Background Extracellular vesicles (EVs) play important roles in intercellular communication by delivering RNA, lipid, and proteins to neighboring or distant cells. Identification and classification of EVs secreted from diverse cell types are essential for understanding their signaling properties. Methods In this study, EVs from the culture media were isolated by ultracentrifugation and analyzed by electron microscopy (EM) and nanoparticle tracking analyses. Conditioned media (CM) from HEK293 cells culture grown either in serum-free (SF) or 10% fetal bovine serum (FBS) containing media were centrifuged at 100,000×g to separate the SN Δ supernatant and the P100 pellet in which exosomes are enriched. Then, the SN Δ fraction was centrifuged at 200,000×g to yield the P200 pellet fraction containing novel EVs smaller than exosomes. The exosomal markers in the EV subgroups were examined by western blotting and immune-EM, and the functional analyses of EVs were conducted on HEK293 and THP-1 cell culture. Results We identified a new group of EVs in the P200 fraction that was smaller than exosomes in size. Typical exosome markers such as Hsp70, TSG101, and CD63 were found in both P100 exosomes and the P200 vesicles, but CD81 was highly enriched in exosomes but not in the P200 vesicles. Furthermore, chemicals that inhibit the major exosome production pathway did not decrease the level of P200 vesicles. Therefore, these small EVs indeed belong to a distinguished group of EVs. Exosomes and the P200 vesicles were found in CM of human cell lines as well as FBS. Addition of the exosomes and the P200 vesicles to human cell cultures enhanced exosome production and cell proliferation, respectively. Conclusions Our study identifies a novel population of EVs present in the P200 fraction. This EV population is distinguished from exosomes in size, protein contents, and biogenesis pathway. Furthermore, exosomes promote their own production whereas the P200 vesicles support cell proliferation. In sum, we report a new group of EVs that are distinct physically, biologically and functionally from exosomes. Electronic supplementary material The online version of this article (10.1186/s12964-019-0401-z) contains supplementary material, which is available to authorized users.
Reliable measurement of the amount of melanin produced by melanocytes is essential to study various skin disorders and to evaluate the efficacy of candidate reagents for such disorders or for whitening purposes. Conventional melanin quantification methods are based on absorption spectroscopy, which measures the melanin from lysed cells grown on two-dimensional (2D) surfaces. The 2D culture environment is intrinsically different from in vivo systems though, and therefore cells often lose their original phenotypes. Melanocytes in particular lose their ability to synthesize melanin, thereby requiring melanogenesis stimulators such as alpha-melanocyte stimulating hormone (α-MSH) to promote melanin synthesis. In this study, we compared melanin synthesis in B16 murine melanoma cells grown in 2D and three-dimensional culture environments. B16 cells instantly formed an aggregate in a hanging-drop culture, and synthesized melanin efficiently without treatment of α-MSH. We were able to measure the melanin secreted from a single melanocyte aggregate, indicating that our method enables non-invasive long-term monitoring of melanin synthesis and secretion in a high-throughput format. We successfully tested the developed platform by quantifying the depigmenting effects of arbutin and kojic acid.
Quantitative and reliable measurement of cellular invasion is important to understand a range of biological processes such as cancer metastasis and angiogenesis. Spheroid invasion assays are an attractive in vitro platform because they effectively mimic the tumor cell invasion of solid tissues. Here, we developed an image analysis-based method to quantify the invasiveness of HT1080 human fibrosarcoma tumor cell spheroids. We segmented a cell-covered area into three subareas using objectively set threshold pixel intensities and calculated invasion indices using these subareas. comparison with conventional parameters for spheroid invasion assays, such as area, length, and detached cells, showed that our indices present the invasion event at an early time and without being convoluted by proliferation. As an application, we then examined paracrine interactions between LLC1 mouse lung carcinoma cells and Raw264.7 mouse macrophage cells with our developed analysis method. We found that the invasion of tumor spheroids was increased by a macrophage-conditioned medium, concomitantly with a decrease in tumor cell proliferation. importantly, invasion was further enhanced by a conditioned medium from activated macrophages by co-culture with tumor cells. thus, our indices reveal that tumor cell invasion is facilitated in a feed-forward manner by communication between tumor cells and macrophages in the tumor microenvironment.Cellular migration and invasion are essential in many biological processes such as cancer metastasis 1-3 and angiogenesis by endothelial cells 4 . Several assays have been developed to measure cellular invasion 3 ; basically, the invasiveness of cells is measured by the distance or the path that cells travel in different setups such as Boyden chambers or wound healing assays. However, the cells are generally grown in two-dimensional (2D) in vitro space, which does not fully recapitulate the behavior of cells grown in three-dimensional (3D) in vivo tissue. Recently, spheroid-based 3D cultures have been widely employed in many applications, particularly drug discovery, as an in vivo-mimicking cellular platform 5-9 . In spheroid culture, cellular interactions among cells or with the extracellular matrix are better preserved 10,11 , and in addition, the physiological gradients inside of the tumor spheroid, such as oxygen, pH, and nutrients, create an environment that better mimics in vivo.To date, most analyses of the invasiveness of cells grown in spheroids have been qualitative and descriptive rather than quantitative [12][13][14] . Raw data of cellular invasion usually takes the form of micrographs that are taken from bright field microscopy, with subsequent quantitative analysis of the micrographs relying on computational image processing accompanied by an impartial determination of the key parameters. Although there are good examples of image analysis of bright field micrographs, such as yeast growth 15 and stem cell proliferation 16 analyses, accurate quantitation based on bright field micrographs is cha...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2025 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.