Hostname: page-component-cd9895bd7-hc48f Total loading time: 0 Render date: 2024-12-26T18:08:30.741Z Has data issue: false hasContentIssue false
  • English
  • Français

Noninvasive Continuous Monitoring of Adipocyte Differentiation: From Macro to Micro Scales

Published online by Cambridge University Press:  04 February 2019

Maayan Lustig Open the ORCID record for Maayan Lustig [Opens in a new window] ,
Qingling Feng ,
Yohan Payan ,
Amit Gefen  and
Dafna Benayahu
Maayan Lustig
Affiliation:
Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 6997801, Israel
Qingling Feng
Affiliation:
Key Laboratory of Advanced Materials of Ministry of Education of China, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China
Yohan Payan
Affiliation:
CNRS, Grenoble INP, TIMC-IMAG, University of Grenoble Alpes, Grenoble F-38000, France
Amit Gefen
Affiliation:
Department of Biomedical Engineering, Faculty of Engineering, Tel Aviv University, Tel Aviv 6997801, Israel
Dafna Benayahu*
Affiliation:
Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel
*
*Author for correspondence: Dafna Benayahu, E-mail: dafnab@tauex.tau.ac.il
Get access

Abstract

3T3-L1 cells serve as model systems for studying adipogenesis and research of adipose tissue-related diseases, e.g. obesity and diabetes. Here, we present two novel and complementary nondestructive methods for adipogenesis analysis of living cells which facilitate continuous monitoring of the same culture over extended periods of time, and are applied in parallel at the macro- and micro-scales. At the macro-scale, we developed visual differences mapping (VDM), a novel method which allows to determine level of adipogenesis (LOA)—a numerical index which quantitatively describes the extent of differentiation in the whole culture, and percentage area populated by adipocytes (PAPBA) across a whole culture, based on the apparent morphological differences between preadipocytes and adipocytes. At the micro-scale, we developed an improved version of our previously published image-processing algorithm, which now provides data regarding single-cell morphology and lipid contents. Both methods were applied here synergistically for measuring differentiation levels in cultures over multiple weeks. VDM revealed that the mean LOA value reached 1.11 ± 0.06 and the mean PAPBA value reached >60%. Micro-scale analysis revealed that during differentiation, the cells transformed from a fibroblast-like shape to a circular shape with a build-up of lipid droplets. We predict a vast potential for implementation of these methods in adipose-related pharmacological research, such as in metabolic-syndrome studies.

