不同冻存复苏条件影响K562细胞的生物学特性

Effects of different cryopreserved and resuscitative conditions on biological characteristics of K562 cell

目的:观察不同冻存复苏条件对白血病K562细胞株生物学特性的影响,并优化培养条件与方法。方法:实验于2006-02/2006-04在吉林医药学院临床检验实验室进行。①将欲冷冻保存的细胞调整到良好的生长状态,即对数生长期。将细胞分成密度相同的4个组。离心收集后分别加入冻存保护液二甲基亚砜,其终浓度依次为5%、10%、15%、20%分装于冻存管。冻存15d后,每组取出1支复苏并接种于细胞培养板培养。培养12h后吸取少量实验细胞,加入等体积的0.1%台盼蓝染液,5min后在血球计数板上计数,被染成深蓝色的细胞为死细胞;染色很淡、边缘光滑且胞体透明的细胞为活细胞。每个标本计数500个细胞,计数2次取平均值,计算细胞的复苏率。②选择冻存条件和冻存细胞密度相同的2支冻存细胞,采用2种不同的方法进行复苏。第1组:立即将冻存管置37℃水浴中,待融化后1000r/min离心5min,吸去上清液,加入含10%小牛血清的RPMI-1640培养基,混匀后接种于细胞培养板继续培养。第2组:将冻存细胞于40℃水浴中迅速溶解后转入37℃水浴箱,融化后离心、加培养基等操作同第1组。复苏细胞培养12h后采用台盼蓝染色计算细胞的平均存活率。③用无血清RPMI-1640培养基制备3×104/mL的K562细胞悬液接种于细胞培养板中,每孔1mL,然后分别加入5%、10%、12%、15%、20%的灭活小牛血清,每种血清浓度设置4个试验孔并于培养12,24,36,48,60,72,84,96h后每种浓度各取1孔计数细胞量,绘制细胞生长曲线。结果:①5%、15%、20%二甲基亚砜组冻存K562细胞复苏率分别与10%二甲基亚砜组比较,差异显著(86.70%,82.03%,63.09%,88.13%,P均<0.01)。②传统37℃水浴方法复苏后的细胞平均存活率,与40℃溶解后37℃恒温方法比较,差异非常显著[(69.13±1.01)%,(87.50±2.24)%,P<0.01]。③在5%血清含量培养基中,K562细胞基本不生长,且有逐渐死亡的趋势;在10%血清含量培养基中K562细胞生长趋势明显,但生长速度相对较慢;在12%、15%、20%血清含量培养基中K562细胞生长迅速,但3种血清浓度之间细胞生长趋势无明显差异。结论:以10%二甲基亚砜作为K562细胞冻存保护剂并采用改良复苏方法可使细胞保持最佳生物学特性,12%血清为K562细胞常规培养的最适浓度。

AIM： To observe the influence of different frozen and recovery conditions on the biological characteristics of K562 cell, and optimize the culture techniques. METHODS： The experiment was carried out at the Department of Clinical Laboratory, Jilin Medical College from February to April 2006. （1）The ceils were adjusted to good growth condition： Logarithm growth phase before cryopreserved, and divided into 4 groups with the same density. The cells were put into cryopreserved tubes containing 5%, 10%, 15% and 20% dimethyl sulfoxide （DMSO） after centrifugatian and collection. One tube from eaeh group was taken out and inoculated to cell culture board after cryopreserved for 15 days. Twelve hours later, a few cells were extracted and 0.1% trypan blue in the.same volume was added, The died cells stained for 5 minutes were in blue-black, while the alive cells were stained lightly with smooth borderline and transparent cell body, The average recovery rates of these ceils were calculated by counting 500 ceils of each sample twice. （2） Two tubes cells in the same cryopreservative and density condition were selected, and then resuscitated by two different methods. The first group： The frozen tube was placed into 37 ℃ water, centrifuged with 1 000 r per minute for 5 minutes after thawing, then the supernatant was removed and RPMI1640 culture medium containing 10% fetal bovine serum （FBS） was added, which was inoculated to cell culture board after mixed, The second group： The frozen tube was shifted to 37 ℃ water after melted rapidly in 40 ℃ water following by the other step the same as the first group, The cell average survival rate was counted by trypan blue exclusion staining after the recovery cell was cultured for 12 hours. （3）3×l0^4/mL K562 cell suspension was prepared with free serum RPMI1640 culture medium and inoculated to cell culture board, 1 mL/pore, and 5%, 10%, 12%, 15%, 20% FBS were added, respectively. The cell of one pore was counted after cultured for 12, 24, 36, 48, 60, 72, 84 and 96 hours, and the cell growth curve was drawn. RESULTS： （1）The recovery rate of K562 cells in 10% DMSO freezing solutions had significant differences compared with 5%, 15% and 20% DMSO freezing solutions （86.70%, 82.03%, 63.09%, 88.13%, P 〈 0.01）. （2）The survival rate of cells with the traditional 37 ℃ water resuscitation method significantly differed from that with dissolved in 40 ℃ water following by 37 ~C resuscitation method [（69.13±1.01）%, （87.50±2.24）%, P 〈 0.01]. （3）K562 cells hardly grew in culture medium containing 5% FBS, and trended to die; while grew slowly in RPMI1640 with 10% FBS. Although K562 cells grew rapidly in RPMI1640 with 12%, 15% and 20% FBS, respectively, no remarkable difference was found in growth condition among each other. CONCLUSION： The better biological characteristics of K562 cell can be kept after cryopreservation by using 10% DMSO as cryoprotective and adopting the improved resuscitation method. Moreover, the RPMI1640 culture medium containing 12% FBS is. the best concentration.