Differentiation and Induction of Lipid Droplet Accumulation in Human Adipocytes as a Model to Study Obesity In Vitro

Summary

The presented protocol aims to develop a cellular model that mimics human obesity. To achieve this, human adipose-derived stem cells are differentiated into adipocytes, which are next fattened using a mixture of selected fatty acids.

Abstract

Obesity-related changes in adipose tissue alter local secretory profiles and influence interactions with other surrounding cells, including cancer cells. The continued delivery of nutrients shifts the metabolism of fat cells, making them act as a lipid reservoir. Typically, research on obesity mechanisms relies on animal models, which, however, do not allow analysis of metabolic processes at the cellular level. Hence, there is a need to develop a reliable in vitro model of human obesity that can help explore changes in the function of obese adipocytes compared to their lean counterparts. This work presents a protocol for the differentiation of human adipocytes from their precursors (adipose-derived stem cells) using a mixture of insulin, isobutyl-1-methylxanthine, dexamethasone, and indomethacin. Subsequently, differentiated adipocytes were incubated with a set of fatty acids, including linoleate, palmitate, and oleate, to promote lipid accumulation. As a result, a valuable model mimicking the hypertrophic cells under obesity conditions was achieved, which can be used for further studies.

Introduction

Obesity is thought to be a major risk factor for a wide range of diseases, such as musculoskeletal disorders, cardiovascular diseases, insulin resistance, metabolic syndrome, diabetes, and selected types of cancer¹. Obesity is one of the most perceptible and under-estimated public health problems, with causes that are multi-elemental. Briefly, obesity is triggered by an imbalance between calories consumed and expended, resulting in enhanced fat storage². The caloric excess leads to pathological expansion and dysfunction of adipose tissue (AT), the key element in the body's control of energy homeostasis.

Adipocytes are the main cellular component of AT³. The main contributor to the growth of AT under obesity conditions is cell hypertrophy, i.e., the increase in the volume of adipocytes by a rise in the amount of fat stored in cytoplasm in the form of lipid droplets (LDs)⁴. Even if obesity is a complex pathological disorder, studies on the biological functioning of adipocytes can serve as a model for analyzing its causes and exploring new treatment strategies. This highlights the need for a deeper understanding of the molecular mechanisms driving alterations in adipocyte function to develop new therapeutic strategies targeting obesity-related diseases.

The animal models are the closest to reflecting physiological conditions, which makes them widely used for in vivo studies of obesity⁵. However, drawing conclusions about fundamental biological processes at the cellular level remains highly challenging in such a multilayer system, which favors the use of in vitro adipocyte models. The in vitro research is most often based on the mouse 3T3-L1 cell model, which, under appropriate conditions, acquires an adipocyte-like phenotype⁶. Nevertheless, due to its origin, this model cannot be used for more complex analyses, such as co-culture conditions with human cells, and the results cannot always be directly translated into human adipocytes. From all these reasons, a model mimicking obesity in humans is being searched, and the usage of human adipose-derived stem cells (ADSCs) with their subsequent differentiation seems to be rational. This model is therefore most suitable for studying human adipocyte hypertrophy in vitro due to its low complexity. It allows comparison of parameters of a single cell culture type under different conditions, which is unattainable in vivo due to the presence of multiple cell types in adipose tissue.

Human preadipocytes and adipose-derived stem cells isolated from AT are the most common examples for studying the dysfunction of this tissue⁶. These cells, after differentiation, are an attractive model for modulating LD levels in adipocytes by additional fattening. Nevertheless, there are relatively few studies examining hypertrophy in human cells^7,8,9, and most use only one type of fatty acid (FA) to induce LD accumulation.

This protocol proposes supplementing human adipocytes with a mixture of FAs (palmitate, oleate, and linoleate) to mimic obesity environments. This allows for the achievement of a tool based on human adipocytes overloaded with lipids, which can be directly compared to control (naïve) adipose cells. This model can be subsequently used to study the phenotypic and functional changes among normal and hypertrophic adipocytes, or, if needed, experiments with co-culture conditions.

Protocol

Use sterile techniques to perform all the steps of the protocol in a laminar flow cell culture hood. See Table of Materials for details about all reagents and equipment. The differentiation protocol described below is suitable for human adipose-derived stem cells and may differ among other cell types and their origin. The reagents and the equipment used are listed in the Table of Materials.

