Scalable manufacturing system for MSC-EV generation

Abstract

Background & Aim Due to their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. Manufacturing that generates enough EVs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. Moreover, a complementary approach that increases the EV productivity, i.e. the number of EVs produced per cell, could further help to accelerate the development of MSC-EVs as a therapy. Methods, Results & Conclusion We developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of MSC-EVs. Briefly, human bone marrow- or umbilical cord-derived MSCs were rapidly expanded under xeno-free conditions, i.e. >150X expansion within 10 days. Cultures were then switched to our proprietary EV collection medium and EVs were harvested for up to three additional days. To increase the productivity of MSCs, two medium supplements were developed that increased EV yield by either increasing the number of EVs generated per cell in a shortened culture process or increasing the number of collected EVs by lengthening the EV collection culture period. At the end of culture, the EVs in the conditioned media were concentrated using a tangential flow filtration (TFF) system. This scalable MSC-EV manufacturing method was implemented in both 2D flask and 3D bioreactor culture and generated over 2,000 particles per cell in 2D and over 4,000 particles per cell in 3D. With the addition of a medium supplement to increase EVs produced per cell, the EV productivity was increased >2x after 24hrs. Alternatively, EV productivity was also increased >2x by addition of the medium supplement that extended EV collection culture period. MSC-EV success in clinical translation will be reliant on a manufacturing method that can scalably and reliably generate large amounts of EVs. These results present one such solution. Furthermore, increasing EV productivity, for instance by medium supplements that increase EVs per cell or lengthen culture times could further address the limitation of generating the EVs required for development and translation of clinical therapies. Due to their potential as a key bioactive agent in regenerative medicine applications, MSC-derived extracellular vesicles (MSC-EVs) are increasingly being investigated as a clinical therapy. Manufacturing that generates enough EVs for product development and clinical doses is currently a limitation in the field and clearly a scalable manufacturing solution will be necessary for successful translation. Moreover, a complementary approach that increases the EV productivity, i.e. the number of EVs produced per cell, could further help to accelerate the development of MSC-EVs as a therapy. We developed a process that leverages a series of new cell culture reagents to couple to our established cell-media system for scalable manufacturing of MSC-EVs. Briefly, human bone marrow- or umbilical cord-derived MSCs were rapidly expanded under xeno-free conditions, i.e. >150X expansion within 10 days. Cultures were then switched to our proprietary EV collection medium and EVs were harvested for up to three additional days. To increase the productivity of MSCs, two medium supplements were developed that increased EV yield by either increasing the number of EVs generated per cell in a shortened culture process or increasing the number of collected EVs by lengthening the EV collection culture period. At the end of culture, the EVs in the conditioned media were concentrated using a tangential flow filtration (TFF) system.