Keywords

adipogenesismorphologylipid dropletsimage processingmapping
Type
Biological Science Applications
Information
Microscopy and Microanalysis , Volume 25 , Issue 1 , February 2019 , pp. 119 - 128
Copyright
Copyright © Microscopy Society of America 2019 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Ben-Or Frank, M, Shoham, N, Benayahu, D & Gefen, A (2015). Effects of accumulation of lipid droplets on load transfer between and within adipocytes. Biomech Model Mechanobiol 14, 15–28. http://link.springer.com/10.1007/s10237-014-0582-8.Google Scholar
Choi, JH, Gimble, JM, Lee, K, Marra, KG, Rubin, JP, Yoo, JJ, Vunjak-Novakovic, G & Kaplan, DL (2010). Adipose tissue engineering for soft tissue regeneration. Tissue Eng Part B Rev 16, 413–426. http://www.liebertonline.com/doi/abs/10.1089/ten.teb.2009.0544.Google Scholar
Choi, SS, Cha, BY, Lee, YS, Yonezawa, T, Teruya, T, Nagai, K & Woo, JT (2009). Magnolol enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells. Life Sci 84, 908–914. http://dx.doi.org/10.1016/j.lfs.2009.04.001.Google Scholar
Deutsch, MJ, Schriever, SC, Roscher, AA & Ensenauer, R (2014). Digital image analysis approach for lipid droplet size quantitation of Oil Red O-stained cultured cells. Anal Biochem 445, 87–89. http://dx.doi.org/10.1016/j.ab.2013.10.001.Google Scholar
Doerner, JF, Delling, M & Clapham, DE (2015). Ion channels and calcium signaling in motile cilia. eLife 4, 1–19. http://elifesciences.org/lookup/doi/10.7554/eLife.11066.Google Scholar
Ejaz, A, Wu, D, Kwan, P & Meydani, M (2009). Curcumin inhibits adipogenesis in 3T3-L1 adipocytes and angiogenesis and obesity in C57/BL mice. J Nutr 139, 919–925. http://jn.nutrition.org/cgi/doi/10.3945/jn.108.100966.Google Scholar
Finlayson, AE & Freeman, KW (2009). A cell motility screen reveals role for MARCKS-related protein in adherens junction formation and tumorigenesis. PLoS ONE 4, 1–8.Google Scholar
Furukawa, S, Fujita, T, Shumabukuro, M, Iwaki, M, Yamada, Y, Makajima, Y, Nakayama, O, Makishima, M, Matsuda, M, Shumomura, I, Shimabukuro, M, Iwaki, M, Yamada, Y, Nakajima, Y, Nakayama, O, Makishima, M, Matsuda, M & Shimomura, I (2004). Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 114, 1752–1761. http://www.jci.org/articles/view/21625.Google Scholar
Harris, LA, Skinner, JR & Wolins, NE (2013). Imaging of neutral lipids and neutral lipid associated proteins. In Methods in Cell Biologyvol, vol. 116, Yang, H and Li, P (Eds.), pp. 213–226. Amsterdam, Netherlands: Elsevier.Google Scholar
Katzengold, R, Shoham, N, Benayahu, D & Gefen, A (2015). Simulating single cell experiments in mechanical testing of adipocytes. Biomech Model Mechanobiol 14, 537–547. http://dx.doi.org/10.1007/s10237-014-0620-6.Google Scholar
Kraus, MJ, Seifert, J, Strasser, EF, Gawaz, M, Schäffer, TE & Rheinlaender, J (2016). Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy. Platelets 27, 541–546. http://www.tandfonline.com/doi/abs/10.3109/09537104.2016.1158400.Google Scholar
Levy, A, Enzer, S, Shoham, N, Zaretsky, U & Gefen, A (2012). Large, but not small sustained tensile strains stimulate adipogenesis in culture. Ann Biomed Eng 40, 1052–1060. http://link.springer.com/10.1007/s10439-011-0496-x.Google Scholar
Lin, Q, Gao, Z, Alarcon, RM, Ye, J & Yun, Z (2009). A role of miR-27 in the regulation of adipogenesis. FEBS J 276, 2348–2358. http://doi.wiley.com/10.1111/j.1742-4658.2009.06967.x.Google Scholar
Lustig, M, Gefen, A & Benayahu, D (2018 a). Adipogenesis and lipid production in adipocytes subjected to sustained tensile deformations and elevated glucose concentration: A living cell-scale model system of diabesity. Biomech Model Mechanobiol 17, 903–913. http://link.springer.com/10.1007/s10237-017-1000-9.Google Scholar
Lustig, M, Moldovan Mor Yossef, L, Gefen, A & Benayahu, D (2018 b). Adipogenesis of 3T3L1 cells subjected to tensile deformations under various glucose concentrations. In Computer Methods in Biomechanics and Biomedical Engineering, Gefen, A and Weihs, D (Eds.), pp. 171–174. Cham, Switzerland: Springer.Google Scholar
Macias-Romero, C, Zubkovs, V, Wang, S & Roke, S (2016). Wide-field medium-repetition-rate multiphoton microscopy reduces photodamage of living cells. Biomed Opt Express 7, 1458. https://www.osapublishing.org/abstract.cfm?URI=boe-7-4-1458.Google Scholar
Mor-Yossef Moldovan, L, Lustig, M, Naftaly, A, Mardamshina, M, Geiger, T, Gefen, A & Benayahu, D (2018). Cell shape alteration during adipogenesis is associated with coordinated matrix cues. J Cell Physiol 234, 3850–3863. doi: 10.1002/jcp.27157.Google Scholar
Murphy, S, Martin, S & Parton, RG (2010). Quantitative analysis of lipid droplet fusion: Inefficient steady state fusion but rapid stimulation by chemical fusogens. PLoS ONE 5, 15030.Google Scholar
