JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Semi-Automated Isolation of the Stromal Vascular Fraction from Murine White Adipose Tissue Using a Tissue Dissociator

Published: May 19th, 2023

DOI:

10.3791/65265

1Division for Health Service Promotion, The University of Tokyo, 2Department of Diabetes and Metabolic Diseases, Graduate School of Medicine, The University of Tokyo, 3The University of Tokyo Excellent Young Researcher Program, The University of Tokyo

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, an....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

  1. 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.
  2. .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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, optimi.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 ....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

NameCompanyCatalog NumberComments
100 mm dishCorning430167
12 well plateCorning3513
60 mm dishIWAKI3010-060
Adipose Tissue Dissociation Kit, mouse and ratMiltenyi Biotec130-105-808contents: Enzyme D, Enzyme R, Enzyme A and Buffer A
Cell strainer 70 µmBD falcon#352350
Collagen coated dishes, 100 mmBD#356450
Collagen coated dishes, 60 mmBD#354401
Collagen I Coat Microplate 6 wellIWAKI4810-010
DexamethasoneWako041-18861
Dissecting ForcepsN/AN/Aautoclave before use
Dissecting Scissors, blunt/sharpN/AN/Aautoclave before use
Dissecting Scissors, sharp/sharpN/AN/Aautoclave before use
DMEM/F-12, GlutaMAX supplementGibco10565-042
Fetal Bovine Serum (FBS)N/AN/A
gentleMACS C TubesMilteny Biotec130-093-237
gentleMACSOcto Dissociator with HeatersMiltenyi Biotec130-096-427
Humulin R Injection U-100Eli Lilly872492
IndomethacinSigmaI7378-5G
Isobutylmethylxanthine (IBMX)Sigma17018-1G
Lipofectamine 2000Life Technologies11668-019
Neomycin SulfateFujifilm146-08871 
Opti-MEMInvitrogen 31985-062
pBABE-neo largeTcDNA (SV40)Add gene#1780
PBS tabletsTakaraT900
Platinum-E (Plat-E) Retroviral Packaging Cell Linecell biolabRV-101
PolybreneNacalai Tesque12996-81
Power Sybr Green Master MixApplied Biosystems4367659
ReverTra Ace qPCR RT Master MixTOYOBO#FSQ-201
RNeasy Mini Kit (250)QIAGEN74106
RosiglitazoneWako180-02653
T3SigmaT2877-100mg
Trypsin-EDTA (0.05%)Gibco25200-056