1. Preparation of ADSCs for differentiation

1. Prepare ADSCs growth medium (ADSCs GM): Supplement Dulbecco's Modified Eagle Medium (DMEM) containing 1 g/L glucose with 10% fetal bovine serum (FBS), 2 mM glutamine and antibiotics - antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B).
2. Warm the ADSCs GM, phosphate-buffered saline (PBS), and trypsin/EDTA solution to 37 °C.
   NOTE: ADSCs are obtained from commercial sources. Use the cells at early passages. Multiple passages lead to a decrease in the differentiation potential of ADSCs¹⁰.
3. Aseptically remove the ADSCs GM from the ADSCs growing in a T75 flask dedicated for cell culture.
4. Wash out the residual ADSCs GM by rinsing the cell culture dish with 5 mL of PBS.
5. Remove the whole volume of PBS from the flask.
6. Add 2 mL of trypsin/EDTA to the culture dish and incubate it at 37 °C till cells are detached. Monitor this process with an inverted microscope.
   NOTE: Tapping the culture dish will expedite cell detachment.
7. Block the trypsin activity by the addition of 6 mL of fresh ADSCs GM.
8. Relocate the whole volume of cell suspension from the culture dish to a tube appropriate for centrifugation.
9. Centrifuge the cells at 300 x g for 5 min at room temperature (RT).
10. Remove the supernatant.
11. Suspend cells in 2 mL of the newly added ADSCs GM.
12. Calculate the cell density and viability with a hemocytometer or an automatic cell counter.
    NOTE: Cell viability can be assessed using trypan blue dye staining.
13. Seed the cells in the desired number of wells, 15,000 cells/well, 96-well plastic plate in 100 µL of ADSCs growth medium.
    NOTE: Seed cells always in duplicate to obtain adipocytes intended for control and incubation with FAs conditions. Depending on the type of experiment, ADSCs can be seeded on different types of plastic plates or dishes. Recommended seeding density is 30,000-60,000 cells per cm². Depending on the cell number needed for the subsequent analyses or downstream experiments, adjust the format of the cell culture dish for 6- or 12-well plates.
14. Culture the cells at 37 °C in a humidified atmosphere containing 5% CO₂.
15. When cells have reached 100% confluence (typically 24-48 h after plating), induce their differentiation into adipocytes following the instructions below.
    NOTE: It is important to reach a high level of cell confluency before induction of differentiation. In other cases, it may affect the efficiency of differentiation. If for plated cells it takes longer than 48 h to reach high confluency, change the medium every 3-4 days for the fresh ADSCs growth medium.

2. Induction of adipogenic differentiation

1. Prepare differentiation medium (DM): Supplement DMEM/F12 medium with 10% FBS, 2 mM glutamine, antibiotics - antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B), 5 µg/mL insulin, 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX), 5 µM dexamethasone, and 200 µM indomethacin.
   NOTE: Prepare DM only in the volume necessary for direct use. Do not store it for a long period, and always use freshly prepared DM. Before use, warm the DM in a water bath to 37 °C. IBMX and indomethacin may precipitate if they are added to the medium at a lower temperature than 37 °C. To solubilize it, warm up the liquid in a water bath to 37 °C.
2. Aseptically remove and discard the whole volume of the medium from the confluent ADSCs.
   NOTE: The ADSCs growth medium should be removed completely before adding DM.
3. Carefully add 100 µL/well of DM to cells growing in a 96-well plate.
   NOTE: Do not let the cells dry out when changing medium.
4. Incubate the cells in an incubator at a temperature of 37 °C, 5% CO₂ for 7 days.
5. Remove and discard the whole volume of DM from the ADSCs.
6. Carefully add 100 µL/well of fresh DM to cells growing in a 96-well plate.
7. Incubate the cells in the incubator at a temperature of 37 °C, 5% CO_2, for the next 7 days.
   NOTE: The progress of adipogenic differentiation can be detected by brightfield observation of lipid vacuoles appearing in the induced cells.

