Single-Cell Analysis of CHO Cells Reveals Clonal Heterogeneity in Hyperosmolality-Induced Stress Response

Area of Science:

• Biotechnology
• Cell Biology
• Bioprocess Engineering

Background:

• Fed-batch cultivation of Chinese hamster ovary (CHO) cells can lead to hyperosmolality due to concentrated feed and base additions.
• Previous research identified effects like increased cell size and growth inhibition from hyperosmolality, but disentangling reagent-specific impacts is limited.

Purpose of the Study:

• To investigate the distinct effects of hyperosmolality and specific osmotic-active reagents on CHO cell behavior.
• To analyze single-cell responses to varying osmotic conditions and identify cellular damage markers.

Main Methods:

• CHO cells were cultured under four osmotic conditions (300-530 mOsm/kg) using high-glucose feed or mannitol.
• Single-cell cultivation data was collected to analyze mass gain and cell division.
• Mitochondrial activity, cell cycle, and DNA integrity were assessed.

Main Results:

• Hyperosmolality and specific reagents induced heterogeneity in cell mass gain and division.
• Extreme hyperosmolality (530 mOsm/kg) from mannitol or glucose overfeeding led to cell cycle arrest.
• Mitochondrial membrane depolarization and mtDNA damage were observed, suggesting premature senescence.

Conclusions:

• Both mannitol-induced hyperosmolality and glucose overfeeding significantly impair CHO cell growth and mitochondrial function.
• These findings highlight the critical need to manage osmotic conditions during bioprocess development for commercial production.