Ultrahigh‐Uniformity Nanopore Size Filter for Extracellular Vesicle Isolation and In Vitro Dermatological Assessment

ABSTRACT Extracellular vesicles (EVs), including exosomes, are abundant in bovine milk and Lactobacillus culture media but difficult to isolate with high efficiency and purity. In response, a micro‐electro‐mechanical systems (MEMS)‐based membrane filter was developed to address these limitations. Under equivalent conditions, the developed filter outperformed commercial filters, achieving a 2.17‐fold higher EV recovery rate compared to the commercial polyethersulfone (PES) membrane from a 5 mL high‐concentration sample, and yielding a total of 50 mL of EV solution at a concentration of 5.52 × 10 10 particles/mL. The membrane was engineered to achieve a minimum pore size of 32 nm and a minimum thickness of 290 nm through separate fabrication processes. Among these, the MEMS160 membrane, which features uniformly distributed 168 nm pores on a 318 nm thick structure, demonstrated enhanced performance by effectively reducing fouling, as confirmed by blocking‐model assessments. Biological evaluations showed that EVs isolated using the developed filter retained notable purity and bioactivity. Specifically, milk‐derived EVs increased the proliferation of human fibroblasts (Hs68) and human follicle dermal papilla cells (HFDPCs) by up to 25% and 50%, respectively, while Lactobacillus‐derived EVs increased proliferation by up to 11% and 53% at certain concentrations. Furthermore, co‐treatment with an anti‐aging peptide (AIMP1‐derived peptide) had a synergistic effect on both cell types. Similar trends were seen in canine and feline fibroblasts. Milk‐derived EVs boosted proliferation by up to 25% in canine and 31% in feline cells, while Lactobacillus‐derived EVs increased it by up to 46% and 34%, respectively. These effects reached statistical significance. These results show the filter's potential for large‐scale EV isolation and dermatological applications, requiring high purity and yield.