ITRAQ-Based Quantitative Proteomic Comparison of 2D and 3D Adipocyte Cell Models Co-cultured with Macrophages Using Online 2D-nanoLC-ESI-MS/MS.
ABSTRACT: The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Among them, 48 proteins involved in carbohydrate metabolism (e.g., PDH?, MDH1/2, FH) and the mitochondrial fatty acid beta oxidation pathway (e.g., VLCAD, ACADM, ECHDC1, ALDH6A1) were relatively up-regulated in the 3D co-culture model compared to those in 2D and 3D mono-cultured cells. Conversely, 12 proteins implicated in cellular component organisation (e.g., ANXA1, ANXA2) and the cell cycle (e.g., MCM family proteins) were down-regulated. These quantitative assessments showed that the 3D co-culture system of adipocytes and macrophages led to the development of insulin resistance, thereby providing a promising in vitro obesity model that is more equivalent to the in vivo conditions with respect to the mechanisms underpinning metabolic syndromes and the effect of new medical treatments for metabolic disorders.
Project description:The demand for novel three-dimensional (3D) cell culture models of adipose tissue has been increasing, and proteomic investigations are important for determining the underlying causes of obesity, type II diabetes, and metabolic disorders. In this study, we performed global quantitative proteomic profiling of three 3D-cultured 3T3-L1 cells (preadipocytes, adipocytes and co-cultured adipocytes with macrophages) and their 2D-cultured counterparts using 2D-nanoLC-ESI-MS/MS with iTRAQ labelling. A total of 2,885 shared proteins from six types of adipose cells were identified and quantified in four replicates. Using iTRAQ-based quantitative assessments, we found that the primary proteins involved in carbohydrate and fatty acid metabolism, adipogenesis and the electron transport chain were highly expressed in 3D cell culture system when compared to those of 2D-cultured cells. Furthermore, it was also shown that the expression levels of proteins associated with metabolic pathways, carbon metabolism and glycolysis/gluconeogenesis were up-regulated, whereas proteins implicated in both DNA replication and the cell cycle were expressed at lower levels compared to those of the 2D mono-cultured cells. Based on these results, the 3D adipocyte model can help elucidate the mechanisms underpinning metabolic syndromes and aid the development of new medical treatments for metabolic disorders.
Project description:Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis. To better understand and quantify how microenvironmental cues influence VIC phenotype and myofibroblast activation, we compared expression profiles of VICs cultured on poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. In general, VICs cultured in hydrogel matrices had lower levels of activation (<10%), similar to levels seen in healthy valve tissue, while VICs cultured on TCPS were ?75% activated myofibroblasts. VICs cultured on TCPS also exhibited a higher magnitude of perturbations in gene expression than soft hydrogel cultures when compared to the native phenotype. Using peptide-modified PEG gels, VICs were seeded on (2D), as well as encapsulated in (3D), matrices of the same composition and modulus. Despite similar levels of activation, VICs cultured in 2D had distinct variations in transcriptional profiles compared to those in 3D hydrogels. Genes related to cell structure and motility were particularly affected by the dimensionality of the culture platform, with higher expression levels in 2D than in 3D. These results indicate that dimensionality may play a significant role in dictating cell phenotype (e.g., through differences in polarity, diffusion of soluble signals), and emphasize the importance of using multiple metrics when characterizing cell phenotype.
Project description:Recently there has been a growing interest in three-dimensional (3D) cell culture systems for drug discovery and development. These 3D culture systems better represent the in vivo cellular environment compared to two-dimensional (2D) cell culture, thereby providing more physiologically reliable information on drug screening and testing. Here we present the quantitative profiling of a drug-induced proteome in 2D- and 3D-cultured colorectal cancer SW480 cells using 2D nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS) integrated with isobaric tags for relative and absolute quantitation (iTRAQ). We identified a total of 4854 shared proteins between 2D- and 3D-cultured SW480 cells and 136/247 differentially expressed proteins (up/down-regulated in 3D compared to 2D). These up/down-regulated proteins were mainly involved in energy metabolism, cell growth, and cell-cell interactions. We also investigated the XAV939 (tankyrase inhibitor)-induced proteome to reveal factors involved in the 3D culture-selective growth inhibitory effect of XAV939 on SW480 cells. We identified novel XAV939-induced proteins, including gelsolin (a possible tumor suppressor) and lactate dehydrogenase A (a key enzyme of glycolysis), which were differentially expressed between 2D- and 3D-cultured SW480 cells. These results provide a promising informative protein dataset to determine the effect of XAV939 on the expression levels of proteins involved in SW480 cell growth.
