Isolation and Culture of White and Brown Preadipocytes from Mouse Embryos

Summary

We present a novel, robust, and reproducible approach for isolating and culturing white and brown preadipocytes from mouse embryos, establishing a powerful in vitro strategy for dissecting the cellular and molecular mechanisms governing adipocyte lineage commitment and differentiation, and providing a framework for investigating the impact of adipose tissue development.

Abstract

Adipose tissue plays a central role in metabolic homeostasis, and its properties are shaped during embryonic development through adipocyte differentiation. Embryonic preadipocytes, therefore, represent a relevant model to study early events in adipose tissue formation and lineage specification. This article describes a reproducible protocol for the isolation and in vitro differentiation of white and brown preadipocytes isolated from mouse embryos at embryonic day 15.5 (E15.5), a developmental stage at which adipose depots begin to form. The method provides detailed guidance for tissue microdissection, enzymatic digestion, primary culture, and lineage-specific differentiation conditions that support cell viability and adipogenic maturation. Representative results include lipid droplet accumulation and lineage-associated marker expression, confirming successful differentiation under defined culture conditions. Using this approach, embryonic preadipocytes can be directed toward white or brown adipocyte fates, enabling comparative analyses of developmental timing, lineage characteristics, and gene expression profiles. This protocol offers a practical and developmentally relevant tool for investigating adipose tissue formation and perinatal programming mechanisms in a controlled experimental setting.

Introduction

Adipose tissue plays a central role in maintaining whole-body homeostasis by regulating energy storage, endocrine signaling, and adaptive thermogenesis, while interacting extensively with both local and distant organs^1,2,3. Two major types of adipose tissue can be distinguished: the white adipose tissue (WAT) and the brown adipose tissue (BAT), which differ markedly in cellular lineage, physiological function, anatomical distribution, and metabolic activity⁴.

WAT constitutes the primary energy reservoir of the organism. White adipocytes are highly specialized for lipid storage and are characterized by the presence of a single large lipid droplet (LD) that occupies most of the cytoplasmic space⁵. In contrast, BAT is specialized in non-shivering thermogenesis. Brown adipocytes contain multiple small LDs and exhibit a high mitochondrial density^3,6. The thermogenic capacity of BAT relies on Uncoupling Protein 1 (UCP1), an inner mitochondrial membrane protein that facilitates proton leak across the membrane⁷. By uncoupling oxidative phosphorylation from ATP synthesis, UCP1 dissipates the proton gradient as heat, thereby contributing to the maintenance of body temperature⁸. Given their diverse roles, WAT and BAT have attracted considerable interest not only as key regulators and potential therapeutic targets in metabolic diseases, but also in developmental and evolutionary biology, immunology, and aging, owing to their complex and far-reaching systemic interactions^9,10. Accumulating evidence also indicates that fetal metabolic programming is closely associated with adipose tissue development, as perturbations in the intrauterine environment can influence adipocyte number, distribution, and function, thereby predisposing individuals to obesity and metabolic diseases later in life¹¹.

To investigate adipose tissue biology, the availability of mature adipocytes is essential. In vitro culture systems provide a controlled environment that allows the assessment of specific factors influencing adipogenesis and adipocyte function. Several protocols have been established for the isolation and differentiation of adipocytes from adult mice or neonatal pups^12,13,14,15. However, these adipocyte progenitors may differ from the original cell populations that give rise to adipose tissues during development. In mice, BAT depots are the first to develop during embryogenesis, providing neonates with non-shivering thermogenic capacity that is critical for postnatal cold adaptation. Depots of brown adipocytes can be detected in the interscapular region as early as embryonic day 14.5 (E14.5)¹⁶. LDs begin to form at E15.5, and UCP1 expression is initiated around E16.5. In the mouse embryo, preadipocytes of the inguinal WAT (iWAT) are also already present at this stage¹⁶.

The protocol described here focuses on preadipocytes isolated at E15.5. This stage corresponds to the embryonic period during which progenitor cells initiate differentiation in vivo¹⁶, thereby representing the primary progenitors that give rise to WAT and BAT. In contrast, adipogenic progenitors isolated at adult or neonatal stages may already have been influenced by environmental factors and are likely to exhibit more restricted plasticity. Therefore, using embryonic progenitors at E15.5 enhances the ability to faithfully recapitulate the in vivo adipogenic program, as these cells retain a high degree of developmental plasticity and are more inclined to follow physiological differentiation trajectories. This approach is of particular interest for developmental biology questions, notably for investigating the effects of environmental factors on preadipocyte differentiation during the perinatal period, providing a valuable model for studying how early-life exposures may shape adipose tissue development and long-term metabolic outcomes. However, users should be cautious about this stage-specificity: the method is optimized for E15.5 embryos, and deviations in embryonic age or dissection precision may impact cell yield or viability. Additionally, the microdissection technique requires practice to avoid contamination with non-adipose tissues, particularly given the small size of embryonic depots. This protocol summarizes all the steps and procedures for isolating, culturing, amplifying, and differentiating embryonic preadipocytes into mature brown and white adipocytes.

