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

Summary

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.

Abstract

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic β-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to β-cells in culture and in transplanted tissue models. These technologies allow the study of candidate β-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating β-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 β-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 β-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Introduction

Pancreatic β-cells are the only insulin-producing cells in the body and are critical to maintain blood glucose homeostasis. While healthy individuals have sufficient β-cell mass and function to properly regulate blood glucose, individuals with diabetes are characterized by insufficient β-cell mass and/or function^1,2. It has been proposed that inducing β-cell proliferation can ultimately increase β-cell mass and restore glucose homeostasis in individuals with diabetes³. However, evaluation and validation of potential β-cell proliferative compounds in intact islets is necessary before effective therapies can be develop....

Protocol

All procedures were approved and performed in accordance with the Vanderbilt Institutional Animal Care and Use Committee.

1. Labeling COI with Fluorophore (Optional)

1. 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 µl of dimethyl sulfoxide (DMSO).
2. Resuspend 50 mg of COI up to a final volume of 800 µl in a vehicle solution (final concentration of 62.5 ng/ µl). The vehicle solution will vary depen....

Results

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 µm 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, surfactant con.......

Discussion

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

Materials

List of materials used in this article

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