摘要
目的:采用不同方法对白血病HL60细胞株进行冻存和复苏,观察生物学特性改变,筛选最佳冻存复苏方案。方法:实验于2006-04/06在吉林医药学院临床检验实验室(国家三级标准)进行。①HL60细胞株由中国科学院上海生物细胞研究所提供。将欲冷冻保存的HL60细胞调整到良好的生长状态(对数生长期),随机数字表法分成4组,离心收集后在含体积分数为0.1小牛血清的RPMI-1640培养基中分别加入二甲基亚砜,使其终浓度依次为50,100,150,200g/L。冻存15d后,每组取1支冻存管复苏并接种于细胞培养板,培养12h后用锥虫蓝拒染法计算细胞复苏率。②选择冻存条件和冻存细胞密度相同的两支冻存管,按不同方法进行HL60细胞复苏。37℃水浴传统复苏法:立即将冻存管置于37℃水浴中,待融化后800r/min离心5min,吸去上清液,加入含体积分数为0.1小牛血清的RPMI-1640培养基,混匀后接种于细胞培养板,1mL/孔,9孔/组,置于体积分数为0.05的CO2培养箱37℃继续培养。40℃溶解后再37℃恒温改良复苏法:将冻存细胞于40℃水浴中迅速溶解,转入37℃水浴箱,融化后离心、加培养基等操作同传统复苏法。复苏细胞培养12h后采用锥虫蓝染色计算细胞存活率。③用无血清RPMI-1640培养基制备HL60细胞悬液,按3×107L-1密度接种于细胞培养板中,1mL/孔,然后分别加入体积分数为0.05,0.1,0.12,0.15,0.2的灭活小牛血清,每种血清浓度设置8个试验孔,并分别于培养12,24,36,48,60,72,84,96h后计数每孔细胞数量,并绘制细胞生长曲线。结果:①不同浓度二甲基亚砜冻存后HL60细胞复苏率的比较:二甲基亚砜200g/L组的细胞复苏率明显低于二甲基亚砜50,100,150g/L组[(64.6±2.8)%,(87.0±1.4)%,(86.4±2.1)%,(85.7±2.8)%;t=25.44,P<0.01],二甲基亚砜50,100,150g/L组间差异无显著性意义(t=0.82～1.44,P>0.05)。②不同复苏方法HL60细胞存活率的比较:与37℃水浴传统复苏法比较,40℃溶解后再37℃恒温改良复苏法的细胞存活率显著升高[(69.5±1.5)%,(87.4±1.8)%,t=23.24,P<0.01]。③RPMI-1640培养基不同血清含量对HL60细胞生长情况的影响:在体积分数为0.05的血清含量培养基中,HL60细胞基本不生长,且有逐渐死亡的趋势;在体积分数为0.1的血清含量培养基中,HL60细胞生长趋势明显,但生长速度相对较慢;在体积分数为0.12,0.15,0.2的血清含量培养基中,HL60细胞生长迅速,3种血清浓度间细胞生长趋势无明显差异。结论:以50g/L二甲基亚砜作为HL60细胞冻存保护剂、联合40℃溶解后再37℃恒温改良复苏法可使细胞保持最佳生物学特性,体积分数为0.12的血清含量为HL60细胞常规培养的最适浓度。
AIM： To discuss the influence of different frozen and recovery conditions to the biologic characteristics of HL60, and optimize the culture technique. METHODS： The experiment was carded out at Department of Clinical Laboratory （National Level Three） at Jilin Medical College from April to June of 2006. ①HL60 cell strain, offered by Shanghai Institute of Biology and Cell in Chinese Academy of Sciences, were adjusted to the logarithm growth phase and divided into 4 groups. Then the centrifugedcells were collecting and cultured in RPMI-1640 medium containing 10% fetal bovine serum （FBS） added with 5%, 10%, 15% and 20% dimethyl sulphoxide （DMSO）, respectively. One tube from each group was taken out and inoculated to cell culture board after 15 days of cryopreservation. Twelve hours later, the average recovery rates of these cells was calculated by the method of trypan blue dye exclusion.②Two tubes in the same condition of cryopreservation and cellular density were collected, and HL60 cells were resuscitated by different methods. First group： Frozen tube was placed into 37 % water and centrifuged 800 r/min for 5 minutes after thawing, followed by supernatant removal and adding RPMI-1640 culture medium containing 10% FBS. The cells were mixed and inoculated to cell culture board containing 5% CO2, 1 mL per well, 9 wells as a group. Second group： Frozen tube was shifted to 37 % water after melting rapidly in 40 % water. The other step was the same as first group. The cell average livability was counted by trypan blue exclusion staining after the recovery cell was cultured for 12 hours. ③HL60 cell suspension was prepared with free serum RPMI-1640 culture medium, the cells were inoculated to cell culture board at the density of 3×10^7 L^-1, 1 mL/well. Then 5%, 10%, 12%, 15%, 20% FBS were added respectively, each concentration of FBS was set in 8 pores. The cell was counted after being cultured for 12, 24, 36, 48, 60, 72, 84 and 96 hours. The cell growth curve was drawn. RESULTS： ①The recovery rate of HL60 cells in freezing solutions with 20% DMSO was significantly lower than those of cells in 5%, 10% and 15% DMSO [（64.6±2.8）%, （87.0±1.4）%, （86.4±2.1）%, （85.7±2.8）%; t =25.44, P 〈 0.01]. There was no significant difference among the 5%, 10% and 15% DMSO groups （t =0.82-1.44, P 〉 0.05）.②The livability of cells dissolving in 40 % water plus 37 % resuscitation method was significantly higher than that of the traditional 37 % water resuscitation method [（69.5±1.5）%, （87.4±1.8）%, t =23.24, P 〈 0.01]. ③HL60 cells hardly grew in culture medium containing 5% FBS while slowly grew in RPMI-1640 with 10% FBS. Although HL60 cells grew rapidly in RPMI-1640 with 12%, 15% and 20% FBS, there was no remarkable difference among the cells. CONCLUSION： The biologic characteristics of HL60 cells will be better after the cryopreservation using 5% DMSO as cryoprotective agent combined with the improved resuscitation method, and RPMI-1640 culture medium containing 12% FBS is the optimal culture medium of HL60 cells.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第24期4714-4717,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
