A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

Summary

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Abstract

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

Introduction

The extracellular matrix (ECM) not only provides a mechanical scaffold for tissues, but also engages in complex crosstalk with cells that reside within it, regulating diverse processes necessary for tissue homeostasis, including cell proliferation, differentiation, signaling, and metabolism¹. While healthy ECM plays an essential role the maintenance of normal tissue function, dysfunctional ECM has been implicated in multiple diseases².

Adipose tissue plays an important role in the pathogenesis of metabolic disease. Obesity is associated with excessive adipocyte hypertrophy and cellular hypoxia....

Protocol

Adipose tissues are procured from human subjects undergoing elective bariatric surgery under institutional review board approval.

1. Preadipocyte isolation and culture reagent preparation

1. Prepare 2% bovine serum albumin (BSA) in 1x phosphate buffered saline solution (PBS). Filter sterilize, and store at 4 °C.
2. Prepare Type II collagenase: 2 mg/mL in 2% BSA in 1x PBS. Prepare immediately before use.
3. Prepare Red Blood Cell (RBC) Lysing Solution: 1.5 M NH_4.......

Discussion

The ECM-adipocyte culture model provides a valuable tool for dissecting the individual roles of ECM and cells in dictating ultimate tissue phenotype. The ECM isolation protocol is quite reproducible, but variability in the decellularization process may be observed. The Day 3 delipidation step is a critical point in the protocol. At the completion of the overnight extraction, delipidation of the matrix should be evidenced by the Polar Solvent Solution turning yellow, while the matrix should transition from a yellow-orange.......

Materials

Name	Company	Catalog Number	Comments
0.25% trypsin-EDTA	Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA	Cat#25200056
1.5 mL cryovial tube	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#02-682-557
10% Neutral Buffered Formalin	VWR International LLC., Radnor, PA, USA	Cat#89370-094
100 µm nylon mesh filter	Corning Inc., Corning, NY, USA	Cat#352360
2-Deoxy-D-glucose	Sigma-Aldrich, Inc., St Louis, MO, USA	Cat#D8375
2 nM 3,3’-5,Triiodo,L-thyronine sodium salt (T3)	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#T6397
24-well tissue culture plates	VWR International LLC., Radnor, PA, USA	Cat#10861-700
3-Isobutyl-1-methylxanthine (IBMX)	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#I5879
96-well tissue culture plates	VWR International LLC., Radnor, PA, USA	Cat#10861-666
Antibiotic-Antimycotic Solution (ABAM)	Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA	Cat#15240062
Biotin	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#B4639
Bovine Serum Albumin (BSA)	Sigma-Aldrich, Inc., St Louis, MO, USA	Cat#A8806
Buffer RLT	Qiagen, Hilden, Germany	Cat#79216
Ciglitizone	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#C3974
Deoxy-D-glucose, 2-[1,2-3H (N)]-	PerkinElmer Inc., Waltham, MA, USA	Cat#NET328A250UC
Deoxyribonuclease I from bovine pancreas, type II-S	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#D4513
Dexamethasone	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#D4902
Dimethyl Sulfoxide	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#BP231	Flammable, caustic
Disodium EDTA	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#BP118
D-pantothenic acid hemicalcium salt	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#21210
Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12	Gibco, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#11320033
Ethanol	Decon Labs, Inc., King of Prussia, PA, USA	Cat#DSP-MD.43	Flammable
EVE Cell Counting Slides, NanoEnTek	VWR International LLC., Radnor, PA, USA	Cat#10027-446
Fetal bovine serum (FBS)	Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA	Cat#10437028
Glutaraldehyde	Sigma-Aldrich, Inc., St Louis, MO, USA	Cat#G5882	Caustic
Hexamethyldisalizane	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#440191	Flammable, caustic
Human insulin solution	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#I9278
Isopropanol	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#A415	Flammable
Isoproterenol	Sigma-Aldrich, Inc., St Louis, MO, USA	Cat#I5627	Flammable
KCl	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#S25484
KH2PO4	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#P5655
Lipase from porcine pancreas, type VI-S	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#L0382
MgSO4*7H2O	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#230391
Na2HPO4	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#S5136
NaCl	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#S3014
NaHCO3	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#S233
NH4Cl	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#A661
Optimal cutting temperature (OCT) compound	Agar Scientific, Ltd., Stansted, Essex, UK	Cat# AGR1180
Oil Red-O Solution (ORO)	Sigma-Aldrich, Inc., St Louis, MO, USA	Cat#O1391
Oil Red-O Stain Kit	American Master Tech Scientific Inc., Lodi, CA, USA	Cat#KTORO-G
Osmium tetroxide	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#201030	Caustic
Phenylmethylsulfonyl fluoride (PMSF)	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#93482	Caustic
Phosphate Buffered Saline Solution (PBS)	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#SH3025601
Ribonuclease A from bovine pancreas, type III-A	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#R5125
RNAEasy Fibrous Tissue MiniKit	Qiagen, Hilden, Germany	Cat#74704
Scintillation Fluid	Fisher Scientific, ThermoFisher Scientific Inc., Waltham, MA USA	Cat#SX18
Scintillation Counter
Scissors, forceps, sterile
Sorensen's phosphate buffer	Thomas Scientific, Inc., Swedesboro, NJ	CAS #: 10049-21-5
T-150 culture flask	VWR International LLC., Radnor, PA, USA	Cat#10062-864
TaqMan Gene Expression Master Mix	ThermoFisher Scientific Inc., Waltham, MA USA	Cat#4369016
Temperature-controlled orbital shaker
Tissue Homogenizer, BeadBug Microtube Homogenizer	Benchmark Scientific	Cat#D1030
Transferrin	Sigma-Aldrich, Inc. St Louis, MO, USA	Cat#T3309
Triglyceride Determination Kit	Sigma-Aldrich, Inc., St Louis, MO, USA	Cat#TR0100
Trypan blue stain, 0.4%	VWR International LLC., Radnor, PA, USA	Cat#10027-446
Type II collagenase	Gibco, ThermoFisher Scientific Inc., Waltham, MA, USA	Cat#17101015
Whatman Reeve Angel filter paper, Grade 201, 150mm	Sigma-Aldrich, Inc., St Louis, MO, USA	Cat#WHA5201150