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 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.
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.
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
2. Enzymatic digestion of visceral adipose tissue and isolation of mature adipocytes and stromal vascular fraction cells
3. RNA extraction from mature adipocytes
4. Purification of total RNA, including microRNAs
NOTE: A column-based total RNA purification method is used to obtain high-quality total RNA.
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.
6. Count and viability of stromal vascular fraction cells
7. Characterization of macrophage subsets from stromal vascular fraction
8. Gating strategy
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 (Figure 1). The RNA integrity assessed by an RNA quality control analyzer resulted in excellent values for the samples of adipocytes isolated with the protocol (RIN = 9.7).
The total of nucleated cells present in VAT-SVF was approximately 2.8 x 106 cells/gram of VAT (2.8 x 106 ± 1.7 x 106), with 64% viability (63.9 ± 2.0) using the trypan blue exclusion test. SVF cells were labeled with fluorophore-conjugated primary antibodies to identify and characterize AT macrophages by fluorescence-activated cell sorting analysis. Flow cytometry analysis generate plots showing different cell populations based on cellular markers (Figure 2). Initially, by plotting the Forward Scatter-area (FSC-A) vs Forward Scatter-height (FSC-H), cell aggregates were easily eliminated from analysis (Figure 2A). Then, cellular debris was excluded by gating the cells based on correct size and complexity using Forward Scatter-area (FSC-A) and the Side Scatter-area (SSC-A) (Figure 2B). Next, to analyze macrophages, it was not necessary to exclude other immune cells. First, monocyte/macrophage lineage cells were selected by the use of CD45 and CD14 markers (Figure 2C,D). Subsequently, the CD45/CD14 double positive cells were separated based on macrophage marker HLA-DR expression, where two subsets macrophages were identified: HLA-DR– and HLA-DR+ (Figure 2E).
Figure 1: RNA quality control from mature adipocytes. (A) RNA concentration, 260/280, and 260/230 ratio; (B) Integrity analysis. Electropherogram, gel image, and RNA integrity number were obtained using an RNA quality control analyzer and an RNA analyzer kit; (C) microRNA quantification. Electropherogram, gel image, and microRNA percent were obtained using an RNA quality control analyzer and a small RNA kit. Please click here to view a larger version of this figure.
Figure 2: Identification of macrophages from visceral adipose tissue. Representative flow cytometry plots of stromal vascular fraction macrophages isolated from visceral adipose tissue collected during a cesarean section using collagenase digestion. (A) Identification of singlets using a first Forward Scatter-area (FSC-A) vs FSC-height (FSC-H) gate to remove doublets. (B) A second Forward Scatter-area (FSC-A) vs Side Scatter-area (SSC-A) gate was used to eliminate debris based on size and density. (C,D) SSC-A vs CD45 gate identified cells from hematopoietic origin, and SSC-A vs CD14 gate identified macrophages. (E) Total CD45+/CD14+ macrophages were gated based on HLA-DR marker and two subsets of macrophages were identified: HLA-DR– and HLA-DR+. Plots illustrate representative data from individual subjects. Please click here to view a larger version of this figure.
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 and SVF cells. Therefore, we implemented this protocol that provides a detailed description to isolate viable mature adipocytes and SVF cells present in a VAT biopsy. It differs from previous reports in the time of tissue digestion and collagenase concentration, as well as time and speed of centrifugation for cell separation, enabling adequate adipocyte RNA yields and detailed macrophage subset characterization.
A large amount of enzymatic and non-enzymatic isolation techniques for AT-derived cells have been proposed, with different effects on the biological characteristics and functional properties of isolated cells17,21,22,23,24, so it is necessary to choose the most appropriate strategy according to the goal pursued. Frequently, mature adipocytes and SVF isolation from adipose tissue is achieved using tissue dissociation enzymes25,26,27. Although different enzymes can be used to dissociate AT, the enzymatic digestion with collagenase remains as the gold standard to digest this tissue27,28. Since VAT is composed of a soft matrix, it can be easily digested with Collagenase Type II. This procedure avoids improper tissue dissociation because this enzyme disrupts the extracellular matrix native collagen, releasing many more cells from the fibrous stroma, with a negligible impact on cell viability, cell yield, and cluster differentiation expression19,27,28,29.
In terms of enzymatic digestion, it is also essential to consider the enzyme concentration, the digestion period as well as the centrifugation parameters to design a proper protocol for mature adipocyte and SVF cell isolation from VAT because these factors may impact cell phenotype, recovery, and viability in the final cell suspension27,29. The use of collagenase as a proteolytic enzyme is ideal at low concentration [range 0.075% a 0.3% (w/v)], with an incubation period of less than 2 h, so the phenotypic and functional SVF characteristics such as proliferation rate, differentiation capacity, and frequency of specific cellular lineages, remain unaltered30,31. We use a maximum digestion period of 60 min and 0.25% collagenase, reducing the concentration and length of collagenase exposure to obtain a negligible impact on cellular viability and SVF cells surface markers expression, avoiding skewed results in the flow cytometry analysis. We also include gentle centrifugation steps and shorter centrifugation times of 200 g/5 min to improve cell recovery during fat cell separation, representing an advantage for the protocol, since long and intense centrifugation periods [above 400 g/1 min] increase the death of adipocytes, limiting the cellular yield32,33,34. The SVF cellular recovery achieved through the collagenase-based digestion protocol is similar to the typical yields of 2–6 x 106 cells/g AT reported in the literature, whereas the SVF viability of 63.9 ± 2.0 is moderately lower than the minimum proposed threshold of 70%–80% for these type of cells35,36,37,38. This is likely because we do not use culture media to resuspend the SVF cells as standard protocols, since we characterize the macrophage population through their surface markers, and it has been reported that these cells exhibit remarkable plasticity in response to environmental conditions, even changing their phenotype39,40,41.
Additionally, it is also important to mention that the pregnant women included in this protocol as VAT donors were healthy, with no clinical evidence of metabolic comorbidities, normal weight gain during pregnancy, average age 20–25 years, and normal pregestational Body Mass Index (BMI 18.5–24.9 kg/m2; n = 7), because some authors have described that different donors, age, BMI and gender or ethnicity have an influence on the cell number, and the expression of SFV surface markers42,43,44.
On the other hand, gene expression profile analysis requires reasonable amounts of intact RNA, but its isolation from mature adipocytes is particularly difficult because these cells have a higher lipid content and in some instances, the cell number is low45,46,47,48. We optimized an RNA isolation procedure from mature adipocytes based on a standard guanidine isothiocyanate/phenol method49,50, implementing easy steps that enable eliminate lipids and cellular debris. After sample preparation, column-based RNA extraction allows us to obtain a high-quality DNA-free RNA, including microRNAs, which is quite unusual for AT samples. The purity and integrity verification of these RNAs through a microfluidics-based RNA quality control analyzer ensures that isolated RNA quality is appropriate for downstream applications such as RT-qPCR assays and expression microarrays.
Besides the advantages mentioned before, AT digestion using collagenase allows liberating adipocytes and resident immune cells simultaneously, maintaining the integrity of cell surface receptors; this is an important fact during SVF cell isolation to get reliable and interpretable flow cytometry data27. Additionally, cells isolated from lipid-laden tissues such as VAT tend to be more prone to aggregation, reducing the sorted cell yield, and increasing autofluorescence51. In the protocol, the elimination of these cell aggregates by filtration through three layers of gauze before sorting and their exclusion with the described gating strategy, minimize background fluorescence, improving sample quality for cell sorting.
Previous studies have been conducted to identify the cells present in the SVF using a single set of CD markers characteristic for these cells52,53,54,55,56,57,58,59. For more in-deep characterization of macrophage subtypes in VAT SVF, we categorized these cells by expression of specific cell surface markers. According to CD45 and CD14 markers expression used to identify monocyte/macrophage linage cells in VAT60, we found that this tissue is composed of a typical CD45+CD14+ macrophage population from hematopoietic origin. Using the same protocol, in a previous study we were able to characterize M1 (CD11c) and M2 (CD163 and CD206) macrophage populations20.
The macrophage phenotyping within AT has been categorized as a spectrum from pro-inflammatory macrophages (classically activated-M1) to anti-inflammatory macrophages (alternatively activated-M2) according to the presence of different activation markers on its surface61,62. HLA-DR macrophage marker reflects the macrophage activation degree63,64, and was incorporated in the protocol to establish the M1 or M2 phenotype: macrophage populations expressing HLA-DR+ are inflammatory and HLA-DR– represent non-inflammatory macrophages. So, based on HLA-DR marker expression, two subsets of macrophages were defined: CD45+CD14+HLA-DR– and CD45+CD14+HLA-DR+, which originate from circulating monocytes and better known as monocyte-derived macrophages within the adipose tissue65,66.
Additionally, we quantified CD11c (M1 marker), as well as CD163 and CD206 (M2 marker) on each macrophages subtype, determining that AT macrophages display all evaluated activation markers on their membrane surface, demonstrating that macrophages present in VAT from pregnant women have more complex characteristics than those described for the overly simplified classifications of M1 and M2 macrophage populations. So, applying basic flow cytometry with multicolor antibody panel and stringent cell gating makes it possible to identify and characterize the macrophages from the heterogeneous pool of cells in the SVF. The refined macrophage phenotyping obtained with the protocol can be useful in studies conducted to elucidate the dynamic changes of macrophage populations in the AT that lead to disturbance in AT homeostasis.
Although this protocol was designed to characterize VAT macrophages, adjustments made in the antibody panel could expand its applications to facilitate sorting of other immune cells residing in the AT, since numerous fluorescent antibodies are available to identify specific markers of different lineage. Moreover, the method allows that cell populations purified by cell sorting can be used for post-sort analyses such as DNA/RNA/protein extraction or ex vivo treatments, obtaining the cells directly into an appropriate media with sterility techniques.
In summary, we consider that the protocol detailed herein represents the most efficient tradeoff between time, resources, cellular yield, and cell viability, besides being highly efficient for RNA and miRNA extraction from fat cells, and for characterization of macrophage population in AT.
The authors have nothing to disclose.
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).
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 | – |