Protocol

All animal procedures were done according to local regulations and under the guidelines defined by the European Union Council Directive of September 22, 2010 (2010/63/EU) (APAFIS#2617-2015110517317420).

All biological waste and enzymatic solutions must be handled and disposed of according to institutional biosafety guidelines and regulations for biohazardous materials. Specifically:
- Biological waste: collect in approved biohazard containers and dispose of via autoclaving or as directed by the institution’s biosafety office.
- Enzymatic solutions: inactivate residual enzymatic activity by adding 10% bleach for 30 min and dispose of as chemical waste according to local regulations.
- Contaminated consumables (pipette tips, tubes, etc): place in biohazard bags and autoclave before disposal.
- Fixation reagents: dispose of as hazardous chemical waste in designated containers.

Always wear appropriate PPE when handling waste.

1. Harvesting mouse embryos

1. Euthanize the pregnant mice (C57Bl/6J) in accordance with the guidelines of the Institutional Animal Care and Use Committee.
2. Disinfect the abdominal area of pregnant mice with 70% ethanol and make a midline incision to expose the uterus.
3. Incise the abdominal cavity to remove the yolk sacs containing the embryos at E15.5 and place them in an agarose box containing cold (4 °C) 1x PBS.
4. Remove the outer membrane of each yolk sac and the placenta, and cut the umbilical cord of the embryos.
5. Place the embryos in a new agarose box containing cold 1x PBS.
   NOTE: Although the central nervous system structures underlying conscious pain perception are not fully developed at this gestational stage, peripheral nociceptors begin to form between E13 and E15. In accordance with institutional animal care guidelines, to minimize potential nociceptive responses, we, however, recommend that embryos be euthanized by decapitation prior to any experimental manipulation.

2. Dissection of embryonic adipose tissues (Figure 1)

1. Dissection of interscapular BAT (Figure 1A)
   1. Cut a rectangle out of a 5% agarose plate and place the embryo inside it to immobilize it. Fill the plate with cold 1x PBS. Place the agarose box with the embryo under a binocular microscope.
   2. Remove the skin from the back of the embryo.
   3. Dissect the interscapular BAT using fine forceps and microscissors.
   4. Carefully separate the BAT from the surrounding tissue (connective tissue and muscle) using microscissors to obtain a BAT sample measuring approximately 4–6 mm x 2–3 mm.
   5. Place the BAT in a 2 mL tube containing 1 mL of cold (4 °C) Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) glutamine with 100 U/mL penicillin/streptomycin (P/S).
2. Dissection of iWAT (Figure 1B)
   1. Place the embryo on its side (on one side and then on the other) on the agarose plate.
   2. Dissect the iWAT using fine forceps and microscissors.
   3. Separate the iWAT from surrounding tissue (connective tissue and skin) carefully until having two depots measuring approximately 2–4 mm x 0.5–0.8 mm.
   4. Place the two WAT samples in a 2 mL tube containing 1 mL of cold DMEM/F-12 glutamine with 100 U/mL penicillin/streptomycin (P/S).
      NOTE: All dissections must be performed under a binocular microscope in the most sterile environment possible, using tools that have been disinfected beforehand.

3. Digestion of adipose tissues

1. Remove the DMEM/F12 with a pipette and add in the tubes 200 µL of DMEM/F12 glutamine with 100 U/mL P/S, 0.2% bovine serum albumin (BSA), 0.5 U Collagenase D, and 0.5 U Dispase II.
2. Cut the tissues with microscissors in the tubes to reduce the pieces of tissue as much as possible and help the digestion process by pipetting up and down 10x.
   NOTE: Fully digested samples should appear as a cloudy suspension without visible tissue clumps.
3. Incubate at 37 °C (in a water bath) for 15 min.
4. Stop the reaction by adding 600 µL of cold DMEM/F-12 glutamine with 100 U/mL P/S, 20% fetal bovine serum (FBS). Resuspend evenly by pipetting up and down 10x.

4. Primary cell culture

NOTE: From this step onward, all procedures must be performed under strict sterile conditions in a Biological Safety Cabinet (BSC) to prevent contamination. Wear sterile gloves, a lab coat, and safety goggles to maintain sterility and protect against potential hazards. Use sterile disposable plasticware, aseptic technique, and ensure all reagents and media are sterile. Avoid contact with non-sterile surfaces to prevent contamination.

