Closed-System Cell Washing and Concentration for MSCs

0:00Hello, everyone. I'm Beatriz Menéndez Yeves, and I work for the past four years at the R&D department at Takeda's Madrid site. I will present our work on the optimization of automated cell washing, volume reduction, and centrifugation technologies for the manufacturing of mesenchymal stromal cells, or MSCs, in a bioreactor-based production environment. This study was conducted at the Cell Therapy Technology Center in Takeda, Madrid, where we are focused on development of scalable and standardized manufacturing processes for advanced cell therapies. The cell therapy industry is rapidly evolving as therapies move from research laboratories to commercial manufacturing. There is an increasing need for processes that are automated, closed, reproducible, scalable, and GMP compliant.

1:01One of the critical challenges in cell therapy manufacturing occurs after cell expansion. Once cells have been grown in bioreactors, they need to be harvested, washed, concentrated, formulated, and cryopreserved. Traditionally, many of these operations have relied on manual centrifugation steps. However, conventional centrifugation becomes increasingly difficult when processing large-scale cultures and introduce risks associated with open handling and operator variability. Therefore, implementing closed-system technologies capable of washing and concentrating cells efficiently is essential for industrial-scale manufacture. To understand the relevance of this work, let's look at the overall manufacturing processes. Typically, the workflow includes cell thawing, cell expansion, harvesting, washing and volume reduction, formulation, cryopreservation, and storage. The focus of this study is the washing and volume reduction step, which directly impacts in product recovery, cell viability, final cell concentration, and cryopreservation efficiency.

2:25Improving this stage can significantly enhance overall manufacturing performance. The main objective of this work was to optimize and evaluate two closed-system technologies for MSCs processing. The first one was the CTS Rotea Counter Flow Centrifuge from Thermo Fisher Scientific, and the second one was the Unifuge Mini or UF Mini from CARR Biosystems. More specifically, we aim to optimize operating parameters for MSCs processing, evaluate recovery and viability, assess concentration performance, compare processing efficiency, determine which applications are best suited for each technology. Let's start with the Rotea system. Rotea operates using the technology called counter flow centrifugation or CFC. In this approach, cells are separated according to their sedimentation properties, mainly size and density.

3:40A balance between centrifugal force and fluid flow allows cells to be retained while washing and concentration occur within the same disposable set. One important advantage of this technology is its ability to perform elutriation. This means that lighter particles and dead cells can be selectively removed from the final product. As a result, Rotea offers not only concentration but also potential improvements in product quality. The second technology evaluated was UF Mini. It uses tubular bowl centrifugation. In this system, cells accumulate inside a rotating chamber while the supernatant is continuously removed. Media exchange and washing can be performed within the same closed environment. When the chamber reaches capacity, the rotor stops, and the concentrated cells are collected out automatically.

4:51This cycle repeats until the entire volume has been processed. The major strength of the UF Mini is its ability to process very large volumes quickly. Before discussing the experimental data, let's compare some key technical characteristics. Rotea offers, as I said earlier, elutriation of dead cells, defined harvest volumes, control of the final cell concentration, regulatory features such as 21 CFR Part 11 compliance, and database connectivity. UF Mini offers higher processing throughput, compatibility with bioreactor tubing systems, very large volume handling, less dependence on initial cell concentration, and each platform presents unique strengths depending of the manufacturing objective. To evaluate both systems, we performed three independent manufacturing runs. MSCs were expanded in bioreactors and subsequently processed using the wash and concentration technologies.

6:12The optimization include adjusting flow rates, centrifugation conditions, washing procedures, concentration parameters, and the primary endpoints were cell recovery, viability, processing time, and final cell concentration. Let's now review the results obtained with Rotea. Across three independent runs, we achieved an average recovery of approximately 91.9%. This level of recovery demonstrates that the system is highly effective at preserving cells during downstream processing. Input cell concentration ranges from approximately 0.12 to 0.39 million cells per milliliter. After processing, output concentration reached values between approximately 42 and 70 million cells per milliliter. This represents a dramatic increase in concentration and highlights one of the major advantages of the Rotea platform. Processing times range from 76 to 89 minutes.

7:31These results demonstrate that MSCs expanded in bioreactors can be successfully harvested, washed, and concentrate using this technology. Recovery rates remained close to 90% throughout the study. Some values exceed 100%. These were not due to actual cell expansion during processing, but rather to expected variability associated with the cell counting methods. Regarding viability, we observe a trend toward improvement after processing, although a statistical significance was not achieved in this specific data set because of the limited sample size. Previous two-dimensional studies have shown a significant effect. The ability of Rotea to eliminate dead cells through elutriation likely contributes to this observation. One of the most relevant finding was the concentration performance.

8:39Rotea was able to increase cell concentration by approximately fourfold or more, depending on the initial conditions. This capability is particularly important for cryopreservation. Highly concentrated cells suspension reduce storage volume requirements and facilitate preparation of the therapeutic doses. In practical manufacturing settings, this can translate into lower cost and improved logistics. We also evaluate UF Mini using MSCs expanded in bioreactors. Recovery values were comparable to those obtained with Rotea, averaging around 90%. However, UF Mini demonstrates advantages in processing speed and volume capacity. The system can operate at substantially higher flow rates and process larger volumes within shorter time frames. These characteristics make it attractive for larger scale manufacturing environments.

9:56When comparing both platforms, several differences become apparent. For Rotea, advantages include high final cell concentration, dead cell removal, better suitability for cryopreservation, and controlled harvest volume. Limitation for Rotea includes longer processing times, minimum concentration requirements, and potential constraints when handling very dilute cultures. And for UF Mini, the advantages include fast processing, large-scale capacity, and flexibility regarding starting concentrations. And on the other hand, limitations for UF Mini include no dead-cell elutriation, lower final cell concentration, and the harvest volume is determined by kit configuration. These technologies should not necessarily be viewed as competitors. Instead, they may serve different purpose within the manufacturing processes. UF Mini may be particularly useful when extremely large volumes must be processed, throughput is the primary concern, and recovery of all cells from a production batch is the priority.

11:38Rotea may be preferable when the final product is intended for cell therapy administration, high cell concentration is required, cryopreservation efficiency is critical, and product quality and viability are major priorities. To summarize, we successfully optimized two closed system technologies for MSCs processing. Both systems achieve recovery rates of approximately 90%. Rotea demonstrate superior concentration capabilities and the additional benefit of dead cell removal through elutriation. The UF Mini demonstrate higher throughput and the ability to process larger volumes more rapidly. These findings suggest that selecting the appropriate technology should depend on the specific requirements of the manufacturing process and the final therapeutic product. Most importantly, the standardization of these protocols represents another step toward scalable, automated, and industrialized production of advanced cell therapies.

12:55Before finishing, I would like to thank all the members of the Cell Therapy Technology Center and the Cell Therapy Science Department at Takeda Madrid. Their dedication, expertise, and collaboration were essential for the success of this work. Thank you very much for your attention.