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.

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.

Human platelet lysate (hPL) has high levels of fibrinogen and coagulation factors, which can lead to gel and precipitate formation during storage and cell culture. Heparin derived from animals is commonly added to minimize these risks, but cannot completely eliminate them. Thus, this study proposes an alternative method to prepare fibrinogen-depleted hPL (Fd-hPL) that supports heparin-free expansion of mesenchymal stem cells (MSCs). hPL was added to heparin to prepare heparin-hPL (H-hPL), whilst Fd-hPL was prepared by adding calcium salt to hPL to remove the fibrin clot. The concentrations of calcium, fibrinogen, and growth factors in H-hPL and Fd-hPL were compared. The effects of H-hPL and Fd-hPL on umbilical cord-derived MSCs (UC-MSCs) were assessed. The results showed that Fd-hPL possessed a significantly higher calcium concentration and a lower fibrinogen level than H-hPL. The concentrations of BDNF, TGF-β1, and PDGF-BB showed no significant difference between H-hPL and Fd-hPL, but Fd-hPL had a lower VEGF concentration. Fd-hPL retained the characteristics of UC-MSCs, as it did not affect the cell viability, proliferation, multilineage differentiation potential, or surface marker expression. In conclusion, Fd-hPL effectively supported the in vitro expansion of MSCs without compromising their characteristics, positioning it as a potential substitute for FBS in MSC culture.

Introduction: Research has unveiled the remarkable properties of extracellular vesicles derived from mesenchymal stromal cells (MSCs), particularly in promoting wound healing, aiding re-epithelialization, revitalizing aging skin, and inhibiting hyperpigmentation. However, investigations into the potential of small extracellular vesicles from umbilical cord-derived MSCs (UC-MSC-sEVs) in reducing scarring and preventing hyperpigmentation remain limited. Therefore, this study aims to evaluate the impact of UC-MSC-sEVs on the synthesis of the skin’s extracellular matrix (ECM) and pigmentation using in vitro models. Methods: The study investigated the impact of characterized UC-MSC-sEVs on various aspects including the proliferation, migration, antioxidant activity, and ECM gene expression of human dermal fibroblasts (HDF). Additionally, the effects of UC-MSC-sEVs on the proliferation, melanin content, and tyrosinase (TYR) activity of human melanoma cells (MNT-1) were examined. Furthermore, ex vivo models were employed to evaluate the skin permeation of PKH26-labelled UC-MSC-sEVs. Results: The findings indicated that a high concentration of UC-MSC-sEVs positively influenced the proliferation of HDF. However, no changes in cell migration rate were observed. While the expressions of collagen type 1 and type 3 remained unaffected by UC-MSC-sEVs treatment, there were dose-dependent increases in the gene expressions of fibronectin, matrix metallopeptidase (MMP) 1, and MMP 3. Furthermore, UC-MSC-sEVs treatment did not impact the antioxidative superoxide dismutase (SOD) expression in HDF. Although UC-MSC-sEVs did not alter the proliferation of MNT-1 cells, it did result in a dose-dependent reduction in melanin synthesis without affecting TYR activity. However, when it was applied topically, UC-MSC-sEVs failed to penetrate the skin barrier and remained localized within the stratum corneum layer even after 18 hours. Conclusion: These results highlight the potential of UC-MSC-sEVs in stimulating HDF proliferation, regulating ECM synthesis, and reducing melanin production. This demonstrates the promising application of UC-MSC-sEVs in medical aesthetics for benefits such as scar reduction, skin rejuvenation, and skin lightening.

Background: Quality control (QC) is an important element in ensuring drug substances’ safety, efficacy, and quality. The dosing regimen for sEVs can be in the form of protein concentration or the number of particles based on the results of a series of quality controls applied as in-process control. Methods: Wharton’s Jelly Mesenchymal Stem Cells (WJMSCs) were isolated from four independent umbilical cord samples and were characterized following the International Society for Cellular Therapy (ISCT) guidelines. Small extracellular vesicles (sEVs) were isolated separately from these four WJMSCs samples using the Tangential Flow Filtration (TFF) method and were characterized per Minimal Information for Studies of Extracellular Vesicles (MISEV2018) guidelines. Each isolated and concentrated sEV preparation was standardized and its purity was determined by the ratio of the number of particles to protein concentration. Results: All the WJMSCs samples passed the Mesenchymal Stem Cells (MSCs) characterization QC tests. Qualitatively, EVs-positive markers (CD63 and TSG101) and intact bilipid membrane vesicles were detected in all the sEV preparations. Quantitatively, the protein and particle concentrations revealed that all the sEV preparations were “impure” with < 1.5 × 10⁹ particles/μg protein. Albumin was co-isolated in all the sEV preparations. Conclusion: In short, all characterized and standardized individual and pooled sEV preparations were deemed “impure” due to albumin co-isolation using the TFF method. For therapeutic development, it is essential to report protein and particle concentrations in EV preparations based on these QC results.

Background: Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, leading to elevated blood glucose levels. Cellular therapies offer promise for improving hyperglycemia in T2DM. This retrospective study aimed to assess the clinical effectiveness of intravenous allogeneic umbilical cord-derived mesenchymal stem cells (UC-MSCs) infusion in T2DM patients through various clinical evaluations, focusing on systemic inflammation, metabolic dysfunction, and insulin resistance. Methods: The data from a total of 218 T2DM patients who attended for follow-up after 6 months, and 83 patients after 12 months after receiving 50– 100× 10⁶ allogeneic UC-MSCs were analyzed. Blood and urine samples were collected at baseline and follow-up. Key evaluations included changes in anthropometry, diabetes indices, lipids, liver, renal, hormonal, and inflammatory markers. Results: All patients demonstrated satisfactory outcomes, without adverse effects. Significant reductions in HbA1c levels were observed at 6-months (p< 0.001) and 12-months (p=0.016). Insulin (p=0.048) and HOMA-IR (p=0.007) levels significantly reduced within 6-months, with same trend at 12-months. ALT and GGT levels significantly decreased (p< 0.05), indicating a reduction in liver inflammation. hs-CRP level among patients with higher inflammation were also reduced at 6-months (p=0.073) and significantly at 12-months (p=0.016). Testosterone (p=0.050) and estradiol (p=0.043) levels increased in males and females, respectively, during 12-month follow-up. Additionally, estimated glomerular filtration rate (eGFR) and creatinine levels improved in stage 2 chronic kidney disease (CKD) at 6- and 12-month (p< 0.05), indicating recovered renal function for those in early stage of CKD. Conclusion: Allogeneic UC-MSCs infusion is safe for patients with T2DM and is associated with overall health outcomes, with sustained benefits up to 12 months. Notably, the treatment significantly improved metabolic indices including glycemic control, liver and renal profile and systemic subclinical inflammation. These findings provide a basis for further exploration of UC-MSCs in managing T2DM in proper randomized control trial, by addressing both metabolic dysregulation and inflammation.