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. Whit....

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 ....

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