Extraction And Identification Of Human Dental Pulp Cells’ Exosomes

Objectives:Dental pulp cells（hDPCs）were cultured in serum-free or exosome-depleted serum conditions.Human dental pulp cell-derived exosomes was extraced and identified by using differential centrifugation and ExoQuickTC kits methods,the best way to obtain the human dental pulp cells’s exosomes was detected by comparing the cells culture and extraction method.Methods:1.Premolars（from 12²6 years old）extracted by orthodontic treatment were collected.Tissue block adherent method was used for hDPCs culturing.Immunohistochemistry was used to detect the expression of vimentin and keratin.2.The 4^th（P4）to 6^th（P6）generation of hDPCs were cultured by using 10%exosome-depleted serum conditioned medium and serum-free conditioned medium for 48h,respectively.3.The cell culture supernatants were collected and exosomes were extracted using differential centrifugation and ExoQuick TC kits.The experiment was divided into four groups.They were:（1）Kit-exosome-depleted serum Group（Kit-serum-hDPCs-DEs）;（2）Kit-hDPCs-DEsgroup;（3）Differential centrifugation,exosome-depleted serum group（UL-serum-hDPCs-DEs）;（4）Differential centrifugation serum-free group（UL-hDPCs-DEs）;4.The exosomes were identified by（1）Morphological identification,transmission electron microscope observation for the morphology of exosomes;（2）Exosomal marker protein detection,Enzyme-linked immunosorbent assay（ELISA）detection of CD63 expression,western blot（WB）detection the exosomal marker protein of CD9 expression.Results:1.The human dental pulp cells were cultured to the third generation by using tissue blocks adherent method.The cells were long spindle-shaped,uniform in size,fibroblast morphology.Immunohistochemical staining showed that hDPC was positive staining for vimentin and negative staining for keratin,suggesting that the cultured human dental pulp cells were derived from mesenchymal sources.2.Growth of human dental pulp cells in serum-free medium:P4-6 hDPCs were cultured with serum-free conditional medium for 48 hours.The microscope showed that the hDPCs were elongated,loosely arranged and three-dimensional structure of cells.3.Growth of human dental pulp cells in the exosome-depleted serum medium:hDPCs were cultured in 10%exosome-depleted serum conditioned medium for48 hours.The cells morphology were uniform,the cytoplasm was full and the cells proliferated well and were arranged closely with whirlpool-shaped.4.Extraction of exosomes:Differential centrifugation and ExoQuick TC kits can extract exosomes vesicles with a diameter of 50-100 nm and surrounded by membranous structures.However exosomes extractedby differential centrifugation showed vesicle rupture phenomenon.5.Identification and concentration of exosomes:（1）the expression of CD63 was detected by ELISA.The results showed that the cell culture supernatants of the four groups expressed CD63;the concentration of exosomes obtained by differentialcentrifugationwas:（1）exosome-depletedserum group（UL-serum-hDPCs-DEs）culture supernatant was（2.5±0.2）×10⁸ particles;（2）concentrations of serum-free group（UL-h DPCs-DEs）was（0.8±0.1）×10⁸particles;concentrations of UL-serum-hDPCs-DEs group were higher than UL-hDPCs-DEs group,the difference was statistically significant（p<0.05）.The concentration of exosomes obtained by the ExoQuickTC kit was:Kit-exosome-depleted serum Group（Kit-hDPCs-DEs）concentrations were（18.5±4.2）×10⁸ particles;Kit-hDPCs-DEs group were（4.22±2.64）×10⁸ particles.The concentrations of Kit-serum-hDPCs-DEs group were higher than Kit-hDPCs-DEs group,the difference was statistically significant（p<0.05）.The concentrationofKit-serum-hDPCs-DEsgroupwerehigherthan UL-serum-hDPCs-DEs group,the difference was statistically significant（p<0.05）;there was no statistically significant（p>0.05）between the two groups.（2）The results of WB showed that CD9 was positively expressed in the supernatant of supernatant-free serum culture.It showed the strongest expression inUL-serum-hDPCs-DEsgroup,andtheweaklyexpressionin Kit-serum-hDPCs-DEs group.CD9 could not be detected in the serum-free group（Kit-hDPCs-DEs and UL-hDPCs-DEs）.Conclusion:Compared with serum-free culture conditions,10%exosome-depleted serum medium was more conducive to the growth and secretion of hDPCs’exosomes.ExoQuickTC kit can extract a lot of exosomes with a smaller amount of supernatant.However,the purity of extracted exosomes by ExoQuickTC kit were lower than by differential centrifugation.Differential centrifugation can extract highly pure exosomes,however,vesicle ruptured exosomes can be found’.The results suggested that cultured with 10%exosome-depleted serum medium can improve secretion of exosomes by human dental pulp cell.The exosomes’extraction method can be selected according to the purpose of study.