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Method Article

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

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DOI:

10.3791/61884

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October 27th, 2020

 ,  ,  ,  ,  ,  ,  ,  , 
1Research Division, Instituto Nacional de Perinatologia, 2Department of Immunobiochemistry, Instituto Nacional de Perinatologia, 3Department of Obstetrics, Instituto Nacional de Perinatologia, 4Department of Academic Programs and Continuing Education, Instituto Nacional de Perinatologia, 5Department of Infectology and Immunology, Instituto Nacional de Perinatologia, 6Clinical Research Branch, Instituto Nacional de Perinatologia, 7Department of Nutrition and Bioprogramming, Instituto Nacional de Perinatologia, 8Department of Human Genetics and Genomics, Instituto Nacional de Perinatologia, Mexico City, Mexico

In This Article

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 tissue1,2. 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, paracrine, and endocrine effects3,4. In humans, white adipose tissue comprises the subcutaneous adipose tissue (SAT) and the visceral adipose tissue (VAT), with important anatomic, molecular, cellular, and physiological differences between them2,5. SAT represents up to 80% of human AT, while VAT is located within the abdominal cavity, mainly in the mesentery and omentum, being metabolically more active6. Moreover, VAT is an endocrine organ that secretes mediators with a substantial impact on body weight, insulin sensitivity, lipid metabolism, and inflammation. Consequently, VAT accumulation leads to abdominal obesity and obesity-related diseases such as type-2 diabetes, metabolic syndrome, hypertension, and cardiovascular disease risk, representing a better predictor of obesity-associated mortality6,7,8,9.

In homeostatic conditions, adipocytes, macrophages, and the other immune cells cooperate to maintain the VAT metabolism through the secretion of anti-inflammatory mediators10. However, excessive VAT expansion promotes the recruitment of activated T cells, NK cells, and macrophages. In fact, in lean VAT, the ratio of the macrophages is 5%, while this ratio rises up to 50% in obesity, with macrophage polarization from anti-inflammatory to pro-inflammatory phenotype, generating a chronic inflammatory environment10,11.

As a consequence of the obesity pandemic, an astonishing number of reports have arisen addressing different VAT research topics, including adipocyte biology, epigenetics, inflammation, endocrine properties, and emerging areas as extracellular vesicles, among others8,10,12,13. However, although the VAT environment is defined by crosstalk between adipocytes and the resident or arriving macrophages, most studies have focused on only one cell population, and there is scarce information about the interaction of these cells in VAT and their pathophysiological consequences11,14. Moreover, valuable studies addressing the adipocyte-macrophage interplay in AT were performed using cell lines, lacking the in vivo priming conditions11,14,15. A suitable strategy to dissect the interaction or the particular contribution of these cells in VAT requires the isolation of both cell types from the same fat biopsy to perform in vitro assays that mirror as similar as possible the in vivo properties that regulate VAT metabolism.

Although the non-enzymatic dissociation methods based on mechanical forces to break the AT ensure minimal manipulation, these methods cannot be used if the aim is to study SVF cells, as they have lower efficiency in cell recovery and low cell viability compared to enzymatic methods, and a larger volume of tissue is needed16,17. Enzymatic digestion using collagenase is a gentle method that allows adequate digestion of collagen and extracellular matrix proteins of fibrous tissues such as WAT18 and is frequently used when trypsin is ineffective or damaging19. The protocol provides fundamental troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF cells from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique, giving information to ensure high yields (quantity, purity, and integrity) of total RNA from mature adipocytes, including microRNAs, for downstream expression applications. Simultaneously, the protocol is optimized to identify macrophage subsets from SVF cells through the staining of multiple membrane-bound markers for further analysis by flow cytometry20.

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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.
  4. Identify an avascular zone and use Kelly forceps to drill the outer side of VAT.
  5. Use Ochsner forceps to clamp the proximal and distal side in the zone where VAT was drilled and use Metzenbaum scissors to cut the tissue.
  6. Tie greater omentum with black silk number 1.
  7. Remove Ochsner forceps and evaluate the hemostasis.
  8. Place the VAT biopsy into a sterile container and transport it to the lab immediately.

2. Enzymatic digestion of visceral adipose tissue and isolation of mature adipocytes and stromal vascular fraction cells

  1. Weigh the VAT biopsy and rinse with 1x PBS pH 7.4.
  2. Cut 4 g of VAT and use scissors to mince the tissue into small pieces in a dissection tray.
  3. Transfer minced VAT to a sterile 50 mL centrifuge tube, add 20 mL of PBS, and shake gently to remove the excess of red blood cells.
  4. Discard PBS and repeat the procedure twice.
  5. Transfer VAT to a new sterile 50 mL centrifuge tube and add 25 mL of digestion solution (0.25% collagenase type II, 5 mM glucose, 1.5% albumin in PBS).
  6. Incubate at 37 °C for 60 min in an orbital shaker at 125 rpm.
  7. Filter the digested tissue through three layers of gauze into a new sterile 50 mL centrifuge tube.
  8. Centrifuge at 200 x g for 5 min at 4 °C.
  9. Two phases are obtained, an upper phase that corresponds to mature adipocytes, while SFV cells remain in the pellet. Gently transfer the mature adipocytes into a new sterile 50 mL centrifuge tube using a transfer pipette.
  10. Add 20 mL of cold PBS, shake gently, and centrifuge at 200 x g for 5 min at 4 °C.
  11. Discard PBS and repeat the procedure twice.
  12. Use a transfer pipette to gently transfer the mature adipocytes into a 1.5 mL DNase-RNase free microcentrifuge tube.
  13. Centrifuge at 200 x g for 1 min at 4 °C and remove the excess PBS using a P200 micropipette. Repeat this step if it is necessary.
  14. Gently transfer 300 μL of mature adipocyte suspension into a 1.5 mL DNase-RNase free microcentrifuge tubes. Store mature adipocytes at -80 °C until RNA extraction.
    NOTE: Adipocytes do not form a pellet.
  15. Once adipocytes are separated (step 2.9), aspirate most of the digestion solution with a transfer pipette, keeping the SVF pellet at the bottom of the tube.
  16. Transfer the pellet with SVF cells into a new 50 mL centrifuge tube using a transfer pipette.
  17. Wash cell pellet gently, resuspending in 20 mL of cold 1x PBS by pipetting up and down.
  18. Centrifuge at 800 x g for 5 min at 4 °C and rapidly discard the supernatant by decantation.
  19. Perform a second wash by repeating steps 2.17 and 2.18.
  20. Add 5 mL of red blood cell lysis buffer equilibrated to room temperature (RT) to the SVF pellet and suspend by repeated pipetting. Do not vortex.
  21. Incubate for 5 min at RT and centrifuge for 5 min at 800 x g. Carefully remove the supernatant using a transfer pipette and dispose of properly.
  22. To neutralize the lysis buffer, add 10 mL of cold 1x PBS and gently mix until the cell pellet disaggregates.
  23. Centrifuge at 800 x g for 5 min to obtain SVF pellet and discard PBS. A white pellet should be visible at the bottom of the tube.
  24. Resuspend cells in 5 mL of 1x PBS at RT by repeated pipetting and filtered through three layers of gauze into a new 50 mL conical tube. Subsequently, these cells will be used for macrophage subpopulation characterization by flow cytometry.

3. RNA extraction from mature adipocytes

  1. Prepare a clean area, using RNase decontamination spray to avoid RNA degradation. Use a set of pipettes reserved for RNA procedures. All tubes and tips employed should be RNase-free.
  2. Thaw the mature adipocyte suspension (section 2.14) at 4 °C if it was stored to -80 °C.
    NOTE: Avoid multiple freeze-thaw cycles.
  3. Lyse cells by adding 1,000 μL of acid-guanidinium-phenol based reagent, and mix thoroughly with a P1000 micropipette to homogenize.
  4. Incubate for 5 min at RT to boost dissociation of nucleoprotein complexes.
  5. Centrifuge cell lysate for 5 min at 12,000 x g at RT. Three phases will be obtained: an upper phase (yellow) corresponding to adipocyte lipids, a middle phase (pink) corresponding to nucleic acids, and a pellet (cellular debris).
  6. Carefully remove the lipid layer, using a P200 micropipette.
  7. Transfer the middle phase into a new 2 mL tube, avoiding lipid remnant and pellet disturbing (approximately 700 μL).

4. Purification of total RNA, including microRNAs

NOTE: A column-based total RNA purification method is used to obtain high-quality total RNA.

  1. Add an equal volume of ethanol (95%–100%) to the sample from section 3.7, approximately 700 μL, and mix by hand inverting the tube for 10 s.
  2. Transfer 700 μL of the mixture into a column inserted in a collection tube, centrifuge and discard the flow-through.
  3. Reload the column and repeat step 4.2.
  4. Transfer the column into a new collection tube.
  5. Add 400 µL of pre-wash buffer to the column and centrifuge. Discard the flow-through and repeat this step.
  6. Add 700 µL of wash buffer to the column and centrifuge for 2 min.
  7. Transfer the column carefully into a nuclease-free tube.
  8. Add 50 µL of nuclease-free water directly to the column matrix and elute the RNA by centrifugation. Prepare aliquots of 10 µL using 200 µL microfuge tubes.
  9. Immediately put aliquots on ice and use one of them to determine RNA concentration and purity.
  10. Prepare an aliquot of 5 µL of total RNA adjusted to 100 ng/µL to measure RNA integrity and microRNA concentration.
  11. Store at -80 °C until use.

