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.
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.
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 function1,2. 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 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 β-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 β-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 β-cell mitogens to islets ex vivo is critical before transplantation protocols are explored.
Currently, there is no technique to measure β-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 β-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 β-cell lines are also characterized as having a much higher degree of replication than endogenous β-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 β-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 β-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 α-cells, may also be analyzed.
All procedures were approved and performed in accordance with the Vanderbilt Institutional Animal Care and Use Committee.
1. Labeling COI with Fluorophore (Optional)
2. COI-loaded Microsphere Preparation via the Water-in-oil-in-water Emulsion Solvent Evaporation Method
3. Preparation of Islet Culture Media and Pre-assay Media
4. Culturing of Intact Mouse Islets
5. Re-suspension of COI-loaded and Control PLGA Microspheres
6. Treatment of Islets with COI-loaded or Control PLGA Microspheres
7. Dispersing Intact Islets onto Microscope Slides
8. Immunofluorescence Labeling of Dissociated Islets for Insulin and the Cell Proliferation Marker, Ki67
9. Image Acquisition and Analysis
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 concentration used, and relative volumes of each water/oil/water phase10.
Following dispersal of intact islets treated with PLGA microspheres and subsequent immunolabeling, it is common to see regions of the sample devoid of any labeling in between cells (Figure 3). While most of the microspheres that are still intact after the culture treatment period are removed during the wash steps, some remain after the islets are spun onto the microscope slides. These microspheres cause the hole-like structures visualized during imaging. These residual microspheres typically do not interfere with subsequent quantification.
After imaging, the percentage of Ki67-positive/insulin-positive cells can be quantified by manually counting the total number of labeled cells, or using software image analysis. Previously, we have demonstrated that recombinant human Connective Tissue Growth Factor (rhCTGF) can stimulate mouse β-cell proliferation in intact islets ex vivo11. Using the aforementioned protocol, we generated PLGA microspheres containing rhCTGF (rhCTGF-PLGA). Treating intact islets with rhCTGF-PLGA microspheres for 3 days resulted in a similar increase in β-cell proliferation as previously reported with the raw protein, demonstrating that the protein did not lose any functionality during microsphere generation (Figure 4).
Figure 1: Visual Representation of PLGA Microspheres. By incorporating a fluorescent dye into the manufacturing protocol, PLGA microspheres can be visualized using an epifluorescent microscope. Scale bar represents 200 μm. Please click here to view a larger version of this figure.
Figure 2: Size Distribution of PLGA Microspheres. Although size distribution may vary, most microspheres will be less than 10 µm in diameter. Please click here to view a larger version of this figure.
Figure 3: Immunofluorescent Visualization of Dispersed Proliferating β-cells. Dispersed islets are immunolabeled for the proliferation marker Ki67 (red) and insulin (green) to mark proliferating β-cells. Nuclei are labeled with DAPI (blue). Arrows indicate Ki67-positive/insulin-positive cells. Asterisks (*) indicate undegraded microspheres. Scale bar represents 100 μm. Please click here to view a larger version of this figure.
Figure 4: Quantification and Analysis of β-cell Proliferation. Islets treated with different amounts of rhCTGF-PLGA microspheres. The number of Ki67-positive/insulin-positive cells was manually counted from all samples and expressed as a percentage of the total number of insulin-positive β-cells. X-axis corresponds to the final concentration of rhCTGF present in each treatment. Statistical significance was determined using a one-way ANOVA followed by Tukey's multiple comparison test. Statistical significance was set at p ≤ 0.05. Error bars represent standard error of the mean. Please click here to view a larger version of this figure.
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 islets. Our described protocol, which uses freshly isolated intact mouse islets, circumvents these issues as the islet architecture is maintained and β-cell proliferation is comparable to that found in vivo.
One significant concern when culturing intact islets is the possibility that cells within the islet core will undergo hypoxia-induced necrosis or will not be exposed to rhCTGF. For this reason, a final concentration of 0.1 mM EGTA is added to the media to loosen cell-cell contacts without disrupting islet architecture. We have previously published that the addition of EGTA alone can increase β-cell proliferation, presumably due to increased access of nutrients and mitogens in the media to the islet core12. The increase in β-cell proliferation in response to EGTA could also be due to the decrease in cell-cell contact itself13,14. The media described in this protocol is also supplemented with horse serum instead of the more traditional fetal bovine serum. This substitution is made due to the presence of placental lactogen in fetal bovine serum, which can stimulate β-cell proliferation on its own, potentially complicating analysis in the proliferation assay.
Determination of the relative toxicity of the PLGA microspheres (with or without the COI) to the islet cells can be assessed through immunofluorescence analysis of markers of cell death or DNA damage, including TUNEL or γ-H2AX15,16. Although the addition of PLGA microspheres has not shown any obvious detrimental effects to islets ex vivo, users should be aware of the effects the microspheres can have on image analysis. As demonstrated in Figure 3 and mentioned in the results section, undegraded microspheres can be apparent as dark spots within immunofluorescent images, with more microspheres appearing when increasing concentrations are utilized.
It should be noted that the described protocol has been tailored to work with isolated mouse islets. As such, it is unclear if identical conditions will work with islets harvested from alternative organisms, such as rat and humans. Notably, islets harvested from different organisms often have different optimal culturing conditions and differing degrees of β-cell proliferation17,18.
Numerous biomaterials are potentially available to administer rhCTGF to isolated islets, including poly(thioketal-urethane) and poly(propylene sulfide)19. We chose to focus on PLGA due to several notable qualities that it possesses. First, PLGA is a relatively affordable reagent and the microspheres can be generated with standard equipment (centrifuge, homogenizer, lyophilizer) available at most research institutions. PLGA is an artificial polymer that has precedent for successful use in FDA approved devices owing to its biocompatibility, biodegradability, and its ability to modulate compound release rates20. PLGA degrades by hydrolysis in the presence of water, releasing its cargo in the process. The chemical composition of PLGA particles can be tailored such that a compound is released in vivo over the period of several weeks. PLGA has also been used in preclinical animal trials and human clinical therapies to locally administer compounds to various organs and tissues21,22. Other hydrophobic polymers can also be used in the generation of biodegradable microspheres. These polymers can respond to specific environmental stimuli, such as pH, temperature, and the presence of reactive oxygen species23,24. Thus, researchers should carefully consider which biomaterial is most appropriate for their studies.
Any researcher utilizing PLGA, or other biomaterials, to study β-cell proliferation ex vivo should be aware of potential differences between the effects of encapsulated COI compared to unencapsulated COI. For example, the delivery of rhCTGF via PLGA could change the degree of and timing of β-cell proliferation induction compared to treatment with unencapsulated protein. Ongoing studies in our lab are currently examining these potential differences.
The analysis of β-cell proliferation in cultured islets is a powerful model for the identification and mechanistic analysis of β-cell mitogens. Using the assay described here we show a relatively quick and cost-effective method for administering a COI to isolated cultured islets with an extended release delivery vehicle. This assay should be applicable to nearly any COI of interest, and our choice to focus on CTGF is purely based on its previously published ability to stimulate β-cell proliferation in vivo and ex vivo11. Additionally, this assay can validate the effectiveness and safety of using PLGA as a delivery method before advancing to models where islets are transplanted in living organisms. Overall, the described protocol provides a novel way to administer compounds to cultured islets with a broader impact on measuring the safety of PLGA to transplantable tissue.
The authors have nothing to disclose.
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.
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 |