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.
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.
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.
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
2. Isolation of adipose tissue
3. Mincing and digestion of adipose tissue
4. Filtration of cell suspension
5. Cell plating
6. Passaging of preadipocytes
7. Preparation of retrovirus expressing SV40 large T antigen for immortalization of preadipocytes (optional)
8. Immortalization of preadipocytes with SV40 large T antigen (optional)
9. Adipocyte differentiation
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.
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.
The authors have nothing to disclose.
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.
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 |