5. Determination of mature adipocyte RNA concentration, purity, and integrity; microRNA quantification assay

NOTE: RNA concentration and purity determinations are performed using a UV-Vis spectrophotometer; RNA integrity and miRNA quantification are performed using an RNA quality control analyzer.

  1. Wash the sample reader with molecular grade water and wipe.
  2. Load 2 µL of elution water (blank), change the setting to RNA, and click on the Blank button.
  3. Load 2 µL of sample and click on the Measure button.
  4. After the reading is complete, record the A260/A280 and A260/A230 ratios as well as the amount of RNA (ng/µL) (Figure 1A).
    NOTE: RNA with OD260/OD280 and OD260/OD230 ratio around 2.0 is considered pure.
  5. Measure the RNA integrity number (RIN) using an RNA integrity kit following the manufacturer´s instructions (Figure 1B).
    NOTE: A RIN =10 corresponds to intact RNA, whereas a RIN ≤ 3.0 indicates a strongly degraded RNA. For expression microarrays or RT-qPCR, use RNA with RIN ≥ 6.0.
  6. Use a small RNA kit to determine the concentration and percent of microRNAs, following the manufacturer´s instructions (Figure 1C).
    NOTE: To avoid the overestimation of small and micro RNAs, use RNA samples with RIN ≥ 6.0.

6. Count and viability of stromal vascular fraction cells

  1. Dilute 10 μL of SVF cell suspension (section 2.24) into 90 μL of 0.4% Trypan Blue Solution (final dilution 1:10) and apply 10 μL to a standard hemocytometer.
  2. Count the viable cells carefully, excluding the dead cells, in four squares at the corner of the counting chamber.
  3. Determine the cell concentration present in the original suspension: Cell concentration = Total cell count/4 x dilution factor (10) x 10,000 = Cells/mL
  4. To calculate the cellular yield (cells per gram of tissue) obtained, multiply the cellular concentration by the original total sample volume (in mL) and divide by the weight of tissue digested (in grams).
  5. Evaluate cell viability as the percentage of the living cells as follows: % Viability = (Number of viable cells / Total number of cells) x 100.

7. Characterization of macrophage subsets from stromal vascular fraction

  1. Transfer a total of 1 x 106 SVF cells/mL to a 5 mL round-bottom polypropylene test tube for flow cytometry and pellet cells by centrifugation at 800 x g for 5 min at 4 °C.
  2. Vortex carefully to loosen the pellet and resuspend the cells in 100 μL of 1x PBS at RT.
  3. Add pre-titrated optimal concentration of each fluorochrome-conjugated monoclonal antibody specific for a cell surface antigen; mix gently and incubate for 15 min in the dark at RT.
  4. Add an excess of cold 1x PBS (≈ 1 mL) and centrifuge the SVF at 400 x g for 5 min at 4 °C.
  5. Discard supernatants rapidly by decantation. Be careful not to disturb the pellet.
  6. Add 500 μL of 1x lysing solution and incubate 15 min at RT, protecting the tubes from direct light.
  7. Remove the solution after centrifuge at 400 x g for 5 min at 4 °C, and store at 2–8 °C in the dark until data acquisition.
  8. Vortex the cells thoroughly at low speed to reduce aggregation before acquiring.
  9. Resuspend the SVF pellet in 5 mL of sheath fluid at RT, and subsequently filter through three layers of gauze into a new 5 mL round-bottom polypropylene test tube before analysis by flow cytometry, to reduce clogging the cell sorter lines.
  10. Vortex each tube briefly before analysis.
  11. Acquire data from the samples of interest. For flow analysis, count a minimum of 10,000 events.
    NOTE: If using a cell sorting flow cytometer, separate the macrophages for application in subsequent studies.

8. Gating strategy

  1. Plot the height or width against the area for Forward Scatter (FSC) to determine the singlet population (Figure 2A).
  2. Select cells plotting the area for Side Scatter (SSC) against FSC, discarding the cellular debris (Figure 2B).
  3. From selected cells, identify the populations expressing CD45 as hematopoietic cells (Figure 2C), and CD45/CD14 double positive cells as macrophages (Figure 2D).
  4. From CD45+/CD14+ macrophages, detect negative and positive HLA-DR cells (Figure 2E).
    NOTE: Include the compensation controls for the antibody panel and adjust spectral overlap on a multicolor flow cytometer adequate for the panel. We perform flow cytometry analysis using a cytometer equipped with three lasers (405 nm violet laser, 488 nm blue laser, and 640 nm red laser), and detectors for the indicated fluorochromes.

