Name: isolation of viable adipocytes and stromal vascular fraction from human visceral adipose tissue suitable for rna analysis and macrophage phenotyping
Uploaded: 2020-10-27T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping at JoVE.c

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).

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
<ol>
	<li>Obtain VAT biopsies through partial omentectomy during cesarean section from healthy adult women with singleton pregnancies at term without labor.</li>
	<li>After the uterine closure and hemostasis, proceed to identify greater omentum and extend it o...

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...

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.

<table>
	<tbody>
		<tr>
			<td>0.2 mL PCR tubes</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td>RNase, DNase free and nonpyrogenic</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>RNase, DNase free and nonpyrogenic</td>
		</tr>
		<tr>
			<td>10 mL serological pipettes</td>
			<td>Corning</td>
			<td>CLS4101-50EA</td>
			<td>Individually plastic wrapped</td>
		</tr>
		<tr>
			<td>10 &#181;L universal pipet tip</td>
			<td>Axygen</td>
			<td>T-300-L-R</td>
			<td>RNase, DNase free and nonpyrogenic</td>
		</tr>
		<tr>
			<td>10 &#181;L universal pipet tip</td>
			<td>Axygen</td>
			<td>T-300-R-S</td>
			<td>RNase, DNase free and nonpyrogenic</td>
		</tr>
		<tr>
			<td>1000 &#181;L universal pipet tip</td>
			<td>Axygen</td>
			<td>T-1000-B-R</td>
			<td>RNase, DNase free and nonpyrogenic</td>
		</tr>
		<tr>
			<td>2.0 mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-200-C</td>
			<td>RNase, DNase free and nonpyrogenic</td>
		</tr>
		<tr>
			<td>200 &#181;L universal pipet tip</td>
			<td>Axygen</td>
			<td>T-200-Y-R</td>
			<td>RNase, DNase free and nonpyrogenic</td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer Instrument</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2101 Bioanalyzer PC</td>
			<td>Agilent</td>
			<td>G2953CA</td>
			<td>2100 Expert Software pre-installed in PC</td>
		</tr>
		<tr>
			<td>5 ml Round Bottom Polystyrene Test Tube</td>
			<td>Corning</td>
			<td>352003&#160;</td>
			<td>Snap cap, sterile</td>
		</tr>
		<tr>
			<td>50 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>CLS430828-100EA</td>
			<td>Polipropilene, conical bottom and sterile</td>
		</tr>
		<tr>
			<td>Acid-guanidinium-phenol based reagent</td>
			<td>Zymo Research</td>
			<td>R2050-1-200</td>
			<td>TRI Reagent or similar</td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Nano Kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Agilent Small RNA Kit</td>
			<td>Agilent</td>
			<td>5067-1548</td>
			<td>-</td>
		</tr>
		<tr>
			<td>APC/Cy7 anti-human CD14 Antibody</td>
			<td>BioLegend</td>
			<td>325620</td>
			<td>0.4 mg/106 cells, present on monocytes/macrophages, clone HCD14</td>
		</tr>
		<tr>
			<td>Baker</td>
			<td>-</td>
			<td>-</td>
			<td>250 ml, non sterile</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A3912-100G</td>
			<td>Heat shock fraction, pH 5.2, &#8805;96%</td>
		</tr>
		<tr>
			<td>Chip priming station</td>
			<td>Agilent</td>
			<td>5065-9951</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Gibco</td>
			<td>17101-015</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Direct-zol RNA Miniprep</td>
			<td>Zymo Research</td>
			<td>R2051</td>
			<td>Supplied with 50 mL TRI reagen</td>
		</tr>
		<tr>
