Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Summary

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Abstract

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

Introduction

White adipose tissue is composed not only of fat cells or adipocytes, but also of a non-fat cell fraction known as stromal vascular fraction (SVF), which contains a heterogeneous cell population consisting in macrophages, other immune cells as regulatory T cells (Tregs), and eosinophils, preadipocytes, and fibroblasts, surrounded by vascular and connective tissue^1,2. Adipose tissue (AT) is now considered an organ that regulates physiological processes related to metabolism and inflammation through adipokines, cytokines, and microRNAs produced and released by different cells into the tissue, with autocrine, par....

Protocol

This protocol was approved by the IRB of the Instituto Nacional de Perinatologia (212250-3210-21002-06-15). Participation was voluntary, and all the enrolled women signed the informed consent form.

1. Visceral adipose tissue collection

1. Obtain VAT biopsies through partial omentectomy during cesarean section from healthy adult women with singleton pregnancies at term without labor.
2. After the uterine closure and hemostasis, proceed to identify greater omentum and extend it on a wet compress. The AT exposed is VAT.
3. Locate the largest blood vessel and trace an imaginary line of 7 x 5 cm to the greater omentum base....

Results

This protocol describes an enzymatic method using collagenase digestion followed by differential centrifugation to isolate, in a single process, viable mature adipocytes and SVF cells from VAT biopsies obtained from healthy pregnant women after partial omentectomy. In this case, we use the adipocytes for RNA extraction and the SVF for macrophage phenotyping.

The RNA extraction protocol enabled to obtain RNA with an adequate purity high integrity, and microRNAs from mature adipocytes (

Discussion

VAT plays a crucial role in metabolic regulation and inflammation. Increasing interest in the role of adipocytes and immune cells in the chronic inflammation associated with obesity has led to the development of different techniques to separate the SVF and fat cells present in AT. However, most techniques do not allow to obtain these two different sets of cells viable for downstream applications from the same VAT biopsy in a single procedure, which could be crucial for studies regarding interactions between adipocytes an.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 mL PCR tubes	Axygen	PCR-02-C	RNase, DNase free and nonpyrogenic
1.5 mL microcentrifuge tubes	Axygen	MCT-150-C	RNase, DNase free and nonpyrogenic
10 mL serological pipettes	Corning	CLS4101-50EA	Individually plastic wrapped
10 µL universal pipet tip	Axygen	T-300-L-R	RNase, DNase free and nonpyrogenic
10 µL universal pipet tip	Axygen	T-300-R-S	RNase, DNase free and nonpyrogenic
1000 µL universal pipet tip	Axygen	T-1000-B-R	RNase, DNase free and nonpyrogenic
2.0 mL microcentrifuge tube	Axygen	MCT-200-C	RNase, DNase free and nonpyrogenic
200 µL universal pipet tip	Axygen	T-200-Y-R	RNase, DNase free and nonpyrogenic
2100 Bioanalyzer Instrument	Agilent	G2939BA	-
2101 Bioanalyzer PC	Agilent	G2953CA	2100 Expert Software pre-installed in PC
5 ml Round Bottom Polystyrene Test Tube	Corning	352003	Snap cap, sterile
50 mL centrifuge tubes	Corning	CLS430828-100EA	Polipropilene, conical bottom and sterile
Acid-guanidinium-phenol based reagent	Zymo Research	R2050-1-200	TRI Reagent or similar
Agilent RNA 6000 Nano Kit	Agilent	5067-1511	-
Agilent Small RNA Kit	Agilent	5067-1548	-
APC/Cy7 anti-human CD14 Antibody	BioLegend	325620	0.4 mg/106 cells, present on monocytes/macrophages, clone HCD14
Baker	-	-	250 ml, non sterile
Bovine serum albumin	Sigma-Aldrich	A3912-100G	Heat shock fraction, pH 5.2, ≥96%
Chip priming station	Agilent	5065-9951	-
Collagenase type II	Gibco	17101-015	Powder
D-(+)-Glucose	Sigma-Aldrich	G8270-100G	Powder
Direct-zol RNA Miniprep	Zymo Research	R2051	Supplied with 50 mL TRI reagen
Dissecting forceps	-	-	Steel, serrated jaws and round ends
Dissection tray	-	-	Stainless steel
Ethyl alcohol	Sigma-Aldrich	E7023-500ML	200 proof, for molecular biology
FACS Flow Sheath Fluid	BD Biosciences	342003	-
FACS Lysing Solution	BD Biosciences	349202	-
FACSAria III Flow Cytometer/Cell Sorter	BD Biosciences	648282	-
FASCDiva Software	BD Biosciences	642868	Software v6.0 pre-installed
Hemacytometer	Sigma	Z359629-1EA	-
Manual cell counter	-	-	-
Mayo dissecting scisors	-	-	Stainless steel
Microcentrifuge	-	-	Adjustable temperature
Nanodrop spectrophotometer	Thermo Scientific	ND2000LAPTOP	-
Orbital shaker	-	-	Adjustable temperature and speed
P10 variable volume micropipette	Thermo Scientific-Finnpipette	4642040	1 to 10 μL
P1000 variable volume micropipette	Thermo Scientific-Finnpipette	4642090	100 to 1000 μL
P2 variable volume micropipette	Thermo Scientific-Finnpipette	4642010	0.2 to 2 μL
P200 variable volume micropipette	Thermo Scientific-Finnpipette	4642080	20 to 200 μL
PCR tube storage rack	Axygen	R96PCRFSP	-
PE/Cy5 anti-human HLA-DR Antibody	BioLegend	307608	0.0625 mg/106 cells, present on macrophages, clone L243
PE/Cy7 anti-human CD45 Antibody	BioLegend	304016	0.1 mg/106 cells, present on leukocytes, clone H130
Phosphate buffered saline	Sigma-Aldrich	P3813-10PAK	Powder, pH 7.4, for preparing 1 L solutions
Pipette controller	-	-	-
Red Blood Cells Lysis Buffer	Roche	11 814 389 001	For preferential lysis of red blood cells from human whole blood
Refrigerated centrifuge	-	-	Whit adapter for 50 mL conical tubes
Sterile Specimen container	-	-	-
Transfer pipette	Thermo Scientific-Samco	204-1S	Sterile
Trypan Blue	Gibco	15250-061	0.4% Solution
Tube racks	-	-	For different tube sizes
Vortex Mini Shaker	Cientifica SENNA	BV101	-