Name: sustained administration of &#946;-cell mitogens to intact mouse islets ex vivo using biodegradable poly(lactic-co-glycolic acid) microspheres
Uploaded: 2016-11-05T15:00:00
Description: Watch this Scientific Journal Video about Sustained Administration of Î²-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Polylactic-co-glycolic acid Microspheres at JoVE.com

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 ...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="819">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Oregon Green 488 Carboxylic Acid, Succinimidyl Ester, 6-isomer</td>
			<td style="width:179px;">ThermoFisher Scientific</td>
			<td style="width:119px;">O6149</td>
			<td style="width:307px;">For labeling COI with fluorophore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO Dimethyl Sulfoxide</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">BP231-1</td>
			<td>For dissolving fluorophore in step 1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable PD-10 Desalting Columns</td>
			<td style="width:179px;">GE Healthcare</td>
			<td style="width:119px;">17-0851-01</td>
			<td>Desalting column used in step 1</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Resomer RG 505, Poly(D,L-lactide-co-glycolide), ester terminated, molecular weight 54,000-69,000</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">739960</td>
			<td>Used in generation of microspheres in step 2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly(vinyl alcohol) molecular weight 89,000-98,000</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">341584</td>
			<td>Used in generation of microspheres in step 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI 1640</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">11879-020</td>
			<td>For culturing islets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dextrose Anhydrous</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">200-075-1</td>
			<td>Supplement for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin-Streptomycin</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4333</td>
			<td>Antibiotics for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal horse serum</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">008-000-121</td>
			<td>Supplement for islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">96-well tissue culture plate</td>
			<td style="width:179px;">Corning</td>
			<td align="right" style="width:119px;">3603</td>
			<td>For culturing islets</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethylene glyco-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">E4378</td>
			<td>Supplement for pre-assay islet media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytospin 4 Cytocentrifuge</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">A78300003</td>
			<td>For spinning cells onto microscope slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EZ Single Cytofunnel</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td style="width:119px;">A78710020</td>
			<td>For spinning cells onto microscope slides</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td>BP118-500</td>
			<td>Used in dissociating islets</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paraformaldehyde</td>
			<td style="width:179px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>For fixing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton&#160; X-100</td>
			<td style="width:179px;">Fisher BioReagents</td>
			<td style="width:119px;">BP151</td>
			<td>For permeabilizing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Normal donkey serum</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">017-000-121</td>
			<td>Blocking reagents for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anti-Ki67 antibody</td>
			<td style="width:179px;">abcam</td>
			<td style="width:119px;">ab15580</td>
			<td>For Ki67 immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Polyclonal Guinea Pig Anti-Insulin</td>
			<td style="width:179px;">Dako</td>
			<td style="width:119px;">A0564</td>
			<td>For insulin immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cy3 AffiniPure Donkey Anti-Rabbit</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">711-165-152</td>
			<td>For Ki67 immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cy5 AffiniPure Donkey Anti-Guinea Pig</td>
			<td style="width:179px;">Jackson ImmunoResearch</td>
			<td style="width:119px;">706-175-148</td>
			<td>For insulin immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:179px;">ThermoFisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>For nuclei visualization in immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Aqua-Mount</td>
			<td style="width:179px;">Lerner Laboratories</td>
			<td align="right" style="width:119px;">13800</td>
			<td>Fast drying mounting media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">FreeZone -105&#176;C 4.5 Liter Cascade Benchtop Freeze Dry System</td>
			<td style="width:179px;">Labconco</td>
			<td align="right" style="width:119px;">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...

Watch this Scientific Journal Video about Sustained Administration of Î²-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Polylactic-co-glycolic acid Microspheres at JoVE.com

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 ...

