microcentrifuge tube.
	<ol>
		<li>Wash the microspheres of excess PVA by adding 1 ml deionized water to each microcentrifuge tube. Centrifuge at 7,500 x g for 8 min, and again carefully remove 900 &#181;l of aqueous solution from the microcentrifuge tubes with a micropipette, pipetting so as to not disturb the microspheres on the bottom of the microcentrifuge tube. 
		NOTE: The remaining 100 &#181;l aqueous solution contains the generated microspheres.</li>
	</ol>
	</li>
	<li>Freeze the aqueous microsphere solution generated in step 2.7 at -80 &#176;C.</li>
	<li>Lyophilize microspheres using a lyophilizer according to manufacturer&#39;s instructions and store at -20 &#176;C. 
	NOTE: Measure loading efficiency of the COI by completely dissolving PLGA microspheres and determining the protein concentration relative to a standard curve of the free protein7.</li>
	<li>As a negative control, generate a batch of &#34;blank&#34; microspheres (hereafter referred to as control microspheres). 
	NOTE: The generation of control microspheres is identical to the generation of hydrophilic COI-loaded microspheres, with no COI addition to 800 &#181;l of the vehicle solution in step 1.2. Match control particles in mass concentration of PLGA relative to COI-loaded PLGA microspheres for &#946;-cell mitogen assay.</li>
</ol>
3. Preparation of Islet Culture Media and Pre-assay Media
<ol>
	<li>Prepare 200 ml media for culture of intact mouse islets: RPMI 1640 media supplemented with 11 mM glucose, 10% horse serum, 100 U/ml penicillin G and 100 &#181;g/ml streptomycin (hereafter referred to as islet culture media). 
	NOTE 1: Unlike fetal bovine serum (FBS), horse serum lacks placental lactogen, an inducer of &#946;-cell proliferation, which confounds analysis in the proliferation assay. 
	NOTE 2: As the PLGA microspheres are not sterilized prior to use, the addition of antibiotics to the culture media is crucial to avoid microbiological contamination.</li>
	<li>Remove 25 ml of islet culture media with an electronic pipettor and place in a 50 ml conical tube. Supplement the 25 ml of islet culture media in the 50 ml conical tube with a final concentration of 0.2 mM EGTA to generate the pre-assay media. 
	NOTE: The addition of EGTA mildly loosens cell-cell contacts in the islets without altering islet architecture. This helps ensure the COI can reach the inner cells of the islet and helps prevent necrosis of the central islet.</li>
</ol>
4. Culturing of Intact Mouse Islets
<ol>
	<li>Isolate intact islets from a pre-selected strain, sex, age, and genotype of mice following a routine collagenase digestion of the pancreas8,9. Pool together islets from like samples.</li>
	<li>Aliquot 40 islets into one well of a 96-well tissue culture plate in 200 &#181;l of islet culture media. Visually size-match islets per well (a precise assessment of islet equivalency (IEQ) between samples is not necessary). Fill empty wells immediately adjacent to wells with islets with 200 &#181;l sterile water to provide an evaporation buffer. Culture islets in media overnight at 37 &#176;C under an atmosphere of 95% air and 5% CO2.</li>
</ol>
5. Re-suspension of COI-loaded and Control PLGA Microspheres
<ol>
	<li>After overnight islet recovery, resuspend microspheres by adding pre-assay media to one aliquot of lyophilized COI-loaded PLGA microspheres such that the final concentration of COI in the microcentrifuge tube is 10 ng/&#181;l (the amount of COI present in a given amount of the generated microspheres was determined in step 2.9). Sonicate the resuspended COI-loaded microspheres in an ice water bath for 10 min at 160 W with 10 sec long pulses at 4 &#176;C.</li>
	<li>Resuspend control PLGA microspheres by adding the same volume of pre-assay media to an equal mass of lyophilized control PLGA microspheres. Sonicate the resuspended control PLGA microspheres in an ice water bath for 10 min at 160 W with 10 sec long pulses at 4 &#176;C.</li>
	<li>Visually confirm microspheres are dispersed by pipetting 2 &#181;l of resuspended microspheres between two glass coverslips. Visualize microspheres using a 40X objective on an epifluorescence or brightfield microscope.</li>
	<li>If significant aggregation of microspheres is still visible, sonicate in an ice water bath for an additional 10 min at 160 W with 10 sec long pulses at 4 &#176;C and repeat visualization of resuspended microspheres.</li>
</ol>
6. Treatment of Islets with COI-loaded or Control PLGA Microspheres
<ol>
	<li>For each well to be treated with COI-loaded microspheres, prepare assay media by diluting the 10 ng/&#181;l of the resuspended COI microspheres with pre-assay media to a final volume of 100 &#181;l and a pre-determined final concentration. 
	NOTE: The optimal final concentration of the protein will vary for each COI used for this assay. Ideally, a range of final concentrations are tested.</li>
	<li>For each well to be used as a control, prepare control assay media by diluting the resuspended control PLGA microspheres generated in step 5.2 with the same dilution volume used to dilute the COI-loaded microspheres in step 6.1. 
	NOTE: Islets treated with control assay media will serve as a negative control to enable detection of any effect the blank microspheres may have on the experimental outcomes.</li>
