In‐Drop Thermal Cycling of Microcrystal Assembly for Senescence Control with Minimal Variation in Efficacy

Abstract

The secretome from mesenchymal stem cells (MSCs) have recently gained attention for new therapeutics. However, clinical application requires in vitro cell manufacturing to attain enough cells. Unfortunately, this process often drives MSCs into a senescent state that drastically changes cellular secretion activities. Antioxidants are used to reverse and prevent the propagation of senescence; however, their activity is short‐lived. Polymer‐stabilized crystallization of antioxidants has been shown to improve bioactivity, but the broad crystal size distribution (CSD) significantly increases the efficacy variation. Efforts are made to crystalize drugs in microdroplets to narrow the CSD, but the fraction of drops containing at least one crystal can be as low as 20%. To this end, this study demonstrates that in‐drop thermal cycling of hyaluronic acid‐modified antioxidant crystals, named microcrystal assembly for senescence control (MASC), can drive the fraction of microdrops containing crystals to >86% while achieving significantly narrower CSDs (13 ± 3 µm) than in bulk (35 ± 11 µm). Therefore, this approach considerably improves the practicality of CSD‐control in drops. In addition to exhibiting uniform release, MASC made with antioxidizing N‐acetylcysteine extends the release time by 40%. MASC further improves the restoration of reactive oxygen species homeostasis in MSCs, thus minimizing cellular senescence and preserving desired secretion activities. It is proposed that MASC is broadly useful to controlling senescence of a wide array of therapeutic cells during biomanufacturing.

Delivery-mediated exosomal therapeutics in ischemia–reperfusion injury: advances, mechanisms, and future directions

“…Furthermore, the standardization of exosome manufacturing including production, isolation, and storage is vital. To increase natural exosome production, many strategies have been adopted including inducing hypoxia [ 147 ], increasing the volume of cell culture from flasks to containers, bioreactors, or hollow fibers [ 148 , 149 ], and reversing quiescence and prevent senescence of MSCs [ 150 ]. In this context, efforts for reliable extraction and purification of exosomes are being made by combining differential ultracentrifugation, precipitation, and filtration methods [ 151 ].…”