Summary
This protocol describes the semi-automated isolation of the stromal vascular fraction (SVF) from murine adipose tissue to obtain preadipocytes and achieve adipocyte differentiation in vitro. Using a tissue dissociator for collagenase digestion reduces experimental variation and increases reproducibility.
Abstract
The in vitro study of white, brown, and beige adipocyte differentiation enables the investigation of cell-autonomous functions of adipocytes and their mechanisms. Immortalized white preadipocyte cell lines are publicly available and widely used. However, the emergence of beige adipocytes in white adipose tissue in response to external cues is difficult to recapitulate to the full extent using publicly available white adipocyte cell lines. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is commonly executed to obtain primary preadipocytes and perform adipocyte differentiation. However, mincing and collagenase digestion of adipose tissue by hand can result in experimental variation and is prone to contamination. Here, we present a modified semi-automated protocol that utilizes a tissue dissociator for collagenase digestion to achieve easier isolation of the SVF, with the aim of reducing experimental variation, reducing contamination, and increasing reproducibility. The obtained preadipocytes and differentiated adipocytes can be used for functional and mechanistic analyses.
Introduction
Adipose tissue biology has been attracting ever-increasing attention because of the growing prevalence of obesity and type 2 diabetes globally1. Adipocytes store excess energy in the form of lipid droplets, which are released upon starvation. Moreover, adipose tissue maintains systemic energy homeostasis by serving as an endocrine organ and communicating with other tissues2,3. Intriguingly, both excess adipose tissue (obesity) and adipose loss (lipodystrophy) are linked to insulin resistance and diabetes1. Adipocytes are divided into three types: white, brown, and beige1. White adipocytes mainly store excess energy as lipids, whereas brown and beige adipocytes dissipate energy in the form of heat via mitochondrial uncoupling protein-1 (Ucp1)1,4. Notably, beige adipocytes (also called "inducible" brown adipocytes) appear in white adipose tissue in response to cold or sympathetic stimulation and exhibit gene expression patterns that overlap with but are distinct from those of "classical" brown adipocytes5. Recently, brown and beige adipocytes have been anticipated as potential targets of anti-obesity and anti-diabetes treatments aimed at "enhancing energy dissipation" rather than "suppressing energy intake"4. Supportively, the risk allele of the FTO obesity variant rs1421085 in humans, which exhibits the strongest association with higher body mass index (BMI) among common variants6,7 and exhibits various gene-environment interactions8,9, is reported to negatively regulate beige adipocyte differentiation and function10. Peroxisome proliferator-activated receptor γ (PPARγ) is known as a master transcriptional regulator of adipogenesis and is necessary and sufficient for adipocyte differentiation11. Transcriptional regulators, such as PRD1-BF1-RIZ1 homologous domain containing 16 (PRDM16), early b cell factor 2 (EBF2), and nuclear factor I-A (NFIA), are crucial for brown and beige adipocyte differentiation and function12,13,14,15,16,17,18. On the other hand, white adipocyte gene programming requires transcriptional regulators, such as transducin-like enhancer protein 3 (TLE3) and zinc finger protein 423 (ZFP423)19,20,21.
In vitro model systems enable molecular studies to be performed that aim to improve the understanding of the mechanism(s) underlying the functions and dysfunctions of adipocytes. Although publicly available and immortalized preadipocyte cell lines such as 3T3-L1 and 3T3-F442A exist22,23,24, the culture of primary preadipocytes and differentiation into adipocytes would be a more suitable model for studying in vivo adipogenesis. Isolation of the stromal vascular fraction (SVF) from murine adipose tissue is a well-known method for obtaining primary preadipocytes25,26. However, collagenase digestion of adipose tissue, which is commonly performed using a bacterial shaker with a tube rack, can result in experimental variation and is prone to contamination27,28. Here, we describe an alternative protocol that uses a gentle magnetic-activated cell sorting (MACS) tissue dissociator for collagenase digestion to achieve easier isolation of the SVF.
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Protocol
All animal experiments described in this protocol were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Tokyo and performed according to the institutional guidelines of the University of Tokyo.
1. Preparation of enzyme solution and medium
- Put both sides of inguinal white adipose tissue (right and left side, approximately 150 mg) from a 7-8-week-old mouse and 2.5 mL of enzyme solution into tube C of the dissociator.
