Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice

Summary

This report describes a protocol for the simultaneous isolation of primary brown and white preadipocytes from newborn mice. Isolated cells can be grown in culture and induced to differentiate into fully mature white and brown adipocytes. The method enables genetic, molecular, and functional characterization of primary fat cells in culture.

Abstract

The understanding of the mechanisms underlying adipocyte differentiation and function has greatly benefited from the use of immortalized white preadipocyte cell lines. These cultured cell lines, however, have limitations. They do not fully capture the diverse functional spectrum of the heterogenous adipocyte populations that are now known to exist within white adipose depots. To provide a more physiologically relevant model to study the complexity of white adipose tissue, a protocol has been developed and optimized to enable simultaneous isolation of primary white and brown adipocyte progenitors from newborn mice, their rapid expansion in culture, and their differentiation in vitro into mature, fully functional adipocytes. The primary advantage of isolating primary cells from newborn, rather than adult mice, is that the adipose depots are actively developing and are, therefore, a rich source of proliferating preadipocytes. Primary preadipocytes isolated using this protocol differentiate rapidly upon reaching confluence and become fully mature in 4-5 days, a temporal window that accurately reflects the appearance of developed fat pads in newborn mice. Primary cultures prepared using this strategy can be expanded and studied with high reproducibility, making them suitable for genetic and phenotypic screens and enabling the study of the cell-autonomous adipocyte phenotypes of genetic mouse models. This protocol offers a simple, rapid, and inexpensive approach to study the complexity of adipose tissue in vitro.

Introduction

Obesity results from a chronic imbalance between energy intake and energy expenditure. As obesity develops, white adipocytes undergo a massive expansion in cell size that results in hypoxia in the microenvironment, cell death, inflammation, and insulin resistance¹. Dysfunctional, hypertrophied adipocytes cannot properly store excess lipids, which accumulate instead in other tissues where they dampen insulin action^2,3. Agents that improve adipocyte function and restore normal lipid partitioning amongst tissues are predicted to be beneficial for the treatment of obesity-associated conditi....

Protocol

This protocol follows all IACUC guidelines of The Scripps Research Institute and the University of Wisconsin – Madison School of Medicine and Public Health.

1. Collection and digestion of adipose depots (day 1)

1. Prepare two 1.5 mL tubes for each pup: one for brown adipose tissue (BAT) and one for white adipose tissue (WAT). Add 250 µL of phosphate-buffered saline (PBS) + 200 µL of 2x isolation buffer (123 mM NaCl, 5 mM KCl, 1.3 mM CaCl₂, 5 mM glucose, 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), penicillin-streptomycin, and 4% fatty acid-free bovine serum albumin) to each tube. Kee....

Results

Section 1 of the protocol will yield a heterogeneous suspension of cells that are visible under a standard light microscope. Filtering of digested tissues with a cell strainer (section 2) will remove undigested tissue. However, some cellular debris, blood cells, and mature adipocytes will pass through (Figure 1C). Gentle washes 1 h after plating will remove non-relevant cells as preadipocytes attach rapidly to the bottom of the well (Figu.......

Discussion

Adipose tissue is critical for systemic insulin sensitivity and glucose homeostasis²⁰. Obesity-linked adipocyte dysfunction is tightly associated with the onset of type 2 diabetes. Therefore, greater understanding of the basic biology and physiology of adipose tissue may enable the design of new treatments for metabolic disorders. As a complement to direct functional and transcriptional analysis of mature adipocytes isolated from fat depots^21,22.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-Isobutyl-1-methylxanthine (IBMX)	Sigma-Aldrich	I7018
6-well plates	Corning	353046
AdipoRed (Nile Red)	Lonza	PT-7009
Antimycin A	Sigma-Aldrich	A8674
BenchMark Fetal Bovine Serum	Gemini Bioproducts LLC	100-106
CaCl2	Sigma-Aldrich	C4901
Cell strainer	Fisher Scientific	22363549
Collagenase, Type 1	Worthington Biochemical Corp	LS004196
ddH2O	Sigma-Aldrich	6442
Dexamethasone	Sigma-Aldrich	D4902
DMEM	Sigma-Aldrich	D5030	For Bioenergetics studies
DMEM, High Glucose, Glutamax	Gibco	10569010
DPBS, no calcium, no magnesium	Gibco	14190144
Fatty Acid-Free BSA	Sigma-Aldrich	A8806
FCCP	Sigma-Aldrich	C2920
Gelatin	Sigma-Aldrich	G1890
Glucose	Sigma-Aldrich	G7021
HEPES	Sigma-Aldrich	H3375
Hoechst 33342	Invitrogen	H1399
Insulin	Sigma-Aldrich	I6634
KCl	Sigma-Aldrich	P9333
NaCl	Sigma-Aldrich	S7653
Norepinephrine	Cayman Chemical	16673
Oligomycin	Sigma-Aldrich	75351
Pen/Strep	Gibco	15140122
Rosiglitazone	Sigma-Aldrich	R2408
Rotenone	Sigma-Aldrich	557368
Seahorse XFe96 FluxPak	Agilent Technologies	102416-100	For Bioenergetics studies
Surgical forceps	ROBOZ Surgical Instrument Co	RS-5158
Surgical Scissors	ROBOZ Surgical Instrument Co	RS-5880
ThermoMixer	Eppendorf	T1317
triiodothyronine (T3)	Sigma-Aldrich	642511