1. Plate the cells onto well slides and incubate them under standard conditions (37 °C, 5% CO₂) for 1 h; for 8-well plates, put 200 µL/well. After incubation, confirm that the cells have adhered by gently moving the plate while observing under a microscope.
   NOTE: At this stage, different BAT or WAT samples can be pooled into a single tube to obtain homogenized cultures in the different wells (e.g., to test the effect of a treatment on similar cultures).
2. Replace the medium with prewarmed (37 °C) DMEM/F-12 glutamine with 100 U/mL P/S, 10% FBS and incubate the cells overnight at 37 °C, 5% CO₂.
   NOTE: By changing the medium, dead/unattached cells and tissue fragments that could not be broken down are removed, leaving only the attached cells.
3. Differentiation treatment (72 h)
   1. For brown adipocyte differentiation, change the medium using prewarmed (37 °C) DMEM/F-12 glutamine with 100 U/mL P/S, 10 % FBS, 125 nM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 nM triiodothyronine (T3), 1 µM Rosiglitazone, 1 µM dexamethasone, and 850 nM insulin and incubate at 37 °C, 5% CO₂ for 72 h.
   2. For white adipocyte differentiation, change the medium using prewarmed DMEM/F-12 glutamine with 100 U/mL P/S, 10% FBS, 0.5 mM IBMX, 1 µM dexamethasone, and 850 nM insulin and incubate at 37 °C, 5% CO₂ for 72 h.
4. Maintenance treatment (every 48 h)
   1. Change the medium using prewarmed (37 °C) DMEM/F-12 glutamine with 100 U/mL P/S, 10 % FBS, 1 nM T3, 1 µM Rosiglitazone, and 850 nM insulin. Renew the medium every 48 h.
      NOTE: All concentrations listed refer to the final working concentrations of reagents. Adjust the final volumes of differentiation and maintenance media based on the number of wells to be treated, using 200 µL per well as a standard volume for 8-well plates.

5. Fixation and immunostaining (Figure 2A)

1. Aspirate the media and wash the cells with cold 1x PBS.
2. Fix the cells 10 min with cold 4% formaldehyde in 1x PBS.
3. Wash the cells with 1x PBS for 3 x 5 min.
4. Permeabilize the cells with 0.1% Triton in 1x PBS for 10 min.
5. Wash the cells with 1x PBS for 3 x 5 min.
6. Block unspecific sites with 5% normal donkey serum (NDS) in 1x PBS for 1 h at room temperature (RT).
   NOTE: The choice of species for the blocking serum depends on the species in which the secondary antibodies are produced.
7. Incubate primary antibodies diluted in 5% NDS in 1x PBS overnight at 4 °C.
   NOTE: Do not add primary antibodies to one of the wells—this will serve as a negative control.
8. Wash the cells with 1x PBS for 2 x 5 min.
9. Wash the cells with 5% NDS in 1x PBS for 5 min.
10. Incubate with the secondary antibodies, DAPI, and Bodipy for 1 h at RT, covered to protect from light.
11. Wash the wells with 1x PBS for 3 x 5 min.
12. Keep the cells at 4 °C in the dark until acquisition of the images.

6. Cryoconservation of preadipocytes

NOTE: It is possible to stop the culture and freeze the cells before or after the differentiation process to store them and reuse them for subsequent cell cultures.

1. Prepare freezing medium: 50% FBS, 40% DMEM/F-12 glutamine with 100 U/mL P/S, 10% Dimethyl Sulfoxide (DMSO).
   CAUTION: DMSO is a hazardous chemical. It is a skin and eye irritant, can facilitate the absorption of other toxic substances through the skin, and may cause harm if inhaled or ingested. Always handle DMSO inside the BSC to minimize exposure and contamination. Wear appropriate personal protective equipment (PPE) (sterile gloves, a lab coat, and safety goggles to prevent contact). Avoid skin contact. In case of contact, wash immediately with plenty of water. Store DMSO in a tightly sealed container within the BSC when not in use, and ensure proper disposal according to lab safety protocols.
2. Wash the cells with prewarmed (37 °C) 1x PBS.
3. Add prewarmed (37 °C) 0.05% trypsin to detach the cells chemically and incubate 5 min at 37 °C, 5% CO₂. Visually check for cell detachment under a microscope.
4. Stop the reaction by adding 3x the volume of prewarmed (37 °C) DMEM/F-12 glutamine with 100 U/mL P/S, 10 % FBS. Resuspend evenly.
5. Centrifuge 7 min at RT, 300 × g and discard the supernatant.
6. Resuspend the cells in 10 mL of freezing medium. Centrifuge 7 min at RT, 300 × g and discard the supernatant, leaving approximatively 3 mL.
   NOTE: Measure the exact volume to calculate the total number of cells after counting.
7. Dilute a small volume 1:2 with Trypan Blue and count live cells using a Neubauer chamber.
8. Adjust volume with freezing medium to achieve 5 × 10⁴ – 1 × 10⁵ cells/vial (final volume: 1 mL/vial).
9. Transfer vials to a freezing container (cooling rate: -1 °C/min) and store at -80 °C overnight.
10. Transfer to liquid nitrogen for long-term storage.
    NOTE: This protocol allows for viability greater than 90% after thawing.
11. Thaw the cells when needed by thawing the vial quickly in a 37 °C water bath.
12. Transfer the vial contents to 4 mL of prewarmed (37 °C) DMEM/F-12 glutamine with 100 U/mL P/S, 10% FBS. Resuspend evenly by gently pipetting up and down 10x. Incubate overnight at 37 °C, 5% CO₂.
13. Replace the medium with fresh DMEM/F-12 glutamine with 100 U/mL P/S, 10 % FBS and renew every 48 h.
    NOTE: It is critical to do this change of medium to remove DMSO remnants, as prolonged exposure can compromise cell viability, differentiation, and function. Ensure complete medium replacement to avoid cytotoxic effects.
14. Proceed with experiments or passage cells when they reach 80% confluency.

