The establishment of in vitro models of the BBB is significant for the evaluation of the mechanism and permeability of drugs and their sustained-release formulations across the BBB. Among the different models, the immortalized human cell line hCMEC/D3 has the potential to be used for a standardized in vitro BBB model due to its high throughput, reproducibility, homology and low cost. The high permeability of the paracellular pathway and the low expression of both certain transporters and metabolic enzymes in this model lead to low physiological levels of physical, transport and metabolic barriers, thus limiting the application of these cells. The barrier properties of this model have been improved in different studies by various means. However, no systematic review has been conducted on the optimization of model-building conditions or on the regulation and expression of transporters in the models. Some existing reviews focus on the overall description of the entire field of blood-brain barrier in vitro models, lacking in-depth and systematic reviews on the experimental details and model evaluation methods based on hCMEC/D3.This paper deals with a detailed review on the optimization of multiple aspects and modalities of the hCMEC/D3 cell culture process, including initial medium, optimal serum levels, Transwell membrane materials, supra-membrane supports, inoculum density, endogenous growth factor, exogenous drug levels, co-culture and transfection methods, to provide references for the establishment and evaluation of hCMEC/D3 cell models.
Keywords: Barrier prosperity; Blood-brain barrier; In vitro models; Transwell; hCMEC/D3 cells.
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