有关利用普通冰箱-18℃冷冻保存组织及细胞,迄今未见报道.本实验对-18℃冷冻保存人肝癌细胞株 Bel-7402进行实验研究,现报告如下:1 材料和方法人肝癌细胞株 Bel-7402引自中山医科大学实验动物中心,置于含100 mL/L新生牛血清的 RPMI 1640培养液中,在37℃,50 mL/L CO_2,饱湿条件下传代培养,细胞贴壁生长.将处于对数生长期的细胞经 D-Hank’s 液洗2次,后用2.5 g/L 胰蛋白酶消化,再用含100mL/L 新生牛血清的1640液洗涤1次.后分别用含0mL/L,5mL/L,10mL/L,15mL/L,20mL/L DMSO的完全培养基(含100mL/L 新生牛血清1640)重新制成细胞悬液.细胞浓度保持在1×10~6/mL 左右.将细胞悬液置于2mL的硬塑料冻存管中,加盖密封,做好标记.直接迅速地放入-18℃冰箱中冻存.每种浓度各分装12支冻存管,每管装1 mL 悬液.于1 wk,1 mo,2 mo,3 mo 后分别进行融冻.将上述的冻存管,直接从-18℃冰箱中取出,迅速投入40℃水浴中使之迅速完全融化(约1min 左右).迅速倒入已备好的RPMI 1640营养液中,并用 RPMI 1640洗涤冻存管2次.后离心,再用 RPMI 1640洗涤细胞2次,置瓶培养;同时制成一定量的细胞悬液,取其中0.5mL 的细胞悬液加入4mL/L 台盼蓝染液0.5 mL,混匀,取少许滴于计数板的计数池内,静置2min 后计算细胞存活率(不着蓝色的细胞为活细胞).细胞存活率=(活细胞数)/(活细胞数+死细胞数)×100%观察指标:①存活率:台盼蓝拒染试验计数;②冻存后复苏的细胞接种入培养瓶,置于含100mL/L 新生牛血清的 RPMI1640培养基中,于37℃,50 mL/L CO_2,饱湿培养箱中培养,24 h 后半保留换液,每天观察.2 结果-18℃条件下用不同浓度的 DMSO 为冷冻保护剂,冻存Bel-7402 1 wk,1 mo,2 mo 及3 mo 后细胞存活率及镜下观察到的生长状况见表1.表1 不同浓度 DMSO 对 Bel-7402的影响DMSO 浓度细胞存活率(%) 接种后见细胞贴壁生长天数(mL/L) 1 wk 1 mo 2 mo 3mo 1 wk 1 mo 2 mo 3 mo0 22.1 17.2 8.2 5.6 ————50 84.3 82.3 78.1 70.1 ————100 90.3 86.5 84.1 79.2 1 2 3 5150 89.8 85.3 83.9 78.9 1 2 3 4200 85.1 81.6 78.3 74.0 1 3 4 6 There is no report about the use of ordinary refrigerators to cryopreserve tissues and cells at -18°C. In this experiment, the human hepatoma cell line Bel-7402 cryopreserved at -18°C was studied experimentally. The report is as follows: 1 Materials and methods Human liver cancer cell line Bel -7402 from the Experimental Animal Center of Sun Yat-sen University of Medical Sciences, placed in RPMI 1640 medium containing 100 mL/L newborn calf serum, subcultured at 37 °C, 50 mL/L CO 2 , satiety, cells adherent growth. The cells in the logarithmic growth phase were washed twice with D-Hank’s solution, then digested with 2.5 g/L trypsin, and washed once with a solution of 1640 containing 100 mL/L newborn calf serum. After that, 0 mL/L was used respectively. , 5mL/L, 10mL/L, 15mL/L, 20mL/L DMSO complete medium (containing 100mL/L newborn calf serum 1640) was reconstituted into cell suspension. The cell concentration was maintained at about 1×10~6/mL Place the cell suspension in a 2mL hard plastic cryovial, cover and seal it, mark it, store it directly and quickly in a freezer at -18°C, and dispense 12 cryovials at each concentration. Pipette 1 mL suspension. After 1 week, 1 month, 2 months, and 3 months respectively, thaw and freeze. The above freezing tube was directly taken out of the -18°C freezer, and quickly put into a 40°C water bath to make it Completely melted (about 1 min or so) quickly. Pour quickly into prepared RPMI 1640 nutrient solution and wash the cryovials twice with RPMI 1640. Centrifuge and centrifuge the cells twice with RPMI 1640. To a certain amount of cell suspension, take 0.5 mL of the cell suspension and add 4 mL/L of trypan blue dye to 0.5 mL. Mix well, take a few drops into the counting cell, and let it stand for 2 min before calculating cell viability ( Blue cells are living cells.) Cell viability = (live cell number)/(viable cell number + number of dead cells) × 100% Observation index: 1 Survival rate: Trypan blue exclusion test count; 2 Cryopreservation The resuscitated cells were inoculated into culture flasks, placed in RPMI1640 medium containing 100 mL/L newborn calf serum, incubated at 37°C, 50 mL/L CO 2 in a humidified incubator, and kept in the medium for 24 hours. Observed .2 Results Different concentrations of DMSO were used as cryoprotectants at -18°C. Cell viability and microscopic growth observed after cryopreservation of Bel-7402 for 1 week, 1 month, 2 months, and 3 months are shown in Table 1. Table 1 Effects of Different Concentrations of DMSO on Bel-7402 DMSO Concentration Cell Survival (%) Number of Cell Adherent Growth Days After Inoculation (mL/L) 1 wk 1 mo 2 mo 3 mo 1 wk 1 Mo 2 mo 3 mo0 22.1 17.2 8.2 5.6 ————50 84.3 82.3 78.1 70.1 ————100 90.3 86.5 84.1 79.2 1 2 3 5150 89.8 85.3 83.9 78.9 1 2 3 4200 85.1 81.6 78.3 74.0 1 3 4 6