Extracellular vesicles (EVs) are minute vesicles with lipid bilayer membranes. EVs are secreted by cells for intercellular communication. Recently, EVs have received much attention, as they are rich in biological components such as nucleic acids, lipids, and proteins that play essential roles in tissue regeneration and disease modification. In addition, EVs can be developed as vaccines against cancer and infectious diseases, as the vesicle membrane has an abundance of antigenic determinants and virulent factors. EVs for therapeutic applications are typically collected from conditioned media of cultured cells. However, the number of EVs secreted by the cells is limited. Thus, it is critical to devise new strategies for the large-scale production of EVs. Here, we discussed the strategies utilized by researchers for the scalable production of EVs. Techniques such as bioreactors, mechanical stimulation, electrical stimulation, thermal stimulation, magnetic field stimulation, topographic clue, hypoxia, serum deprivation, pH modification, exposure to small molecules, exposure to nanoparticles, increasing the intracellular calcium concentration, and genetic modification have been used to improve the secretion of EVs by cultured cells. In addition, nitrogen cavitation, porous membrane extrusion, and sonication have been utilized to prepare EV-mimetic nanovesicles that share many characteristics with naturally secreted EVs. Apart from inducing EV production, these upscaling interventions have also been reported to modify the EVs’ cargo and thus their functionality and therapeutic potential. In summary, it is imperative to identify a reliable upscaling technique that can produce large quantities of EVs consistently. Ideally, the produced EVs should also possess cargo with improved therapeutic potential.

Exosomes are the small extracellular vesicles secreted by cells for intercellular communication. Exosomes are rich in therapeutic cargos such as microRNA (miRNA), long non-coding RNA (lncRNA), small interfering RNA (siRNA), DNA, protein, and lipids. Recently, many studies have focused on miRNAs as a promising therapeutic factor to support cartilage regeneration. Exosomes are known to contain a substantial amount of a variety of miRNAs. miRNAs regulate the post-transcriptional gene expression by base-pairing with the target messenger RNA (mRNA), leading to gene silencing. Several exosomal miRNAs have been found to play a role in cartilage regeneration by promoting chondrocyte proliferation and matrix secretion, reducing scar tissue formation, and subsiding inflammation. The exosomal miRNA cargo can be modulated using techniques such as cell transfection and priming as well as post-secretion modifications to upregulate specific miRNAs to enhance the therapeutic effect. Exosomes are delivered to the joints through direct injection or via encapsulation within a scaffold for sustained release. To date, exosome therapy for cartilage injuries has yet to be optimized as the ideal cell source for exosomes, and the dose and method of delivery have yet to be identified. More importantly, a deeper understanding of the role of exosomal miRNAs in cartilage repair is paramount for the development of more effective exosome therapy.

Facial aesthetics involve the application of non-invasive or minimally invasive techniques to improve facial appearance. Currently, extracellular vesicles (EVs) are attracting much interest as nanocarriers in facial aesthetics due to their lipid bilayer membrane, nanosized dimensions, biological origin, intercellular communication ability, and capability to modulate the molecular activities of recipient cells that play important roles in skin rejuvenation. Therefore, EVs have been suggested to have therapeutic potential in improving skin conditions, and these highlighted the potential to develop EV-based cosmetic products. This review summarizes EVs’ latest research, reporting applications in facial aesthetics, including scar removal, facial rejuvenation, anti-aging, and anti-pigmentation. This review also discussed the advanced delivery strategy of EVs, the therapeutic potential of plant EVs, and clinical studies using EVs to improve skin conditions. In summary, EV therapy reduces scarring, rejuvenates aging skin, and reduces pigmentation. These observations warrant the development of EV-based cosmetic products. However, more efforts are needed to establish a large-scale EV production platform that can consistently produce functional EVs and understand EVs’ underlying mechanism of action to improve their efficacy.