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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 (

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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...

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

This study was supported from the Instituto Nacional de Perinatologia (grant numbers: 3300-11402-01-575-17 and 212250-3210-21002-06-15) and CONACyT, Fondo Sectorial de Investigacion en Salud y Seguridad Social (FOSISS) (grant number 2015-3-2-61661).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
0.2 mL PCR tubesAxygenPCR-02-CRNase, DNase free and nonpyrogenic
1.5 mL microcentrifuge tubesAxygenMCT-150-CRNase, DNase free and nonpyrogenic
10 mL serological pipettesCorningCLS4101-50EAIndividually plastic wrapped
10 µL universal pipet tipAxygenT-300-L-RRNase, DNase free and nonpyrogenic
10 µL universal pipet tipAxygenT-300-R-SRNase, DNase free and nonpyrogenic
1000 µL universal pipet tipAxygenT-1000-B-RRNase, DNase free and nonpyrogenic
2.0 mL microcentrifuge tubeAxygenMCT-200-CRNase, DNase free and nonpyrogenic
200 µL universal pipet tipAxygenT-200-Y-RRNase, DNase free and nonpyrogenic
2100 Bioanalyzer InstrumentAgilentG2939BA-
2101 Bioanalyzer PCAgilentG2953CA2100 Expert Software pre-installed in PC
5 ml Round Bottom Polystyrene Test TubeCorning352003 Snap cap, sterile
50 mL centrifuge tubesCorningCLS430828-100EAPolipropilene, conical bottom and sterile
Acid-guanidinium-phenol based reagentZymo ResearchR2050-1-200TRI Reagent or similar
Agilent RNA 6000 Nano KitAgilent5067-1511-
Agilent Small RNA KitAgilent5067-1548-
APC/Cy7 anti-human CD14 AntibodyBioLegend3256200.4 mg/106 cells, present on monocytes/macrophages, clone HCD14
Baker--250 ml, non sterile
Bovine serum albuminSigma-AldrichA3912-100GHeat shock fraction, pH 5.2, ≥96%
Chip priming stationAgilent5065-9951-
Collagenase type IIGibco17101-015Powder
D-(+)-GlucoseSigma-AldrichG8270-100GPowder
Direct-zol RNA MiniprepZymo ResearchR2051Supplied with 50 mL TRI reagen
Dissecting forceps--Steel, serrated jaws and round ends
Dissection tray--Stainless steel
Ethyl alcoholSigma-AldrichE7023-500ML200 proof, for molecular biology
FACS Flow Sheath FluidBD Biosciences342003-
FACS Lysing SolutionBD Biosciences349202-
FACSAria III Flow Cytometer/Cell SorterBD Biosciences648282-
FASCDiva SoftwareBD Biosciences642868Software v6.0 pre-installed
HemacytometerSigmaZ359629-1EA-
Manual cell counter---
Mayo dissecting scisors--Stainless steel
Microcentrifuge--Adjustable temperature
Nanodrop spectrophotometerThermo ScientificND2000LAPTOP-
Orbital shaker--Adjustable temperature and speed
P10 variable volume micropipette Thermo Scientific-Finnpipette46420401 to 10 μL
P1000 variable volume micropipette Thermo Scientific-Finnpipette4642090100 to 1000 μL
P2 variable volume micropipette Thermo Scientific-Finnpipette46420100.2 to 2 μL
P200 variable volume micropipette Thermo Scientific-Finnpipette464208020 to 200 μL
PCR tube storage rackAxygenR96PCRFSP-
PE/Cy5 anti-human HLA-DR AntibodyBioLegend3076080.0625 mg/106 cells, present on macrophages, clone L243
PE/Cy7 anti-human CD45 AntibodyBioLegend3040160.1 mg/106 cells, present on leukocytes, clone H130
Phosphate buffered salineSigma-AldrichP3813-10PAKPowder, pH 7.4, for preparing 1 L solutions
Pipette controller---
Red Blood Cells Lysis BufferRoche11 814 389 001For preferential lysis of red blood cells from human whole blood
Refrigerated centrifuge--Whit adapter for 50 mL conical tubes
Sterile Specimen container---
Transfer pipetteThermo Scientific-Samco204-1SSterile
Trypan BlueGibco15250-0610.4% Solution
Tube racks--For different tube sizes
Vortex Mini ShakerCientifica SENNABV101-

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Adipose Tissue IsolationMature AdipocytesCollagenase DigestionRNA ExtractionFlow CytometryCell Viability AssayCentrifugation ProtocolHuman Visceral Fat
Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping
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