Obesity-related changes in adipose tissue alter local secretory profiles and influence interactions with other cells, including cancer cells. The prolonged caloric excess shifts the metabolic activity of adipocytes from energy supply to lipid storage. The in vitro conditions-based studies might help to explore changes in the functioning of obese adipocytes. This work presents a protocol for the differentiation of human adipocytes from their precursors with their subsequent incubation with fatty acids, leading to the achievement of a valuable model mimicking the hypertrophic cells under obesity conditions, which can be used for further studies. 

The article was funded by the National Science Centre, Poland, with grants received by Dorota Nowak (OPUS 22, No. 2021/43/B/NZ3/01458) and Aleksandra Simiczyjew (SONATA, 2020/39/D/NZ5/02330).

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
<ol>
	<li>Prepare ADSCs growth medium (ADSCs GM): Supplement Dulbecco&#39;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 &#181;g/mL streptomycin, 0.25 &#181;g/mL amphotericin B).</li>
	<li>Warm the ADSCs GM, phosphate-buffered saline (PBS), and trypsin/EDTA solution to 37 &#176;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.</li>
	<li>Aseptically remove the ADSCs GM from the ADSCs growing in a T75 flask dedicated for cell culture.</li>
	<li>Wash out the residual ADSCs GM by rinsing the cell culture dish with 5 mL of PBS.</li>
	<li>Remove the whole volume of PBS from the flask.</li>
	<li>Add 2 mL of trypsin/EDTA to the culture dish and incubate it at 37 &#176;C till cells are detached. Monitor this process with an inverted microscope. 
	NOTE: Tapping the culture dish will expedite cell detachment.</li>
	<li>Block the trypsin activity by the addition of 6 mL of fresh ADSCs GM.</li>
	<li>Relocate the whole volume of cell suspension from the culture dish to a tube appropriate for centrifugation.</li>
	<li>Centrifuge the cells at 300 x g for 5 min at room temperature (RT).</li>
	<li>Remove the supernatant.</li>
	<li>Suspend cells in 2 mL of the newly added ADSCs GM.</li>
	<li>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.</li>
	<li>Seed the cells in the desired number of wells, 15,000 cells/well, 96-well plastic plate in 100 &#181;L 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.</li>
	<li>Culture the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	<li>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.</li>
</ol>
2. Induction of adipogenic differentiation
<ol>
	<li>Prepare differentiation medium (DM): Supplement DMEM/F12 medium with 10% FBS, 2 mM glutamine, antibiotics - antimycotic solution (100 U/mL penicillin, 100 &#181;g/mL streptomycin, 0.25 &#181;g/mL amphotericin B), 5 &#181;g/mL insulin, 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX), 5 &#181;M dexamethasone, and 200 &#181;M 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 &#176;C. IBMX and indomethacin may precipitate if they are added to the medium at a lower temperature than 37 &#176;C. To solubilize it, warm up the liquid in a water bath to 37 &#176;C.</li>
	<li>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.</li>
	<li>Carefully add 100 &#181;L/well of DM to cells growing in a 96-well plate. 
	NOTE: Do not let the cells dry out when changing medium.</li>
	<li>Incubate the cells in an incubator at a temperature of 37 &#176;C, 5% CO2 for 7 days.</li>
	<li>Remove and discard the whole volume of DM from the ADSCs.</li>
	<li>Carefully add 100 &#181;L/well of fresh DM to cells growing in a 96-well plate.</li>
	<li>Incubate the cells in the incubator at a temperature of 37 &#176;C, 5% CO2, 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.</li>
</ol>
3. Adipocytes&#39; fattening
<ol>
	<li>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 &#181;g/mL streptomycin, 0.25 &#181;g/mL amphotericin B), 0.33 mM linoleate, 0.17 mM palmitate, and 0.17 mM oleate.</li>
	<li>Prepare control adipocyte culture medium: Supplement DMEM/F12 medium with 10% FBS, 2 mM glutamine, antibiotics - antimycotic solution (100 U/mL penicillin, 100 &#181;g/mL streptomycin, 0.25 &#181;g/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 &#176;C.</li>
	<li>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.</li>
	<li>Carefully add 100 &#181;L/well of adipocyte culture medium supplemented with FAs to half of the wells with cells growing in a 96-well plate.</li>
	<li>Carefully add 100 &#181;L/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.</li>
	<li>Incubate the cells at 37 &#176;C in a humidified atmosphere containing 5% CO2 for the next 48 h.</li>
	<li>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.</li>
</ol>
4. Lipid droplet stains
<ol>
	<li>Prepare adipocyte culture medium: Supplement DMEM/F12 medium with 10% FBS, 2 mM glutamine, and antibiotics - antimycotic solution (100 U/mL penicillin, 100 &#181;g/mL streptomycin, 0.25 &#181;g/mL amphotericin B).</li>
	<li>Dilute fluorescent dye to stain LDs in adipocyte culture medium in a dilution of 1:1000.</li>
	<li>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.</li>
	<li>Carefully add 50 &#181;L/well of adipocyte culture medium with fluorescent dye. 
	NOTE: Do not let the cells dry out when changing medium.</li>
	<li>Incubate cells at 37 &#176;C in a humidified atmosphere containing 5% CO2 for 15 min.</li>
	<li>Wash out the fluorescence dye by replacing it with the 100 &#181;L/well adipocyte culture medium.</li>
	<li>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.</li>
</ol>

