Intravenous Risk Study Of Mouse Bone Marrow Mesenchymal Stem Cell Suspension Feasibility And Short-term Storage Of DNA Enzyme Ⅰ Pretreatment Reduced

Objective: This study is an exploratory experimental, our laboratory pre injection ofallogeneic bone marrow mesenchymal stem cell suspension through goat jugular, led to acutepulmonary embolism. aims to compare the influence of cell activity and biologicalcharacteristics of mouse bone marrow mesenchymal stem cells (BMMSCs) at4℃duringdifferent preservation times, thus to provide a reference for safe and effective short-termpreservation of BMMSCs. investigating a novel method to reduce the occurrence of pulmonaryembolism after bone marrow mesenchymal stem cells (BMSCs) injection by using DNase Ipretreatment. providing security information in clinical or oral tissue engineering applications infuture.Methods:1）Mouse BMSCs were isolated, cultured and purified to the3rdgeneration invitro. Identification BMSCs to the bone, fat differentiation capacity, expressed their cell surfacemolecular markers by flow cytometry detection;2）mouse BMMSCs were directly stored at4℃using phosphate-buffered saline (PBS) as short-term preservation solution. After0h,4h,12h,24h and48h, morphology, living rate, proliferation and differentiation capacity ofBMMSCs were detected;3）Proliferation and differentiation ability along with aggregationstatus in cell suspension of BMSCs before and after DNase I treatment were detected in vitro;4）Mouse BMSCs were isolated, cultured and purified to the3rdgeneration in vitro. C57BL/6mice were divided into4groups: PBS injection group (n=30),10U/ml DNase I-PBS injectiongroup (n=30), BMSCs injection group (n=30) and DNase I pretreated BMSCs injection group(n=30);5）PBS injection group and BMSCs injection group were injected5m/kg PBS or lethaldose (3×106) of BMSCs, acute death rate were calculated and different organs of dead micewere examined by histological analysis, compared with PBS injection group as a negativecontrol;6）DNase I pretreated BMSCs injection group were injected5ml/kg DNase Ipretreated BMSCs (3×106/ml) and the acute death rate were calculated.Result:1) After0h,4h,12h and24h of4℃storage, the morphology of the mouseBMMSCs is normal, with a living rate of80%, while there was no significant differences inproliferation and differentiation capacities among these groups(P>0.05). When stored for48h,an increasing number of dead cells and cell debris were observed in the medium of BMMSCs, the proportion of living cells declined to20%, the proliferation and differentiation capacitiesrevealed significant differences compare with0h (P<0.05);2) DNase I pretreatment did notaffect the proliferation and differentiation ability of BMMSCs in vitro(p＞0.05), but theproportion of aggregated cells declined significantly (p＜0.05);3) The acute death rate ofBMSCs injection group is40%lower than PBS injection group, histological examinationrevealed cardiopneumatic abnormality. Some deeply stained masses were found in pulmonicvessel, suggesting acute pulmonary embolism was occurred;4) The acute death rate of DNase Ipretreated BMSCs injection group was23.3%, which was significantly lower than BMSCsinjection group(p＜0.05).Conclution:1) It is a simple and feasible method for short-term preservation of mouseBMMSCs at4℃within24h before further manipulation. But the activities and biologicalcharacteristics of BMMSCs would be lost gradually with the elongation of storage time;2)DNase I pretreatment would not affect the biological character of BMSCs but could inhibitsingle cell aggregation, thus reduce the risk of BMSCs intravenous injection caused acutepulmonary embolism. providing security information in clinical or oral tissue engineeringapplications in future.