  1. Rosen, E. D., Spiegelman, B. M. What we talk about when we talk about fat. Cell. 156 (1-2), 20-44 (2014).
  2. Hotamisligil, G. S., Shargill, N. S., Spiegelman, B. M. Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science. 259 (5091), 87-91 (1993).
  3. Zhang, Y., et al. Positional cloning of the mouse obese gene and its human homologue. Nature. 372 (6505), 425-432 (1994).
  4. Kajimura, S., Saito, M. A new era in brown adipose tissue biology: Molecular control of brown fat development and energy homeostasis. Annual Review of Physiology. 76, 225-249 (2014).
  5. Wu, J., et al. Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human. Cell. 150 (2), 366-376 (2012).
  6. Loos, R. J. F., Yeo, G. S. H. The genetics of obesity: from discovery to biology. Nature Reviews Genetics. 23 (2), 120-133 (2022).
  7. Loos, R. J. F., Yeo, G. S. H. The bigger picture of FTO-the first GWAS-identified obesity gene. Nature Reviews Endocrinology. 10 (1), 51-61 (2014).
  8. Hiraike, Y., Yang, C. T., Liu, W. J., Yamada, T., Lee, C. L. FTO obesity variant-exercise interaction on changes in body weight and BMI: The Taiwan biobank study. The Journal of Clinical Endocrinology and Metabolism. 106 (9), e3673-e3681 (2021).
  9. Li, S., et al. Physical activity attenuates the genetic predisposition to obesity in 20,000 men and women from EPIC-Norfolk prospective population study. PLoS Medicine. 7 (8), e1000332 (2010).
  10. Claussnitzer, M., et al. FTO obesity variant circuitry and adipocyte browning in humans. The New England Journal of Medicine. 373 (10), 895-907 (2015).
  11. Tontonoz, P., Hu, E., Spiegelman, B. M. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. 79 (7), 1147-1156 (1994).
  12. Seale, P., et al. Transcriptional control of brown fat determination by PRDM16. Cell Metabolism. 6 (1), 38-54 (2007).
  13. Seale, P., et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature. 454 (7207), 961-967 (2008).
  14. Rajakumari, S., et al. EBF2 determines and maintains brown adipocyte identity. Cell Metabolism. 17 (4), 562-574 (2013).
  15. Shapira, S. N., et al. EBF2 transcriptionally regulates brown adipogenesis via the histone reader DPF3 and the BAF chromatin remodeling complex. Genes and Development. 31 (7), 660-673 (2017).
  16. Hiraike, Y., et al. NFIA co-localizes with PPARγ and transcriptionally controls the brown fat gene program. Nature Cell Biology. 19 (9), 1081-1092 (2017).
  17. Hiraike, Y., et al. NFIA differentially controls adipogenic and myogenic gene program through distinct pathways to ensure brown and beige adipocyte differentiation. PLoS Genetics. 16 (9), e1009044 (2020).
  18. Hiraike, Y., et al. NFIA determines the cis-effect of genetic variation on Ucp1 expression in murine thermogenic adipocytes. iScience. 25 (8), 104729 (2022).
  19. Villanueva, C. J., et al. Adipose subtype-selective recruitment of TLE3 or Prdm16 by PPARγ specifies lipid storage versus thermogenic gene programs. Cell Metabolism. 17 (3), 423-435 (2013).
  20. Pearson, S., et al. Loss of TLE3 promotes the mitochondrial program in beige adipocytes and improves glucose metabolism. Genes and Development. 33 (13-14), 747-762 (2019).
  21. Shao, M., et al. Zfp423 maintains white adipocyte identity through suppression of the beige cell thermogenic gene program. Cell Metabolism. 23 (6), 1167-1184 (2016).
  22. Green, H., Kehinde, O. Sublines of mouse 3T3 cells that accumulate lipid. Cell. 1 (3), 113-116 (1974).
  23. Green, H., Kehinde, O. An established preadipose cell line and its differentiation in culture II. Factors affecting the adipose conversion. Cell. 5 (1), 19-27 (1975).
  24. Green, H., Kehinde, O. Spontaneous heritable changes leading to increased adipose conversion in 3T3 cells. Cell. 7 (1), 105-113 (1976).
  25. Aune, U. L., Ruiz, L., Kajimura, S. Isolation and differentiation of stromal vascular cells to beige/brite cells. Journal of Visualized Experiments. (73), e50191 (2013).
  26. Galmozzi, A., Kok, B. P., Saez, E. Isolation and differentiation of primary white and brown preadipocytes from newborn mice. Journal of Visualized Experiments. (167), e62005 (2021).
  27. Priya, N., Sarcar, S., Majumdar, A. S., SundarRaj, S. Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate. Journal of Tissue Engineering and Regenerative Medicine. 8 (9), 706-716 (2014).
  28. Lockhart, R. A., Aronowitz, J. A., Dos-Anjos Vilaboa, S. Use of freshly isolated human adipose stromal cells for clinical applications. Aesthetic Surgery Journal. 37, S4-S8 (2017).
  29. Morita, S., Kojima, T., Kitamura, T. Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Therapy. 7 (12), 1063-1066 (2000).
  30. Li, Y., Fromme, T., Klingenspor, M. Meaningful respirometric measurements of UCP1-mediated thermogenesis. Biochimie. 134, 56-61 (2017).
  31. Hiraike, Y. Chromatin immunoprecipitation with mouse adipocytes using hypotonic buffer to enrich nuclear fraction before fixation. STAR Protocols. 4 (1), 102093 (2023).
  32. Rodeheffer, M. S., Birsoy, K., Friedman, J. M. Identification of white adipocyte progenitor cells in vivo. Cell. 135 (2), 240-249 (2008).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved