It should be noted that this calculation is limited to evaluation of the cost of cells alone and does not account for the additional time and complexity introduced by some 3D cell culture approaches. For example, 2D cultures of primary human hepatocytes can be plated and ready for use in experiments in approximately 24 h, whereas 3D cultures require up to 7 days to enable complete aggregation of hepatocytes. Moreover, while many vendors are moving towards qualifying specific lots of primary human hepatocytes for spheroid formation, other cell subtypes may be less characterized and require evaluation of several lots of primary cell material and/or additional time in optimizing culture protocols to successfully generate 3D cell culture models for use in downstream assays.
Additionally, while many endpoints are easily translatable from 2D to 3D culture, assays on 3D cell culture models may require some optimization and/or additional assay time. For example, while 2D labeling is often quite straightforward, small molecule dyes and antibody labels can require optimization of permeabilizing protocols and additional incubation times to ensure complete penetration of these labels into 3D cell culture models. Moreover, analysis of such markers in an entire 3D model requires the acquisition of many more images (i.e. for a 200 µm spheroid, 20 separate images would need to be acquired if confocal imaging is used with a 10 µm z-step, whereas only a single image is required to capture cells in 2D cultures). However, this increased time can be bypassed in a higher throughput screening approach by only analyzing cells on the exterior of 3D models, such that only one image is required for analysis.
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