3. Adipocytes' fattening

1. Prepare adipocyte culture medium supplemented with FAs: Supplement DMEM/F12 medium with 10% FBS, 2 mM glutamine, antibiotics - antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B), 0.33 mM linoleate, 0.17 mM palmitate, and 0.17 mM oleate.
2. Prepare control adipocyte culture medium: Supplement DMEM/F12 medium with 10% FBS, 2 mM glutamine, antibiotics - antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B), 0.44% bovine serum albumin (BSA), and 0.26% ethanol (the latter two compounds were also used as FAs solvents).
   NOTE: Prepare control and supplement with FAs adipocyte culture media only in the volume necessary for direct use. Do not store it for a long period, and always use freshly prepared media. Before use, warm the media in a water bath to 37 °C.
3. After a total of 14 days of differentiation, carefully remove and discard the whole volume of DM medium from adipocytes differentiated on a 96-well plate.
4. Carefully add 100 µL/well of adipocyte culture medium supplemented with FAs to half of the wells with cells growing in a 96-well plate.
5. Carefully add 100 µL/well of control adipocyte culture medium to the second half of the wells with cells growing in a 96-well plate.
   NOTE: Do not let the cells dry out when changing medium.
6. Incubate the cells at 37 °C in a humidified atmosphere containing 5% CO₂ for the next 48 h.
7. After 48 h, use the control cells and cells incubated with FAs for subsequent experiments.
   NOTE: At this stage, achieved fattened adipocytes can be used for multiple experimental purposes, like cell staining, analysis of cellular metabolism, or co-culture with other cell types.

4. Lipid droplet stains

1. Prepare adipocyte culture medium: Supplement DMEM/F12 medium with 10% FBS, 2 mM glutamine, and antibiotics - antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, 0.25 µg/mL amphotericin B).
2. Dilute fluorescent dye to stain LDs in adipocyte culture medium in a dilution of 1:1000.
3. Remove and discard the whole volume of the control adipocyte culture medium and adipocyte culture medium supplemented with FAs from cells growing in a 96-well plate.
4. Carefully add 50 µL/well of adipocyte culture medium with fluorescent dye.
   NOTE: Do not let the cells dry out when changing medium.
5. Incubate cells at 37 °C in a humidified atmosphere containing 5% CO₂ for 15 min.
6. Wash out the fluorescence dye by replacing it with the 100 µL/well adipocyte culture medium.
7. Image acquisition in the green fluorescence detection channel (Ex 427 nm/Em 585 nm) with the fluorescent microscope.
   NOTE: This is the protocol for live cell staining. Staining can also be performed after formaldehyde fixation of the cells.

Results

The method described herein allows differentiation of human ADSCs into mature adipocytes, followed by enhanced lipid accumulation, mimicking the hypertrophic cells observed under obesity conditions. The time course of the differentiation of human ADSCs is shown in Figure 1. At day 0, when cells reach 100% confluency (Figure 1A), ADSCs exhibit elongated, spindle-like morphology. At this time point, the differentiation protocol can be applied using DM containing a...

Discussion

The present protocol is a replicable two-step procedure for the differentiation of ADSCs, followed by incubation with FAs to achieve a cellular model mimicking obesity. The critical step determining its effectiveness is the number of passages of ADSCs after its isolation. Multiple passages lead to a decrease in the differentiation potential of these cells¹⁰.

During adipocyte differentiation, ADSCs are shifted from dividing cells to growth-arrested adipocytes. Growth arr...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-Isobutyl-1-methylxanthine (IBMX)	Merck	I5879
0.25% trypsin/0.05% EDTA pH 7.2	IITD PAN, Wrocaw, Poland	20-500
96-well plate	Thermo	130188
antibiotics-antimycotic solution	Sigma-Aldrich	A5955
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A3294
Cell counter	N/A	N/A
Centrifuge	N/A	N/A
Dexamethasone	Sigma-Aldrich	D4902
Dulbecco's Modified Eagle Medium (DMEM)	IITD PAN, Wrocaw, Poland	10-500	containing 1 g/L glucose
Dulbecco's Modified Eagle Medium:Nutrient Mixture F12	Gibco	11320-033
Ethanol	Chempur	113964200#1L
Fetal bovine serum (FBS)	Gibco	A5256701
hADSC Human Adipose-Derived Stem Cells	Lonza	PT-5006
Incubator	N/A	N/A
Indomethacin	Sigma-Aldrich	I8280-5G
Insulin	Sigma-Aldrich	I9278
Inverted microscope	N/A	N/A
L-glutamine	Sigma-Aldrich	25030-024
Linoleic acid sodium salt	Sigma-Aldrich	L8134
LipidSpot fluorescent	Biotium	70065
Phosphate-buffered saline (PBS)	Thermo	10010023
Soduim oleate	Sigma-Aldrich	O7501
Soduim palmitate	Sigma-Aldrich	P9767
T75 cell culture flask	Thermo	130190
Trypan blue	Sigma-Aldrich	95896HJ