Investigation On Scheduled Expansion Of Umbilical Cord Blood Cells In Vitro

Stem cells are rich in umbilical cord blood., which has been widely used in clinicsince recently. To remedy the relatively low number of stem cells in UCB, all kinds ofexpansion methods has arisen. But experts found that differentiation concured. So theusage of UCB stem cells transplantation is limited. In this study we use a new method-scheduled expansion. That is UCB MNC is added into the culture system in smallerdosage for several times. There are two advantages. One is the "niche" of Stroma couldbe more reasonably used. The other is former added cells provide a goodmicroenvironment for the latter. At the same time, former added cells can persistedlysecrete some kinds of cell factors which is indispensable for the growth of blood cells.This method not only keeps the "quantity" but also the "quality" of stem cells. Wehope that by this way, single UCB could provide enough original cells for adulttransplantation, so could make short- or long-term blood reconstitution after chemical-or radio-therapy in patients suffering from hematopoietic malignant disease, ovaryneoplasm,marrow tumor, and many kinds of immunodeficiency and so on. At thesame time, now in clinic using cytokines treating malignant tumor also have maderapid progress. But many problems exist. Such as dose, patten and time. Thehematopoietic injure of mice received lethal or second-lethal irradiation has similarpathobiological process with that of the patients received chemical- or radiotherapy.This provides a good animal model. In the second part of this study, for the first timewe used RSP-CM and LP3-CM treaing⁶⁰C_oγ ray irradiated mice. We found thatRSP-CM could prolong the living time and accelerate the PBWBC of irradiated mice.And exists dose- and time-effect. LP3-CM had no effect. This will lead to a newinsight into the clinical treatment of ovary cancer.The results show:1. Effect of UCB MNC cryopreservationJudging from trypan blue dye exclusion, viable UCB MNC counts weresignificantly different frOm pro- and post-cryopreservation. But it was not differentafter freezing in liquid nitrogen. Flowing cytometry assayed that CD34 antigenexpression was same pro- and poSt-cryopreservation.2. Expansion resuIts2.l Expansion results of UBC MNC; Groups with stroma (Group A B)comparing with Groups without Stroma(Group C D),the totle count of MNC were ofsignificant difference. Also in Group A and Group B cell groWth present an increasingtendency. The peak was at the 2nd and 3rd week. In Group C and D from the 3rd weekon, cell count began decreasing. At last, MNC counts in scheduled group were muchmore than that in one-time group.2.2 Elpansion results of UCB CD34+ cellIn the l" week, CD34 cell groWth in Group A and B aPpear a similar tendency.From 2,d week on the former has grown faster. Till 3ru week, CD34 antigen never hasexpressed in one-time groups. There were similar results in Group C and D.2.3 CoIony assaysComparing with fresh cord blood, colony forming ability of cells recovered fromcultUred for diffeent weeks reduced .But in scheduled groups there were still colonyfOrming cells till 4th week.3. Tbe effect of R8P--CM trating mice received different doses of eeCO Yirradiation.3. l We use RSP--CM and LP3-CM twting mice received different doses 60C. yirradiation. The result was that RSP-CM could markedly enhance the vitality of micereceived 7.5 Gy.3.2 Comparing with control group, RSP-CM, prevent group, prevent and curegroup, and cure group were of no diffeence.3.3 GM-CSF has weaker effed on higher dose irradiated mice. But judgin8 fromproteCt quoient, it has effeCt on all groups, and there exiSt dose- and time-effect.ConcIusion:l. Cryopreservation UCB in liquid nitrogen is an effeCtive method. By this wayit can keep for several years and the proliferation ability would not be harmed.2. Puttin8 MNC in scheduled process in liquid cultUre system is a good way forexpanding stem cells. It c...