Project description:MSCs in vivo have a markedly different three-dimensioanal (3D) niche compared to the traditional two-dimensional (2D) culture in vitro. We used microarrays to detail the global difference of gene expression between MSCs cultured on 3D and 2D matrixes Overall design: Total RNA from each group (MSCs in 2D surface culture, MSCs in 3D collagen, chitosan, and PLGA matrixes) was analysed using gene microarrays
Project description:A genomic expression comparison was done among neural progenitor cells cultured on 2D substrates, 3D porous polystyrene scaffolds, and as 3D neural spheres (in vivo surrogate), with the goal of assessing the feasibility of establishing the meaning of 3D and associated physiological relevance at the molecular level Neural progenitor cells were cultured on 2D surfaces, in 3D scaffolds and as 3D neural spheres. Chemical cues are controlled by coating. Only spacial properties of the culture systems were compared.
Project description:Analysis of 2D (transwell) and 3D (collagen type I) cultured MDCK cells and HGF (a MAPK activator). Traditional 2D cultures are fast and inexpensive but do no mimic natural niche/cell environment as well as the more laborious and costly 3D-cultures. 3D cultures, arguably, are better models for the study of developmental processes, such as tubulogenesis. Epithelial organs (such as kidney) develop via tubulogenesis, a process, at least in part, regulated by MAPK signaling. Therefore, 2D and 3D cells also treated with HGF plus MAPK inhibitors. Results provide insights into differential response to HGF-induced tubulogenesis depending on cell culture conditions (2D vs. 3D). 29 samples total: 2D and 3D control (untreated) in quadruplicate, respectively; 2D and 3D + HGF in quadruplicate, respectively; 2D + HGF + PD-98059 in quadruplicate; 3D + HGF + PD-98059 in triplicate; 2D + HGF + U0126 in triplicate; and 3D + HGF + U0126 in triplicate.
Project description:Live-cell assays to measure cellular function performed within 3D cultures have the potential to elucidate the underlying processes behind disease progression and tissue formation. Cells cultured in 3D interact and remodel their microenvironment and can develop into complex structures. We have developed a transcription factor (TF) activity array that uses bioluminescence imaging (BLI) of lentiviral delivered luminescent reporter constructs that allows for the non-invasive imaging of TF activity in both 2D and 3D culture. Imaging can be applied repeatedly throughout culture to capture dynamic TF activity, though appropriate normalization is necessary. We investigated in-well normalization using Gaussia or Renilla luciferase, and external well normalization using firefly luciferase. Gaussia and Renilla luciferase were each unable to provide consistent normalization for long-term measurement of TF activity. However, external well normalization provided low variability and accounted for changes in cellular dynamics. Using external normalization, dynamic TF activities were quantified for five TFs. The array captured expected changes in TF activity to stimuli, however the array also provided dynamic profiles within 2D and 3D that have not been previously characterized. The development of the technology to dynamically track TF activity within cells cultured in both 2D and 3D can provide greater understanding of complex cellular processes.
Project description:Cell culture is an important and necessary process in drug discovery, cancer research, as well as stem cell study. Most cells are currently cultured using two-dimensional (2D) methods but new and improved methods that implement three-dimensional (3D) cell culturing techniques suggest compelling evidence that much more advanced experiments can be performed yielding valuable insights. When performing 3D cell culture experiments, the cell environment can be manipulated to mimic that of a cell in vivo and provide more accurate data about cell-to-cell interactions, tumor characteristics, drug discovery, metabolic profiling, stem cell research, and other types of diseases. Scaffold based techniques such as hydrogel-based support, polymeric hard material-based support, hydrophilic glass fiber, and organoids are employed, and each provide their own advantages and applications. Likewise, there are also scaffold free techniques used such as hanging drop microplates, magnetic levitation, and spheroid microplates with ultra-low attachment coating. 3D cell culture has the potential to provide alternative ways to study organ behavior via the use of organoids and is expected to eventually bridge the gap between 2D cell culture and animal models. The present review compares 2D cell culture to 3D cell culture, provides the details surrounding the different 3D culture techniques, as well as focuses on the present and future applications of 3D cell culture.
