CHO-K1 cells were chosen to generate EPO and EPO-HBHAc fusion proteins, and stable clones expressing EPO and EPO-HBHAc were selected and cryopreserved. Furthermore, the target proteins were purified under a two-step purification process. Chemical conjugation is another approach to generate EPO-HBHAc; however, it is challenging to control the specific site of conjugation between EPO and HBHAc. A heterogeneous mixture would be generated, and EPO bioactivity could be affected if critical sites on EPO were occupied by HBHAc. The production of proteins in mammalian cells is an important tool not only for basic research but also in the biotech industry. Mammalian proteins, including tissue plasminogen activator and EPO, require a mammalian cell production system for better bioactivity, and this could be attributed to the ability of mammalian cells to generate proteins with appropriate molecular structures and biochemical properties [15]. Furthermore, compared with transient transfection, stable expression of the transgene is generally more desirable for large-scale production, demonstrating higher protein quality and homogeneity [16].
The adherent expression system is a traditional method for protein expression and was selected to generate EPO-HBHAc on a bench-scale in this study. However, the surface area of the flask might limit the yield of target proteins [17]. This is a proof-of-concept study, evaluating the protein production and purification process for a novel CPP-modified protein, and bench-scale production was sufficient for this purpose. However, a scale-up protein expression system for target proteins is necessary for further in vivo evaluations, with the suspension-adapted cell culture system providing an alternative to increasing the yield of this novel CPP-modified protein. In previous study investigating EPO production, the conditioned medium was collected every 2 days [18]; however, in the present study, the highest protein expression was observed after 4 days of incubation. Protein expression levels can vary among the different groups, and thus, we suggest that evaluating the protein expression profile is crucial when setting up a new protein production platform.
In our in vitro co-culture BBB model, the TEER value was significantly higher than that observed in the mono-culture model, and the penetration of 70K-dextran was significantly blocked from the upper chamber to the basolateral chamber. It remains a challenge to design an optimized in vitro experimental model to mimic the physiological and functional characteristics of the BBB. High junctional tightness measured as TEER is an important feature for an appropriate model mimicking the BBB in vitro. However, the optimal value of TEER for experiments could vary when obtained from different studies. This could be attributed to differences in measuring equipment and temperature, as well as the handling of the cells [14]. Hence, the tightness of in vitro BBB models was validated using permeability studies with hydrophilic tracers [19].
Additionally, our mono-culture model showed lower TEER values and higher paracellular diffusion when compared with the co-culture model, which is consistent with previous studies. Owing to inadequate tight junctions in endothelial cell mono-culture models, several research groups have attempted to reduce paracellular diffusion in these mono-culture models. One approach is to co-culture the endothelial cells with astrocytes [20, 21]. Previous studies have reported that cultured astrocytes implanted into areas with normally leaky vessels were able to induce the tightening of the endothelium, demonstrating that astrocytes play a major role in inducing barrier properties [22]. To better mimic the physiological structure of the BBB, endothelial cells are co-cultured with astrocytes, and the interaction between endothelial cells and astrocytes increases the expression of transporters, as well as that of tight junctions in endothelial cells. Furthermore, this interaction induces the formation of cell polarity in endothelial cells. Collectively, these advantages reveal that the endothelium-astrocyte co-culture model is more representative of the BBB [23–27].
A large amount of 4K-dextran was transported from the upper chamber to the basolateral chamber in both the monoculture and co-culture BBB models. This demonstrates that our models failed to afford sufficient barrier properties to small peptides/proteins with a molecular weight of approximately 4,000 Dalton. In contrast, only a limited amount of 70K-dextran was transported from the upper chamber to the basolateral chamber in both the monoculture and co-culture BBB models. Based on our results, the extents of EPO and EPO-HBHAc transportation differed in the co-culture model, suggesting that our co-culture model could be applied in BBB penetrating investigations for substances with a molecular weight larger than that of EPO.