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<li>Abbasi, K., Mehdizadeh, A., Hamishehkar, H., Nouri, M., Darabi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fatty+acid+composition+of+lipid+fractions+in+white-+and+brown-like+adipocytes+derived+from+human+mesenchymal+stem+cells%5BTitle%5D)+AND+%22Adipocyte%22%5BJournal%5D)">Fatty acid composition of lipid fractions in white- and brown-like adipocytes derived from human mesenchymal stem cells.</a> Adipocyte. 14 (1), 2566481(2025).</li>
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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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 arrest in preadipocytes is a prerequisite step for differentiation induction12. Only in growth-arrested cells, which can be achieved by contact-inhibition at 100% confluence, does the hormonal regimen (insulin, indomethacin, dexamethasone, and IBMX) induce adipocyte differentiation. IBMX in combination with dexamethasone stimulates expression of PPAR&#947; (peroxisome proliferator-activated receptor gamma), a key regulator of adipogenesis13. IBMX is a competitive, nonselective phosphodiesterase inhibitor that raises intracellular cAMP levels and activates protein kinase A (PKA). The PKA signaling pathway is required for the transcriptional activation of PPAR&#947; and, consequently, adipogenic gene expression. In addition, both dexamethasone and IBMX are inducers of C/EBP&#948; (CCAAT/enhancer-binding protein delta) and C/EBP&#946;, which are transcription factors that promote cell growth and differentiation14. Similarly, indomethacin promotes adipogenesis by increasing C/EBP&#946; and PPAR&#947; expression15. Insulin is a critical hormone that drives adipocyte differentiation by activating key transcription factors, such as PPAR&#947; and SREBP-1c (sterol regulatory element-binding protein-1c), which orchestrate the cellular changes from preadipocytes to mature fat cells. Insulin signaling, involving Ras GTPases, leads to the activation of CREB, a protein that then triggers the expression of genes responsible for the adipocyte phenotype. Additionally, insulin promotes glucose uptake via the GLUT4 transporter and lipid storage, while inhibiting lipolysis, supporting adipocyte lipid storage requirements16.
The described protocol is based on human ADSCs. It is worth noting that different cell types exhibit distinct characteristics during adipogenic differentiation, including variations in induction time, stimulatory compounds, and lipid droplet morphology14,17. Moreover, recently, Abbasi et al. identified that white-like (WLAs) and brown-like adipocytes (BLAs) differentiated from human mesenchymal stem cells (MSCs) present distinct lipid profiles. While WLAs are enriched in long-chain monounsaturated fatty acids, favoring lipid storage, BLAs exhibit a more diverse fatty acid composition across lipid fractions associated with greater membrane fluidity, metabolic flexibility, and thermogenic potential18. These findings underscore adipocyte heterogeneity and indicate that further studies are needed to better understand it.
A number of investigational approaches have been proposed to analyze the mechanisms of obesity. A commonly used method is treating cells with FAs, which can be used in various configurations and concentrations. Commonly, using only one fatty acid (palmitic or oleic) in studies does not accurately reflect the diversity of fatty acids in a patient&#39;s plasma. Moreover, the application of each of the listed acids has diverse consequences on fat cell biology. Both acids contribute to the occurrence of such obesity features as LDs hypertrophy, while only palmitic acid increased adipocyte basal lipolysis and induced an adipocyte secretome capable of altering macrophage gene expression19. Palmitate frequently evokes cytotoxic cell reactions that are opposite to those of most other fatty acids. Multiple reports indicate that supplementation with an identical concentration of oleate averts the opposing action of palmitate. Therefore, it is imprecise to incubate cells only with palmitate without other unsaturated FAs. Though Watt et al. indicated that plasma contains a variety of long-chain fatty acids, with about 35% saturated and 65% unsaturated20. This is why, to avoid inaccurate conclusions, the experiments should be performed with a mixture of saturated and unsaturated FAs delivered in a molar ratio and at concentrations that reflect those found in blood (e.g., 1:1 or 1:2 palmitate/oleate; 1:2:1 palmitate/oleate/linoleate)20.
Another issue is how fatty acids are delivered to cells. The &#34;carrier&#34; of the acids added into the culture medium also seems to be important. Due to their low solubility, FAs are commonly analyzed as complexes with albumins, such as bovine serum albumin (BSA). In the described protocol, fatty acids added to the cells are complexed with BSA. In the organism, non-esterified FAs are transported mainly with serum albumin. Moreover, studies using primary hepatocyte cultures show that, in the presence of albumin, palmitate uptake is more efficient21. BSA is an appropriate way to distribute FAs to cells because of its similarity to the biological system of non-esterified FAs carriage. In the case of in vitro systems, albumin-promoted FAs delivery follows the &#34;pseudo facilitation model&#34; in which FAs are delivered as a function of their unbound concentration in the medium at a physiological concentration of albumin (~200 &#181;M), while at lower concentrations (&#60; 150 &#181;M), delivery will depend on albumin concentration21.
