Suspension Culture of Mammalian Cells Using Thermosensitive Microcarrier that Allows Cell Detachment without Proteolytic Enzyme Treatment

Abstract

Microcarriers are used to expand anchorage-dependent cells in large-scale suspension bioreactors. Proteolytic enzyme treatment is necessary to detach cells cultured on microcarriers for cell harvest or scale-up, but the enzyme treatment damages the cells and extracellular matrices and complicates the culture process. Here, we fabricated thermosensitive microcarriers from which cells can be detached by temperature change without proteolytic enzyme treatment. A thermosensitive polymer, poly-N-isopropylacrylamide (pNIPAAm), was incorporated on the surface of Cytodex-3 microcarriers. pNIPAAm-grafted microcarriers allowed human bone marrow-derived mesenchymal stem cells (hBMMSCs) to adhere, spread, and grow successfully on the microcarriers as nongrafted microcarriers did. By dropping temperature below 32°C, more than 82.5% of hBMMSCs were detached from pNIPAAm-grafted microcarriers. The trypsin treatment for cell detachment induced apoptosis and death of some of the detached cells, but cell detachment from pNIPAAm-grafted microcarriers by temperature change significantly reduced the apoptosis and cell death. pNIPAAm-grafted microcarriers can significantly reduce cell extracellular matrix damage in the cell detachment process and simplify the cell detachment process by avoiding proteolytic enzyme treatment. pNIPAAm-grafted microcarriers would be valuable to a variety of potential fields demanding a large amount of cells without cell damage, such as cell therapy, tissue engineering, and other biological and clinical applications.

Dynamic cell culture on calcium phosphate microcarriers for bone tissue engineering applications

“…In addition, having intermittent rotating culture regimes as opposed to constant rotating regimes has also been shown to enhance cell adhesion in dynamic cultures. 1,23,24 …”

Biomanufacturing of Therapeutic Cells: State of the Art, Current Challenges, and Future Perspectives

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Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

“…Scaled production of iPSCs has focused on suspension culture 20–25 or suspension with the use of microcarrier substrates 26–28 partly because these techniques are successfully deployed for large scale, non-iPSC, eukaryotic based production. Several groups have demonstrated suspension culture systems that yield pluripotent stem cells 20,21,23,24,29 .…”