Hyperthermic shift and cell engineering increase small extracellular vesicle production in HEK293F cells

Area of Science:

• Biotechnology
• Cell Biology
• Bioprocess Engineering

Background:

• Small extracellular vesicles (sEVs) show therapeutic potential due to properties like BBB permeability and low immunogenicity.
• Economic manufacturability is critical for the clinical application of sEVs.
• Bioprocess optimization and cell line engineering are key strategies for improving biologic production.

Purpose of the Study:

• To identify optimal bioprocess conditions for enhancing sEV production in HEK293F cells.
• To investigate the molecular mechanisms behind hyperthermia-induced increases in sEV productivity.
• To identify genetic targets for cell line engineering to further improve sEV yield.

Main Methods:

• Design of experiments (DoE) screening for bioprocess optimization.
• Short-term hyperthermia treatment at 40°C.
• Transcriptomics and proteomics analyses to study cellular responses.
• Genetic screening of genes involved in exosome biogenesis and heat shock response.

Main Results:

• Short-term hyperthermia (40°C) increased volumetric productivity 5.4-fold with maintained cell viability (>90%).
• Hyperthermia induced a heat-shock response and upregulated molecules related to exosome biogenesis and vesicle trafficking.
• Overexpression of six genes (CHMP1A, CHMP3, CHMP5, VPS28, CD82, EZR) significantly enhanced sEV secretion and titer.

Conclusions:

• Short-term hyperthermia is an effective strategy to enhance sEV production.
• Heat shock proteins and upregulated exosome-related pathways contribute to increased sEV yield.
• Specific gene targets identified offer potential for developing engineered cell lines for improved therapeutic sEV manufacturing.