Moreover, the dissolution of long-chain FAs is problematic due to their poor solubility in water-based solutions. The approaches used to address this problem often involve the use of organic solvents and heating. In this protocol, ethanol was used to dissolve FAs. However, organic solvents can affect cell viability when the final concentration used is too high. Additionally, it is worth pointing out that control conditions should always be treated with the same concentrations of solvent and the carrier of FAs as the fattened cells.
Adipose tissue is a multicellular system. It is worth noting that, despite the mechanisms of AT enlargement, in most cases, long-lasting, disproportionate caloric intake ultimately contributes to adipocyte dysfunction. This is accompanied by quantitative and qualitative modifications in the composition of AT at the cellular level. Immune cells are of specific significance in this respect. Chronic, low-grade inflammation is a major hallmark of obese adipose tissue, and it is now known that, at least in mice, almost every immune cell type can be found in adipose tissue under one experimental condition or another22. The presented model does not account for these aspects of obesity-related modifications in adipocytes. However, even if one uses mature adipocytes isolated from patients with obesity, it would still be difficult to directly compare these cells with adipocytes from AT of lean patients due to the large inter-individual variability. Therefore, it is necessary to develop a method to stimulate adipocytes that mimics the conditions of obesity as closely as possible. It is worth underlining that, in fattened adipocytes generated as described in the presented model, we previously detected multiple secretory and metabolic alterations11.
In conclusion, the described procedures provide a simple, effective protocol for the differentiation of human ADSCs and their subsequent treatment with a combination of fatty acids. The protocol enables the generation of a high number of adipocytes, does not require complex manipulations or advanced infrastructure, and can be readily implemented in most cell culture laboratories. The application of the described model facilitates reproducing the human obesity conditions. This model can be used to evaluate the molecular mechanisms driving hypertrophy in human adipocytes. Moreover, it is applicable for investigating hypertrophy-associated processes such as insulin resistance and inflammation. Given the central endocrine role of adipose tissue, the presented model provides a relevant platform for examining hypertrophy-related alterations in the adipocyte secretome and their regulatory mechanisms. It is also well-suited for studying the effects of fatty acids on intracellular signaling, organelle function, and cellular metabolism. The use of physiologically relevant fatty acids enables the assessment of compound uptake and storage in lipid droplets, especially for substances that modulate lipid metabolism.
Troubleshooting 
It may occur that preadipocyte differentiation proceeds with low efficiency, which may be caused by their lower-than-100% confluence before the addition of differentiation medium. Subconfluent cultures typically show reduced responsiveness to adipogenic cues. Additionally, it is crucial to prepare fresh differentiation media immediately before use, as components such as IBMX, dexamethasone, or insulin lose activity during storage. The poor quality and storage conditions of reagents may also limit the efficiency of lipid droplet accumulation in differentiated adipocytes. It is important to test multiple FBS lots, as serum composition may influence differentiation efficiency.
During the preparation of fatty acids, several solubility issues may arise. To improve it, the solutions of FAs should be warmed up gently. Moreover, it is important to dissolve fatty acids with appropriate solvents (e.g., ethanol) and aliquot FA stocks to minimize freeze-thaw cycles. If cytotoxicity is observed during adipocyte fatty acid loading, the fatty acid concentrations should be titrated to identify a non-toxic working range. Additionally, the proper FA:BSA molar ratio and complete conjugation before application should be maintained. If small lipid droplets are detected despite prolonged differentiation, the fattening period should be extended, with concomitant verification that FAs remain stable throughout treatment.
When a high background signal or weak staining during lipid droplet visualization is observed, the dye dilution should be optimized with extended washing steps, as needed. This procedure will help avoid nonspecific staining. It is also important to protect dye solutions from light and prepare working dilutions fresh to prevent photobleaching. If lipid loss during staining occurs, cells should be fixed with paraformaldehyde-based solutions to preserve lipid droplets. Moreover, during the staining procedure, cells should not be exposed to the organic solvents.
Fully differentiated and fattened adipocytes are sensitive to mechanical disturbances; therefore, all media changes should be performed slowly to avoid applying strong aspiration forces that could cause lipid loss.

