Cryopreservation of Hybrid Artificial Organ

在本研究中，我阐明了细胞密度对冷冻保存人工组织(模仿杂交人工器官内部)细胞解冻后活力的影响，以及微囊化细胞(混合人工器官之一)在冷冻保存中是否比悬浮细胞具有优势。首先，将人成纤维细胞在以胶原海绵(直径20 mm，厚度1 mm)为细胞外基质中三维培养2天。4个人工组织首先堆积在试验室中，然后以0.3~50℃/℃的降温速度在含有10%二甲基亚砜的培养液中冷冻，然后在-196 min的条件下冷冻2 h，最后解冻。溶解胶原基质后，用台盼蓝拒染法检测成纤维细胞解冻后的活性。结果表明，随着细胞密度的增加，解冻后的活力下降，最适宜的高活力降温速率向低速方向移动。当细胞密度较高时，细胞间的接触或脱水受阻可能会导致细胞内冻结。其次，将大鼠嗜铬细胞瘤(PC12)细胞微囊化在海藻酸盐-聚赖氨酸-海藻酸膜中。用差示扫描量热仪以0.5~10℃/m in的降温速率冷冻4~-80℃的微囊化PC12细胞，并测量其潜热。用多巴胺释放法评价解冻后的存活率。结果表明，微囊化细胞的潜热在0.5和1℃/m in时明显低于悬浮细胞。与悬浮细胞相比，微囊化PC12细胞的冻存活力在0.5min和1min/℃时均有不同程度的提高。因此，在微囊化的PC12细胞中，在冷冻过程中达到胶囊内解冻状态会降低溶解效果，提高解冻后的活力。

In this study, I clarified the effect of cell density on the post-thaw viability of cells in cryopreserved artificial tissue (Imitation of the inside of the hybrid artificial organ), and whether microencapsulated cells (one of the hybrid artificial organ) have an advantage over suspended cells in cryopreservation.Firstly, human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix. Different cell densities for the artificial tissue were used, from 10^4 to 10^7 cells/cm^3. Four artificial tissues were first stacked in a test chamber, then frozen at a cooling rate of 0.3 to 50℃/min in a solution (culture medium) containing 10% dimethylsulfoxide, then kept frozen at -196℃ for 2 hours, and finally thawed. After dissolving the collagen matrix, post-thaw viability of fibroblasts was evaluated by using a trypan blue exclusion assay. Results show that with increasing cell density, the post-thaw viability decreased, and the most suitable cooling rate for high viability shifted to the low cooling rate side. When the cell density was high, cell-to-cell contact or an obstruction to dehydration seemed to induce intracellular freezing.Secondly, rat pheochromocytoma (PC12) cells were microencapsulated in alginate-polylysine-alginate membranes. Microencapsulated PC12 cells were frozen with differential scanning calorimetry at a cooling rate of 0.5 to 10℃/min over 4 to -80℃, and their latent heat was measured. The post-thaw viability was evaluated with dopamine release. As a result, latent heat of encapsulated cells was lower than that of suspended cells at 0.5 and 1℃/min. The post-thaw viability of microencapsulated PC12 cells was improved at 0.5 and 1℃/min compared with that of suspended cells. Therefore, in microencapsulated PC12 cells, achievement of intra-capsules unfrozen condition during freezing leads to reducing the solution effect and improving the post-thaw viability.