無傷のマウスの膵島のβ細胞のマイトジェンの持続的投与

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.