GMP‐compliant manufacturing of biologically active cell‐derived vesicles produced by extrusion technology

Abstract

Extracellular vesicles (EVs) released by a variety of cell types have been shown to act as a natural delivery system for bioactive molecules such as RNAs and proteins. EV therapy holds great promise as a safe and cell‐free therapy for many immunological and degenerative diseases. However, translation to clinical application is limited by several factors, including insufficient large‐scale manufacturing technologies and low yield. We have developed a novel drug delivery platform technology, BioDrone™, based on cell‐derived vesicles (CDVs) produced from diverse cell sources by using a proprietary extrusion process. This extrusion technology generates nanosized vesicles in far greater numbers than naturally obtained EVs. We demonstrate that the CDVs are surrounded by a lipid bilayer membrane with a correct membrane topology. Physical, biochemical and functional characterisation results demonstrate the potential of CDVs to act as effective therapeutics. Umbilical cord mesenchymal stem cell (UCMSC)‐derived CDVs exhibit a biological activity that is similar to UCMSCs or UCMSC‐derived EVs. Lastly, we present the establishment of a GMP‐compliant process to allow the production of a large number of UCMSC‐CDVs in a reproducible manner. GMP‐compliant manufacturing of CDVs will facilitate the preclinical and clinical evaluation of these emerging therapeutics in anti‐inflammatory or regenerative medicine. This study also represents a crucial step in the development of this novel drug delivery platform based on CDVs.

Formation and Investigation of Cell‐Derived Nanovesicles as Potential Therapeutics against Chronic Liver Disease

“…[ 61 ] Additionally, a switch from harsh purification techniques such as ultracentrifugation to more gentle and scalable techniques, such as tangential flow filtration needs to progress, which has already started in the EV and EV‐mimetic field. [ 34,62,63 ]…”

“…Also, the addition of more extrusion cycles as well as extrusion through membranes with smaller pores should be considered, as it has already successfully been done by other groups. [34,40,60] TEM microscopy as well as physicochemical characterization revealed a spherical shape of the particles with a nanoranged diameter and narrow size distribution. The cdNVs, derived from all tested conditions, induced a concentration-dependent deactivation in naïve and TGF-𝛽 1 activated LX-2 cells, restoring parts of the lost lipid droplets, and decreasing COL1A1, as well as SPARC mRNA transcription.…”

“…[55][56][57] Artificial EVs have been previously described to preserve the targeting capacity of natural EVs. [34,58] To evaluate if cdNVs show an enhanced uptake in LX-2 cells and to determine whether it is attributed to the membrane components and structure, a comparison was made between the uptake of cdNV, cdNV -L, PL, PL_200 by LX-2 cells after 24 h. The particles were labeled with the hydrophobic nonexchangeable dye DiR and the uptake was measured via flow cytometry. The labeling efficiency for cdNVs and cdNV-Ls was 65-70% (Figure 7a).…”

“…[ 30 ] Inspired by the biological characteristics of natural EVs, engineered nanovesicles have demonstrated the ability to retain key biological and therapeutic features while addressing production, scalability, and standardization issues. [ 31–34 ]…”

“…[30] Inspired by the biological characteristics of natural EVs, engineered nanovesicles have demonstrated the ability to retain key biological and therapeutic features while addressing production, scalability, and standardization issues. [31][32][33][34] In a prior study conducted by our group, we demonstrated that EVs released from quiescent-like LX-2 cells [35] induced an antifibrotic shift in LX-2 cells. [36] Inspired by these findings, we explored the potential of generating particles from quiescent LX-2 cells as a therapeutic approach against chronic liver disease.…”