## 2 Biogenesis of Exosomes  
According to the International Society for Extracellular Vesicles (ISEV), extracellular vesicle (EV) is the generic name used to refer to particles naturally released from a cell that is composed of a lipid bilayer and is unable to replicate (lack of functional nucleus). It is difficult to assign an EV to a particular pathway of biogenesis, except that the process was observed using live imaging techniques [5]. The nomenclature of exosomes is derived in 3 different ways: based on their biogenesis, physiological functions within cells, and lastly, empirical definition based on isolation [14]. In this review, exosomes will be defined by their biogenesis. Cells are professionals in producing and exporting molecular products, for instance, transporting insulin to the bloodstream [15]. Before transportation, these molecules are sent first for cell packaging, and they are the exosomes that are first released intracellularly. Multivesicular endosomes (MVEs) also encompass endosomes which are then formed. There are three ways in which exosome biogenesis is formed. First, vesicular budding into discrete endosomes with multivesicular body (MVB) maturation, which releases exosomes after plasma membrane fusion. Second, their immediate release from the plasma membrane by vesicular budding, and lastly, its release is delayed due to budding at the intracellular plasma membrane-connected compartments (IPMCs) after which there is a deconstruction of IPMC necks [16], as shown in Figure 1. Several molecules--such as endosomal sorting complexes required for transport (ESCRT) machinery, tetraspannis, and lipids--were involved in intraluminal vesicle (ILV) biogenesis, [17]. Whereas two different mechanisms, namely inducible release and trans-Golgi network, were assumed to participate in exosome secretion [18]. Intracellular exosomes are fused into MVEs in the cytoplasm. The mechanisms for exosome generation and release have been well described [19]. Interestingly, exosomes are released into the extracellular matrix after the fusion of the MVEs with the cell membrane [20]. The peripheral membrane proteins (Rab GTPases) were found to facilitate the fusion of the MVEs with plasma membranes including RAB11 and RAB35 after which flotillin- and other cell-specific protein-rich exosomes are released [21], while the second mechanism facilitating the release of exosomes loaded with CD63, TSG1010, and ALIX involves RAB27A and RAB27B [19]. Microvesicles undergo a different mode of biogenesis in contrast to exosomes. They are formed by direct blebbing from the plasma membrane. In summary, diverse complex pathways are used to generate exosomes from endosomes. The formation of the exosomes also varies considerably depending on the type as well as the physiological state of the origin of the cell [22]. Figure 1: Biogenesis of exosomes. Limited MVB is subjected to internal budding leading to late endosome formation, which in turn generates exosomes in a constitutive way. The formation of intraluminal vesicles (ILVs) within MVBs from late endosomal membranes is caused by invagination [23]. During invagination, some proteins are incorporated into the membrane and the cytosolic components are engulfed and enclosed in ILVs. The release of ILVs is indicated in the extracellular space after fusion with the exosome's plasma membrane. Degradation of the components occurs in the lysosomes. Canonical exosomes are biconcave in shape produced by artificial drying, but they appeared spherical when viewed using transmission electron microscopy [4]. Recent scientific evidence indicates the role of the alternative pathway in exosomal cargo into MVBs. This process occurs in an ESCRT-independent manner dependent upon microdomains based on raft. These microdomains are enriched by sphingomyelinases that form ceramides [24]. Hydrolytic removal of phosphocholine moiety results in the formation of ceramides which is known to promote lateral phase separation and microdomains coalescence.