dc.description.abstract | Exosomes are small vesicle (30-200 nm) encapsulated by a lipid bilayer which contains biomarkers such protein, DNA, and RNA inside. They play a crucial role in the detection of diseases, cancer progression and metastasis. Due to this, exosome isolation techniques have gained considerable attention, especially when liquid biopsy has played a profound role in diagnosis nowadays. Different isolation methods include ultracentrifugation, polymer precipitation, immunoaffinity capture, ultrafiltration and so on have been proposed and adapted in applications. However, most of these methods require large and expensive instruments. Moreover, centrifugation technique can potentially destroy the exosomes, generating lower recovery. In this regard, we aim to develop an easy and efficient technique by means of membrane filtration to isolate exosomes from HCT116 cell culture medium, and human platelet poor plasma (PPP). Herein, we develop a dual layer membrane filtration process endowed with antifouling property to increase the yield and purity of exosome isolation.
The first layer is a porous membrane made from a blend of polyvinylidene fluoride (PVDF) and zwitterionic polystyrene poly 4-Vinylpyridine (PS-r-zP4VP) that was used to filter out large contaminants. The obtained permeate passed through the second layer based on sulfobetaine methacrylate (SBMA) hydrogel composite cellulose triacetate (CTA) membrane which was utilized to remove smaller contaminants. The final retentate was collected to analyze exosomes. Size distribution and concentration of the retentate were evaluated using NTA, morphology was assessed by FE-SEM, and immunoaffinity of the exosomes were measured by ELISA. Furthermore, protein concentration of the isolate samples was determined through BCA assay to analyze the purity of the exosomes.
The membrane filtration system was able to provide more than 90% exosome recovery with increase exosome concentration by 1.8-fold by comparing with input PPP. In contrast, conventional method using centrifugal ultrafiltration and ultracentrifugation only yielded 60% recovery, 1.2-fold concentrate and 30% recovery, 0.8-fold concentrate. Hence, these results indicate that dual membrane filtration method can provide a promising approach to isolate exosomes in a more economical and efficient manner. | en_US |