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

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.

Adipocytes Migration is Altered Through Differentiation

“…Consistent with our previous work, remarkable morphological differences are apparent between the preadipocytes and mature adipocytes (Lustig et al, 2018 a , 2019; Mor-Yossef Moldovan et al, 2019); the preadipocytes are elongated with a fibroblast-like shape, and are lower in their three-dimensional (3D) cell profile compared to mature adipocytes which are packed with LDs and are hence greater in size, in addition to their more circular projected shape (Fig. 2).…”

“…As shown previously by our group, adipogenesis is influenced by mechanical stimulations; it is accelerated in cultures subjected to static tensile deformations and adipocyte stiffness increases with the accumulation of LDs (Levy et al, 2012;Shoham et al, 2012Shoham et al, , 2014Shoham et al, , 2015Shoham & Gefen, 2012a, 2012bBen-Or Frank et al, 2015;Katzengold et al, 2015;Lustig et al, 2018aLustig et al, , 2019Mor-Yossef Moldovan et al, 2019). The aforementioned work led us to apply a proteomic approach using high-resolution mass spectrometry to reveal the molecular alterations occurring during differentiation; we revealed that the cells' microenvironment changed from one which contained fibrillary collagens of types I, III, and V to one containing collagens of types IV and VI.…”

“…Hence, research at a single-cell level will have an important contribution to expanding our knowledge of the mechanisms by which cells' motility is affected as the cells undergo differentiation. 3T3-L1 adipocyte cells are used widely as a model system to address many aspects regarding adipocyte differentiation (Shoham & Gefen, 2011; Levy et al, 2012; Shoham et al, 2012, 2014, 2016; Ben-Or Frank et al, 2015; Katzengold et al, 2015; Lustig et al, 2018 a , 2018 b , 2019; Mor-Yossef Moldovan et al, 2019). Despite this, no study has thus far addressed the motility of differentiating adipocytes.…”

In Utero Exposure to Simulated Complex Urban Air Pollution Disturbs Intestinal Suckling-to-Weaning Transition in Mice

“…At least 100 well-oriented mucosa, villi, and crypts were measured in at least 5 individual mice from each group. For vacuole morphologic analyses, an image processing algorithm developed by Lustig et al was adapted [57]. This algorithm converts a microscope image from a red-green-blue image to a grayscale image: the vacuoles are colored white and all other components are blackened.…”

Murine in vitro cellular models to better understand adipogenesis and its potential applications

“…These cells were also served as a model system to develop a visual difference mapping (VDM) platform, a new method used to determine the program of adipogenesis. The system analyses the conversion process of fibroblast-like cells into a rounded shape with formation of lipid droplets (Lustig, Feng, Payan et al, 2019). These advanced techniques and high-throughput screening could assist in finding effective and potent therapies to combat obesity and its related metabolic disorders.…”