- Reconstitute Enzyme D with 3 mL of Dulbecco's modified eagle medium (DMEM)/F12 without fetal bovine serum (FBS) or antibiotics, Enzyme R with 2.7 mL of DMEM/F12 without FBS or antibiotics, and Enzyme A with 1 mL of buffer A.
- Prepare 2.5 mL of enzyme solution by adding 100 µL of Enzyme D, 50 µL of Enzyme R, 12.5 µL of Enzyme A, and 2.35 mL of DMEM/F12 without FBS or antibiotics into tube C. Set tube C with enzyme solution on ice.
2. Isolation of adipose tissue
- Euthanize 7-8-week-old male C57BL/6J mice (approximately 20 g) by cervical dislocation.
- At a clean bench, to isolate inguinal white adipose tissue, cut the skin with blunt/sharp scissors at the abdomen and toward the lower extremities. Isolate the adipose tissue from the inside of the thighs using sharp/sharp scissors. One side of inguinal adipose tissue wieghs ~75 mg.
- Put the isolated adipose tissue into tube C with enzyme solution on ice, and keep the tube within the clean bench to maintain sterility.
3. Mincing and digestion of adipose tissue
- At the clean bench, add 2.5 mL of enzyme solution to tube C.
- Mince the adipose tissue into small pieces by cutting with sharp/sharp scissors 50 times. Cut the adipose tissue into ~2 mm2 pieces.
- Tightly close the cap of tube C, turn the tube upside down, and attach it to the sleeve of the tissue dissociatorwith the cap on. Then, digest the sample for ~40 min at 37 °C.
NOTE: This study used gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec 130-096-427), and the preinstalled program "37C_mr_ATDK_1" was followed.
4. Filtration of cell suspension
- Pre-warm DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin to 37 °C.
- Detach tube C from the tissue dissociator and stop digestion by adding 5 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and gently pipetting four times.
- Centrifuge at 700 x g for 10 min at 20 °C.
- Carefully aspirate the supernatant without disturbing the cell pellet.
- Resuspend the pellet by adding 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and gently pipetting five times.
- Filter the cell suspension through a 70 µm diameter cell strainer placed on a new 50 mL tube.
- Centrifuge at 250 x g for 5 min and resuspend the pellet in 10 mL of phosphate-buffered saline (PBS).
5. Cell plating
- Centrifuge at 500 x g for 5 min.
- Remove the supernatant and resuspend the pellet by adding 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and pipetting ten times.
- Plate the cell suspension on a 10 cm collagen-coated dish and place the dishes in a cell culture incubator (37 °C, 5% CO2) for 1-2 h.
NOTE: Collagen-coated dishes are not be absolutely required. However, based on experience, collagen-coated dishes do help the adherence of preadipocytes and thus increase the survival of the cells. - Aspirate the medium and wash the cells twice with 3 mL of PBS per wash.
- Add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and return the dishes to the cell-culture incubator (37 °C, 5% CO2).
6. Passaging of preadipocytes
- The next day, aspirate the medium (the cells will be adherent, and thus the medium can be aspirated), wash the cells twice with 3 mL of PBS per wash, and add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin.
- When the cells reach 80% confluence (usually 4 days after SVF isolation), split the cells into four 10 cm collagen-coated dishes.
- Specifically, aspirate the medium, wash the cells with PBS (room temperature [RT]), add 1 mL of pre-warmed 0.05% trypsin, and incubate for 5 min in the cell-culture incubator.
- Add 10 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin to quench the trypsin activity. After pipetting, centrifuge at 440 x g for 5 min.
- Aspirate the supernatant, resuspend the cells by adding 40 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin, and plate the cells in four 10 cm collagen-coated dishes.
- Passage the cells again when they reach 80% confluence.
7. Preparation of retrovirus expressing SV40 large T antigen for immortalization of preadipocytes (optional)
- At least 3 days before immortalization, plate Platinum-E (Plat-E) packaging cells29 at a density of 3.0 x 105 cells/mL in 2 mL of DMEM with 10% FBS without antibiotics. Use a hemacytometer to count the cells.
- The next day, transfect 4 µg of pBABE-neo largeTcDNA (add gene plasmid #1780) using Lipofectamine 2000, according to the manufacturer's instructions.
