Presentation Information
[P01-150]Nanofibrous shear protectant suppress cell cycle arrest at G1 phase induced by mechanical stress
○Eiichiro Kaneko1, Yasushi Sato2, Masashi Fujiwara3, Masatoshi Maeki4, Hirofumi Tani4, Kenji Tajima4 (1. Graduate School of Chemical Science and Engineering, Hokkaido University (Japan), 2. Advanced Medical Engineering Research Center, Asahikawa Medical University (Japan), 3. Institute for Chemical Reaction Design and Discovery (WPI-ICReDD), Hokkaido University (Japan), 4. Faculty of Engineering, Hokkaido University (Japan))
Keywords:
CHO cells,suspension culture,mechanical stress,cell cycle,cellulose nanofiber
[Purpose]
The growing demand for antibody therapeutics, regenerative medicine, and cultivated meat means there is an increasing need to advance large-scale animal cell culture technologies. However, mammalian cells are highly sensitive to the mechanical stress generated by medium flow, which restricts the ability to further increase scale and density of culture [Al-Rubeai et al., Biotechnol. Bioeng., 1995]. Previous studies have shown that adding bacterial cellulose nanofiber (NFBC), which has an exceptionally long fiber length of approximately 15 μm, increases medium viscosity. This reduces flow velocity and shear rate, resulting in suppressed cell death and increased cell numbers [Kose et al., Cellulose, 2013; Tajima et al., Biomacromolecules, 2017; Kaneko et al., Int. J. Biol. Macromol., 2024]. However, the underlying biological mechanism remains unclear. Therefore, this study aimed to clarify how NFBC sustains cell proliferation under conditions of high hydrodynamic stress, with particular focus on cell cycle regulation.
[Methods]
CHO-DP12 clone #1934 cells were seeded at a concentration of 2.0 × 105 cells/mL in a total volume of 20 mL of Ham’s F-12 medium supplemented with 10% NBS, and then HP-NFBC, a form of NFBC whose surface is coated with hydroxypropyl cellulose, was supplemented at the concentration of 0.10 w/v%. The cells were cultured in suspension in a 100 mL Erlenmeyer flask at rotating speeds ranging from 60 to 125 rpm. Viable and dead cell numbers were quantified using trypan-blue exclusion method and LDH assay, respectively. Cell cycle distribution was analyzed by flow cytometer (CytoFLEX, Beckman Coulter).To synchronize the cell cycle, the cells were subjected to either serum starvation or treatment with 10 μM palbociclib for 24 h. The synchronized cells were then cultured in suspension at 125 rpm for 24 h, after which cell cycle progression was evaluated.
[Results and Discussion]
Increasing the rotating speed reduced the number of viable cells and increased the number of dead cells in the absence of HP-NFBC. In contrast, in the presence of HP-NFBC, high viability and low cell death were maintained across the entire range of 60 to 125 rpm. At lower rotating speeds, cell cycle distributions were similar regardless of the presence of HP-NFBC. However, under the 125 rpm condition without HP-NFBC, the proportion of cells in the G0/G1 phase increased, whereas this increase was not observed in the presence of HP-NFBC. These results suggest that HP-NFBC mitigates hydrodynamic stress-induced impairment of cell cycle progression induced by hydrodynamic stress and suppresses the accumulation of cells in G0/G1.To explore the stage at which HP-NFBC may influence cell cycle progression, cell synchronization experiments were performed. Serum starvation was used to arrest cells in G0 or early G1, while palbociclib treatment arrested cells by inhibiting cyclin D-CDK4/6 activity. Significant differences in cell cycle progression were observed in serum-starved cells, whereas only minor differences were detected in palbociclib-arrested cells. These observations suggest that the effect of HP-NFBC is exerted before, or around, the cyclin D-CDK4/6-dependent restriction point.Overall, these findings suggest that HP-NFBC mitigates the detrimental effects of hydrodynamic stress in rotated suspension culture, thereby suppressing cell death and maintaining cell cycle progression under high-stress conditions.
The growing demand for antibody therapeutics, regenerative medicine, and cultivated meat means there is an increasing need to advance large-scale animal cell culture technologies. However, mammalian cells are highly sensitive to the mechanical stress generated by medium flow, which restricts the ability to further increase scale and density of culture [Al-Rubeai et al., Biotechnol. Bioeng., 1995]. Previous studies have shown that adding bacterial cellulose nanofiber (NFBC), which has an exceptionally long fiber length of approximately 15 μm, increases medium viscosity. This reduces flow velocity and shear rate, resulting in suppressed cell death and increased cell numbers [Kose et al., Cellulose, 2013; Tajima et al., Biomacromolecules, 2017; Kaneko et al., Int. J. Biol. Macromol., 2024]. However, the underlying biological mechanism remains unclear. Therefore, this study aimed to clarify how NFBC sustains cell proliferation under conditions of high hydrodynamic stress, with particular focus on cell cycle regulation.
[Methods]
CHO-DP12 clone #1934 cells were seeded at a concentration of 2.0 × 105 cells/mL in a total volume of 20 mL of Ham’s F-12 medium supplemented with 10% NBS, and then HP-NFBC, a form of NFBC whose surface is coated with hydroxypropyl cellulose, was supplemented at the concentration of 0.10 w/v%. The cells were cultured in suspension in a 100 mL Erlenmeyer flask at rotating speeds ranging from 60 to 125 rpm. Viable and dead cell numbers were quantified using trypan-blue exclusion method and LDH assay, respectively. Cell cycle distribution was analyzed by flow cytometer (CytoFLEX, Beckman Coulter).To synchronize the cell cycle, the cells were subjected to either serum starvation or treatment with 10 μM palbociclib for 24 h. The synchronized cells were then cultured in suspension at 125 rpm for 24 h, after which cell cycle progression was evaluated.
[Results and Discussion]
Increasing the rotating speed reduced the number of viable cells and increased the number of dead cells in the absence of HP-NFBC. In contrast, in the presence of HP-NFBC, high viability and low cell death were maintained across the entire range of 60 to 125 rpm. At lower rotating speeds, cell cycle distributions were similar regardless of the presence of HP-NFBC. However, under the 125 rpm condition without HP-NFBC, the proportion of cells in the G0/G1 phase increased, whereas this increase was not observed in the presence of HP-NFBC. These results suggest that HP-NFBC mitigates hydrodynamic stress-induced impairment of cell cycle progression induced by hydrodynamic stress and suppresses the accumulation of cells in G0/G1.To explore the stage at which HP-NFBC may influence cell cycle progression, cell synchronization experiments were performed. Serum starvation was used to arrest cells in G0 or early G1, while palbociclib treatment arrested cells by inhibiting cyclin D-CDK4/6 activity. Significant differences in cell cycle progression were observed in serum-starved cells, whereas only minor differences were detected in palbociclib-arrested cells. These observations suggest that the effect of HP-NFBC is exerted before, or around, the cyclin D-CDK4/6-dependent restriction point.Overall, these findings suggest that HP-NFBC mitigates the detrimental effects of hydrodynamic stress in rotated suspension culture, thereby suppressing cell death and maintaining cell cycle progression under high-stress conditions.
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