			<td>Dissecting forceps</td>
			<td>-</td>
			<td>-</td>
			<td>Steel, serrated jaws and round ends</td>
		</tr>
		<tr>
			<td>Dissection tray</td>
			<td>-</td>
			<td>-</td>
			<td>Stainless steel</td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023-500ML</td>
			<td>200&#160;proof, for molecular biology</td>
		</tr>
		<tr>
			<td>FACS Flow Sheath Fluid</td>
			<td>BD Biosciences</td>
			<td>342003</td>
			<td>-</td>
		</tr>
		<tr>
			<td>FACS Lysing Solution</td>
			<td>BD Biosciences</td>
			<td>349202</td>
			<td>-</td>
		</tr>
		<tr>
			<td>FACSAria III Flow Cytometer/Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>648282</td>
			<td>-</td>
		</tr>
		<tr>
			<td>FASCDiva Software</td>
			<td>BD Biosciences</td>
			<td>642868</td>
			<td>Software v6.0 pre-installed</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma</td>
			<td>Z359629-1EA</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Manual cell counter</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Mayo dissecting scisors</td>
			<td>-</td>
			<td>-</td>
			<td>Stainless steel</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>-</td>
			<td>-</td>
			<td>Adjustable temperature</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND2000LAPTOP</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>-</td>
			<td>-</td>
			<td>Adjustable temperature and speed</td>
		</tr>
		<tr>
			<td>P10&#160;variable volume&#160;micropipette&#160;</td>
			<td>Thermo Scientific-Finnpipette</td>
			<td>4642040</td>
			<td>1 to 10 &#956;L</td>
		</tr>
		<tr>
			<td>P1000&#160;variable volume&#160;micropipette&#160;</td>
			<td>Thermo Scientific-Finnpipette</td>
			<td>4642090</td>
			<td>100 to 1000 &#956;L</td>
		</tr>
		<tr>
			<td>P2&#160;variable volume&#160;micropipette&#160;</td>
			<td>Thermo Scientific-Finnpipette</td>
			<td>4642010</td>
			<td>0.2 to 2 &#956;L</td>
		</tr>
		<tr>
			<td>P200&#160;variable volume&#160;micropipette&#160;</td>
			<td>Thermo Scientific-Finnpipette</td>
			<td>4642080</td>
			<td>20 to 200 &#956;L</td>
		</tr>
		<tr>
			<td>PCR tube storage rack</td>
			<td>Axygen</td>
			<td>R96PCRFSP</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PE/Cy5 anti-human HLA-DR Antibody</td>
			<td>BioLegend</td>
			<td>307608</td>
			<td>0.0625 mg/106 cells, present on macrophages, clone L243</td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-human CD45 Antibody</td>
			<td>BioLegend</td>
			<td>304016</td>
			<td>0.1 mg/106 cells, present on leukocytes, clone H130</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P3813-10PAK</td>
			<td>Powder, pH 7.4, for preparing 1 L solutions</td>
		</tr>
		<tr>
			<td>Pipette controller</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Red Blood Cells Lysis Buffer</td>
			<td>Roche</td>
			<td>11 814 389 001</td>
			<td>For preferential lysis of red blood cells from human whole blood</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>-</td>
			<td>-</td>
			<td>Whit adapter for 50 mL conical tubes</td>
		</tr>
		<tr>
			<td>Sterile Specimen container</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Thermo Scientific-Samco</td>
			<td>204-1S</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td>0.4% Solution</td>
		</tr>
		<tr>
			<td>Tube racks</td>
			<td>-</td>
			<td>-</td>
			<td>For different tube sizes</td>
		</tr>
		<tr>
			<td>Vortex Mini Shaker</td>
			<td>Cientifica SENNA</td>
			<td>BV101</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