- After 24 h, aspirate the medium and add 2 mL of DMEM with 10% FBS and 1% penicillin-streptomycin.
- The next day, harvest the retroviral supernatant. The retrovirus can be used for immortalization immediately or stored at -80 °C.
8. Immortalization of preadipocytes with SV40 large T antigen (optional)
- Passage and expand the preadipocytes twice after SVF isolation.
- Plate the cells in 6-well plates at a density of 0.5-1.0 x 104 cells/mL in 2 mL of DMEM/F12 with 10% FBS and 1% penicillin-streptomycin. Use a hemacytometer to count the cells.
- On the next day, prepare 250 µL of fresh or thawed retrovirus expressing SV40 large T antigen per well of the 6-well plates. Centrifuge at 440 x g for 5 min and place the supernatant in a new tube.
- Add 750 µL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin and 1 µL of polybrene per 250 µL of retroviral supernatant to make the working virus.
- Aspirate the medium and add 1,000 µL of the working retrovirus to each well containing preadipocytes.
- Add 1 mL of DMEM/F12 with 10% FBS and 1% penicillin-streptomycin 4 h later.
- The next day, separate the infected preadipocytes into a 10 cm dish and select the infected cells with DMEM/F12 containing 10% FBS, 1% penicillin-streptomycin, and 500 µg/mL neomycin. To monitor the antibiotics selection, prepare a dish of uninfected cells with neomycin.
- Continue to passage the infected cells with neomycin until all uninfected cells in the monitor dish are dead.
9. Adipocyte differentiation
- Plate the cells. For 12-well plates, plate the cells at a density of 4.0 x 104 cells/mL in 1 mL of DMEM/F12 containing 10% FBS and 1% penicillin-streptomycin.
- When the preadipocytes become confluent (48-72 h after plating), aspirate the medium and replace it with differentiation medium (see Table 1 for composition).
- At day 2 of differentiation (i.e., 48 h after inducing differentiation), replace the medium with maintenance medium (see Table 1 for composition). Afterward, change the maintenance medium every 2 days. Terminal differentiation is achieved at day 7 of differentiation.
- Oil red o staining
- For oil red o staining, aspirate the medium and rinse the cells with 1 mL of PBS per well. Fix the cells by adding 1 mL of 4% formaldehyde per well, and incubate the cells for 15 min at RT.
- During the incubation, dilute the 0.5% oil red o stock solution (in isopropyl alcohol) to 0.3% using ddH2O and filter the working solution using a filter paper.
- After the 15 min of incubation, remove the formaldehyde, rinse the cells with 60% isopropyl alcohol, add 1 mL of filtered oil red o working solution, and incubate for 1 h at RT.
- After the incubation, wash the cells with running water. Then, observe lipid-laden adipocytes under an inverted microscope (magnification: 20x objective and 10x eyepiece).
- mRNA expression analysis
NOTE: Refer to Table 2 for the list of primers used in this study.- Aspirate the medium and add 1 mL/well of trizol to the well.
- Incubate for 15 min at RT, detach the cells by pipetting, and collect the samples in 1.5 mL tubes. The samples can be stored at -80 °C until RNA extraction.
- Thaw the frozen sample to RT, add 200 µL of chloroform to the sample, and shake vigorously for 15 s.
- Incubate the sample at RT for 3 min and then spin at 20,000 x g for 15 min at 4 °C.
- Carefully remove the aqueous phase (top, colorless) and transfer to new tubes. Never transfer the protein phase (middle, white) or the phenol/chloroform phase (bottom, pink).
- Slowly add an equal volume of 70% EtOH and mix gently. Do not vortex.
- Load the sample (up to 700 µL) into an RNA spin column seated in a collection tube. Be sure to include any precipitate that may have formed.
- Spin at 9,000 x g for 30 s at 4 °C , and then discard the flow-through.
- Add 700 µL of buffer RW1, spin at 9,000 x g for 30 s at 4 °C , and then discard the flow-through.
- Transfer the column into a new collection tube and add 500 µL of buffer RPE.
- Spin at 9,000 x g for 30 s at 4 °C and then discard the flow-through.
- Add 500 µL of buffer RPE, spin at 9,000 x g for 2 min at 4 °C, and then discard the flow-through.
- Transfer the column to a new collection tube, and spin (columns without buffer) at 9,000 x g for 1-2 min at 4 °C.