Results

As illustrated in Figure 1, intact BAT and iWAT depots can be efficiently isolated from embryos at E15.5. While BAT is easily detectable in E15.5 embryos, detecting iWAT may require training. In both cases, it is essential to microdissect all surrounding tissue after rough dissection to avoid culturing other cell types.

The initial stages of digestion and culture of white and brown preadipocytes are identical (Figure 2A,B

Discussion

The culture of preadipocytes has been extensively described in a variety of experimental contexts. However, existing protocols predominantly rely on adult tissues or embryonic fibroblasts, and no standardized methods are currently available for isolating and culturing embryonic preadipocytes directly from developing adipose tissues. Studying these cells is particularly important, as they represent the authentic preadipocyte populations residing within their native embryonic microenvironment. Such analyses are critical fo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3,5,3'-triiodothyronine	Sigma	709719	T3
3-isobutyl-1-methylxanthine	Sigma	I7018	IBMX
Agarose	Sigma	A0169
Alcohol	VWR	1.59010.0500
Anti-UCP1 antibody	Abcam	AB10983
Binocular microscope	Leica	MZ9.5
Biological safety cabinet	Thermo Scientific	51022482	BSC, HeraSafe KS Class II Safety Cabinet
Bodipy	Invitrogen	D3922	Excitation/Emission 493/504 nm
Bovine serum albumin	Euromedex	04-100-812-C	BSA
Centrifuge	Eppendorf	5430 R
Clamps	FST (Germany Stainless)
Collagenase D	Sigma	11088882001
Cryotubes	Sarstedt	72.379
Culture plates (8-wells)	Ibidi	80826
Culture plates (12-wells)	Corning Costar	CLS3513	polystyrene plate, flat bottom, sterile
Dexamethasone	Sigma	D2915
Dispase II	Sigma	4942078001
DMEM/F12	Gibco	31331	glutamine added
DMSO	Sigma	D2438
Donkey anti-rabbit antibody	Invitrogen	A32795	Alexa Fluo Plus 647
Eppendorfs 1,5 mL	Eppendorf	15625367
FBS	Gibco	A5256701
Formaldehyde 37 %	Thermo Scientific	119690010
Freezing container	Thermo Scientific	5100-0001
Hoechst	Invitrogen	H3570
Incubator	Thermo Scientific	50116048	Heracell 150i CO2 incubator
Indomethacin	Sigma	I7378
Insulin	Sigma	I9278
inverted microscope equipped with a 10× objective and halogen illumination	Carl Zeiss Axio Vert.A1
Mice C57BL/6J	Charles River
Microscissors	FST (Germany Stainless)
NDS	Sigma	D9663
Neubauer Chamber	Thermo Scientific	10195580
PBS 10x	Gibco	14200
PBS 1x	Gibco	14190
Penicilin/Streptomicin	Gibco	15140-122
Petri dishes	Sarstedt	83.3902
qPCR plates	Thermo Scientific	AB-0600
qPCR probe for Fabp4	Thermo Scientific	4331182	TaqMan assays, Mm00445878_m1
qPCR probe for Pparg	Thermo Scientific	4331182	TaqMan assays, Mm00440940_m1
qPCR probe for Ucp-1	Thermo Scientific	4331182	TaqMan assays, Mm01244861_m1
Reverse transcription kit	Thermo Scientific	4368813	High capacity cDNA kit
RNA extraction kit	Omega Bio-Tek	R6934-02	E.Z.N.A. Total RNA Kit II
Rosiglitazone	Sigma	R2408
Triton X-100	Thermo Scientific	A16046
Trypan Blue	Sigma	T8154
Trypsin-EDTA	Gibco	25300054
Water bath	Grant	SAP12	12 L capacity