Isolation Of Rat Bone Marrow Mesenchymal Stem Cell-derived Exosome And The Study Of The Uptake Of Exosome By H9C2

Objective(1) To explore whether we can successfully isolate the hypoxia preconditioning rat bone marrow mesenchymal stem cells (BMSCs)-derived exosomes with the combination of step-by-step centrifugation and ultracentrifugation with sucrose/D2O.(2) To study the feature such as the diameter distribution and average radius of the hypoxia preconditioning conditioned BMSCs-derived exosomes.(3) To elucidate whether rat BMSCs-derived exosomes can be uptaken by myocardial cell line H9C2in order to confirm that rat BMSCs-derived exosomes are able to target cardiomyocytes at a high efficiency.(4) To explore the feature of time-dependent uptake of rat hypoxia preconditioning BMSCs-derived exosomes by H9C2.Methods(1) We explore a well-established method to isolate rat BMSCs primary, we culture them with15%FBS α-MEM and gradually purify them, we use the3to6generations for the following studies.(2) Then, we collect exosomes from the supernatant of hypoxia preconditioning BMSCs by the combination of step-by-step centrifugation and ultracentrifugation.(3) We observe the extracts by transmission electron microscopy (TEM) to intuitively determine whether they are exosomes.(4) we use SDS-PAGE system western blot to perform the characterization of exosomes by detecting the qualitative expression of CD63and CD9.(5) By the result from transmission electron microscopy, we statistically analyze the messages of the diameter distribution and average radius of isolated exosomes in order to get the knowledge of the unique characterization of BMSCs-derived exosomes.(6) We track exosomes with membrane fluorescence dye PKH-67, co-culture PKH-67-dyed exosomes with H9C2to observe the location of exosomes within H9C2for the observation whether rat BMSCs-derived exosomes are able to target cardiomyocytes at a high efficiency. Further, the incubated the PKH-67-labeled exosomes with H9C2for different periods to explore the feature of time-dependent uptake of rat hypoxia preconditioning BMSCs-derived exosomes by H9C2.Results(1) According to the result of Transmission electron microscopy, we observed that the extract are of micro-capsule structures, approximately spherical or ellipsoidal shapes and homogeneous sizes, they scattered neatly in the vision, so the extract can intuitively be excluded the possibility of proteins.(2) Apoptotic bodies are reported to be cell debris from the apoptotic cells, their diameters range from100to1000nm, however, the diameters of extract are approximately in the range of20nm to60nm, therefore, we can obviously confirm that the extract are not apoptotic bodies.(3) The results of SDS-PAGE system western blot showed that, the extracts were both positive for expression of CD63and CD9molecules, in addition, compared to CD63molecule, the hypoxic preconditioning rat BMSCs-derived exosomes contained more CD9molecules. Therefore, according to the result of western blot, our extract are neither microvesicle nor micropaticle, in short, our extract are exosomes.(4) By image analysis software Image-Pro Plus6.0and Excel, we found that rat BMSCs-derived exosomes are of diameters from20nm to60nm or about, and of highly homogeneous shapes.(5) By Graph Pad5.0, we proved that, the minimum radius of exosome is2nm, the maximum radius is31nm, the25%quantile of radius is13nm, the75%quantile radius is21nm, the median radius is17nm, the mean±standard deviation of radius is17.03±0.4016nm.(6) In the incorporation experiment, we could only observed green fluorescent-flashing exosomes pellets within the cytoplasm of H9C2in the dyed exosomes positive group.(7) By the control groups and experimental subgroups of0+2h,0+3h and0+4h, we excluded the phenomenon of fluorescence quenching which may have impact on the experiment result, in other words, the numbers of exosomes in H9C2cytoplasm only depend on the periods of incubation. The experimental groups indicate that, we can observe PKH-67-dyed exosomes1hour after the co-culture of exosome and H9C2, during the incubation period of1.5h to2.5h, the numbers are more and the fluorescence intensity are more stronger than any other experimental subgroups.ConclusionIn our experiences, we successfully isolated exosomes by the combination of step-by-step centrifugation and ultracentrifugation with sucrose/D2O. In evidence, the diameter of hypoxia preconditioning rat BMSCs-derived exosomes ranges from20nm to60nm, and the average is34.06nm. Moreover, we confirm that hypoxia preconditioning BMSCs-derived exosomes have the ability to incorporate into Cytoplasm of H9C2efficiently so that myocardial are the biological target of BMSCs-derived exosomes. Further, we find that, the peak of the uptake of exosomes by H9C2appears1.5h to2.5h after incubating them together.