Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

All experiments were carried out according to the approved guidelines, and all procedures were approved by the Ethical Committee of the Faculty of Pharmacy, The British University in Egypt (BUE), Cairo, Egypt. The Ad-MSC isolation protocol was adopted from Lopez and Spencer, with modifications¹⁵.

1. Isolation of Ad-MSCs from rat epididymal fat pads

1. Use male Sprague-Dawley rats weighing 250-300 g that are not more than 1 month of age (two per isolation). Anesthetize the animals, then euthanize them by cervical dislocation. Spray the euthanized animal with 70% ethanol. Excise the skin and the muscle at the lower part of the abdomen, and then pull out the two testes to expose the epididymal fat pads.
2. Gently cut the epididymal fat pads and be careful not to cut the blood vessels.
3. Isolate the fat tissue surrounding the epididymis, then place it in a Petri dish containing phosphate-buffered saline (PBS).
4. In the biosafety cabinet, cut these fat pads into small pieces using forceps and scalpel. Transfer these cut adipose tissue pieces into a 50 mL centrifuge tube containing 10 mL of sterile PBS.
5. Wash the minced fat tissues 5 times with 10 mL of PBS each time to remove the contaminating blood. The following washing procedures must be meticulously carried out.
   1. Add 10 mL of PBS to the tissue and mix thoroughly for 45 s. Then, allow the tissue to settle via gravity by keeping the tube standing for 5 min to separate into two layers.
   2. Aspirate the PBS from the infranatant using a 10 ml syringe and replace with fresh 10 mL of PBS for another wash.
   3. Repeat this washing step 4-5 times until the PBS is clear of blood. This indicates that most, if not all, of the blood is removed.
6. After washing, resuspend the adipose tissue in 10 mL of collagenase solution (0.1% collagenase type 1 in PBS). Seal the tube tightly with parafilm, then incubate it in a shaking water bath (37 °C, 80 rpm, for 45 min) until the tissue is almost homogenous.
   NOTE: Avoid the complete digestion of the tissue (when the solution becomes completely homogenous with complete disappearance of all tissue residues/pieces), because it adversely affects the culturing of cells afterward. Usually, the digestion takes 30-45 min.
7. Once the collagenase digestion is done, vortex the tube for 15 s, then centrifuge for 5 min at 300 x g. Vortex the tube again for 10 s, followed by another 5 min centrifugation. Three layers will then be observed. Carefully discard the oil layer, followed by the aqueous layer, without disturbing the stromal vascular fraction (SVF) pellet.
   NOTE: Remove the supernatant containing adipocytes and the collagenase solution. First, remove the adipocytes and the accompanying oil layer with a 10 mL pipette to ensure complete removal of the oil droplets, then remove the underneath aqueous layer.
8. Resuspend the SVF pellet at the bottom of the tube in 10 mL of sterile BSA solution (1% albumin, bovine serum Fraction V solution in PBS), then centrifuge the tube for 5 min at 300 x g.
9. After centrifugation, discard the supernatant and suspend the SVF pellet in 8.5 mL of complete media for Ad-MSCs (composed of Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 [DMEM/F12] media supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, and 2 mM L-glutamine).
10. Culture the cells in a 25 cm² flask at 37 °C under 5% CO_2. On the following day, check the attached cells, remove the suspended cells, and replace the media. Afterward, change the media every other day.
11. Passage the cells every 5-7 days when they are 80%-90% confluent using Trypsin-EDTA. Use cells between passages 3-5 for the subsequent experiments described in this protocol. A schematic presentation of all the isolation steps is provided in Figure 1.
    NOTE: Usually, Trypisn-EDTA potency may vary among different suppliers, so try to minimize the time of trypsinization to avoid toxic effects on the cells.

2. Characterization of Ad-MSCs by immunophenotyping using flow cytometry analyses

1. At passage 3, split the cells using Trypsin-EDTA. Wash with PBS 2 times and then count the cells with a hemocytometer.
2. Incubate 100,000 cells at 4 °C in the dark for 20 min with either mouse anti-rat CD90 or CD105 (mesenchymal markers) or CD34 (hematopoietic marker) monoclonal antibody labeled with fluorescein-isothiocyanate (FITC).
3. Wash the cells and suspend them in 500 µL of FACS buffer. Analyze by flow cytometry²⁵.

