The diameter of exosomes usually ranges from 30 nm to 150 nm, and they possess a lipid bilayer membrane structure similar to that of the cell membrane. The interiors of exosomes contain various bioactive molecules, including proteins, nucleic acids (such as mRNA, miRNA, lncRNA, etc.), lipids, and metabolites. Most cells produce exosomes, and different sources of exosomes carry different specific substances, thereby exerting different specific functions.
Exosomes have demonstrated remarkable effects in anti-aging, skin regeneration, and scar repair, and have gradually become a new favorite in the field of medical aesthetics in recent years. The bioactive substances carried by exosomes, such as growth factors and cytokines, can activate cell growth, repair, and exert anti-inflammatory effects. Stem cell culture medium is the main source of exosomes, and commonly used stem cells include adipose-derived mesenchymal stem cells (ADSCs), umbilical cord-derived mesenchymal stem cells (UC-MSCs), and induced pluripotent stem cells (iPSCs). In addition, exosomes derived from lactic acid bacteria and plant cell culture media (such as thyme and schizandra) have also gradually entered the research and application stage.
The separation and purification of exosomes mainly include the clarification and filtration of small exosomes, the concentration of exosomes, the size classification of exosomes, and the desalting and decolorization of exosomes.
The methods for isolating and purifying exosomes commonly include ultracentrifugation, immunoadsorption, ultrafiltration and chromatography, etc.
For small-scale experiments, the use of capsule filters or microfiltration membranes can be employed to retain cells and debris, resulting in a clarified exosome filtrate; then, through ultracentrifugation, an exosome concentrate can be prepared. By repeatedly washing and centrifuging, a more purified exosome can be obtained. Alternatively, size separation can be achieved through gel chromatography, and specific exosomes with higher purity can be obtained through immunoadsorption chromatography.
However, the pilot-scale process has limitations when applied on a large scale. Specifically, it is characterized by low feasibility for process scaling up and a lengthy operation procedure.
Large-scale production can achieve the aforementioned separation and purification purposes by using membrane filtration methods.
Clarification and filtration of exosomes
Usually, the majority of cells are removed by centrifugation first, and then the residual cells and debris in the centrifugation supernatant are removed using a cartridge filter, tangential flow membrane packaging or hollow fiber membranes to obtain a clear exosome filtrate.
Dead-end filters usually have a relatively good initial clarification effect, but they are prone to rapid clogging, which leads to a decrease in filtration flux, membrane pore blockage, and ultimately prevents exosomes from passing through.
Counter-flow filter membrane packs and hollow fiber membranes can achieve control of membrane surface fouling. For the centrifugation supernatant of cell culture solutions with high turbidity, the flow channels of the hollow fiber membranes are wider, which have better pollutant-capturing capacity and are a better choice.
Concentration and purification of exosomes
The exosome filtrate is filtered using ultrafiltration membranes or ultrafiltration hollow fiber membranes to achieve high-magnification concentration of low-concentration exosomes, and to remove inorganic salts and phenolphthalein from the stem cell culture medium.
The pore size distribution of polyethersulfone ultrafiltration membranes is uniform, and they have little adsorption effect on specific proteins, thereby improving the purification yield of exosomes; they have no adverse effects on the structure and function of exosomes, and can ensure the integrity and biological activity of exosomes; the polyethersulfone material has high mechanical strength, good chemical stability such as resistance to acids, alkalis, and cleaning agents, which can ensure the service life and reusability of the membrane, and guarantee the stability of the process.
Opt for ultrafiltration membranes, which can achieve the retention and concentration of exosomes, as well as the filtration and passage of small-molecule impurities, pigments and inorganic salts, thereby improving the purity of exosomes.
Optimize the membrane filtration parameters (shear force, filtration pressure, temperature) to maintain the integrity of exosome morphology and ensure that the particle size remains unchanged.
The membrane filtration method for purifying exosomes has the advantages of large processing capacity, high filtration efficiency, stable process, and the ability to simultaneously scale up the membrane and equipment.
Wuhan Bona has extensive experience in membrane filtration purification engineering. It has successfully achieved continuous clarification filtration, concentration, and removal of small molecule components from various sources of exosomes, earning recognition and praise from industry customers.
