(580d) Scalable Induced Pluripotent STEM Cell (IPSC)-Based Platform to Produce Tissue-Specific Extracellular Vesicles (EVs)

Extracellular vesicles (EVs) are naturally produced nanosized phospholipid bilayer bound vesicles that have the potential to target currently undruggable locations such as the brain because of their unique ability to cross the blood brain barrier (BBB)1. EVs are membrane derived particles that can carry proteins, lipids, nucleic acids, and other biomolecules throughout the body2. They are secreted by a broad range of cell types and play a crucial role in numerous physiological and pathological processes in the body and provide a safer alternative to cell-based therapies. While EVs exhibit massive potential, collecting enough for even a single dose of a treatment is resource-, cost-, and labor-intensive because many clinically relevant cell lines are typically grown in a 2D adherent monolayer culture rather than a 3D suspension culture3. Getting enough EVs from these cell types for new treatments targeting a variety of diseases poses a major problem for the field. Therefore, there is an urgent need to develop a platform for growing adherent cells in suspension culture which will increase EVs per volume and eventually be scaled up further into bioreactors for therapeutic treatments.

Mesenchymal stem cells (MSCs) naturally produce therapeutically relevant EVs that have potential to treat many diseases such as graft versus host disease, rheumatoid arthritis, and Crohn’s disease. However, primary MSCs vary extensively between different tissues and donors, have limited replicative potential, and are typically grown in 2D monolayers. MSCs differentiated from induced pluripotent stem cells (iPSCs) can provide a reproducible non-invasive source of MSCs that avoids obstacles due to donor-to-donor variability and scarcity of human tissue. This study aims to develop a scalable platform to produce EVs from tissue-specific cell types, using iPSC-derived MSCs as a proof of concept. We are optimizing EV production by adapting MSCs into a pseudo-suspension culture using GelMA-Cad hydrogel microspheres. GelMA-Cad is gelatin with an N-cadherin attached to it to support cell growth by mimic cell adhesion to the extracellular matrix. We have shown that GelMA-Cad supports cell growth in both 2D and 3D culture. By seeding MSCs into hydrogel microspheres, adherent cells can be grown in a pseudo-suspension culture allowing for increased cell density and EV production per volume of culture. Consistently sized microspheres are formed using a custom microfluidic device to support efficient cell growth and EV production. MSC growth and EV production has been confirmed in GelMA-Cad layers, and EV yield and particles size was compared to traditional 2D adherent cultures. Here, we found that MSCs seeded in GelMA-Cad produced a similar number of small EVs but produced a greater number of large EVs than MSCs grown in a 2D adherent culture. A key objective of this study is to determine if the production of EVs from MSCs embedded in GelMA-Cad leads to higher volumetric productivity and greater reproducibility than adherent cultures.

Future work will expand this platform to other iPSC-derived cell types beyond MSCs to neurons. By using EVs derived from neurons, the EVs will be able to cross the BBB and home back to their cell line of origin. Therefore, the long-term goal is to develop novel strategies to collect sufficient EVs for clinical treatments from any cell line of interest.