3. Assessment of the differentiation potential of isolated Ad-MSCs into various mesenchymal lineages

1. Adipogenic differentiation
   1. Resuspend the cells in complete media (as described in step 1.9.), then culture the cells at a density of 3.7 x 10⁴ cells/well in a 24-well tissue culture plate. Incubate at 37 °C in a humidified atmosphere of 5% CO₂. Next,change the media every 3 days until the cells reach almost 100% confluency, which usually takes 3 days.
   2. When the cells reach the desired confluency, replace the proliferation media with the adipogenic differentiation media (consisting of LG-DMEM media supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, and 2 mM L-glutamine with 1x adipogenic supplement, supplied with the Mesenchymal Stem Cells Functional Identification Kit) to induce adipogenesis. Then, change the media every 3 days (0.5 mL/well). Take care to perform the media replacement gently so as not to disrupt the lipid vacuoles.
   3. After 21 days, assess the adipogenic differentiation by microscopic examination of the lipid vacuoles appearance and by Oil Red staining.
   4. Prepare a stock solution of Oil Red by dissolving 30 mg of Oil Red stain in 10 mL of isopropanol, and then keep it for at least 20 min at room temperature. Afterward, prepare a working solution by mixing 3 parts stock solution with 2 parts double-distilled water (ddH₂O), mix them well, and keep it for 10 min at room temperature, then filter afterward by filter paper.
   5. To document the adipogenic differentiation, wash the cells 2 times with PBS, then fix them using neutral-buffered formalin 10% (0.75 ml/well) for 15 min. After that, wash the cells 2 times with PBS using 1 mL/well and incubate the cells for 5 min each time. It is important to completely aspirate the PBS before adding the Oil Red working solution.
   6. After the last wash, add the Oil Red working solution to the cells and incubate them for 60 min at 37 °C. Afterward, remove the stain solution and gently wash the cells 2 times with PBS. Observe the stained lipid droplets under the microscope.
2. Osteogenic differentiation
   1. Seed 7.4 x 10³ cells/well (0.5 mL medium/well) in a 24-well plate and incubate them at 37 °C in a humidified atmosphere of 5% CO₂ in complete media (as described in step 1.9.) for 3 days to reach almost 70% confluency.
   2. Induce the cells with the osteogenic supplement (provided with the kit) in LG-DMEM media supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, and 2 mM L-glutamine.
   3. Continue the osteogenic induction for 21 days, changing the differentiation media every 3 days.
   4. Prepare a working solution of Alizarin Red S stain by dissolving 200 mg of Alizarin Red S stain in 9 mL of ddH₂O, and then adjust pH to 4.1-4.3 using ammonium hydroxide and HCl. Next, make up the volume to 10 mL by ddH₂O. Following this, remove any precipitates by filtration using filter paper.
   5. Wash the cells 2 times with PBS, then fix them using 10% buffered formalin (0.75 mL/well) for 15 min. Afterward, wash the cells 2 times using PBS (1 mL/well). Each time, incubate the cells for 5 min.
   6. Incubate the fixed cells with the prepared 2% Alizarin-Red S solution for 30 min in a 37 °C incubator.
   7. After that, remove the stain solution, wash the cells 2 times with ddH₂O and once with PBS, and then observe the stained calcium-rich extracellular matrix to assess osteogenic differentiation under the microscope.
3. Chondrogenic differentiation
   1. Seed 7.4 x 10³ cells/well (0.5 mL medium/well) in a 24-well plate and incubate these at 37 °C in a humidified atmosphere of 5% CO₂ in complete media (as described in step 1.9.) for 3 days to reach almost 70% confluency.
   2. When the desired confluency is reached, induce the chondrogenic differentiation of the cells with serum-free DMEM/F12 containing insulin-transferrin selenium (ITS), chondrogenic supplement (provided with the kit), 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, and 2 mM L-glutamine²¹.
   3. Incubate the cells with the chondrogenic media for 21 days, while changing the chondrogenic differentiation media every 3 days.
   4. Prepare a working solution of Alcian Blue 8GX stain as follows: First, prepare a 3% glacial acetic acid solution in ddH₂O by adding 3 mL of glacial acetic acid to 97 mL of ddH₂O. Then, prepare a working solution of Alcian Blue 8GX stain by mixing 0.1 g in 100 mL of 3% glacial acid solution, and remove any precipitates by filtration using filter paper.
   5. Wash the cells 2 times with PBS, then fix them using 10% buffered formalin (0.75 mL/well) for 15 min. Afterward, wash the cells 2 times using PBS (1 mL/well). Each time, incubate the cells for 5 min.
   6. The next step is to incubate the cells in the prepared 0.1% Alcian Blue 8GX stain in 3% glacial acetic acid for 60 min at 37 °C. Wash the stained cells 2 times with ddH₂O and once with PBS, and then observe the stained sulfated proteoglycans under the microscope.

