Novel surface tagging technology for selection of complex proliferation-controlled mammalian cell phenotypes

Area of Science:

• Biotechnology
• Cell Biology
• Bioprocessing

Background:

• Biphasic production processes require cell cycle arrest for enhanced product yield.
• Genetic drift can lead to the emergence of unwanted proliferation-competent mutants during arrested phases.
• Cell surface markers offer potential for selecting specific cell phenotypes.

Purpose of the Study:

• To evaluate cell surface markers for ex vivo selection of growth-arrested phenotypes.
• To develop an engineered Chinese hamster ovary (CHO) cell line for improved biphasic production.
• To assess the efficacy of p27 and a novel surface marker (Hook) in cell cycle control and selection.

Main Methods:

• Multigene metabolic engineering of CHO cells to express secreted alkaline phosphatase (SEAP), p27, and Hook.
• Utilizing a tricistronic tetracycline-repressible system for controlled gene expression.
• Employing FACS- or magnetic-based cell sorting for selection of engineered cells.

Main Results:

• The engineered CHO-SS101(5) cell line exhibited p27-mediated G1-phase cell-cycle arrest in the absence of tetracycline.
• SEAP production increased fivefold in arrested CHO-SS101(5) cells compared to proliferation-competent controls.
• Concomitant expression of Hook enabled efficient selection of engineered cells from mixed populations using FACS or magnetic sorting.

Conclusions:

• Regulated p27 expression and surface marker display provide a robust strategy for biphasic bioproduction.
• Ex vivo selection of engineered cells is crucial for maintaining desired phenotypes in biopharmaceutical manufacturing.
• This approach holds promise for applications in gene therapy and tissue engineering requiring in vivo cell maintenance.