Cryopreservation of Hybrid Artificial Organ

在本研究中，我阐明了细胞密度对低温保存人工组织（杂交人工器官内部的模拟）中细胞解冻后活力的影响，以及微囊化细胞（杂交人工器官之一）在低温保存中是否比悬浮细胞有优势。首先，将人成纤维细胞在胶原海绵（直径20mm，厚度1mm）中三维培养2天，作为细胞外基质。人造组织采用不同的细胞密度，从10^4到10^7个细胞/cm^3。4个人工组织先在实验箱中堆叠，然后在含有10%二甲亚砜的溶液（培养基）中以0.3 ~ 50℃/min的冷却速率冷冻，然后在-196℃下冷冻2小时，最后解冻。溶解胶原基质后，用台盼蓝排除法评估解冻后成纤维细胞的活力。结果表明：随着细胞密度的增加，解冻后活力降低，高活力的最适宜冷却速率向低冷却速率侧转移；当细胞密度高时，细胞间接触或阻碍脱水似乎会诱导细胞内冻结。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-海藻酸盐膜中。采用差示扫描量热法，以0.5 ~ 10℃/min的冷却速率在4 ~ -80℃下冷冻PC12细胞，测定其潜热。用多巴胺释放评价解冻后细胞活力。结果表明，在0.5℃/min和1℃/min时，包被细胞的潜热低于悬浮细胞。在0.5和1℃/min条件下，微囊化PC12细胞的解冻后活力较悬浮细胞明显提高。因此，在微囊化PC12细胞中，在冷冻过程中达到囊内解冻状态，可以降低溶液效应，提高解冻后细胞活力。

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.