Abstract

Bulk production of recombinant proteins and therapeutic vaccines largely depends on cellculture techniques. Suspension culture using microcarrier is a common manufacturing practice, despite its production limitations caused by shear force damage. Here we report a simple and scalable method to overcome this and improve the healthy cell mass. We hypothesized to curb the cell damage and increase the yield of cellular products through unique functionalization of microcarrier with an analog of peptide that is known to induce collagen and fibronectin like extracellular matrix (ECM) molecules. To test the hypothesis polystyrene beads (75-150 μm) were covalently grafted with HPLC purified peptide through spacer in a manner that produces ~ 4.2 micromole/g substitution. After UV and FTIR characterization, the functionalized beads (BSP), were evaluated by culturing Vero (African green monkey kidney) and MDCK (Madin-Darby canine kidney) cells. Microscopic study after 24 hours of culture exhibited cells in clusters over the beads and 81% of functionalized versus 41% unmodified beads (UB) were observed to be occupied by the cells. Alamar-blue assay confirmed that functionalization facilitates microcarrier to sustain more viable cells. Even with the present density of functional moiety, BSP sustained significantly (p<0.001) more viable cells in 24-30 hours in comparison to Cytodex, the most commonly used microcarrier. Thus, present technique provides an option to improve the number of healthy cells by stimulating a native mechanism in situ. Accomplished at room temperature, similar yet customized functionalization with defined moieties can potentially produce differentiated stem cells for tissue engineering and regenerative medicine.

Keywords: Alamar blue, extracellular matrix (ECM), functional peptides, MDCK, solid microcarrier, suspension culture, vero.

Graphical Abstract