4. Differentiation of Ad-MSCs into IPCs

1. At passage 3, split the Ad-MSCs using Trypsin- EDTA. First, remove the media, then wash the cells while still attached in the culture flask with 5 mL of PBS. Next, add 5 mL of Trypsin- EDTA to the 75 cm² flask and incubate at 37 °C for 3-10 min.
   NOTE: Try to induce the cells at early passages, usually between P3-P5, as late passages adversely affect the cells' properties and differentiation abilities^17,24.
2. Afterward, inspect the cells for detachment and for being a single-cell suspension. Add 5 mL of complete DMEM/F12 media to the flask to inhibit the trypsin action. Collect the cell suspension and transfer it to a 15 mL tube, and then add another 5 mL of complete DMEM/F12 media to the tube.
3. Centrifuge the cells at 300 x g for 2 min to pellet the cells.
4. Wash the cells once with PBS, centrifuge again at 300 x g for 2 min, and then resuspended the cell pellet in 3-5 mL of complete DMEM/F12 media.
5. Count the cells using trypan blue exclusion dye and a hemocytometer.
6. Then, seed the cells in 6-well plates (1 x 10⁶ cells/plate) and a 12-well plate (for GSIS assay; 1 x 10⁶ cells/plate) and incubate in a 37 °C, 5% CO₂ incubator in complete media (as described in step 1.9.) for 3 days to reach almost 90% confluency.
7. On the day of induction, remove the media, wash the cells with PBS, and then pre-induce the cells for 2 days by pre-induction media (DMEM/F12 supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, 2 mM L-glutamine, 10 mM nicotinamide [NA], and 1mM β-mercaptoethanol [β-ME]).
8. Induce the cells for another 1 day using high glucose DMEM (HG-DMEM, 4.5g/L glucose) supplemented with 2.5% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, 2 mM L-glutamine, 10 mM NA, and 1mM β-mercaptoethanol. Collect cell pellets at the end of this stage (designated D3 cells in this protocol).
9. Incubate the cells for an additional 7 days in the final differentiation induction media composed of HG-DMEM supplemented with 2.5% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, 2 mM L-glutamine, 10 mM NA, 1mM β-mercaptoethanol, and 10 nM exendin-4.
10. At the end of the specified period, collect cell pellets (named Final in this protocol), and assess the successful differentiation of the cells by DTZ staining and GSIS assay, as follows in this protocol.
11. Assess the generated IPCs by following steps 5 and 6 below.

5. Dithizone staining

1. Prepare dithizone (DTZ) stain stock solution as follows: Completely dissolve 25 mg of DTZ in 2.5 mL of dimethyl sulfoxide (DMSO), divide into aliquots, and store at −20 °C in the dark until use.
2. For staining, prepare a working solution 1:100 (volume/volume) by diluting the stock solution in complete culture medium (HG-DMEM supplemented with 5% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B, and 2 mM L-glutamine). Then, filter the working solution through a 0.2 µm syringe filter.
3. Next, for the specified DTZ staining culture cells in a 6-well plate and, after aspirating the culture media, wash the cells once with PBS, and then add 2 mL of DTZ working solution into each well. Incubate for at least 2 h at 37 °C in the CO₂ incubator.
4. Carefully wash the cells 2 times with PBS. After the last wash, add 2 mL of PBS into each well. Then, observe the crimson red-stained IPCs under an inverted microscope. Use uninduced Ad-MSCs initially cultured in complete growth medium (10% FBS – DMEM/F12) as a control.

