Toxicology Division, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, Kerala, India.
Cryobiology. 2011 Dec;63(3):273-8. doi: 10.1016/j.cryobiol.2011.09.139. Epub 2011 Oct 2.
The feasibility of an indigenously developed ELISA method to determine cytokine response to wide spectrum of pyrogenic stimuli utilizing fresh human whole blood is limited by the availability of healthy donors. The possibility of using cryopreservation of pooled human blood for detection of cytokine response to lipopolysaccharide is explored in this study. The effect of cryopreservation on blood parameters, cellular morphology and cytokine response were compared with that of the pooled fresh blood and cryopreserved blood from single and multiple donors. In vitro pyrogenic stimulation with 0.5 and 5 EU of LPS was monitored on fresh and cryopreserved pooled blood from single and multiple donors. The release of IL-1β was quantitated by Sandwich ELISA (1, 10, 25, 45 and 75 days) after storage. The results indicated that the cryopreserved blood displayed enhanced IL-1β release on stimulation with LPS, when compared to fresh blood. The maximum release of IL-1β level was observed at 2h when 5 EU of LPS was treated with pooled fresh blood which is similar to that of fresh blood. After 75 days storage of pooled cryopreserved blood the IL-1β release was maximum at 9 and 15 h when treated with 5 and 0.5 EU of LPS. Observations of the study suggest that cryopreserved pooled blood is an economically and experimentally viable alternative to fresh blood. This investigative study promises short term storage and regular supply of non-allergic, pathogen free human blood for the detection of interleukin-1β for the evaluation of in vitro pyrogenicity.
利用新鲜人全血来测定对广谱热原刺激的细胞因子反应的本土化酶联免疫吸附测定（ELISA）方法的可行性受到健康供体可用性的限制。本研究探讨了使用冷冻保存混合人血来检测对脂多糖的细胞因子反应的可能性。比较了冷冻保存对血液参数、细胞形态和细胞因子反应的影响，与新鲜混合血液和来自单个和多个供体的冷冻保存血液进行比较。用 0.5 和 5 EU 的 LPS 在体外对新鲜和冷冻保存的来自单个和多个供体的混合血液进行热原刺激。在储存后 1、10、25、45 和 75 天，通过夹心 ELISA 定量测定 IL-1β 的释放。结果表明，与新鲜血液相比，冷冻保存的血液在 LPS 刺激下显示出增强的 IL-1β 释放。当用 5 EU 的 LPS 处理新鲜混合血液时，观察到最大的 IL-1β 水平释放发生在 2 小时，与新鲜血液相似。在储存 75 天后，当用 5 和 0.5 EU 的 LPS 处理时，最大的 IL-1β 释放分别发生在 9 和 15 小时。研究观察结果表明，冷冻保存的混合血液是新鲜血液的一种经济和实验可行的替代品。这项研究为检测白细胞介素-1β提供了短期储存和定期供应非过敏、无病原体的人类血液，用于评估体外热原性。
