Protocol for the in vitro isolation and culture of mature adipocytes and white adipose tissue explants from humans and mice

Summary

White adipose tissue (WAT) explants culture allows the study of this tissue ex vivo, maintaining its structure and properties. Concurrently, isolating mature adipocytes facilitates research into fat cell metabolism and hormonal regulation. Here, we present a protocol for obtaining, isolating, and processing mature adipocytes, alongside the cultivation of WAT explants from humans and mice. We describe steps for WAT retrieval, culturing of WAT explants, WAT digestion, and adipocytes separation. We then detail procedures for culturing isolated mature adipocytes.

For complete details on the use and execution of this protocol, please refer to Villanueva-Carmona et al. (2023).¹

Graphical abstract

Highlights

• •Isolation and culture of mature adipocytes and white adipose tissue explants
• •Protocol applicable to white adipose tissue from rodents and humans
• •Step-by-step details on adipose tissue digestion and adipocyte separation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

White adipose tissue (WAT) explants culture allows the study of this tissue ex vivo, maintaining its structure and properties. Concurrently, isolating mature adipocytes facilitates research into fat cell metabolism and hormonal regulation. Here, we present a protocol for obtaining, isolating, and processing mature adipocytes, alongside the cultivation of WAT explants from humans and mice. We describe steps for WAT retrieval, culturing of WAT explants, WAT digestion, and adipocytes separation. We then detail procedures for culturing isolated mature adipocytes.

Before you begin

The following protocol outlines the detailed procedures for the isolation and culture of mature adipocytes and WAT explants from adult mice and humans. It has been successfully utilized with various types of WAT, including subcutaneous, epididymal, and retroperitoneal, in both lean and diet-induced obese mice, as well as in a variety of genetically modified mouse models. As such, this approach can be customized for different WAT sites and rodent species, and it is also applicable to humans, both in lean and obese conditions. For the collection of WAT from mice, please follow steps 1-6; and for the collection of human WAT, please follow step 7, in before you begin section.

Institutional permissions

All animal experiments involved in these studies were overseen and approved by the Universitat Rovira i Virgili Animal Welfare and Governmental Ethics Committee (ref. 10970). All animal procedures adhered to the guidelines provided by the European Union Directive 86/609/EEC and recommendation 2007/526/EC pertaining to the protection of animals used for experimental and other scientific purposes, which are enacted under Spanish law 1201/2005.

All study participants were recruited from University Hospital Joan XXIII and Hospital de Santa Tecla, both located in Tarragona, Spain. Each individual provided informed consent, with the study receiving approval from the respective hospitals' ethics and research committees (references CEIm 177/2018 approved on the 29th of November 2018 and CEIm 41p/2015 approved on the 31st of August 2015). The study’s procedures adhered to the ethical guidelines stated in the Declaration of Helsinki.

Mouse euthanasia and white adipose tissue retrieval

Timing: 1 h

• 1.Humanely euthanize the mouse via cervical dislocation or other institutionally approved methods.

Note: Depending on the experimental approach, mice may need to undergo a fasting period.

• 2.Sterilize the abdominal region with 70% ethanol.
• 3.Make an incision in the abdomen to expose the internal organs.
• 4.Using sterile scissors and forceps, harvest the desired adipose tissue depots.
• 5.Rinse each collected tissue with phosphate-buffered saline (PBS) supplemented with 1% antibiotic/antimycotic solution.
• 6.Pool the tissues of at least three mice from the same group, sorted by tissue type, into a 50 mL Falcon tube containing PBS supplemented with 1% antibiotic/antimycotic solution.

CRITICAL: The harvested adipose tissue can be stored in a PBS solution supplemented with antibiotics until it is ready for use (should not exceed 2–3 h). This solution aids in preserving the tissue's viability while guarding against bacterial or microbial contamination (Figure 1A).