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&#39;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 cells7,8,9, 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 (na&#239;ve) 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.

<table><tbody><tr><td>3-Isobutyl-1-methylxanthine (IBMX)</td><td>Merck</td><td>I5879</td><td></td></tr><tr><td>0.25% trypsin/0.05% EDTA pH 7.2</td><td>IITD PAN, Wroc&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/69759/69759eq1.jpg&quot; style=&quot;margin: auto;&quot;/&gt;aw, Poland</td><td>20-500</td><td></td></tr><tr><td>96-well plate</td><td>Thermo</td><td>130188</td><td></td></tr><tr><td>antibiotics-antimycotic solution</td><td>Sigma-Aldrich</td><td>A5955</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A3294</td><td></td></tr><tr><td>Cell counter</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Centrifuge</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>IITD PAN, Wroc&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/69759/69759eq1.jpg&quot; style=&quot;margin: auto;&quot;/&gt;aw, Poland</td><td>10-500</td><td>containing 1 g/L glucose</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium:Nutrient Mixture F12</td><td>Gibco</td><td>11320-033</td><td></td></tr><tr><td>Ethanol</td><td>Chempur</td><td>113964200#1L</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>A5256701</td><td></td></tr><tr><td>hADSC Human Adipose-Derived Stem Cells</td><td>Lonza</td><td>PT-5006</td><td></td></tr><tr><td>Incubator</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Indomethacin</td><td>Sigma-Aldrich</td><td>I8280-5G</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I9278</td><td></td></tr><tr><td>Inverted microscope</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma-Aldrich</td><td>25030-024</td><td></td></tr><tr><td>Linoleic acid sodium salt</td><td>Sigma-Aldrich</td><td>L8134</td><td></td></tr><tr><td>LipidSpot fluorescent</td><td>Biotium</td><td>70065</td><td></td></tr><tr><td>Phosphate-buffered saline (PBS)</td><td>Thermo</td><td>10010023</td><td></td></tr><tr><td>Soduim oleate</td><td>Sigma-Aldrich</td><td>O7501</td><td></td></tr><tr><td>Soduim palmitate</td><td>Sigma-Aldrich</td><td>P9767</td><td></td></tr><tr><td>T75 cell culture flask</td><td>Thermo</td><td>130190</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma-Aldrich</td><td>95896HJ</td><td></td></tr></tbody></table>

