Mammalian cells can produce complex biological biopharmaceuticals like recombinant monoclonal antibodies. The three phases of cell growth include the exponential phase, stationary phase, and death phase. The mechanisms governing these phases in cell culture depends on extrinsic factors such as availability of nutrients and growth factors, and intrinsic factors like cell cycle regulators and stress responses.
Researchers at Manchester Institute of Biotechnology at the University of Manchester (Manchester, UK) engineered mammalian cell growth dynamics for biomanufacturing precision. They achieved control via a ‘gas and brake’ system to control cell growth. By tailoring the growth phases, they developed a versatile platform for predictable and high-level production. This is the first time multiplex-engineering of mammalian cell lines has been used to enable accessible fine-tune control of three culture phases simultaneously.
First they used CRISPR/Cas9 to knock out pro-apoptotic proteins Bax and Bak, proteins involved in cell death to render a death-resistant cell line. Apoptosis is the major cause of cell death in mammalian cell cultures. Secondly, they incorporated a growth acceleration system which the authors referred to as a ‘gas pedal’ based on an Abscisic acid-inducible system regulating the expression of the transcriptional regulator cMYC which results in rapid cell density increase and control of the cell cycle. Lastly, they used a stationary-phase inducing system similar to a ‘brake pedal’. Forr this, they used a Tetracycline-inducible genetic circuit based on the transcriptional regulator BLIMP1 gene that led to the cessation of cell growth and the arrest of the cell cycle. The gene of interests (GOIs) are actively repressed by the tTS protein in the absence of tetracycline while they are robustly activated by the presence of rtTA. Authors noticced a gradual decline in cell densities with increasing tetracycline concentrations.
Suspension-adapted CHO–K1 and HEK293 cells were used to generate the death-resistant CHO cell line. Resistance genes for Hygromycin and Blasticidin were used during plasmid design for the project. Cell line development incorporated Puromycin as a selection agent.
This dual controllable system ie the combination of “gas and brake pedals” (ABA-MYC and TET-BLIMP1 accordingly) enabled dynamic and precise control of mammalian cell growth dynamics. Their work shows how synthetic biology tools and cell engineering can manipulate mammalian cell growth to enhance the manufacture of biopharmaceuticals.
Reference
Torres M et al (2024) Engineering mammalian cell growth dynamics for biomanufacturing. Metabolic Eng. 82:89-99 Link
Product Reference
Authors used Abscisic Acid (ABA) (TOKU-E) in their study. Link.