The overall goal of this protocol is to use Poly(lactic-co-glycolic Acid)or PLGA, microspheres, to deliver compounds of interest to cultured intact pancreatic islets to study the effects of individual compounds on adult pancreatic beta cell proliferation. This method can be used to test a variety of compounds delivered locally in a sustained release manner to intact islets. The main advantage of this method is that it can be easily adapted for alternative delivery biomaterials, compounds of interest, and endpoint assays.This method also has the potential for delivering compounds of interest in conjunction with the adopted transfer of islets in vivo. Demonstrating how to make the PLGA microshperes will be Taylor Kavanaugh, the graduate student in my laboratory. Begin by adding one milligram of the fluorescently labeled compound of interest to 100 microliters of deionized water to form the first water phase.Next, dissolve 65 milligrams of PLGA in 750 microliters of dichloromethane in a scintillation vial, and ultrasonicate the mixture for 10 to 30 seconds at 160 watts to completely dissolve the PLGA. This forms the oil phase. Add all of the first water phase to the oil phase in a dropwise manner.Then use a handheld homogenizer to emulsify the solution at 20, 000 rpm for 30 seconds, forming the first water-oil phase. Then add all of the first water-oil phase dropwise into 15 milliliters of 1%PVA solution and emulsify as just demonstrated. Next, transfer the entire volume of the emulsion into a 200 milliliter round-bottom flask and use a rotary evaporator to remove the solvent for generation of the aqueous phase at 635 millimeters of mercury vacuum for one hour.Aliquot one milliliter of the aqueous phase into each of 14 microcentrifuge tubes, and pellet the microspheres by centrifugation. At the end of the spin, use a micropipette to remove 900 microliters of aqueous solution from each tube, taking care not to disturb the pellets. Wash the microspheres in one milliliter of de-ionized water, followed by the careful removal of 900 microliters of the supernatant.Then add 900 microliters of deionized water to the remaining 100 microliters of the supernatants. Then briefly vortex and sonicate to resuspend the pellets. After isolating the islets from the experimental animals of interest, seed 40 visually size-matched islets into one well of a 96 well tissue culture plate in 200 microliters of islet culture medium.The next morning, resuspend one aliquot of microspheres in pre-assay medium, such that the final concentration of the compound of interest in the tube is 10 nanograms per microliter and sonicate the microspheres in an ice water bath for 10 minutes at 160 watts with 10 second long pulses at four degrees Celsius. Next, carefully remove 100 microliters of islet culture medium from each well without dislodging the islets, and incubate the cultures with 100 microliters of the microsphere containing assay medium. After three days, carefully discard the assay medium from all of the wells without disturbing the islets, and gently wash the islets in 200 microliters of PBS two times.Islets only loosely adhere to a 96 well plate. Therefore, it is critical that medium and PBS are gently removed from each well so that the islets are not accidentally dislodged and discarded. After the second wash, gently immerse the islets in 100 microliters of trypsin EDTA solution for three minutes at room temperature, with gentle pipetting after two minutes.Transfer the entire volume of each well into individual microcentrifuge tubes and add 400 microliters of islet culture medium to each tube to stop the dissociation. After collecting the microsphere treated islets by centrifugation, use a micropipette to gently remove the supernatants and resuspend the pellets in 200 microliters of fresh islet culture medium. Then spin the dissociated islets onto charged microscope slides on a cytocentrifuge.After air drying, draw a box around the islets with a hydrophobic marking pen and fix the cells in 75 microliters of four percent paraformaldehyde at room temperature for 10 minutes, followed by two washes with 75 microliters of PBS. After the second watch, permeabolize the cells with 75 microliters of 0.2%Triton X-100 in PBS for 10 minutes, followed by a final wash with 75 microliters of PBS alone. To immunolabel the islets, begin by placing the slides cell side up in a humid chamber and remove the PBS.Next, block the non-specific binding with 75 microliters of normal donkey serum at room temperature. After one hour, replace the serum with 75 microliters of the primary antibody of interest for another hour at room temperature. At the end of the incubation, carefully aspirate the primary antibody solution and wash the samples with 375 microliter PBS washes.Then incubate the islets with 75 microliters of the appropriate immunofluorescent tagged secondary antibody for a one hour incubation at room temperature protected from light. At the end of the incubation, replace the secondary antibody solution with 75 microliters of Dappy for three minutes, followed by a five minute rinse in deionized water. At the end of the wash, spot a 100 microliter drop of fast-drying mounting solution containing anti-fade reagent onto a glass cover slip and invert one microscope slide, sample side down, onto the mounting solution.The largest fraction of microspheres will be between 1-10 microns in diameter, although some microspheres may be larger. Following the dispersal of the intact PLGA microsphere treated islets and their subsequent immunolabeling, it is common to observe regions of the sample devoid of any labeling between the cells, caused by intact microspheres that have not been completely removed during the wash steps. After imaging, the percentage of Ki67 insulin positive cells can be quantified using software image analysis, or by manually counting the total number of labeled cells.Treating the intact islets with recombinant human connective tissue growth factor PLGA microspheres for three days as just demonstrated results in an increased beta-cell proliferation, demonstrating that the protein does not lose any functionality during the microsphere generation. Once mastered, this technique can yield results in less than one week if it is performed properly. While attempting this procedure, it is important to remember that the impact on beta-cell proliferation in your experiment will vary depending on the compound of interest and the exact release kinetics of the generated microspheres.Following this procedure, other methods, such as cotransplantation of compound-loaded microspheres with intact pancreatic islets, can be performed to address other questions such as can the compound of interest induce beta-cell proliferation in vivo. PLGA has a history of use in devices and drug delivery systems that have been approved by the FDA, thus it represents a good choice for researchers and clinicians looking to deliver therapeutic cargo to defied tissues over an extended period of time. After watching this video, you should have a good understanding of how to generate compound-loaded PLGA microspheres and administer them to cultured intact islets.Don't forget that working with paraformaldehyde can be extremely hazardous and that precautions, such as wearing the appropriate personal protective equipment, should always be taken while performing this procedure.

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