Cryopreservation of collagen-based tissue equivalents. I. Effect of freezing in the absence of cryoprotective agents
Cryopreservation of collagen-based tissue equivalents. I. Effect of freezing in the absence of cryoprotective agents
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DOI：
10.1089/10763270360728008
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发表时间：
2003-12-01
影响因子：
--
通讯作者：
Tranquillo, RT
中科院分区：
文献类型：
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作者：
Devireddy, RV;Neidert, MR;Tranquillo, RT
在不同的冷却条件下，研究了冷冻对组织等效物（tissue-equivalent, TEs）活力和力学性能的影响，最终目的是优化冷冻保存工艺。将包埋的人包皮成纤维细胞置于胶原凝胶中培养2周，制备出生物人工组织（TEs）。将te从基质上分离出来，使用速冻机（CRF）以不同的冷却速率（0.5、2、5、20和40度/分钟至-80或-160度）或定向凝固阶段（DSS）（5度/分钟至-80度）或快速冷冻（>1000度/分钟）将te冷冻在磷酸盐缓冲盐水中。解冻后立即用乙锭同型二聚体和赫斯特法评估成纤维细胞在TEs中的活力。在MTS (Eden Prairie， MN) Micro Bionix系统上进行单轴拉伸实验，以评估冻融后TEs的解冻后力学性能。CRF的冷却速率为2或5℃/min，对于保存TEs解冻后的即时细胞活力和机械性能都是最佳的。解冻后72 h，在5℃/min至-80℃低温下冷冻的TEs细胞活力略有下降，切线模量和极限拉伸应力显著增加，表明细胞介导的恢复机制。解冻后的机械性能和细胞活力都受到低温-160℃的不利影响。在DSS中冻结会对机械性能产生不利影响。
The effect of freezing on the viability and mechanical properties of tissue-equivalents (TEs) was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. TEs (a class of bioartificial tissues) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. TEs were detached from the substrate and frozen in phosphate-buffered saline using a controlled rate freezer (CRF) at various cooling rates (0.5, 2, 5, 20, and 40degreesC/min to -80 or -160degreesC) or in a directional solidification stage (DSS) (5degreesC/min to -80degreesC) or slam frozen (>1000degreesC/min). Viability of the fibroblasts in the TEs was assessed by ethidium homodimer and Hoechst assays immediately after thawing. Uniaxial tension experiments were also performed on an MTS (Eden Prairie, MN) Micro Bionix system to assess the postthaw mechanical properties of the frozen-thawed TEs. Cooling rates of either 2 or 5degreesC/min using the CRF were optimal for preserving both immediate cell viability and mechanical properties of the TEs, postthaw. By 72 h postthaw, TEs frozen in the CRF at 5degreesC/min to -80degreesC showed a slight decrease in cell viability, with a significant increase in tangent modulus and ultimate tensile stress suggesting a cell-mediated recovery mechanism. Both the postthaw mechanical properties and cell viability are adversely affected by freezing to the lower end temperature of -160degreesC. Mechanical properties are adversely affected by freezing in the DSS.