Yeo, J. H. | Search bioRxiv

bioRxiv2023

Serum albumin maintains Wnt water-solubility and activity

Wnt proteins regulate adult tissue homeostasis and repair by driving stem cell self-renewal and differentiation. High-performance Wnt preparations have enormous therapeutic potential, especially alongside various stem cell technologies. Currently, most of these Wnt preparations contain FBS or the detergent CHAPS to maintain Wnt solubility and activity. Recently, afamin was identified as a serum factor that solubilizes Wnt3a in conditioned media (CM), obviating the requirement for animal sera. Here, we report serum albumin (SA) is required for afamin-mediated solubilization of Wnt3a in CM. Moreover, SA-mediated solubilization of purified Wnt3a in tubes does not require afamin. This means conventional CHAPS-Wnt3a preparations can be modified into SA-purified Wnt3a (SA-pWnt3a) preparations by exchanging CHAPS for SA through dialysis. SA-pWnt3a preparations effectively promote the growth of human stem cell organoids. These data suggest SA as a physiological factor for maintaining Wnt3a activity in therapeutic applications.

biochemistry↗

bioRxiv2021

Nanocharacterisation of extracellular vesicles uncovers the correlation between isolation methods, physicochemical composition and their biological function

Extracellular vesicles (EVs) have been lauded as next generation medicines, but very few EV-based therapeutics have progressed to clinical use. Limited clinical translation is largely due to technical barriers that hamper our ability to mass-produce EVs, i.e. to isolate, purify and characterise them effectively. Technical limitations in comprehensive characterisation of EVs leads to unpredicted biological effects of EVs. Here, using a range of optical and non-optical techniques, we showed that the differences in molecular composition of EVs isolated using two isolation methods correlated with the differences in their biological function. Our results demonstrated that the isolation method determines the composition of isolated EVs at single and sub-population levels. Besides the composition, we measured for the first time the dry mass and predicted sedimentation of EVs. These parameters were shown to correlate well with the biological and functional effects of EVs on single cell and cell cultures. We anticipate that our new multiscale characterisation approach, which goes beyond traditional experimental methodology, will support fundamental understanding of EVs as well as elucidate the functional effects of EVs in in vitro and in vivo studies. Our findings and methodology will be pivotal for developing optimal isolation methods and establishing EVs as mainstream therapeutics and diagnostics. This innovative approach is applicable to a wide range of sectors including biopharma and biotechnology as well as to regulatory agencies.

cell biology↗