Note: Adipose tissue provides a versatile substrate for research studies, particularly in the field of metabolism. It can be utilized as tissue explants or by isolating mature adipocytes, with each approach offering unique advantages and insights. When adipose tissue explants are used, small sections of tissue are cultured ex vivo. This approach retains the native structure and heterogeneity of the tissue, allowing for the investigation of cell-cell and cell-matrix interactions.² On the other hand, mature adipocyte isolation involves processing the adipose tissue to extract these specialized cells. This strategy enables more targeted experiments to be conducted, focusing on the metabolic activities and regulatory mechanisms of the adipocytes themselves. However, it does not offer the complexity of cell interactions present within the tissue. Thus, the choice between adipose tissue explants and mature adipocyte isolation largely depends on the research objectives and the level of cellular complexity required in the experimental design.

Figure 1.

Process of digestion of adipose tissue

(A) Adipose tissue stored in a PBS solution supplemented with 1% Ab/Am.

(B) Tissue placed in a sterile Petri dish containing PBS with 1% Ab/Am.

(C) Adipose tissue minced.

(D and E) Transfer of the minced tissue into a sterile falcon tube containing the collagenase solution.

(F) Incubation for 1 h at 37 ºC on an orbital shaker with the collagenase solution.

Collection of human white adipose tissue

Timing: 30 min

The procedures outlined below are applicable not only to mouse adipose tissue but also to human samples. Human adipose tissue samples were collected from age- and gender-matched donors undergoing non-acute surgical procedures, such as hernia or cholecystectomy, in a scheduled routine surgery. Donors were classified based on their BMI as either lean (BMI < 25 kg/m²) or obese (BMI ≥ 30 kg/m²), following the criteria set by the World Health Organization (WHO).

• 7.Collect adipose tissue samples, and immediately place them into a sterile container with PBS supplemented with 1% antibiotic/antimycotic solution to ensure their integrity and prevent any contamination.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Phosphate-buffered saline (PBS)	Hyclone (GE HealthCare Life Sciences)	Cat.#SH30028.02
2-[4-(2-Hydroxyethyl)Piperazin-1-Yl] Ethanesulfonic Acid (HEPES)	Fisher Scientific	Cat.#BP310-1; CAS ID: 7365-45-9
Bovine serum albumin (BSA)	Sigma-Aldrich	Cat.#A7030; CAS ID: 9048-46-8
Collagenase from Clostridium histolyticum	Sigma-Aldrich	Cat.#C0130; CAS ID: 9001-12-1
Experimental Models: Organisms/Strains
Adult male C57BL/6 wild-type mice	Charles River	C57BL/6
Biological samples
Human adipose tissue	IISPV Biobank (PT17/0015/0029)	N/A
Other
Antibiotic/antimycotic solution	Hyclone (GE HealthCare Life Sciences)	Cat.#SV30079.01
Fetal bovine serum (FBS)	Hyclone (GE HealthCare Life Sciences)	Cat.#SH30071.03
DMEM/F12 medium	Gibco (Life Technologies Limited)	Cat.#31330-038

Step-by-step method details

For culture of white adipose tissue explants, please follow steps 1–6. For mature adipocyte isolation and culture, proceed with steps 7–27.

Culture of white adipose tissue explants

Timing: 4 h

The culture of WAT explants is an essential procedure that allows us to mimic and study the tissue’s natural environment. This method helps preserve the cellular structure and functions, providing valuable insights into adipose tissue biology.² Sterile techniques must be upheld throughout the entire process to reduce the risk of contamination and its potential adverse effects on cell viability.

• 1.With sterile scissors, dissect the adipose tissue into small, uniformly sized pieces of approximately 40 mg each.
• 2.Gently place two of the pre-cut adipose tissue pieces into each well of a 24-well plate containing 350 μL of DMEM/F12 medium, supplemented with 0.2% of BSA. Ensure that the explants are fully immersed in the medium.
• 3.Place the culture dishes with the adipose tissue explants in a cell culture incubator set to 37°C and 5% CO₂ for 1–2 h.

