1 Overview

1.1 Purpose and Scope

This procedure is intended to establish an in vitro adipogenic induction and Oil Red O staining evaluation workflow for mesenchymal stem cells (MSCs). It is applicable to adipogenic differentiation studies using MSCs derived from bone marrow, adipose tissue, umbilical cord, and other sources.

1.2 Principle

Dexamethasone, 3-isobutyl-1-methylxanthine (IBMX), indomethacin, and insulin can promote the differentiation of MSCs into adipocytes. During induction, intracellular lipid droplets rich in triglycerides and neutral lipids gradually form. Oil Red O dissolves in intracellular neutral lipids and stains lipid droplets red.

2 Materials and Reagents

2.1 Cells

(1) Bone marrow-derived MSCs.

(2) Adipose-derived MSCs.

(3) Umbilical cord-derived MSCs.

Cells with good growth status, no mycoplasma contamination, and stable differentiation potential are recommended.

2.2 Main Reagents

(1) 0.1% gelatin coating solution.

(2) MSC complete medium.

(3) High-glucose DMEM medium.

(4) Fetal bovine serum (FBS).

(5) Penicillin-streptomycin solution (P/S).

(6) Dexamethasone.

(7) 3-Isobutyl-1-methylxanthine (IBMX).

(8) Indomethacin.

(9) Insulin.

(10) Trypsin cell dissociation solution.

(11) PBS.

(12) 4% paraformaldehyde fixative.

(13) Oil Red O.

(14) Isopropanol.

(15) Distilled or deionized water.

2.3 Adipogenic Induction Medium A

Use high-glucose DMEM as the basal medium and supplement it with the following components:

(1) 10% FBS.

(2) 1% P/S.

(3) Dexamethasone at a final concentration of 1 μM.

(4) IBMX at a final concentration of 0.5 mM.

(5) Indomethacin at a final concentration of 100 μM.

(6) Insulin at a final concentration of 10 μg/mL.

2.4 Adipogenic Induction Medium B

Use high-glucose DMEM as the basal medium and supplement it with the following components:

(1) 10% FBS.

(2) 1% P/S.

(3) Insulin at a final concentration of 10 μg/mL.

The media should preferably be freshly prepared before use or aliquoted for storage. Repeated freeze-thaw cycles should be avoided.

2.5 Main Equipment

(1) 6-well cell culture plates.

(2) CO₂ incubator.

(3) Inverted microscope.

(4) Centrifuge.

(5) Automated cell counter or hemocytometer.

3 Experimental Groups

3.1 Adipogenic Induction Group

Cells are alternately cultured in adipogenic induction medium A and medium B.

3.2 Uninduced Control Group

Cells are cultured in standard complete medium without dexamethasone, IBMX, indomethacin, or insulin.

3.3 Replicates

At least three replicate wells are recommended for each group. The cell source, passage number, seeding density, and culture duration should be kept consistent among groups.

4 Experimental Procedure

4.1 Coating of Culture Plates

(1) Add an appropriate volume of 0.1% gelatin coating solution to each well of a 6-well plate so that the entire bottom surface is covered.

(2) Incubate at room temperature for 30 min.

(3) Aspirate the coating solution and avoid allowing the well surface to remain dry for an extended period.

4.2 Cell Dissociation and Collection

(1) Select MSCs at approximately 85% confluence.

(2) Remove the original culture medium and gently wash the cells with PBS.

(3) Add an appropriate volume of trypsin cell dissociation solution.

(4) When the cells gradually detach, add MSC complete medium to terminate digestion.

(5) Collect the cell suspension and centrifuge at 300 × g for 5 min.

(6) Discard the supernatant and resuspend the cells in MSC complete medium.

4.3 Cell Seeding

(1) Count the cells and adjust the cell concentration.

(2) Seed the cells into gelatin-coated 6-well plates at 2 × 10⁴-3 × 10⁴ cells/cm².

(3) Add 2 mL of MSC complete medium to each well.

(4) Culture the cells in a humidified incubator at 37°C with 5% CO₂.