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.

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 adipogenic inducers. After 24 h, cells undergo morphological changes, and single cells with small LDs can be visible (Figure 1B). At day 7, when the DM should be replaced by the fresh one, cells with multiple small LDs can be observed (Figure 1C). After the next 7 days, at day 14 of cell differentiation, adipocytes with accumulated multiple LDs can be visible (Figure 1D).
To generate hypertrophic adipocytes, the described fattening protocol should be applied immediately after ADSC differentiation. During incubation in adipocyte culture medium supplemented with FAs, cells accumulate more large LDs, which can be detected with a light microscope (Figure 2). Fattened adipocytes can be considered a model of hypertrophic cells and used for subsequent analysis, such as cell staining (Figure 2). These cells can serve as a model for analyzing the cellular metabolism of control and fattened adipocytes11 or be used in co-culture with other cell types.
<img alt="Cell differentiation over 14 days; microscopy images, days 0, 1, 7, 14; insulin, IBMX, dexamethasone." class="xfigimg" src="/files/ftp_upload/69759/69759fig1.jpg" data-altupdatedat="1776139283000" data-alt="Figure 1"> 
Figure 1: Differentiation of human ADSCs into adipocytes. Representative images of cell morphology showing different stages of ADSCs differentiation at days (A) 0, (B) 1, (C) 7, and (D) 14 following DM treatment. (A) Once the ADSCs cells reached confluency (day 0), adipogenesis was induced with the differentiation medium (DM) containing 5 µg/mL insulin, 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX), 5 µM dexamethasone, and 200 µM indomethacin. (C) On the 7th day of differentiation, this medium was replaced with fresh DM. Scale bars: 100 µm. <a href="https://www.jove.com/files/ftp_upload/69759/69759fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Lipid droplet analysis; phase-contrast, LD staining, merge; microscopy image; cellular lipid study." class="xfigimg" src="/files/ftp_upload/69759/69759fig2.jpg" data-altupdatedat="1776139283000" data-alt="Figure 2"> 
Figure 2: Morphology of adipocytes incubated with fatty acids (FAs). Representative phase-contrast and fluorescence microscopy images of control and hypertrophic adipocytes with accumulated lipid droplets (LDs). Adipocytes were stained for neutral lipids present in LDs with Lipid Spot 488 dye marked in green. Insets in the upper left corner show an enlarged view of the areas boxed in red. Scale bars: 100 µm. <a href="https://www.jove.com/files/ftp_upload/69759/69759fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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

Obesity-related changes in adipose tissue alter local secretory profiles and influence interactions with other surrounding cells, including cancer cells. ...

Differentiation and induction of lipid droplet accumulation in human adipocytes as a model to study obesity in vitro. 

Aliccia Bollig-Fischer

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