Note: Tissue should rest for at least 1 h before proceeding, but not longer than 2 h.

• 4.Check the culture under a microscope to assess the integrity of the adipose tissue explants.
• 5.Perform any necessary experiments or treatments based on your experimental design.

Note: Cell culture incubations or treatments should be performed in a cell culture incubator.

Note: The viability of adipose tissue explants can exhibit variability, typically spanning from several hours up to approximately 24 h under experimental conditions. During this time, various metabolic processes can be studied such as the uptake and release of nutrients or the response to hormones, as well as gene expression analysis.

• 6.After the treatment, you may collect the conditioned medium from the adipose tissue explants. This medium can be utilized in experiments like ELISAs to analyze secreted proteins. Alternatively, you can also collect the adipose tissue itself for further studies, such as RNA expression or Western Blot analyses.

Note: Your decision to collect either the conditioned medium or the adipose tissue depends on your research objectives and experimental requirements.

Note: Performing experiments involving adipose tissue explants are conducted within an incubator, as it provides a controlled environment that mimics the physiological conditions necessary for maintaining the viability and functionality of the tissue explants.

Adipose tissue digestion

Timing: 2 h

In addition to studying WAT explants, isolating mature adipocytes, which are key in fat storage, release, and hormonal regulation,³ can be a focal point of your study. The digestion of adipose tissue allows you to isolate these cells for subsequent investigation. This procedure entails the disruption of the extracellular matrix and connective tissue, resulting in the liberation of individual cells for further investigation.⁴ Using a cell culture hood throughout the entire process is essential to ensure sterility. After euthanizing the animal and harvesting the adipose tissue, the following are the steps for adipose tissue digestion:

• 7.Weigh each adipose tissue separately.
• 8.Place the excised tissue in a sterile petri dish containing PBS with 1% antibiotic/antimycotic solution (Figure 1B).
• 9.Cut the adipose tissue into small pieces using sterile scissors and forceps (Figure 1C).
• 10.Transfer the minced tissue to a new sterile petri dish containing PBS. Repeat this step twice for a total of three consecutive washes.

Note: This step helps eliminate impurities and contaminants, thereby creating a suitable environment for experimentation.

• 11.Adjust the collagenase concentration based on the tissue weight. For 2 g of collected WAT adipose tissue, we use 10 mL of PBS containing 1% BSA and 0.2% collagenase.

Note: The collagenase solution plays a crucial role in enzymatically dissociating cells from tissue fragments, enabling the isolation of target cells.

• 12.Transfer the minced tissue into a sterile 50 mL Falcon tube containing the collagenase solution (Figure 1D).
• 13.Incubate the tube for 30 min–1 h at 37°C on an orbital shaker set at 200 rpm. This allows for the collagenase enzyme to digest the tissue (Figure 1E).

Note: In the context of this particular protocol, subcutaneous WAT may undergo incubation for a maximum duration of 1 h during the digestion process. Conversely, visceral WAT is incubated with collagenase solution for a period not exceeding 30 min. This shorter digestion time for visceral WAT aims to mitigate the risk of excessive adipocyte breakdown.

CRITICAL: Be sure to optimize the digestion time for your specific adipose tissue and experimental needs. Extended digestion may lead to increased adipocyte death, whereas inadequate digestion could result in incomplete tissue dissociation.⁴ Consider running pilot experiments to determine the optimal digestion time for your tissue type.

Adipocyte separation

Timing: 30 min

Upon completion of the digestion process (Figure 2A), the subsequent steps focus on segregating the mature adipocytes from the stromal vascular fraction (SVF) and tissue remnants.

• 14.Centrifuge the digested samples at 300 g for 5 min at 22°C (Figure 2B).
• 15.Invert the tube to facilitate thorough phase mixing (Figure 2C).
• 16.Repeat the centrifugation step under the same conditions (Figure 2D).

