460147 Single High-EDR Exposure of Cultured Human Cells Reveal Effects of Prolonged Doublings and Microcarrier Attachment on Cell Shear Susceptibility
Materials and Methods: Beginning with cells grown in T-flaks, a contractional flow device, called a “torture chamber” was used, flowing hMSC and Vero cells through with flow rates, subjecting them to previously defined hydrodynamic forces6. The Vero and hMSC cells were suspended in their respective media and then were flowed at various flow rates through the torture chamber. Following this the cells were collected and centrifuged. A cytotoxicity assay (Promega) was used to quantify the cell damage due to the shear stress. T-flasks were reseeded using cells that were not passed through the torture chamber. An approximate count of the cells that attached one day after seeding was gathered to determine the generation count. The number of cell divisions was estimated for each hMSC batch run through the torture chamber. These were placed into bins of generation counts for comparison to Vero cells using ANOVA, each bin representing what is traditionally considered a “passage”. Following this microcarrier effects on shear sensitivity were studied. Human Embryonic Kidney cells (HEK-293) were grown on two different types of microcarriers, collogen coated (Solohill) and dextran matrix (GE cytodex). The HEK-293 cells were grown on the carriers in spinner flasks under protocols typically used for growth on the microcarrier, and then the carriers were flowed through the torture chamber at varying concentrations. These different concentrations were formed by collecting different amounts of media with carriers from the spinner flaks and diluting them to a constant volume, with the highest concentration run being 4 times the lowest concentration, and intermediate concentrations run at 2 and 3 times the lowest concentration. The cells and carriers were then collected, centrifuged, and tested using the same cytotoxicity assay to assess the cell damage due to the shear stress. After taking a sample of the supernatant for the cytotoxicity assay, the cells were then lysed, putting the LDH level on a per cell basis.
Results and Discussion: For suspended cells, the amount of LDH released after shear stress exposure for the hMSC cells after 20.5-24.5 and 24.5-28.5 were not significantly different than the vero cells. However, the hMSCs that had generation counts over 28.5 released significantly (p<0.05) more LDH than vero cells at energy dissipation rates above 6.0*106 W/m3. Specifically, the vero cells and the hMSC cells at generation counts below 28.5 show less than 10 percent LDH, compared to around 20 for the higher generation hMSC cells. At EDR rates of around 4.0*107 W/m3, the hMSC cells with a generation count of 32.5 to 36.5 exhibit about 60% LDH, indicating a high amount of cell damage and death. The generational effect could be due to changes in the cytoskeleton that have been previously found, possibly contributing to tears and LDH release7. The work with the micorcarriers is currently ongoing, but preliminary results show that the percent LDH increases with increasing micorcarrier concentration. The percent LDH increases sharply at higher concentrations, indicating a concentration where the shear effects on the cells become a significant factor. In our preliminary results, the LDH levels increase dramatically between the 3X concentration trial 4x concentration trial.
Conclusions: Our results indicate that the hMSCs might be just as tolerant to sheer stress as other cells currently used for manufacturing, as long as the cells are used when they are still young and aren’t entering the process of senescence. The results also indicate that the torture chamber can be used to screen conditions that would allow for optimal growth of cells. Computational modeling has been previously done on the chamber to determine EDR rates for the different flow rates used, and data is available for EDR in spinner flaks and bioreactors based on agitation speed, so the torture chamber could be used to determine the agitation speed that allows for minimal sheer stress effects. The torture chamber can also be used to determine the concentration of carriers that can be used with minimal cell death. More carriers provide more surface area for the cells to grow on, but too many carriers introduces cell damage due to collisions. The use of the chamber allows for the determination of the concentration that would have the most surface area without introducing excessive cell damage. The torture chamber can be used to screen conditions for production of various products without needing to waste production runs to try out conditions to determine viability.
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