Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D‐Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity

Abstract

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV‐associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D‐printed scaffold‐perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow‐derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40‐80‐fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture‐generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D‐printing technologies.

Advances in the application of extracellular vesicles derived from three-dimensional culture of stem cells

“…Although 3D culture can increase the yield of SC-EVs and improve their activity in some aspects to enhance their effects, different 3D culture methods result in different biophysical factors such as shear stress and mechanical structure [ 143 ], which have a significant impact on the properties of EVs and lead to the production of EVs with large differences. However, there are many different 3D culture methods at present, and even the EVs produced by the same culture method still need to be further characterized.…”

“…For instance, UCMSCs cultured in hollow fiber bioreactor have been found to produce 7.5-fold higher amounts of exosomes compared to those produced by 2D culture flasks [ 142 ]. Additionally, Kronstadt et al reported that 3D printed scaffold-perfused bioreactor culture increased the yield of BMMSC-EVs by approximately 40- to 80-fold (depending on the measurement method) compared to conventional 2D cell culture [ 143 ]. The production of exosomes by hBMMSCs in 3D culture using hang-drop and poly (2-hydroxyethyl methacrylate) coating methods was also found to be approximately twofold and 2.4-fold higher, respectively, compared to 2D monolayer culture [ 144 ].…”

“…Furthermore, 3D-EVs showed superior bioactivity [ 143 , 145 ]. Haraszti et al confirmed that exosomes derived from 3D cultured UCMSCs, obtained by tangential flow filtration, exhibited a seven-fold greater ability to transfer siRNA to neurons compared to 2D-Exos obtained by differential ultracentrifugation [ 32 ].…”

“…In a diabetic mouse wound-healing model, BMMSCs were cultured using a 3D-printed scaffold perfusion bioreactor, and it was discovered that their EVs significantly promoted wound healing and neoangiogenesis. In contrast, EVs derived from 2D flask cultured BMMSCs did not have this effect [ 143 ]. Similar results were observed by Yuan et al and Jeske et al, who demonstrated that EVs derived from BMMSCs cultured in 3D aggregation wave reactor and a novel microcarrier-based vertical-wheel bioreactor were able to promote cell growth and accelerate wound closure faster than 2D-EVs in an in vitro wound healing model using primary human dermal fibroblasts [ 139 , 148 ].…”

Mesenchymal stromal cell-derived extracellular vesicles therapy openings new translational challenges in immunomodulating acute liver inflammation

“…For instance, the impact of a 3D-printed scaffold-perfusion bioreactor system on the production and bioactivity of EVs secreted by MSCs has been explored. Findings suggest that culture in a perfusion bioreactor can yield an approximate 40–80-fold increase in MSC EVs production, varying with the measurement technique, compared to traditional cell culture methods [ 79 ]. Although the application of MSC EVs produced via this system in liver disease has not yet been fully evaluated, the approach holds substantial potential for future therapeutic interventions.…”