Project description:The mechanobiological behavior of mesenchymal stem cells (MSCs) in two- (2D) or three-dimensional (3D) cultures relies on the formation of actin filaments which occur as stress fibers and depends on mitochondrial dynamics involving vimentin intermediate filaments. Here we investigate whether human platelet lysate (HPL), that can potentially replace fetal bovine serum for clinical-scale expansion of functional cells, can modulate the stress fiber formation, alter mitochondrial morphology, change membrane elasticity and modulate immune regulatory molecules IDO and GARP in amnion derived MSCs. We can provide evidence that culture supplementation with HPL led to a reduction of stress fiber formation in 2D cultured MSCs compared to a conventional growth medium (MSCGM). 3D MSC cultures, in contrast, showed decreased actin concentrations independent of HPL supplementation. When stress fibers were further segregated by their binding to focal adhesions, a reduction in ventral stress fibers was observed in response to HPL in 2D cultured MSCs, while the length of the individual ventral stress fibers increased. Dorsal stress fibers or transverse arcs were not affected. Interestingly, ventral stress fiber formation did not correlate with membrane elasticity. 2D cultured MSCs did not show differences in the Young's modulus when propagated in the presence of HPL and further cultivation to passage 3 also had no effect on membrane elasticity. In addition, HPL reduced the mitochondrial mass of 2D cultured MSCs while the mitochondrial mass in 3D cultured MSCs was low initially. When mitochondria were segregated into punctuate, rods and networks, a cultivation-induced increase in punctuate and network mitochondria was observed in 2D cultured MSCs of passage 3. Finally, mRNA and protein expression of the immunomodulatory molecule IDO relied on stimulation of 2D culture MSCs with pro-inflammatory cytokines IFN-? and TNF-? with no effect upon HPL supplementation. GARP mRNA and surface expression was constitutively expressed and did not respond to HPL supplementation or stimulation with IFN-? and TNF-?. In conclusion, we can say that MSCs cultivated in 2D and 3D are sensitive to medium supplementation with HPL with changes in actin filament formation, mitochondrial dynamics and membrane elasticity that can have an impact on the immunomodulatory function of MSCs.
Project description:Purpose: the goal of this study is to detect and compare the transcriptome expression of mouse macrophages cultured in 3D and 2D environments, and find the effect of 3D culture on macrophages compared with traditional 2D culture. Methods: Total RNA was extracted from purified and untreated RAW264.7 cells from 3D and 2D culture systems using TRIzol. The RNA samples were analyzed using Whole Genome Oligo Microarrays. After RNA had been hybridized to the microarray, it was washed and scanned, and data were extracted using Agilent Feature Extraction Software. Gene expression data were generated using Affymetrix GeneChip Human Genome U133 Plus 2.0 on an Affymetrix 3000 instrument running Gene‑Chip operating software. Result: RNA-sequencing (RNA-seq) revealed that after 24 h of culture under the same conditions, 3D-cultured macrophages showed significant differences in gene expression. RNA-seq detected 18580 genes in the 2D group and 15777 genes in the 3D group, among which 6762 were differentially expressed (|log2(fold change)| >= 1, padj < 0.05) in both 3D and 2D groups. A total of 5949 genes were downregulated and 813 were upregulated. Conclusion: Our study represents the first detailed analysis of the effect of 3D culture on mouse macrophages, and the results showed that compared with traditional 2D culture, the gene expression of macrophages under 3D culture was significantly changed.These findings are therefore worthy of further investigation and verification, and provide novel avenues for future research in cytology and macrophages. Overall design: the mRNA profiles of mouse macrophages RAW264.7 in 3D and 2D culture