4.4 Adipogenic Induction

(1) When cell confluence reaches 90%-100%, aspirate the MSC complete medium.

(2) Add 2 mL of prewarmed adipogenic induction medium A and culture for 2 d.

(3) Aspirate medium A, add 2 mL of adipogenic induction medium B, and culture for 1 d.

(4) Continue induction by alternating medium A for 2 d and medium B for 1 d.

(5) Replace the medium of the uninduced control group with standard complete medium at the same time.

(6) Continue induction for approximately 14 d and observe lipid droplet formation under a microscope.

(7) Perform Oil Red O staining when an appropriate number and size of intracellular lipid droplets have formed.

5 Preparation of Oil Red O Working Solution

5.1 Oil Red O Stock Solution

(1) Weigh 0.5 g of Oil Red O.

(2) Add 100 mL of isopropanol.

(3) After complete dissolution, store the solution protected from light in a tightly sealed container.

5.2 Oil Red O Working Solution

(1) Mix the Oil Red O stock solution and distilled water at a ratio of 3:2.

(2) Mix thoroughly and allow the solution to stand.

(3) Filter through qualitative filter paper before use to remove precipitates.

(4) The working solution should preferably be freshly prepared before use.

6 Oil Red O Staining

6.1 Cell Fixation

(1) Aspirate the culture medium.

(2) Gently wash the cells once or twice with PBS.

(3) Add 4% paraformaldehyde fixative to completely cover the cell layer.

(4) Fix at room temperature for 30 min.

(5) Aspirate the fixative and gently wash the cells once or twice with PBS.

6.2 Staining

(1) Add an appropriate volume of Oil Red O working solution, approximately 1 mL per well of a 6-well plate.

(2) Stain at room temperature for 15-30 min.

(3) Aspirate the staining solution.

(4) Gently wash three times with PBS or distilled water.

(5) Observe and record the staining results under a microscope.

6.3 Interpretation of Results

Intracellular neutral lipids and lipid droplets appear red after Oil Red O staining. A markedly greater number, area, and staining intensity of lipid droplets in the adipogenic induction group than in the uninduced control group supports successful adipogenic differentiation.

7 Critical Control Points

7.1 Cell Condition

(1) Cells should not be excessively passaged.

(2) Cells should be in good growth condition before induction.

(3) The differentiation capacities of MSCs from different sources should not be directly compared using the same threshold.

7.2 Confluence at the Start of Induction

Induction should begin when cell confluence reaches 90%-100%. Insufficient confluence may reduce adipogenic differentiation efficiency.

7.3 Alternation of Induction Media

The sequence and culture duration of medium A and medium B should remain consistent.

7.4 Oil Red O Working Solution

The working solution must be filtered before use to reduce dye crystals and nonspecific precipitates.

8 Common Problems and Cause Analysis

8.1 Limited Lipid Droplet Formation

(1) Insufficient cell confluence.

(2) Insufficient induction duration.

(3) Inaccurate concentrations or loss of activity of the induction components.

(4) Weak adipogenic potential due to cell source or passage number.

8.2 High Background Staining

(1) The Oil Red O working solution was not sufficiently filtered.

(2) The staining time was too long.

(3) Washing after staining was insufficient.

8.3 Detachment of the Cell Layer

(1) Fixation, medium replacement, or washing was performed too vigorously.

(2) Cell attachment was poor.

(3) Digestion time was too long or the seeding density was inappropriate.

9 Safety and Waste Disposal

(1) Laboratory coats and disposable gloves should be worn during the experiment.

(2) Isopropanol is flammable and should be kept away from ignition sources and handled under well-ventilated conditions.

(3) Paraformaldehyde fixation should be performed under well-ventilated conditions.

(4) Waste liquids containing isopropanol, Oil Red O, or paraformaldehyde should be collected separately and disposed of according to laboratory regulations.

10 Products Related to Adipogenic Differentiation of Mesenchymal Stem Cells and Evaluation by Oil Red O Staining

For more related articles, please see below:

[1] Experimental induction of human mesenchymal stem cell immortalization with telomerase

[2] Mesenchymal Stem Cells (MSCs) Identification