Lidocaine Inhibits Proliferation and Promotes Cluster Formation of Bone Marrow Mesenchymal Stem Cells Cultured in High Glucose Plus Serum Deprivation DMEM.
Authors: Zou Ting , Yang Bei , Lu Xia ying , Xiong Ming Di , Ni Xiu li , Zhang Da Lei , Wang Xin chang , Wang Jing lei , Wu Lei , Xu Si fan , Yu Xiao chun , Gao Jun hong , Wang Zhihong 
Published: October 2019

Abstract
Abstract 4582

Objectives
Bone marrow mesenchymal stem cells (BMSCs) have multilineage differentiation potential and highly proliferative potential in vitro. Cell therapy with BMSCs has great promise in regenerating the damaged tissues, restoring their function and treating ischemic related failures, but the chief limitation may be the poor viability of BMSCs transplanted into the pre-existing ischemic environment, where massive death of transplanted cells has been detected. The serum deprivation (SD) is a component of ischemia. Lidocaine can be used for relief of pain and has been one of the most widely used local anesthetics. Intravenously, it has been used successfully for emergency treatment of ventricular arrhythmias. It reportedly also showed anti- proliferative, anti-inflammatory and protective effects when administered before or after the ischemic insult. Which effects has it on BMSCs during the BMSCs transplanted in the patient with lidocaine treatment? The experimental objective is to investigate the effects of lidocaine on the proliferation of BMSCs cultured in high glucose plus serum deprivation DMEM.

Methods
BMSCs were separated from adult male SD rat bone marrow. The BMSCs were cultured in the high glucose (4.5g/L) DMEM containing 10% fetal bovine serum (FBS). All experiments were performed using BMSCs from the 3rd passage adherent cell fraction. The BMSCs were divided into 10 groups according to the concentrations of lidocaine and FBS: 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml lidocaine, containing 10% FBS; 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml lidocaine, containing 0% FBS. Cell proliferation of the BMSCs was assessed by MTT assay at the time points of 24h, 48h and 72h respectively.

Results
As the MTT colorimetric assay revealed, lidocaine with 2μg/ml, 20μg/ml, 200μg/ml, 2000μg/ml all inhibited the proliferation of the BMSCs at under the condition of 10% FBS and under the condition of 0% FBS. The cells cultured in 0μg/ml lidocaine, 10% FBS displayed a significant proliferation compared to those cultured in 0μg/ml lidocaine,0% FBS (serum deprivation) (P<0.01, n=5) from 24h to 72h. The number of vital cells cultured in 2μg/ml, 20μg/ml, 200μg/ml lidocaine,10% FBS was more significantly compared to that cultured in 2μg/ml, 20μg/ml, 200μg/ml lidocaine, 0% FBS (P<0.01, n=5) at 24 h, 48h, 72 h respectively. But in 2000μg/ml lidocaine, the number of vital cells was similar and nearly zero both the BMSCs cultured in 10% FBS and serum deprivation groups. The number of vital BMSCs of all groups showed having a proliferative potential from 24h to 72h.
BMSCs cultured under serum deprivation with 0μg/ml, 2μg/ml, 20μg/ml, 200μg/ml lidocaine gave rise to cell cluster formation, but all groups of BMSCs cultured in the presence of serum did not form the cell clusters. The phenomenon that the number and the size of the cell clusters were more could be observed in the BMSCs cultured under serum deprivation with 20μg/ml, 200μg/ml lidocaine.

Conclusions
The proliferation capacity of the BMSCs cultured in the high glucose DMEM without serum was significantly decreased compared to those cultured in 10% FBS. Lidocaine inhibited the proliferation capacity both of the BMSCs cultured in the high glucose DMEM without serum and with 10% FBS. The results suggest that lidocaine does not exert protective effects on BMSCs in serum deprivation under the high glucose DMEM. Cytotoxicity to lidocaine was maximum at 2000μg/ml. BMSCs cultured under serum deprivation can induce and lidocaine can promote the formation of cell clusters. It is a fascinating point to investigate the relationship between the protective effects and toxicity of lidocaine on BMSCs in high and low glucose microenvironment. It is interested to explore the function and cell characteristics of the cell clusters in a further step of the research work.
This research was supported by the grant from Jiangxi Provincial Department of Education, P. R. China (No. GJJ09103).

Disclosures:
No relevant conflicts of interest to declare.