	<li>Carefully remove 100 &#181;l of islet culture media from each well with a micropipette such that no islets are dislodged from the wells. Gently add 100 &#181;l of assay media or 100 &#181;l of control assay media to each well.</li>
	<li>Incubate islets at 37 &#176;C under an atmosphere of 95% air and 5% CO2 for three days.</li>
</ol>
7. Dispersing Intact Islets onto Microscope Slides
<ol>
	<li>After three days, use a micropipette to carefully remove and discard assay media from all wells so as not to dislodge islets. Gently add 200 &#181;l PBS to islets to wash them of the assay media. Carefully remove and discard PBS with a micropipette and gently wash islets again with an additional 200 &#181;l PBS.</li>
	<li>Remove and discard PBS with a micropipette and add 100 &#181;l of 0.025% trypsin, 2 mM EDTA solution. Incubate at RT for 3 min. Pipet islets in the trypsin-EDTA solution up and down every 2 min until islets are visually confirmed to be dispersed into single cells (note that some small clumps of cells may still be present) using a light microscope. Transfer the entire volume of each well individually into labeled- microcentrifuge tubes.</li>
	<li>Add 400 &#181;l of islet culture media to each microcentrifuge tube to stop the trypsin-mediated cell dissociation.</li>
	<li>Centrifuge samples for 5 min at 100 x g at 4 &#176;C to pellet islet cells to the bottom of microcentrifuge tubes.</li>
	<li>Gently remove supernatant using a micropipette, and resuspend pellet in 200 &#181;l fresh islet culture media.</li>
	<li>Using a cyto-centrifuge, centrifuge dissociated islets at 140 x g for 3 min onto charged microscope slides.</li>
	<li>Air dry slides at RT for 10 min, then draw a box around the dissociated islets with a hydrophobic marking pen.</li>
	<li>Fix cells with 75 &#181;l 4% paraformaldehyde (PFA) at RT for 10 min. Remove 4% PFA and gently wash cells in 75 &#181;l PBS two times. After washing, permeabilize cells with 75 &#181;l 0.2% Triton X-100 in PBS for 10 min. Then, wash cells with 75 &#181;l PBS. 
	Caution: PFA is known to be allergenic, carcinogenic, and toxic.</li>
</ol>
8. Immunofluorescence Labeling of Dissociated Islets for Insulin and the Cell Proliferation Marker, Ki67
<ol>
	<li>Prepare a humid chamber by placing two wet paper towels in the bottom of a 100 slide capacity microscope slide box.</li>
	<li>Place slides flat down (cells facing up) in humid chamber. Prepare samples for labeling by aspirating the PBS from the previous wash step using a micropipette and adding 75 &#181;l of blocking solution (5% Normal Donkey Serum (NDS) in PBS) to each slide to block non-specific binding of antibodies to the specimens. Incubate slides in blocking solution for 1 hr at RT.</li>
	<li>Gently aspirate blocking solution using a micropipette and add 75 &#181;l of primary antibody solution containing guinea pig anti-insulin and rabbit anti-Ki67 antibodies, diluted 1 &#181;g/ml in 5% NDS in PBS. Incubate at RT for 1 hr. 
	NOTE: Alternative markers of cell proliferation include proliferating cell nuclear antigen (PCNA) and phosphohistone H3 (PH3),</li>
	<li>Gently aspirate primary antibody solution using a micropipette and rinse the cells with 75 &#181;l PBS. Aspirate PBS using a micropipette and rinse cells two more times, each time with 75 &#181;L PBS. Incubate each sample with 75 &#181;l anti-guinea pig Cy5 secondary antibody and anti-rabbit Cy3 secondary antibody diluted to 2 &#181;g/ml in blocking solution for 1 hr at RT. 
	NOTE: Do not use secondary antibodies labeled with the same or spectrally overlapping fluorophore that was already used to label the microspheres.</li>
	<li>Aspirate secondary antibody solution using a micropipette and incubate in 75 &#181;l of 300 nM DAPI in PBS for 3 min at RT. Aspirate DAPI using a micropipette and rinse in deionized water for 5 min.</li>
	<li>Gently aspirate water from samples using a micropipette. Spot a drop (roughly 100 &#181;l) of fast-drying mounting solution containing antifade reagent on a glass coverslip. Invert the microscope slide sample side down, onto the mounting solution. Gently press the coverslip against the slide using a pipet tip to remove bubbles and wipe away excess liquid with a soft cleaning tissue. Allow coverslips to dry overnight at RT.</li>
</ol>
9. Image Acquisition and Analysis
<ol>
	<li>Use an epifluorescence microscope with an attached camera for image acquisition. For each fluorophore, determine the optimal exposure time (typically 20 msec for DAPI, 40 - 80 msec for insulin, and 250 msec for Ki67). Ensure image acquisition parameters are equal for all samples.</li>
	<li>For each sample, manually count 3,000 insulin-positive cells and of those count how many are also Ki67-positive. Only count insulin-positive cells that have a well-defined and clearly visible nucleus. Calculate the percentage of proliferating &#946;-cells for each sample by dividing the number of Ki67-positive/insulin-positive cells by the total number of insulin-positive cells and multiplying by 100.</li>
	<li>After determining the percentage of proliferating &#946;-cells for each sample, determine if a statistically significant difference in &#946;-cell proliferation exists between control and experimental samples by analyzing results with a one-way ANOVA using commercially available statistical software.</li>
</ol>

