Application of hydroxypropyl methylcellulose in separation of human umbilical mesenchymal stem cells

Objective To explore the feasibility and effectiveness of application of hydroxypropyl methylcellulose in the separation of human umbilical mesenchymal stem cells(MSCs).Methods Umbilical blood samples were collected from healthy full-term births(50-100 ml for each birth) at a municipal hospital in Shenyang city and treated with heparin.The experiment was performed within 6 hours of blood collection.The umbilical blood samples were firstly diluted with saline.Hydroxypropyl methylcellulose,gelatin,and hydroxyethyl starch were used as sedimentation factors in erythrocyte sedimentation.The erythrocyte sedimentation rate,the number of umbilical MSCs recovered,and the survival rate of MSCs were determined after the reagents were mixed at the ratio of 2 (blood sample):1(saline):3(sedimentation factor) and settled down for the occurrence of clear interface.The MSCs were further separated with Percoll solution.The MSCs were subcultured and the expressions of CD44,CD90,CD105,CD34,and CD45 were detected with flow cytometry for the 35th generation of MSCs subcultured with the revived cells of 3rd and 20th generation after 6 months' freezing with liquid nitrogen.Results The erythrocyte sedimentation time was significantly shorter using hydroxypropyl methylcellulose compared to that using gelatin or hydroxyethyl starch(15 minutes vs.30 minutes)(P<0.05).The recovery of umbilical MSCs was 6.24×10⁶/ml-6.72×10⁶/ml using hydroxypropyl methylcellulose and higher than that using hydroxyethyl starch(5.12×10⁶/ml-6.88×10⁶/ml) or gelatin (2.08×10⁶/ml-2.56×10⁶/ml),with significant differences(both P<0.05).With the application of hydroxypropyl methylcellulose,positive expressions of CD44,CD90,and CD105 and negative expressions of CD34 and CD45 were observed in 35th generation of MSCs and the expressions were consistent with those of the stem cells.Conclusion Hydroxypropyl methylcellulose can be used for umbilical blood stem cells separation at the appropriate concentration of 0.2%,with better result than that using gelatin or hydroxyethyl starch in cell separation.