A Comparative Study on Mononuclear Cell Separation and Cryopreservation of Human Cord Blood

Lee, Kun Soo; Kim, Ki Ho; Kim, Mi Jung; Choi, Eun Jin

Abstract

BACKGROUND: Although the bone marrow transplantation is now recognized as an effective therapy for some hematologic and malignant diseases, HLA-matched related donor is limited. Human umbilical cord blood contains sufficient numbers of hematopoietic stem cells to provide long-term engraftment in the unrelated recipient with several advantages. The number of hematopoietic stem cells is an important factor for the better result in transplantation. The purpose of this study is the development of technique for the collection, separation and cryopreservation of the cord blood mononuclear cells for transplantation.
METHODS
Forty-four cord bloods were collected during an uneventful delivery from the Kyungpook National University Hospital and Hana Hospital, Taegu, Korea from May to June, 1997. The mononuclear cell separation with Ficoll-Hypaque(Lymphocyte separation medium, Bionetics, USA) or red cell sedimentation with 3% gelatin(Sigma, USA) or 6% hydroxyethyl starch(HES; Fresenius, Germany) was done and the numbers and viabilities were compared with hemocytometry(Technicon H-3 RTX System, Irland). The viabilities of separated cells by different cell separators in the L-15 medium(Gifco, USA) containing 2% FBS(Gifco, USA) according to time were compared. The viabilities of separated cells by different cell separators in the L-15 medium(Gifco, USA) containing 17% FBS according to time and temperature were compared. The cell viabilities according to the different cell separating, freezing(direct, program-A or program-B) and thawing(rapid or slow) methods were compared. The method of direct freezing is putting the cells into the liquid nitrogen chamber(-196oC) directly. The method of program-A is lowering the temperature as 1oC/min to -90oC by programmed cell freezer(Cryomed 1010, USA) and then putting the cells into the liquid nitrogen chamber. The method of program-B is lowering the temperature as 1oC/min, to -4oC and 25oC/min, to -40oC, and raising as 15oC/min, to -12oC, and lowering again as 1oC/min, to -40oC and 10oC/min, to -90oC by programmed cell freezer and then putting the cells into the liquid nitrogen chamber. The method of rapid thawing is thawing the freezed cells in the 37oC incubator and the method of slow thawing is in the room temperature of 20oC. The functional aspect of stem cell after cryopreservation was performed with cell culture in the methylcellulose medium(Sigma, USA) and GM-CSF(LG Pharmacy, Korea) in the 5% CO2 incubator(37oC; Forma, USA). The statistical analysis was done with Analysis of Variance Procedure and T-test.
RESULTS
The mean volume of cord blood was 121 mL(80~155 mL) and was no difference with maternal and fetal conditions, but the large volume could be obtained with rapid collection from early ligation of cord. The hemoglobin was 14.2+/-1.6 g/dL, hematocrit 46+/-6%, red blood cell 4.1+/-0.4x106/microL, platelet 236+/-82x103/microL and total white blood cell 11,893+/-4,010/microL with neutrophil 54.5+/-11.5%, eosinophil 2.6+/-1.3%, basophil 1.4+/-1.1%, lymphocyte 31.7+/-12.0% and monocyte 8.5+/-2.8%. The removal of red blood cell was 98.3% with F-H, 80.7% with gelatin and 54.3% with HES(P<0.0001). The removal of platelet was 64.8% with F-H, 85.2% with gelatin and 80.0% with HES. The removal of mononuclear cell was 73.3+/-15.4% with F-H, 99.1+/-22.4% with gelatin and 94.2+/-22.8% with HES(P<0.0001). The removal of polymorphonuclear cell was 91.0% with F-H, 42.3% with gelatin and 35.5% with HES(P<0.0001). The viabilities of separated cells by gelatin or HES in the L-15 medium containing 2% FBS for 18~24 hours were low compared to that by F-H and these differences were resolved with raising the concentration of FBS up to 17%. The program-B freezing(59.4%) was the best method in recovery of cells compared to the direct(36.9%) or program-A freezing(41.6%). But no differnces were observed according to the different cell separating or thawing methods. The colony formation from cryopreserved cell was observed at 7 th day of cell culture.
CONCLUSION
In summary, the large volume of cord blood could be obtained with rapid collection after ligation of cord. The best way of mononuclear cell separation was the method with 3% gelatin among three the program-B freezing was the best way in recovery of cells after thawing these will be important factors to raise the success rate in hematopoietic stem cell transplantation.