Multipotent mesenchymal stromal cells (MSCs) hold promises for cell therapy and tissue engineering due to their self-renewal and differentiation abilities, along with immunomodulatory properties and trophic factor secretion. Extracellular vesicles (EVs) from MSCs offer similar therapeutic effects. However, MSCs are heterogeneous and lead to variable outcomes. In vitro priming enhances MSC performance, improving immunomodulation, angiogenesis, proliferation, and tissue regeneration. Various stimuli, such as cytokines, growth factors, and oxygen tension, can prime MSCs. Two classical priming methods, interferon-gamma (IFN-γ) and hypoxia, enhance MSC immunomodulation, although standardized protocols are lacking. This review discusses priming protocols, highlighting the most commonly used concentrations and durations, along with mechanisms and in vivo therapeutics effects of primed MSCs and their EVs. The feasibility of up-scaling their production was also discussed. The review concluded that priming with IFN-γ or hypoxia (alone or in combination with other factors) boosted the immunomodulation capability of MSCs and their EVs, primarily via the JAK/STAT and PI3K/AKT and Leptin/JAK/STAT and TGF-β/Smad signalling pathways, respectively. Incorporating priming in MSC and EV production enables translation into cell-based or cell-free therapies for various disorders.

The scarcity of chondrocytes is a major challenge for cartilage tissue engineering. Monolayer expansion is necessary to amplify the limited number of chondrocytes needed for clinical application. Growth factors are often added to improve monolayer culture conditions, promoting proliferation, and enhancing chondrogenesis. Limited knowledge on the biosafety of the cell products manipulated with growth factors in culture has driven this study to evaluate the impact of growth factor cocktail supplements in chondrocyte culture medium on chondrocyte genetic stability and tumorigenicity. The growth factors were basic fibroblast growth factor (b-FGF), transforming growth factor β2 (TGF β2), insulin-like growth factor 1 (IGF-1), insulin-transferrin-selenium (ITS), and platelet-derived growth factor (PD-GF). Nasal septal chondrocytes cultured in growth factor cocktail exhibited a significantly high proliferative capacity. Comet assay revealed no significant DNA damage. Flow cytometry showed chondrocytes were mostly at G0-G1 phase, exhibiting normal cell cycle profile with no aneuploidy. We observed a decreased tumour suppressor genes’ expression (p53, p21, pRB) and no TP53 mutations or tumour formation after 6 months of implantation in nude mice. Our data suggest growth factor cocktail has a low risk of inducing genotoxic and tumorigenic effects on chondrocytes up to passage 6 with 16.6 population doublings. This preclinical tumorigenicity and genetic instability evaluation is crucial for further clinical works.

Mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEVs) have shown great promise in promoting tissue repair, including skin wound healing, but challenges like rapid degradation and short retention have limited their clinical application. Hydrogels have emerged as effective carriers for sustained EV release. Three-dimensional printing enables the development of personalized skin substitutes tailored to the wound size and shape. This study aimed to develop 3D bioprinted gelatin–genipin hydrogels incorporating human umbilical cord MSC-sEVs (hUCMSC-sEVs) for future skin wound healing applications. Gelatin hydrogels (8% and 10% w/v) were crosslinked with 0.3% genipin (GECL) to improve stability. The hydrogels were evaluated for their suitability for extrusion-based 3D bioprinting and physicochemical properties, such as the swelling ratio, hydrophilicity, enzymatic degradation, and water vapor transmission rate (WVTR). Chemical characterization was performed using EDX, XRD, and FTIR. The hUCMSC-sEVs were isolated via centrifugation and tangential flow filtration (TFF) and characterized. The crosslinked hydrogels were successfully 3D bioprinted and demonstrated superior properties, including high hydrophilicity, a swelling ratio of ~500%, slower degradation, and optimal WVTR. hUCMSC-sEVs, ranging from 50 to 200 nm, were positive for surface and cytosolic markers. Adding 75 μg/mL of hUCMSC-EVs into 10% GECL hydrogels significantly improved the biocompatibility. These hydrogels offer ideal properties for 3D bioprinting and wound healing, demonstrating their potential as biomaterial scaffolds for skin tissue regeneration applications.