The Effect Of Polysaccharides On The Expansion Of CD34~+ And CIK Cells

Cell preparation is an important part of cell therapy,which can be achieved by simulating the microenvironment to promote cell proliferation in vitro.The presence of polysaccharides in microenvironment plays a critical role in regulating cell growth,proliferation,apoptosis and differentiation.Therefore,we studied the effects of polysaccharides on the in vitro expansion of hematopoietic stem cells(HSC)and cytokine induced killer(CIK).In this paper,the effect of sodium alginate,hyaluronic acid,pectin and xanthan gum(XG)on the in vitro expansion of CD34+cells in a serum-free culture system were investigated.The results showed that only XG could promote the proliferation of total cells among the four polysaccharides.When 100 ?g/ml XG was added to the culture system,the expansion fold of total cells reached 48.5 ± 28.9 on day 14,which was significantly higher than that of the control group 20.3±9.3(p<0.05).However,when analyzing the cells composition in culture,it was found that the proportion of CD34+cells and CD34+CD38-cells rich in HSPCs was significantly lower than that of the control group(p<0.05),and the proportion of CD48-cells in CD34+cells with bone marrow reconstruction ability decreased while the proportion of lymphoid CD3+cells and CD 19+ cells increased.In conclusion,XG can promote the differentiation of CD34+cells into lymphoid cells.In view of this,PBMCs were set as subjects to investigate the effect of XG on the proliferation of CIK cells in a serum-free culture system.Compared with the control group without adding XG,when the culture system was supplemented with 100 ?g/ml XG,the total cell expansion fold at day 21 reached 4534 ± 886,significantly higher than that of the control group 1299 ± 347(p<0.05);the proportion of CD3+CD56+ cells reached(25.5±4.6)%,significantly higher than that of the control group(17.5 ± 1.9)%(p<95),but there was no significant change in the proportion of CD3+and CD3+CD8b+cells;the expansion fold of main effector CD3+CD56+cells reached 30102 ± 10225,significantly higher than that of the control group 5897±2016(p<0.05),indicating that XG can effectively promote the expansion of CIK cells in vitro.K562 cells were used as target cells and the cytotoxic activity of the expanded CIK cells was evaluated at a 10:1 E/T ratio,the results showed that the cytotoxic activity of CIK cells reached(45.3 ± 3.5)%at day 21,which was significantly higher than that of the control group(36.7±5.1)%(p<0.05),suggested that XG enhanced the cytotoxic activity of expanded CIK cells.The expression of CD 107a,intracellular granzyme B and perforin related to cytotoxicity was further analyzed.It was found that the proportion of cells expressing granzyme B and perforin in the expanded cells reached(53.6±33.8)%and(48.3±11.1)%,respectively,significantly higher than the(27.5 ± 34.2)%and(37.5 ± 11.2)%of control group(p<0.05),but the proportion of cells expressing CD 107a was not significantly different from the control group.Therefore,it is presumed that XG enhanced the cytotoxicity of expanded CIK cells by increasing the proportion of cells expressing granzyme B and perforin in the expanded CIK cells.To explore the reason why XG promotes the expansion of CIK cells,glucose,mannose and glucuronic acid(Gla),which are the three components of xanthan gum,were added to the culture system respectively,as well as a mixture of them(Mix),to examine the effect of xanthan gum on the expansion of CIK cells.The results showed that the total cell expansion fold of the experimental group supplemented with 40?g/ml Glc or Man was similar to that of the control on day 21,the total cells relative expansion fold of the experimental group with 20?g/ml Gla was 1.59±0.30 times compared with control group;while the proportion of CD3+,CD3+CD56+and CD3+CD8+cells in the culture was similar to that of the control group;the addition of Gla increased the main effector cells CD3+CD56+cells amplification to 1.71±0.36 times compared with control group;when adding the Mix,relative expansion fold of total cells and the main effectorcells-CD3+CD56" cells were 1.69±0.41 and 1.89 ± 0.48 times compared with control group,respectively,which was similar to that of the Gla alone.It was speculated that XG promoted the expansion of CIK cells by releasing Gla in the culture.The above results provide technical support for optimizing the in vitro expansion process of CIK cells.