| 摘要: |
| 细胞微团,细胞聚集体所构建的三维细胞模型在肿瘤研究领域中逐渐成为更合适的体外研究模型,但目前对于细胞微团的低温保存方法尚不完善。本文研究了A549肺癌细胞的接种密度以及培养天数对肺癌细胞微团尺寸及生长状态的影响,结果表明:接种密度为2×10个/ml,培养三天的肺癌细胞微团状态良好,可形成致密的结构,微团直径为344.24 um±0.74 um,最适用于低温保存。采用程序降温盒,程控降温仪两种慢速冷冻法低温保存肺癌细胞微团,复苏后细胞微团活力和增殖率远低于新鲜组;使用塑料麦管和玻璃毛细管快速或玻璃化保存肺癌细胞微团,当采用玻璃毛细管为冷冻载体,以含有20%EG+20%Me2SO+Q.5 mol/L海藻糖的IDMEM作为低温保护剂时,复苏后细胞微团相对新鲜组活力为94.59%±9.23%,三天增殖率与新鲜组最为接近。最后,对冷冻载体的降温速率以及保护剂的临界降温速率进行测定,分析了细胞微团低温保存的热力学机理。 |
| 关键词: 肺癌细胞微团 慢速冷冻法 玻璃化 低温保护剂 |
| DOI： |
| 投稿时间：2020-11-25 修订日期：2021-02-06
|
| 基金项目:国家自然科学基金(51376132)资助项目。 |
|
| The Cryopreservation of Lung Cancer Spheroids |
|
Zhou Xinli, li Jiahui, Du Yukun
|
| (Institute of Biothermal Science ,University of Shanghai for Science and Technology) |
| Abstract: |
| Three-dimensional in vitro models constructed using cell spheroids or cell aggregates have gradually become more suitable in the field of tumor research. However, cryopreservation methods for cell spheroids are not ideal. The influence of the seeding density of A549 cells and cell culture days on the size and growth of A549 spheroids was studied. The results showed that when the seeding density was 2×104 cells /mL and the cells were cultured for three days, cell spheroids were in good condition and the microaggregates formed a compact structure with a diameter of 344.24 ± 0.74 μm, which is most suitable for cryopreservation. Then, the cooling box and program control cooling instrument were used to cryopreserve the A549 spheroids. After rewarming, the vitality and proliferation rate of cell spheroids were much lower than those of fresh cell spheroids. Plastic straws and glass capillaries were used to quickly freeze or vitrify the A549 spheroids. When glass capillaries were used as the freezing carrier and 20% EG + 20% Me2SO + 0.5 mol/L trehalose was used as the cryoprotectant, the relative vitality of A549 spheroids to fresh spheroids was 94.59% ± 9.23%, and the three-day culture proliferation rate was closest to that of the fresh group. Finally, the cooling rate of the freezing carrier and the critical cooling rate of the cryoprotectant were measured, and the thermodynamic mechanism of cell spheroid cryopreservation was analyzed. |
| Key words: lung cancer cell spheroids slow freezing method vitrification cryoprotectant |