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The authors have nothing to disclose.

The study of &#946;-cell proliferation in culture is typically hampered by several difficulties. First, immortalized &#946;-cell lines are characterized by higher degrees of proliferation than what is found in endogenous &#946;-cells in live islets. Additionally, these immortalized cell lines lack the normal architecture critical for normal &#946;-cell function. These two facts make it difficult to determine if results obtained using immortalized &#946;-cell lines will hold true when tested in vivo or in whole i...

Pancreatic &#946;-cells are the only insulin-producing cells in the body and are critical to maintain blood glucose homeostasis. While healthy individuals have sufficient &#946;-cell mass and function to properly regulate blood glucose, individuals with diabetes are characterized by insufficient &#946;-cell mass and/or function1,2. It has been proposed that inducing &#946;-cell proliferation can ultimately increase &#946;-cell mass and restore glucose homeostasis in individuals with diabetes3. However, evaluation and validation of potential &#946;-cell proliferative compounds in intact islets is necessary before effective therapies can be developed. Transplantation of cadaveric human islets into individuals with diabetes restores blood glucose homeostasis for some time, but the availability and success of this experimental procedure is hindered by a shortage of human islets available for transplant and by &#946;-cell death in the islets after transplant4. Even with the discovery of factors that induce multiplication of insulin-producing cells, a major challenge still exists in delivering these factors to relevant sites in vivo. One strategy for sustained local delivery of &#946;-cell proliferative compounds is poly(lactic-co-glycolic) acid (PLGA). PLGA has a history of use in FDA approved drug delivery products owing to its high safety, biodegradability, and extended release kinetics5. Specifically, PLGA is a copolymer of lactide and glycolide that degrades via hydrolysis with water either in vivo or in culture into lactic acid and glycolic acid, which are naturally occurring metabolites in the body. The encapsulated drug compound can be released in the surrounding environment by both diffusion and/or degradation-controlled release mechanisms. Encapsulation of COI provides protection against enzymatic degradation, improving the bioavailability of the reagent compared to unencapsulated COI5. We suggest that PLGA microspheres can be used to administer candidate compounds to intact islets in culture, and ultimately in vivo. Testing the efficacy of PLGA to administer &#946;-cell mitogens to islets ex vivo is critical before transplantation protocols are explored.
Currently, there is no technique to measure &#946;-cell proliferation in live animals. Experiments to assess effectiveness of potential proliferative compounds in vivo therefore require administration of these compounds to live animals, with subsequent dissection and processing of pancreata for immunolabeling. Such protocols are expensive and laborious, and require the compound to be administered systemically, without any guarantee that they will reach the islets. Conversely, several immortalized &#946;-cell lines are available for the study of insulin-producing cells in culture, but these cell lines lack the islet architecture and environment found in living organisms6. Immortalized &#946;-cell lines are also characterized as having a much higher degree of replication than endogenous &#946;-cells in vivo, thus complicating analysis of compounds that induce proliferation. In this study, we describe a protocol that uses intact islets isolated from adult mice. Unlike &#946;-cell lines, intact islets retain normal islet architecture. Likewise, in contrast to experiments conducted in vivo, administering proliferative compounds directly to cultured intact islets significantly reduces the quantity of reagents that is necessary to accurately measure &#946;-cell proliferation.
The current study utilizes PLGA to administer a COI, in this example, recombinant human Connective Tissue Growth Factor (rhCTGF). The method described here confers a significant advantage over the administration of raw compound to cultured islets as it allows for a continual release of compound into the media. Notably, this assay can be modified to administer a wide variety of proteins and antibodies of interest to intact islets. Effects on other endocrine cell types, including &#945;-cells, may also be analyzed.

