Establishment And Application Of Blood-brain Barrier In Vitro And Multiple Organ Cell-culture Model

Objective: With the increasing number of chemicals in production and life,it becomes a global concern to evaluate the safety of potential toxicity of chemicals.In the traditional animal experiment,many problems such as long experimental period,complex influencing factors and other aspects of problems exist.In vitro experiments lack the physical barrier and metabolic effect of the body,which affects the extrapolation of results to human.In this study,an alternative method of in vitro cell model is established to evaluate the toxicity of exogenous compounds more quickly and accurately and overcome the shortcomings of in vitro experiments.Methods:1 Establishment and validation of blood brain barrier(BBB)model:The expression of closely related proteins ZO-1,Occludin-1 and Claudins in HUVEC cells was determined by immunoblotting.In vitro BBB model is established through the non-contact co-culture of human umbilical vein endothelial cells(HUVEC)and rat glioma cells(C6).The tightness of BBB is evaluatation through the trans-endotheilal electrical resistance(TEER)determined by Millicell-ERS instrument.The permeability of BBB was evaluated by HRP tracer effect.2 Establishment and validation of the integrate discrete multiple organ cell co-culture(idMOC)model: the HK-2 cells,L-02 cells and SH-SY5 Y cells were inoculated in different orifice of homemade multiple organ cell cocultured plate and the orifice is connected with each other.The growth of HK-2 cells,human L-02 hepatocytes and SH-SY5 Y neurons under co-culture and separated culture was observed.CCK-8 was measured in L-02,HK-2,SHSY5 Y for 4 days co-culture and individual culture.The growth curve was determined to evaluated the establishment of idMOC model.3 Verification and application of BBB and idMOC model: in the control group,SH-SY5 Y cell was directly exposed to 140 ?g/ml and 270 ?g/ml ACR,In the BBB group,SH-SY5 Y cells was exposed to 140 ?g/ml and 270 ?g/ml ACR through BBB.In the idMOC group,SH-SY5 Y cell was exposed to 140?g/ml and 270 ?g/ml ACR at idMOC culture.Compared with the control group,the late apoptotic rate of BBB group was significantly lower(P<0.05).Compared with the control group,the rate apoptosis rate of idMOC group was lower(P < 0.05).ACR prototype and its metabolites cause the common neurotoxicity through the body metabolism and BBB eventually.Results:1 BBB model is successfully established in vitro1.1 Establishment of BBB model in vitroThe structural basis of BBB is protein Tj formed between endothelial cells.In this study,the expression of the Tj-related proteins such as Occludin,claudins and ZO-1 in HUVEC cells showed high expression indicating the HUVEC cells could be used to construct the in vitro BBB model.Co-culture model of HUVEC cells and C6 cells was successfully established by coculture method.1.2 Validation of BBB model in vitroUnder the action of C6 glial cells,HUVEC cells can form a wide range of close-connected complex and then inhibit the external electric field under the cross-endothelial movement,resulting in up to 235 ?/cm2 TEER value.And the mean of Papp was lower than that of endothelial cell culture group at 4,5and 6 days(P<0.05).In general,the greater the TEER value is the smaller the the permeability of BBB,the better the barrier function.HRP reacts with the substrate to produce a color reaction and a good linear relationship between absorbance at 450 nm(D450)and HRP concentration,the performance of each model was stable during 60 min test.The results shows that the permeability of the co-culture group was lower than that of the individual culture group at each time point,the difference was significant(P <0.05),and the results of TEER were confirmed.2 idMOC model in vitro is successfully established:2.1 The establishment of the idMOC modelIn this study,we successfully made a multi-organ co-culture plate.The method was stable.The cells were able to exchange material through the exchange pore of the culture plate.Cell growth was good,no death.A model of co-culture of multiple organ cells in vitro was established.Substances in the body by the liver and other metabolic organs for metabolism,activation or detoxification process in vitro can be achieved.2.2 Validation of iDMOC modelThe absorbance of the fourth day of HK-2 was(0.70 ± 0.032)in single cell culture and(0.78 ± 0.021)in co-culture respectively.The absorbance of L-02 on the fourth day was(0.83 ±0.064)and(0.77 ±0.045)respectively,the absorbance of SH-SY5 Y on the fourth day was(0.60 ± 0.041)and(0.61 ±0.037)respectively.The growth curve of each kind of cell was similar to that of co-culture group,the difference was not statistically significant(P> 0.05).3 Application of BBB and idMOC ModelControl group?ACR was directly exposed to 140?g / ml group?ACR was directly exposed to 270?g / ml group?ACR through BBB of 140?g / ml?ACR through BBB of 270?g / ml?ACR at idMOC culture of 140?g / ml?ACR at idMOC culture of 270?g / ml,late apoptotic rate is(1.90±0.10)%,(23.53±1.80)%,(82.57±2.56)%,(20.27±0.57)%,(73.83±0.76)%,(12.73±1.00)%,(52.73±1.56%).Different concentration of ACR through BBB compared with directly exposed,ACR can through the BBB to cause neurotoxity.Different concentration of ACR at idMOC culture compared with directly exposed,The toxic effect of ACR on SH-SY5 Y cells was significantly decreased by in vivo metabolism.Conclusion:1 Based on HUVEC cells and C6 cells co-culture system,a blood-brain barrier model in vitro was established.2 A co-culture model of co-culture of HK-2 cells,human L-02 hepatocytes and SH-SY5 Y cells was established.3 ACR can induce neurotoxicity,the barrier effect of BBB in vitro and the metabolic activation of in vitro idMOC model can attenuate the intensity of neurotoxicity,and the effect of ACR on neurotoxicity can be induced by ACR in different in vitro conditions.