Cheng T C, La Peyre J F, Buchanan J T, Tiersch T R, Cooper R K
Department of Veterinary Science, Louisiana State University Agricultural Center, Baton Rouge 70803, USA.
In Vitro Cell Dev Biol Anim. 2001 Apr;37(4):237-44. doi: 10.1007/BF02577536.
Conditions were developed to cryopreserve cells from pronase-dissociated atria and ventricles of eastern oysters (Crassostrea virginica). The effect of three concentrations (5, 10, 15%) of the cryoprotectants (dimethyl sulfoxide, glycerol, and propylene glycol), three thawing temperatures (25, 45, 75 degrees C), and three cooling rates (slow, medium, fast) were compared. Cells were frozen at -80 degrees C and plunged in liquid nitrogen. Thawed cells were seeded in 96-well plates and primary cultures were evaluated after 3 d by measuring the metabolic activity using a tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and by comparing the relative spreading of cells between treatments. The best conditions for freezing and thawing of cells for each cryoprotectant were selected and a final study was performed to compare cryoprotectants. For this final study, we measured the number of cells and their viability 3 d after thawing, in addition to determining cell metabolic activity and cell spreading. Primary cultures of cells fozen without cryoprotectant and of nonfrozen cells were used as controls in all studies. Atrial cells were best cryopreserved with glycerol at a concentration of 10%, a medium cooling rate, and thawing at 45 degrees C. After thawing, atrial cells showed 53+/-5% of the metabolic activity, 84+/-5% of the number, and 92+/-2% of the viability of nonfrozen cells. For ventricular cells, 10% glycerol, a medium cooling rate, and thawing at 25 degrees C yielded the best results. The thawed ventricular cells showed 83+/-5% of the metabolic activity, 91+/-5% of the number, and 96+/-2% of the viability of nonfrozen cells.
已制定出用于冷冻保存来自经链霉蛋白酶解离的美国东部牡蛎（弗吉尼亚巨蛎）心房和心室细胞的条件。比较了三种浓度（5%、10%、15%）的冷冻保护剂（二甲基亚砜、甘油和丙二醇）、三种解冻温度（25℃、45℃、75℃）以及三种冷却速率（慢、中、快）的效果。细胞在-80℃下冷冻，然后投入液氮中。解冻后的细胞接种于96孔板中，3天后通过使用四唑化合物3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑测量代谢活性，并比较各处理组细胞的相对铺展情况来评估原代培养物。为每种冷冻保护剂选择了细胞冷冻和解冻的最佳条件，并进行了一项最终研究以比较冷冻保护剂。在这项最终研究中，除了测定细胞代谢活性和细胞铺展情况外，我们还在解冻3天后测量了细胞数量及其活力。在所有研究中，未使用冷冻保护剂冷冻的细胞和未冷冻细胞的原代培养物用作对照。心房细胞用10%浓度的甘油、中等冷却速率以及在45℃下解冻时冷冻保存效果最佳。解冻后，心房细胞显示出非冷冻细胞代谢活性的53±5%、细胞数量的84±5%以及活力的92±2%。对于心室细胞，10%甘油、中等冷却速率以及在25℃下解冻产生了最佳结果。解冻后的心室细胞显示出非冷冻细胞代谢活性的83±5%、细胞数量的91±5%以及活力的96±2%。