isolation of viable adipocytes and stromal vascular fraction from human visceral adipose tissue suitable for rna analysis and macrophage phenotyping

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.

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 (<strong c...

Watch this Scientific Journal Video about Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping at JoVE.c

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

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.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

This protocol allows for isolation and study of viable adipocytes and stromal vascular fraction SVF cells from human visceral fat. Enzymatic digestion using collagenase ensures high yields of total RNA from mature adipocytes, including microRNAs suitable for expression analysis. Simultaneously, the method is optimized to identify macrophages by flow cytometry.This protocol provides fundamental troubleshooting guidelines for easy and efficient isolation of viable mature adipocytes and SVF cells from human VAT biopsies in a single process. After the uterine closure and hemostasis, identify greater omentum and extend it on a wet compress. Identify an avascular zone and use Kelly forceps to drill the outer side of visceral adipose tissue, or VAT.Use Ochsner forceps to clamp the proximal and distal side in the zone where VAT was drilled, and used Metzenbaum scissors to cut the tissue. Tie the greater omentum with black silk number one, then remove the Ochsner forceps and evaluate the hemostasis. To perform enzymatic digestion, cut four grams of VAT and use scissors to mince the tissue into small pieces in a dissection tray.Transfer the pieces to a new sterile 50-milliliter centrifuge tube and add 25 milliliters of digestion solution. Incubate the tube at 37 degrees Celsius for 60 minutes in an orbital shaker at 125 RPM. Then filter the digested tissue through three layers of gauze into a new sterile 50-milliliter centrifuge tube.Centrifuge the tube at 200 G for five minutes at four degrees Celsius. After centrifugation, two phases should be visible. The upper phase corresponds to mature adipocytes while SVF cells remain in the pellet.Transfer the mature adipocytes to a tube. Lyse cells by adding 1000 microliters of acid-guanidinium-phenol-based reagent and mix thoroughly with a P1000 micropipette to homogenize. Incubate the mixture for five minutes at room temperature to boost dissociation of nucleo-protein complexes.Centrifuge the cell lysate for five minutes at 12, 000 G to obtain three phases, an upper yellow phase corresponding to adipocyte lipids, a middle pink phase corresponding to nucleic acids, and a pellet with cellular debris. Carefully remove the lipid layer with a P200 micro pipette. Then transfer the middle phase into a new two-milliliter tube, taking care not to disturb the other phases.After purifying the RNA and measuring the concentration with a UV visible spectrophotometer, use an RNA integrity kit to determine the RNA integrity number. Then use the small RNA kit to determine the concentration and percent of microRNAs. Place the clip into a bioanalyzer.Dilute 10 microliters of SVF cell suspension into 90 microliters of 0.4%trypan blue solution, and apply 10 microliters to a standard hemocytometer. Count the viable cells carefully, excluding the dead cells, in four squares at the corner of the counting chamber. Transfer a total of 1 million SVF cells per milliliter to a five-milliliter round-bottom polypropylene test tube for flow cytometry and pellet the cells by centrifugation at 800 G for five minutes at four degrees Celsius.Vortex carefully to loosen the pellet and resuspend the cells in 100 microliters of PBS. Add the pre-titrated optimal concentration of each fluorochrome-conjugated monoclonal antibody specific for a cell surface antigen and mix gently, then incubate the cells for 15 minutes in the dark. Add an excess of cold PBS.And centrifuge the SVF at 400 G for five minutes at four degrees Celsius. Decant the supernatant, taking care to not disturb the pellet. Resuspend the pellet in five milliliters of sheath fluid.Then filter the cells through three layers of gauze into a new five-milliliter round-bottom polypropylene test tube. Vortex each tube briefly before analysis and count a minimum of 10, 000 events for flow analysis. This RNA extraction protocol was used to obtain adequate purity and high integrity RNA and microRNAs from mature adipocytes.The RNA integrity was assessed by an RNA quality control analyzer. Approximately 2.8 times 10 to the six nucleated cells were present per gram of visceral adipose tissue. The stromal vascular fraction cells were labeled with fluorophore-conjugated primary antibodies to identify and characterize 18 macrophages by fluorescence-activated cell-sorting analysis.Cell aggregates were eliminated from analysis by plotting the forward scatter area versus forward scatter height. Then cellular debris was excluded by gating the cells, based on the correct size and complexity, using forward scatter area and the side scatter area. To analyze macrophages, the monocyte and macrophage lineage cells were selected using CD45 and CD14 markers.Subsequently, the CD45-CD14 double positive cells were separated based on macrophage marker HLA-DR expression. Two subsets of macrophages were identified. In addition to identifying new cell phenotypes, this protocol makes it possible to use purified cell populations for future analyses, such as DNA methylation, post-translational modifications, and signaling pathways.

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