Advancing the Manufacture of Mesenchymal Stromal Cell-Derived Extracellular Vesicles (MSC-EVs) in Stirred-Tank Bioreactors

Dr. Marta Costa, Senior Researcher at the Instituto de Biologia Experimental e Tecnológica
Dr. Marta Costa

Dr. Marta Costa is a Senior Researcher at the Instituto de Biologia Experimental e Tecnológica (iBET) and holds a PhD in Bioengineering (MIT Portugal Program, pursued at Instituto Superior Técnico and Georgia Institute of Technology, 2012 - 2017). Over the last 12 years, Dr. Costa has focused on the development of novel bioprocesses to manufacture cell-based products both in academic (Instituto Superior Técnico, Georgia Institute of Technology) and in industrial settings (Cell and Gene Therapy Catapult, London, UK).

Dr. Marta Costa is working to overcome the challenges of generating extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs), and recently her focus has been scaling up MSC-EV manufacturing. We had the opportunity to talk to Dr. Costa about her latest publication, her journey to this field of research, and the future of MSC-EV manufacturing.


Topics:

  • Mesenchymal Stromal Cell Scale-Up
  • Extracellular Vesicles

A scientist is someone who needs to be free to the point that they need to be able to refute their own ideas and accept that they might be wrong. But we also need to think about solutions to scientific questions. And we always need to be searching for a “why”.

Dr. Marta Costa

Marta as a Cell Therapy Scientist

What inspired you to become a scientist?

I was never one of those kids who knew from the very beginning what they would like to be when they grew up. However, I was into reading and writing, and, at primary school, I had an amazing teacher who exposed me to a poem that said something like, “Studying is not just reading books in school, it is also learning to be free, without foolish ideas”. In summary, the main idea was that it is very important that we are capable of thinking. And I think of scientists in a way to be a reflection of this. A scientist is someone who needs to be free to the point that they need to be able to refute their own ideas and accept that they might be wrong. But we also need to think about solutions to scientific questions. And we always need to be searching for a “why”. And I think this was something that really was very captivating to me. So, when the critical time to make a decision on what to study arrived, my decision was not based on what I would like to be as a professional, but rather what I would like to study, investigate deeper, and I guess probably, in a way, this was already telling that I would like to become a scientist. And so, being fascinated by the mechanisms that are not visible to the naked eye but that explain life, I picked Biochemistry as the discipline that I would like to study.

Can you share your scientific journey and what led you to your current field of research?

I'm currently a Senior Scientist at iBET, based in Portugal. Regarding my background, I started with a bachelor's in biochemistry, then pursued a master's degree in a more applied area: bioengineering and nanosystems. There, I began connecting with the cell therapy world, specifically working with stem cells. I decided to pursue a PhD in this field working in two institutions, Instituto Superior Técnico (Portugal) and the Georgia Institute of Technology (USA). After my PhD, I moved to London to work at Cell and Gene Therapy Catapult, focusing on exploring bioengineering tools to develop cell therapies. A few years later, I returned to Portugal and, about four years ago, joined iBET.

Could you describe the main focus of your lab's work?

I'm working in the Stem Cell Bioengineering lab at iBET, in collaboration with both academic and industrial partners. We're focused on bridging engineering and cell biology to accelerate the development of next-generation cell therapies. Generally, we work with stem cells, so iPSCs, hematopoietic, and mesenchymal stromal cells, but also with immunotherapies. We explore bioengineering strategies to essentially boost the therapeutic features of cells and their derived products, like the extracellular vesicles, which we will probably talk about quite soon so that we can, overall, shorten the gap between scientific research and application in clinical settings. This is, very briefly, what we do in our lab.

What aspects of scientific research do you find most enjoyable and most challenging?

As a researcher, it's the diversity of activities that we are exposed to that makes our job very enjoyable. So, we are not only designing experiments but also performing them and critically analyzing the results to help us better delineate our research questions. But, we are also writers and communicators, as we need to present our results in several meetings and conferences, or in interviews such as this one. But we are also teachers, forming Master's and PhD students. And probably, more importantly, we are also learners. And I think it is this multitude of tasks that's really enjoyable in our life as scientists, and particularly for me.

Regarding the most challenging feature of our daily routine, I'd say it probably is when we need to deal with a lot of paperwork in our job, as it is stealing time away from our main scientific purpose.

Dr. Costa’s Latest Research

Can you summarize the key findings from your recent publication “Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors” and discuss its significance in scaling up MSC-EV production?

In terms of methodology, we have implemented scalable processes, both from the upstream and downstream side, with the potential to be integrated into continuous production of extracellular vesicles. In this case, we developed a process exploring cell culture in stirred tank bioreactors, but also EV isolation by tangential flow filtration, followed by size exclusion chromatography. By integrating scalable methods, we were able to obtain higher EV yields. This was one of the major contributions, but we also explored the role that Process Analytical Technology tools like Raman spectroscopy can play in biomanufacturing cells and cell-derived products. In this case, we were able to highlight that, continuously monitoring cell culture parameters (in our specific case, metabolites such as glucose) could help us decide on optimal times for cell harvest and EV collection. Another important aspect of this work was that, by using a chemically defined medium, we could manufacture, in parallel, not only mesenchymal stromal cells but also their derived EVs without impacting their critical quality attributes. So, it was a win-win.

How long did it take to develop and optimize the workflow described in your recent publication?

It took us roughly one year. But of course, I think we also benefited from the fact that at iBET, we already have strong knowledge in bioprocessing, but also in bioanalytics. So, this really allows us to streamline the approach to many of the research questions that were posed during our work.

What are the main challenges in using suspension culture to generate EVs from MSCs? Are these challenges for the whole field or specific to your research?

I think that when we work at a small scale, it's probably easier to think of performing cell culture in 2D static systems that rely on adherent cultures. But the thing is, although traditional systems are simple, they become a huge disadvantage when we move our process toward the manufacture of cells for therapies. These protocols need to be closer to clinical translation if we really want to impact the field. Suspension cultures to generate EVs from MSCs or other cell types provide us a stronger opportunity to scale up the manufacture of EVs. Additionally, by exploring suspension cultures in bioreactors that are amenable to the incorporation of PAT tools, we can ensure more homogeneous cell culture than in 2D adherent platforms while allowing us to closely monitor critical process parameters. This will, ultimately, contribute to generating safer and more robust cell and cell-derived products. Although it might take a bit longer to establish the bioprocess in suspension cultures, I think that the effort is definitely worth it as down the road, we will see major benefits. The only thing that we need to include as an extra step compared to culturing adherent cells in cell factories, for instance, is implementing technologies that allow us to more easily separate EVs from the cells since both of them will be in suspension.

How do you see the potential applications of your findings contributing to the advancement of the MSC-EV field?

Hopefully, we have contributed to developing scalable bioprocesses, concerning both upstream and downstream methodologies to manufacture clinically relevant EV doses. This is an approach that can be translated into different processes, of course. We have also highlighted the potential that analytical technology tools can play in informing us in real time on how we can take advantage of the higher control we have over cell culture. In this case, real-time feedback on our stirred tank bioreactors to maximize EV production.

What are your future research directions to continue this work?

I think the next steps need to focus on further exploring tools such as Raman spectroscopy to better predict and then minimize potential batch-to-batch variability that might result from small variations in the bioprocesses and in the cell culture conditions. Additionally, developing a fully integrated bioprocess that would allow the continuous isolation of EVs and cells being expanded would certainly represent a valuable step forward in the EV manufacturing field. Importantly, from a more purely biological point of view, we need to understand better the cell mechanisms that drive the secretion of more functional EVs, not only in quantity but also in quality, that will be able to revert or at least ameliorate some conditions such as heart disease, among others. So, in summary, I’d say, continue exploring better tools to have more predictive ways to manage our cell cultures and try to implement a fully integrated bioprocess but also, of course, try to understand how controlling the cell culture parameters can drive the secretion of functional EVs.

MesenCult™ in Mesenchymal Stromal Cell Research

Why did you choose to use MesenCult™-ACF Plus, and how has it contributed to advancing your research?

We were interested in selecting a chemically defined medium. In this specific project, that was very important because in the manufacturing of cell and cell-derived products, we need to rely on highly controllable variables that chemically defined medium provides. In contrast with media that is supplemented with FBS, for instance, or hPL, we know that, from batch to batch, there are always some changes. But also, we wanted to take full advantage of the fact that we could generate both the MSCs and the MSC-derived EVs without having to change the medium to EV culture medium. Other mediums affect cell growth when optimizing for EV production. So, by using this chemically defined medium, we had the chance to manufacture cells and isolate the EVs in parallel.

Did you explore different culture media before choosing MesenCult™?

We tested several chemically defined media before selecting MesenCult™. We performed a screening assay with different types of microcarriers and media to select the best one. Since we were prioritizing a chemically defined medium, and it performed well with Corning® Synthemax® II microcarriers, this was the combination that we ended up selecting.


Bottle of MesenCult™-ACF Plus and vials of supplements or the expansion of mesenchymal stem cells

Reduce variability in your human mesenchymal stromal cell cultures by using this animal component-free (ACF) and extracellular vesicle (EV)-free medium.

MesenCult™-ACF Plus Culture Kit is optimized so you can derive human MSCs from multiple sources, such as bone marrow or adipose tissue, without serum.

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