Note: This centrifugation step will separate the different components of the tissue suspension based on their sedimentation rates.

• 17.Collect the mature adipocytes from the top portion of the centrifuge tube (Figure 3A) and carefully transfer them into a new 15 mL Falcon tube with a Pasteur pipette (Figure 3B).
• 18.Rinse the adipocytes with 8–9 mL of PBS to guarantee the removal of remaining impurities or residual solutions (Figure 3C).
• 19.Centrifuge the Falcon tube containing PBS and adipocytes at 300 g for 5 min at 22°C (Figure 3D).
• 20.Cautiously collect the mature adipocytes from the upper layer using a pipette for subsequent analysis or culture (Figures 3E and 3F).

Note: It's crucial to note that the SVF pellet, derived from the separation procedure, is not only significant for isolating mature adipocytes but also for isolating other essential components, including stem cells, macrophages, and endothelial cells, among others.⁵

Figure 2.

Separation of mature adipocytes

(A) Adipose tissue after incubation with the collagenase solution.

(B) Digested samples after the first centrifugation.

(C) Inverted tube to facilitate complete mixing of phases.

(D) Separation of mature adipocytes (arrow) from SVF and tissue remnants after centrifugation.

Figure 3.

Collection of isolated mature adipocytes

(A) Process of recovering the mature adipocytes from the top portion of the centrifuge tube.

(B) Mature adipocytes transferred into a new 15 mL Falcon tube.

(C) Adipocytes rinsed with PBS.

(D) Falcon tube containing PBS and adipocytes after centrifugation.

(E and F) Collection of mature adipocytes from the upper layer into an Eppendorf tube.

Cultivation of isolated mature adipocytes

Timing: 1 day

This section provides detailed steps for the cultivation and collection of isolated mature adipocytes, enabling further analysis and experimental studies.

• 21.Based on the experimental plan, seed the appropriate quantity of mature adipocytes in culture dishes or plates containing DMEM/F12 supplemented with 10% FBS and 1% antibiotic/antimycotic solution (Figures 4A and 4B).

Note: Owing to the heterogeneous size distribution of mature adipocytes, precise cell counting, and seeding densities become challenging to determine. The number of seeded wells is based on the total weight of the adipose tissue instead. In this specific protocol, using a 12-well plate, we recommend placing the adipocytes isolated from 1 g of tissue into each individual well.

CRITICAL: To achieve uniform dispersion of mature adipocytes across the culture plate, it is imperative to thoroughly homogenize them with the medium before seeding. Adipocytes have a tendency to float, and without adequate mixing, they might not distribute evenly within the plate, potentially affecting the success of the culture establishment.

• 22.Place the adipocytes in a cell culture incubator for 18 h, maintaining an environment of 37°C and 5% CO₂.
• 23.In line with the experimental schedule, continue with the planned assays, treatments, or experiments.
• 24.Collect the mature adipocytes with the medium and centrifuge at 400 g for 5 min at 22°C.
• 25.Post-centrifugation, cautiously collect the adipocytes that have floated to the top (Figure 4C).
• 26.Rinse these with PBS and repeat the centrifugation at 400 g for 5 min at 22°C.
• 27.Recover the mature adipocytes from the uppermost layer using a micropipette to perform the subsequent analysis or freeze at −80°C in an Eppendorf for preservation.

CRITICAL: It is important to note that the viability and functionality of the adipocytes might decrease as the duration of the culture extends. As such, it is standard practice to use mature adipocytes in experiments within the first 48 h post-isolation to ensure optimal functionality and responsiveness of the cells.

Note: While not a mandatory step in the protocol, the visual examination of adipocytes under a microscope can be enriched by investigating the maturity of adipocytes through analyzing typical markers like PPARG, FASN, and GLUT4, among others. The analysis of mature adipocytes can take place either immediately before or after freezing. In this scenario, frozen adipocytes can maintain RNA and protein quality for several months to a year when preserved at −80°C. As a result, RNA and/or protein extraction can be conducted during this timeframe.

