An engineered Madin-Darby canine kidney (MDCK) cell line that is more suitable for human influenza virus isolation and propagation was developed by Takada et al. By altering the expression levels of α-2, 6- sialoglycans and α-2, 3-sialoglycans of the host cell. However, the adherent nature of this humanized MDCK (hCK) cell line limits the scalability of ist use in large-scale production of influenza vaccines. Over the span of 10 months, we adapted this adherent serum dependent hCK cell line to a serum free suspension cell line without altering ist engineered characteristic. The adaptation study was performed in three phases: 1) adaptation from growth in serum–containing media to serum-free media in flatware, 2) adaptation from adherent serum free to suspension serum free in shake flasks, and 3) the generation of a stable (progenitor) cell bank with optimal growth. After testing multiple serum free media and methods of motion (for developing growth in suspension), we successfully generated a Research Cell Bank with ≥ 80% viability, ≤20% aggregation, and ≤ 30 hour doubling time and were able to successfully tech transfer the process to Manufacturing for production of a GMP Master Cell bank. This modified serum free suspension MDCK cell line provides an ideal platform for isolation of human influenza viruses from clinical specimens, and production of influenza vaccines at an industrial scale.