Construction Of A Microfluidic Chip For Liver And Kidney Tissue Culture And Its Appilcation On Organ Tropic Study Of Tumor-Derived Exosomes

Objectives: We aim to construct a liver-kidney-on-a-chip based on the microfluidic technique,on which precision-cut liver slices(PLS)and precision-cut kidney slices(PKS)were co-cultured.Then we used the microfluidic chip to study the organ tropism of cancer exosomes.Materials and Methods: This microfluidic chip is composed of Polydimethylsiloxane(PDMS)membrane and porous membrane.The PDMS membrane was fabricated by replicate molding on a SU8 master that was prepared by spinning SU8 photoresist onto a glass substrate and patterned by photolithography.After the punching,the pre-polymer thermosetting method is used to complete the sealing of the PDMS membrane and the porous membrane.Human Umbilical Vein Endothelial Cell(HUVEC),Breast cancer(BC)cell lines,MDA-MB-231 and MCF7,were used in this study.The tissue culture chambers of the microfluidic chip were coated with 1: 8 Matrigel,then HUVEC cells were seeded in.The barrier function of HUVEC cells was evaluated using FITC-Dextran with a molecular weight of 10 KD.SD rats(1 to 2 weeks old)were anesthetized,then liver and kidneys were harvested.PLS and PKS with the size of 5 mm in diameter and 250 ?m in thickness were sectioned using Leica VT1200 S vibrating blade microtome.Tissue slices were placed in the microfluidic tissue culture chambers and culture medium perfused at a constant flow rate of 0.75 ?L/min for 24 hrs with a syringe pump.Then PLS and PKS were divided into two groups.One group was stained with Hoechst33342,Rh-123 and PI directly to assess the cellular viability.Another group was fixed with 4% paraformaldehyde and 30% sucrose solution overnight and embedded into O.C.T.Sections with 7 ?m thickness were prepared.HE staining was performed to recognize tissue structures.The expressions of Ki67 and CXCL12 in tissue were examined by immunofluorescent staining using O.C.T.sections.Fresh PLS and PKS without in vitro culture were used as control.MDA-MB-231 and MCF7 cells were cultured in media supplemented with 10% exosome-free FBS for 48 hrs.Then cell culture media were collected for exosome isolation using Total Exosome Isolation Reagent according to the manufacturer's instructions.We used transmission electron microscopy to characterize the size and morphology of exosomes.Western blot(WB)was performed to confirm the expression of CD81,CD63,Hsp70,CXCR4 and GAPDH expression in exosomes.PKH67 membrane dye was utilized to label exosomes.Tissue-specific tropism assay of exosomes was performed using the microfluidic device.PLS and PKS were cultured in the microfluidic tissue culture chambers and PKH67-labelled exosomes were added in the microchannels and incubated for 30 or 60 min.Then the exosomes entered into PLS and PKS were evaluated.SD rats were used to assess exosome tissue tropism assay.Exosomes(10 ?g)were labelled with PKH67 and injected into the male SD rat via the tail vein.After 24 hrs,SD rat were euthanized and liver and kidney tissues were harvest.Tissues were fixed with 4% paraformaldehyde and 30% sucrose solution overnight and embedded into O.C.T.Sections of 7 ?m thickness were prepared and stained with DAPI.Exosomes entered into liver and kidney were examined and recorded by a fluorescent microscope.Inhibition of exosome tissue tropism was conducted with AMD3100,an antagonist of CXCR4.BC exosomes were incubated with different concentrations of AMD 3100 before they were adding into the microfluidic chip.Then the liver tropism of BC exosomes was analyzed.Inhibition of liver tropism by AMD3100 using animal models was performed by injecting AMD 3100-treated exosomes(10 ?g)into 2 week-old male SD rat via tail vein.After 24 hrs,SD rats were euthanized.Liver and kidney tissues were removed for analyzing tissue tropism of exosomes.Results: The tissue culture microfluidic chip is composed of four layers,including the first PDMS layer,porous membrane,the second PDMS layer,PDMS cover.PLS and PKS with 250 ?m thickness were cultured in the microfluidic tissue culture chambers for 24 hrs.Then cell viability,architecture integrity,cell proliferation ability,and CXCL12 expression of the liver and kidney PTS were examined.Hoechst33342,Rh-123 and PI staining demonstrated that cellular viability remained over 90%.Typical liver and kidney structures could be observed in the HE stained sections.Scattered positive cells were found in the mmunofluorescent staining sections against Ki67.These results suggest that tissue slices remained good cellular viability,tissue structure,proliferation capability and CXCL12 expression in the microfluidic culture condition at least for 24 hrs.Then tissue tropism of MDA-MB-231-and MCF7-derived exosomes was assessed on the microfluidic chip.Transmission electron microscopy photos typical cup-shaped morphology of these exosomes.WB confirmed the expression of CD81,CD63,Hsp70 and GAPDH in exosomes.Tissue tropism study based on the microfluidic chip demonstrated that both MDA-MB-231-exosome(p<0.001)and MCF-7-exosome(p<0.01)showed liver tropism,compared to kidney.And MDA-MB-231-exosome showed higher liver tropic ability than MCF7-exosome(p<0.01).Similarly,in vivo experiments demonstrated that both MDA-MB-231-exosome(p < 0.0001)and MCF-7-exosome(p<0.0001)showed liver tropism,compared to kidney.And more MDA-MB-231-exosomes were found in the liver than MCF7-exosome(p<0.0001).WB confirmed the expression of CXCR4 in both the cells and exosomes of MDA-MB-231 and MCF-7.The expression of CXCL12 showed significantly higher expression in liver than in kidney(p < 0.05).These results suggest that the CXCL12/CXCR4 biological axis may play a role in the liver tropism of BC exosomes.Inhibition experiment was performed using different concentrations of AMD3100,CXCR4 receptor antagonist.It was found that liver tropism of MDA-MB-231-exosome could be inhibited by AMD3100 in a dose-dependent manner on the microfluidic chip.Inhibition experiments on animal models demonstrated that liver tropism of MDA-MB-231-exosome decreased by AMD3100(p<0.05).Conclusions:(1)In this study,we developed a liver-kidney-on-a-chip based on the microfluidic technique,on which liver and kidney PTS were co-cultured.Cell viability,tissue architecture,proliferation,and chemokine secretion were well preserved in the liver and kidney PTS by perfusion culture.(2)BC exosomes presented liver tropism on both the microfluidic chip and animal models.(3)CXCL12/CXCR4 biological axis might play a role in liver tropism of BC exosomes.