<table><tbody><tr><td>Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer</td><td>ThermoFisher Scientific</td><td>O6149</td><td>For labeling COI with fluorophore</td></tr><tr><td>DMSO Dimethyl Sulfoxide</td><td>Fisher BioReagents</td><td>BP231-1</td><td>For dissolving fluorophore in step 1</td></tr><tr><td>Disposable PD-10 Desalting Columns</td><td>GE Healthcare</td><td>17-0851-01</td><td>Desalting column used in step 1</td></tr><tr><td>Resomer RG 505, Poly(D,L-lactide-co-glycolide), ester terminated, molecular weight 54,000 - 69,000</td><td>Sigma-Aldrich</td><td>739960</td><td>Used in generation of microspheres in step 2</td></tr><tr><td>Poly(vinyl alcohol) molecular weight 89,000 - 98,000</td><td>Sigma-Aldrich</td><td>341584</td><td>Used in generation of microspheres in step 2</td></tr><tr><td>RPMI 1640</td><td>Thermo Scientific</td><td>11879-020</td><td>For culturing islets</td></tr><tr><td>Dextrose Anhydrous</td><td>Fisher BioReagents</td><td>200-075-1</td><td>Supplement for islet media</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td>Antibiotics for islet media</td></tr><tr><td>Normal horse serum</td><td>Jackson ImmunoResearch</td><td>008-000-121</td><td>Supplement for islet media</td></tr><tr><td>96-well tissue culture plate</td><td>Corning</td><td>3603</td><td>For culturing islets</td></tr><tr><td>Ethylene glyco-bis(2-aminoethylether)-&lt;em&gt;N,N,N&#039;,N&#039;&lt;/em&gt;-tetraacetic acid</td><td>Sigma-Aldrich</td><td>E4378</td><td>Supplement for pre-assay islet media</td></tr><tr><td>Cytospin 4 Cytocentrifuge</td><td>Thermo Scientific</td><td>A78300003</td><td>For spinning cells onto microscope slides</td></tr><tr><td>EZ Single Cytofunnel</td><td>Thermo Scientific</td><td>A78710020</td><td>For spinning cells onto microscope slides</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Fisher BioReagents</td><td>BP118-500</td><td>Used in dissociating islets</td></tr><tr><td>paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td>For fixing cells</td></tr><tr><td>Triton&amp;nbsp; X-100</td><td>Fisher BioReagents</td><td>BP151</td><td>For permeabilizing cells</td></tr><tr><td>Normal donkey serum</td><td>Jackson ImmunoResearch</td><td>017-000-121</td><td>Blocking reagents for immunofluorescence</td></tr><tr><td>Anti-Ki67 antibody</td><td>abcam</td><td>ab15580</td><td>For Ki67 immunofluorescence</td></tr><tr><td>Polyclonal Guinea Pig Anti-Insulin</td><td>Dako</td><td>A0564</td><td>For insulin immunofluorescence</td></tr><tr><td>Cy3 AffiniPure Donkey Anti-Rabbit</td><td>Jackson ImmunoResearch</td><td>711-165-152</td><td>For Ki67 immunofluorescence</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Guinea Pig</td><td>Jackson ImmunoResearch</td><td>706-175-148</td><td>For insulin immunofluorescence</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>ThermoFisher Scientific</td><td>D1306</td><td>For nuclei visualization in immunofluorescence</td></tr><tr><td>Aqua-Mount</td><td>Lerner Laboratories</td><td>13800</td><td>Fast drying mounting media</td></tr><tr><td>FreeZone -105 &amp;deg;C 4.5 Liter Cascade Benchtop Freeze Dry System</td><td>Labconco</td><td>7382020</td><td>For lyophilization of microspheres</td></tr></tbody></table>

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.

Figure 1 is a visual representation of the microspheres generated using the above protocol. The protocol described here yields rhCTGF-loaded microspheres of various sizes. The largest fraction of microspheres will be between 1 and 10 &#181;m in diameter, though some microspheres may be larger (Figure 2). If desired, microsphere size can be tuned and optimized based on fabrication parameters such as homogenization speed and time, surfactan...

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

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 ...

sustained-administration-cell-mitogens-to-intact-mouse-islets-ex-vivo

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JoVE Journal

Bioengineering

7.2K Views.  Vanderbilt University Medical Center. 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 β-cell proliferation. This method is suitable for determining the efficacy of candidate β-cell mitogens.

Video: Sustained Administration of β-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

Medicine

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

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.

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.

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

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

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Here we present refined surgical procedures on successfully performing intraportal islet transplantation, a clinically relevant but technically challenging surgical procedure, in mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

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. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

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

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A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

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

Intraportal Transplantation of Pancreatic Islets in Mouse Model

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice