(W1130-11-61) Forced Degradation of Cell-Based Therapeutic Products Using Various Stresses and Use of Flow Imaging Microscopy for Identification of Damaged Cells and Cell Debris

Purpose: Cell-based therapeutic products hold promise for treating various diseases, with cell lines like A20 B lymphoma serving as valuable models for studying the effects of stress conditions, such as freeze-thaw stress, shaking stress, and thawing temperature on cell therapies. The novelty of this research lies in the unique utilization of the Flow Cam 8000 for evaluating total cell concentration, cell viability, symmetry, and cell size distribution before and after exposure to stress conditions. By employing this innovative approach, we aim to enhance our understanding of how these stress factors affect the quality and efficacy of cell-based therapeutic products, ultimately contributing to the optimization of their formulation and storage.

Methods: A20 B lymphoma cell suspensions were subjected to forced degradation studies mimicking conditions encountered during the production and handling of cell-based therapeutic products. Stress conditions included freezing with different DMSO concentrations, thawing at various temperatures, and shaking stress. Cell viability and cell counts were assessed using a hemocytometer and FlowCAM analysis after each stress condition.

Results: The study evaluated the effects of different concentrations of DMSO, Cell suspensions supplemented with DMSO at varying concentrations (0%, 1%, 2.5%, 5%, 10%, and 20% v/v) were subjected to one freeze-thaw cycle. Higher DMSO concentrations (5% and 10% v/v) showed better cell recovery post-thawing compared to lower concentrations (0% and 1% v/v). Cell counts significantly decreased with 0% and 1% DMSO, indicating a loss of cell viability under these conditions. Shaking Stress: Shaking at 185 rpm for 3 hours resulted in increased cellular debris, correlated with reduced cell viability and total cell count. The presence of FBS in cell suspensions led to additional debris particles. Thawing Temperature: Thawing at higher temperatures (37°C) yielded higher recovery of viable cells compared to lower temperatures. Flow imaging microscopy highlighted the impact of thawing temperature on cell quality and recovery.

Conclusion: This study demonstrates that thermal, freeze-thaw, and shaking stresses are critical factors to consider when formulating cell-based medicinal products. By employing a combination of analytical methods such as FlowCAM analysis, hemocytometry, and automated cell counting, it is possible to gain a comprehensive understanding of the effects of stress conditions on cell integrity and functionality. These findings underscore the importance of optimizing storage and handling protocols to maintain the quality of cell-based therapeutic products.

References: Sediq AS, Klem R, Nejadnik MR, Meij P, Jiskoot W. Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability. Pharm Res. 2018 May 30;35(8):150. doi: 10.1007/s11095-018-2422-5. PMID: 29846807; PMCID: PMC5976703.

Fig.1 Particle size distribution of cell suspensions after freezing with different concentrations of DMSO using FlowCam.

Fig.2 Symmetry distribution of cell suspensions after thawing at different temperature with using FlowCam.

Fig.3 Particle size distribution of cell suspensions after exposure to shaking and static conditions using FlowCam.