Figure 4.

Process of cultivation of mature adipocytes

(A and B) Mature adipocytes seeded in culture dishes.

(C) Culture medium and mature adipocytes collected in Eppendorf tubes.

Expected outcomes

The protocol for isolating and culturing mature adipocytes aims for several key outcomes. These encompass the successful isolation of viable and functional mature adipocytes, preservation of their distinct morphology, lipid content during culture, preservation of their metabolic activity, responsiveness to stimuli, and retention of gene expression profiles relevant to adipocyte function. Additionally, culturing mature adipocytes enables the study of adipocyte-specific processes, such as lipid metabolism or adipokine secretion.

With white adipose tissue explants, one primary anticipated result is the preservation of the tissue’s cellular architecture, crucial for examining cell morphology, cell-to-cell communication, and tissue-specific functions.

Limitations

This protocol describes the successful technique of isolating and culturing mature adipocytes and white adipose tissue explants. However, some limitations are noteworthy. Firstly, the presence of multiple cell types, including preadipocytes, immune, and vascular cells, can impact cell growth and overall tissue complexity. While in vitro systems are useful, they may not fully replicate the intricate in vivo microenvironment.

During the culture process, cells may experience phenotypic changes or gene expression alterations, potentially affecting their characteristics and behavior. Careful optimization of culture conditions - incorporating appropriate culture media, supplements, and techniques - is crucial for preserving cell viability and functionality. Furthermore, cell viability and function may decrease over time in long-term culture, requiring close monitoring. Moreover, acquiring large amounts of mature adipocytes or white adipose tissue for experimentation can be challenging due to variations in tissue abundance and accessibility, making standardized and consistent sample collection difficult.

Recognizing these constraints is important when designing experiments, interpreting results, and developing strategies to improve the reliability and relevance of adipose tissue research.

Troubleshooting

Problem 1

Low cell viability - related to step 13.

Potential solution

This can be caused by several factors. Sterile techniques must be upheld throughout the entire process to minimize contamination and its negative impact on cell viability. The digestion time and temperature during tissue dissociation need careful optimization as insufficient or excessive digestion can affect cell viability.

Problem 2

Inconsistencies arising from adipose tissue source - related to step 4 in before you begin section.

Potential solution

Adipose tissue depots vary in their cell yield and adipocyte maturity. Visceral adipose tissue typically contains larger adipocytes, indicative of greater maturity and higher lipid content, compared to subcutaneous adipose tissue. In contrast, subcutaneous adipose tissue, more vascularized than visceral adipose tissue, contains smaller, less mature adipocytes.⁶ This increased vascularization can influence the ease of isolation and subsequent adipocyte culture, potentially leading to a higher yield of viable cells. Furthermore, the procedure is effective for normal adipose tissue function, but its effectiveness might differ in a situation involving a pathological condition, such as obesity. It’s crucial to fine-tune the procedure if the pathological situation impacts adipose tissue. Therefore, choosing the adipose tissue depot for isolation depends on the specific research question and objectives.

Problem 3

Determining the number of mice required for the experiment - related to step 6 in before you begin section.

Potential solution

Striking a balance between scientific objectives and ethical considerations is crucial, aiming to minimize the number of mice utilized while achieving statistically significant results. A strategy known as sample pooling could help minimize the number of mice required for isolating and culturing mature adipocytes. This approach involves combining adipose tissue samples from multiple mice to generate a larger pool of cells or explants for the experiment. This method maximized tissue utilization from each mouse, enabling robust results while minimizing the overall number of mice used in the study.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Sonia Fernández Veledo (sonia.fernandez@iispv.cat).

Materials availability

The present study did not produce any novel or distinct reagents.

Data and code availability

No datasets were generated for this protocol.