- Transfer the column into a new 1.5 mL collection tube and add 30 µL of RNase-free water directly onto the column membrane.
- Incubate the sample at RT for 1-2 min. Then, spin at 9,000 x g for 1 min at 4 °C to elute the RNA.
- Check the RNA concentration using a fluorospectrometer.
NOTE: It is recommended to align the concentration here. - Perform reverse transcription using ~200 ng of RNA template. Use a commercial quantitative real-time polymerase chain reaction (qPCR-RT) kit and follow the manufacturer's protocol. After the reaction, dilute the cDNA 20 times to 200 µL.
- Add 2.0 µL of cDNA, 3.0 µL of Power Sybr Green Master Mix, 0.018 µL of 100 µM qPCR forward primer, 0.018 µL of 100 µM qPCR reverse primer, and 0.96 µL of ddH2O to each well of a 384-well plate.
- Run a qPCR (hold stage: 50°C for 2 min and 95 °C for 2 min; PCR stage: 40 cycles of 95 °C for 15 s and 60 °C for 1 min; melt curve stage: 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s). Use the standard curve method for quantification.
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Representative Results
This protocol yields fully differentiated, lipid-laden adipocytes 7 days after inducing adipocyte differentiation. The degree of adipocyte differentiation can be evaluated by the oil red o staining of triglycerides and lipids (Figure 1A), or mRNA expression analysis using qPCR-RT of adipocyte genes, such as the master regulator of adipogenesis Pparg and its target Fabp4 (Figure 1B). To induce beige adipocyte differentiation in vitro, a thiazolidinediones class of PPARγ agonist, such as rosiglitazone, can be used. Indeed, in experiments with rosiglitazone, we observe a dose-dependent effect of concentrations up to 1 µM on the expression levels of the brown fat-specific genes, such as Ucp1 and Ppargc1a. On the other hand, the effect of rosiglitazone on Fabp4 is saturated at a concentration of 0.1 µM (Figure 1C).
Differentiated adipocytes obtained by this protocol can be used for various functional and mechanistic analyses, such as oxygen consumption rate (OCR) analysis16,30 (Figure 2A) and chromatin immunoprecipitation31 (ChIP; Figure 2B). Immortalized preadipocytes can be stored in a liquid nitrogen cell storage system without loss of viability and can be thawed at any time for use in experiments.
Figure 1: Differentiation of preadipocytes into lipid-laden adipocytes. (A) SVFs from inguinal white adipose tissue (iWAT) of wild-type mice were stained with oil red o at day 0 or 7 of adipocyte differentiation.(B) mRNA expression levels of Pparg and Fabp4 at day 0 and day 7 of adipocyte differentiation (mean ± standard error of the mean [S.E.M.]; N = 3). (C) mRNA expression of the general adipocyte marker Fabp4 and the brown fat-specific genes Ucp1 and Ppargc1a in SVF-derived adipocytes treated with 0.1, 0.2, 0.5, and 1.0 µM rosiglitazone, along with the differentiation and maintenance medium. Please click here to view a larger version of this figure.
Figure 2: Examples of functional and mechanistic analyses with differentiated adipocytes. (A) OCR analysis of iWAT SVF-derived adipocytes (N = 10). (B) ChIP-qPCR for H3K27Ac in Nfiaflox/flox iWAT SVF-derived adipocytes at day 0 and 7 of differentiation. The Ins-0.4 kb and Fabp4-13 kb site are shown as background sites (mean ± S.E.M.; N = 2). For both experiments, 1.0 µM rosiglitazone was added along with the differentiation and maintenance medium to induce beige adipocyte differentiation. Please click here to view a larger version of this figure.
Table 1: Composition of differentiation and maintenance medium. Please click here to download this Table.
Table 2: The list of primers used in this study. Please click here to download this Table.
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Discussion
Here, we described a protocol for isolation of the SVF from murine adipose tissue to obtain preadipocytes and perform adipocyte differentiation in vitro. The use of a tissue dissociator for collagenase digestion decreased experimental variation, decreased the risk of contamination, and increased reproducibility. While this procedure is a critical step within the presented protocol, the process is highly automated and optimization is not needed. However, depending on the mouse age and adipose tissue depot, optimization of mincing, for example size pieces or cutting time, might be required.