6. Gene expression of β-cell markers by RT-qPCR

1. RNA extraction
   1. After differentiation, collect the cell pellets and store them at −80 °C until use. Suspend each cell pellet (derived from the six wells of one 6-well plate pooled) in 1 mL of guanidium thiocyanate RNA extraction reagent in a 1.5 mL nuclease-free tube, along with pipetting up and down several times. Incubate the lysed samples at room temperature for 5 min to permit complete dissociation of the nucleoprotein complexes.
   2. Add 200 µL of chloroform per 1 mL of RNA extraction reagent, and securely cap the tubes to be vigorously shaken by hand for 15 s. Next, incubate for 3 min at room temperature.
   3. Centrifuge the samples at 13,800 x g for 15 min at 4 °C. This will lead to mixture separation into a lower chloroform phase, an interphase, and a colorless upper aqueous phase, with the RNA remaining in the aqueous phase.
   4. Place the upper aqueous phase into a new tube, while carefully avoiding touching the interphase.
   5. Add 600 µL of 100% isopropanol to the aqueous phase (1:1 volume), then mix the samples thoroughly by inversion 25 times, and incubate at room temperature for 10 min.
   6. Centrifuge the samples at 18,800 x g for 30 min at 4 °C. The precipitated RNA forms a small gel-like pellet on the tube side.
   7. Remove the supernatant and wash the pellets with 1 mL of 80% ice-cold ethanol. After adding the ethanol, slowly conduct multiple pipetting of the RNA pellet for 10 s.
   8. Centrifuge the samples for 5 min at 9,600 x g at 4 °C. Discard the supernatant and leave the RNA pellets to air-dry for 5-10 min.
   9. After drying, dissolve the RNA pellets in 25-100 µL of RNase-free water (according to the initial pellet size) by pipetting up and down several times until complete solubilization in 1.5 mL nuclease-free tubes. Avoid excessive drying of the RNA pellet.
      NOTE: Usually, the pellet becomes transparent when it dries. However, once it is clear, promptly resuspend it to avoid excessive dryness of the pellet.
   10. Quantify RNA by determining the optical densities (OD) at 260 nm and 280 nm, using nuclease-free water as blank.
   11. Ensure that the samples have OD260/OD280 = 1.8-2.0.
2. cDNA synthesis
   1. Synthesize cDNA from the isolated total RNA using a cDNA synthesis kit.
   2. Carry out the cDNA synthesis reaction according to the manufacturer's instructions. In a 200 µL PCR tube, add a volume of RNA solution containing 0.5 µg of total RNA to the reaction master mix shown in Table 1.
   3. After adding the RNA to the master mix, enter the mixture through a cycling program of 50 °C for 30 min for one cycle, followed by an inactivation cycle of 95 °C for 2 min, using a thermal cycler.
   4. Dilute the cDNA using nuclease-free water to a final concentration of 2 ng/µL. Store at −20 °C for subsequent RT-qPCR.
3. RT-qPCR using SYBR Green Master Mix
   1. Carry out the RT-qPCR reaction for each target gene using cDNA template according to the reaction mix shown in Table 2. Do all the measures in triplicates.
   2. The sets of primers used are shown in Table 3. Perform all the RT-qPCR procedures on ice.
   3. Carry out the RT-qPCR reaction using a real-time PCR machine on default program settings. The thermal cycling conditions included initial denaturation of 95 °C for 10 min, followed by 45 cycles of denaturation (95 °C, 15 s) and combined annealing/extension (60 °C, 1 min).
   4. Determine the C_t values and detect the expression levels in accordance to 2^-ΔΔCt, with β-actin as an internal control.

Table 1: cDNA synthesis master mix volumes.

Table 2: RT-qPCR reaction mixture.

Table 3: Forward and reverse primer sequences.

7. Glucose-stimulated insulin secretion

1. Preparation of Kreb's Ringer bicarbonate (KRB) buffer
   1. Prepare Kreb's Ringer bicarbonate (KRB) buffer solution with both 2 mM glucose (low glucose) and 20 mM glucose (high glucose) concentrations for the glucose-stimulated insulin secretion (GSIS) assay. The components used for the KRB buffer preparation are given in Table 4.
   2. Adjust the pH of the buffer using 1 N HCl to about 7.25-7.35 (i.e., 0.1-0.2 units below the desired pH 7.4, because it may rise during filtration).
   3. Add bovine serum albumin (BSA) freshly in a concentration of 0.1 % (weight/volume).
   4. Add glucose afterward (18 mg for 50 mL of KRB to prepare the 2 mM low glucose KRB buffer, and 180 mg for 50 mL of KRB to prepare the 20 mM high glucose KRB buffer).
   5. Sterilize the prepared LG and HG KRB buffers by filtration through a 0.2 µm syringe filter to be ready for the GSIS assay.
2. GSIS assay
   1. Initially seed 1 x 10⁵ cells/well in a 12-well cell culture plate and induce these cells by using the exact same differentiation induction protocol.
   2. At the end of the induction protocol, gently wash the generated IPCs (in plate) 2 times with PBS and once with LG-KRB.
   3. Afterward, incubate the IPCs with 300 µL of LG-KRB/well for 1 h, and then discard this buffer.
   4. Next, incubate the IPCs with 300 µL of either 2 mM (LG) or 20 mM (HG) KRB buffer for an additional 1 h. At the end of the incubation period, collect the supernatant and store at −80 °C for the subsequent secreted insulin assay using a commercial ELISA kit and following the manufacturer's instructions.
      NOTE: Try to be as gentle as possible while aspirating or adding the PBS and KRB buffer when performing the GSIS assay.

Table 4: The components used for the KRB buffer preparation.

8. Statistical analysis

1. Express all data as mean ± standard error of the mean (SEM). All comparisons were done using one-way analysis of variance (ANOVA) and Tukey's post hoc test using statistical software. Results with p-values **p ≤ 0.01 and *p ≤ 0.05 were considered statistically significant.