Abstract
The cultivation of cells in 3D systems is commonly regarded to be more physiological than in 2D as it comes much closer to the natural situation in tissues in many different aspects. However, 3D cell culture is much more complex. Cells within the pores of a printed 3D scaffold face a special situation concerning cell-material interaction and cell adhesion, cell proliferation, and supply of medium and oxygen into the core of the scaffolds. Biological assays (for cell proliferation, viability, and activity) have been validated primarily for 2D cell cultures and need to be adapted for 3D cultures. Likewise, in imaging, a number of points need to be taken into account in order to get a clear picture of the cells in 3D scaffolds, preferably with the method of multiphoton microscopy. Here, we describe a method for pretreatment and cell seeding of porous inorganic composite scaffolds (α-TCP/HA) for bone tissue engineering and for cultivation of the cell-scaffold constructs. The analytical methods described are the cell proliferation assay and the ALP activity assay. A step-by-step protocol is provided here that safely tackles typical difficulties that arise with this 3D cell-scaffold setting. In addition, MPM imaging of cells is described both with and without labeling. The combination of biochemical assays and imaging provides valuable insights into the possibilities of analysis with this 3D cell-scaffold system.
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Acknowledgments
We would like to thank Sascha Heinemann (Innotere GmbH, Radebeul, Germany) for discussions on α-TCP/HA scaffolds.
This work was funded by the Deutsche Forschungsgemeinschaft (grant numbers VI 770/3-1 (M.V.), FR 2993/25-1 (O.F.), and TRR 225 “Biofabrication” (project 326998133, subproject Z02) (O.F.)). We acknowledge ongoing support through the Erlangen Graduate School in Advanced Optical Technologies (SAOT) within the German Excellence Initiative.
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Krolinski, A., Sommer, K., Wiesner, J., Friedrich, O., Vielreicher, M. (2023). Optimized Method of 3D Scaffold Seeding, Cell Cultivation, and Monitoring Cell Status for Bone Tissue Engineering. In: Friedrich, O., Gilbert, D.F. (eds) Cell Viability Assays. Methods in Molecular Biology, vol 2644. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3052-5_30
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DOI: https://doi.org/10.1007/978-1-0716-3052-5_30
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