Neubrand, VE, Thomas, C, Schmidt, S, Debant, A & Schiavo, G (2010). Kidins220/ARMS regulates Rac1-dependent neurite outgrowth by direct interaction with the RhoGEF Trio. J Cell Sci 123, 2111–2123. http://jcs.biologists.org/cgi/doi/10.1242/jcs.064055.Google Scholar
Or-Tzadikario, S, Sopher, R & Gefen, A (2010). Quantitative monitoring of lipid accumulation over time in cultured adipocytes as function of culture conditions: Toward controlled adipose tissue engineering. Tissue Eng Part C Methods 16, 1167–1181. http://www.liebertonline.com/doi/abs/10.1089/ten.tec.2009.0755.Google Scholar
Patrick, CW Jr. (2000). Adipose tissue engineering: The future of breast and soft tissue reconstruction following tumor resection. Semin Surg Oncol 19, 302–311.Google Scholar
Poulos, SP, Dodson, MV & Hausman, GJ (2010). Cell line models for differentiation: Preadipocytes and adipocytes. Exp Biol Med 235, 1185–1193. http://journals.sagepub.com/doi/10.1258/ebm.2010.010063.Google Scholar
Qiu, B & Simon, MC (2016). BODIPY 493/503 staining of neutral lipid droplets for microscopy and quantification by flow cytometry. Bio Protoc 6, 417–24.e5. http://www.ncbi.nlm.nih.gov/pubmed/24655651%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC4126411%0Ahttp://www.ncbi.nlm.nih.gov/pubmed/28573161%0Ahttp://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=PMC5448404.Google Scholar
Ramírez-Zacarías, JL, Castro-Muñozledo, F & Kuri-Harcuch, W (1992). Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O. Histochemistry 97, 493–497.Google Scholar
Ross, SE (2000). Inhibition of adipogenesis by Wnt signaling. Science 289, 950–953. http://www.sciencemag.org/cgi/doi/10.1126/science.289.5481.950.Google Scholar
Shoham, N & Gefen, A (2011). Stochastic modeling of adipogenesis in 3T3-L1 cultures to determine probabilities of events in the cell's life cycle. Ann Biomed Eng 39, 2637–2653. http://link.springer.com/10.1007/s10439-011-0341-2.Google Scholar
Shoham, N & Gefen, A (2012 a). Mechanotransduction in adipocytes. J Biomech 45, 1–8. http://linkinghub.elsevier.com/retrieve/pii/S0021929011006622.Google Scholar
Shoham, N & Gefen, A (2012 b). The influence of mechanical stretching on mitosis, growth, and adipose conversion in adipocyte cultures. Biomech Model Mechanobiol 11, 1029–1045. http://link.springer.com/10.1007/s10237-011-0371-6.Google Scholar
Shoham, N, Girshovitz, P, Katzengold, R, Shaked, NT, Benayahu, D & Gefen, A (2014). Adipocyte stiffness increases with accumulation of lipid droplets. Biophys J 106, 1421–1431. http://linkinghub.elsevier.com/retrieve/pii/S0006349514001805.Google Scholar
Shoham, N, Gottlieb, R, Sharabani-Yosef, O, Zaretsky, U, Benayahu, D & Gefen, A (2012). Static mechanical stretching accelerates lipid production in 3T3-L1 adipocytes by activating the MEK signaling pathway. AJP: Cell Physiol 302, C429–C441. http://ajpcell.physiology.org/cgi/doi/10.1152/ajpcell.00167.2011.Google Scholar
Shoham, N, Mor-Yossef Moldovan, L, Benayahu, D & Gefen, A (2015). Multiscale modeling of tissue-engineered fat: Is there a deformation-driven positive feedback loop in adipogenesis? Tissue Eng Part A 21, 1354–1363. http://www.ncbi.nlm.nih.gov/pubmed/25517541.Google Scholar
Steppan, CM, Bailey, ST, Bhat, S, Brown, EJ, Banerjee, RR, Wright, CM, Patel, HR, Ahima, RS & Lazar, MA (2001). The hormone resistin links obesity to diabetes. Nature 409, 307–312. http://www.ncbi.nlm.nih.gov/pubmed/11201732%5Cnhttp://www.nature.com/nature/journal/v409/n6818/pdf/409307a0.pdf.Google Scholar
Strober, W (1997). Monitoring cell growth. In Current Protocols in Immunology, vol. 21 Appendix 3, pp. 2–3. Hoboken, NJ, USA: John Wiley & Sons, Inc. http://onlinelibrary.wiley.com/doi/10.1002/0471142735.ima03as21/full.Google Scholar
The Mathworks, Inc (2016) Matlab User's Manual. Natick, MA: The Mathworks, Inc.Google Scholar
Thompson, GL, Roth, CC, Kuipers, MA, Tolstykh, GP, Beier, HT & Ibey, BL (2016). Permeabilization of the nuclear envelope following nanosecond pulsed electric field exposure. Biochem Biophys Res Commun 470, 35–40. http://dx.doi.org/10.1016/j.bbrc.2015.12.092.Google Scholar
Toneff, MJ, Sreekumar, A, Tinnirello, A, Hollander, P, Den, , Habib, S, Li, S, Ellis, MJ, Xin, L, Mani, SA & Rosen, JM (2016). The Z-cad dual fluorescent sensor detects dynamic changes between the epithelial and mesenchymal cellular states. BMC Biol 14, 1–16. http://dx.doi.org/10.1186/s12915-016-0269-y.Google Scholar
Weinmeister, P, Lukowski, R, Linder, S, Traidl-Hoffmann, C, Hengst, L, Hofmann, F & Feil, R (2008). Cyclic guanosine monophosphate-dependent protein kinase I promotes adhesion of primary vascular smooth muscle cells. Mol Biol Cell 19, 4434–4441. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2555919&tool=pmcentrez&rendertype=abstract.Google Scholar
Supplementary material: File

Lustig et al. supplementary material

Lustig et al. supplementary material 1

Download Lustig et al. supplementary material(File)
File 51.4 KB