The SVF is known as a heterogenous population consisting of preadipocytes, immune cells such as macrophages, endothelial cells, and other cells. Because preadipocytes adhere to culture dishes and are tolerant to washing and medium changes, they are enriched in the cell population during the passages. However, it is reasonable to assume that the "preadipocytes" obtained by this protocol are still heterogeneous. Fluorescent-activated cell sorting (FACS) using antibodies against previously reported surface markers of preadipocytes such as PDGFRα32 would be required to obtain a purer preadipocyte population.
In summary, we described here a protocol for SVF isolation using a tissue dissociator for collagenase digestion. This protocol offers easier isolation of the SVF compared with the conventional protocol using a bacterial shaker with a tube rack, and provides reduced experimental variation, reduced contamination, and increased reproducibility16,17,18.
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Disclosures
None of the authors have a competing financial interest related to this work.
Acknowledgments
The authors would like to thank Takahito Wada and Saiko Yoshida (The University of Tokyo, Tokyo, Japan) for their experimental assistance. This work was funded by the following grants to Y.H.: research grant from the University of Tokyo Excellent Young Researcher Program; Japan Society for the Promotion of Science (JSPS) KAKENHI Grant-in-Aid for Early-Career Scientists, grant number 19K17976; grant for the Front Runner of Future Diabetes Research (FFDR) from the Japan Foundation for Applied Enzymology, grant number 17F005; grant from the Pharmacological Research Foundation; grant from the Mochida Memorial Foundation for Medical and Pharmaceutical Research; grant from the MSD Life Science Foundation; grant from the Daiwa Securities Health Foundation; grant from the Tokyo Biochemical Research Foundation; Life Science Research grant from the Takeda Science Foundation; and grant from the SENSHIN Medical Research Foundation.
Materials
| Name | Company | Catalog Number | Comments |
| 100 mm dish | Corning | 430167 | |
| 12 well plate | Corning | 3513 | |
| 60 mm dish | IWAKI | 3010-060 | |
| Adipose Tissue Dissociation Kit, mouse and rat | Miltenyi Biotec | 130-105-808 | contents: Enzyme D, Enzyme R, Enzyme A and Buffer A |
| Cell strainer 70 µm | BD falcon | #352350 | |
| Collagen coated dishes, 100 mm | BD | #356450 | |
| Collagen coated dishes, 60 mm | BD | #354401 | |
| Collagen I Coat Microplate 6 well | IWAKI | 4810-010 | |
| Dexamethasone | Wako | 041-18861 | |
| Dissecting Forceps | N/A | N/A | autoclave before use |
| Dissecting Scissors, blunt/sharp | N/A | N/A | autoclave before use |
| Dissecting Scissors, sharp/sharp | N/A | N/A | autoclave before use |
| DMEM/F-12, GlutaMAX supplement | Gibco | 10565-042 | |
| Fetal Bovine Serum (FBS) | N/A | N/A | |
| gentleMACS C Tubes | Milteny Biotec | 130-093-237 | |
| gentleMACSOcto Dissociator with Heaters | Miltenyi Biotec | 130-096-427 | |
| Humulin R Injection U-100 | Eli Lilly | 872492 | |
| Indomethacin | Sigma | I7378-5G | |
| Isobutylmethylxanthine (IBMX) | Sigma | 17018-1G | |
| Lipofectamine 2000 | Life Technologies | 11668-019 | |
| Neomycin Sulfate | Fujifilm | 146-08871 | |
| Opti-MEM | Invitrogen | 31985-062 | |
| pBABE-neo largeTcDNA (SV40) | Add gene | #1780 | |
| PBS tablets | Takara | T900 | |
| Platinum-E (Plat-E) Retroviral Packaging Cell Line | cell biolab | RV-101 | |
| Polybrene | Nacalai Tesque | 12996-81 | |
| Power Sybr Green Master Mix | Applied Biosystems | 4367659 | |
| ReverTra Ace qPCR RT Master Mix | TOYOBO | #FSQ-201 | |
| RNeasy Mini Kit (250) | QIAGEN | 74106 | |
| Rosiglitazone | Wako | 180-02653 | |
| T3 | Sigma | T2877-100mg | |
| Trypsin-EDTA (0.05%) | Gibco | 25200-056 |
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