背景:多能间充质干细胞(MSCs)广泛存在于成年生物体中,与组织维持和修复有关,调节造血,和免疫反应。人(h)MSCs在组织工程中的应用,基于细胞的治疗,和医疗设备,但目前尚不清楚它们如何对体内移植后的缺氧或炎症等不利条件作出反应。虽然内毒素检测是评价移植MSCs质量和安全性的必要条件,目前尚无关于内毒素剂量反应的报道,以确定体外MSC培养系统的限度.本研究旨在准确量化细胞培养系统中内毒素污染的风险,以建立可接受的hMSC增殖内毒素限值。
方法:三种类型的骨髓来源的hMSC(hMSC-1:21年,M/B;hMSC-2:36岁,M/B;hMSC-3:43岁,M/C)和脂肪来源的干细胞(ADSC;StemProHuman)在含有各种浓度的内毒素(0.1-1000ng/ml)的商业试剂盒的培养基中培养。使用血细胞计数器通过细胞计数来估计细胞的增殖能力。阐明内毒素对hMSCs增殖影响的分子机制,从培养的细胞中提取细胞蛋白,并进行液相色谱-串联质谱鸟枪蛋白质组学分析。采用酶联免疫吸附试验(ELISA)评价内毒素刺激诱导的hMSCs中Cu/Zn型超氧化物歧化酶(SOD1)和Fe/Mn型超氧化物歧化酶(SOD2)的表达,还估计了SOD2对hMSC增殖的影响。
结果:尽管在培养期间细胞形态没有变化,ADSC的增殖能力随内毒素浓度增加至超过0.1ng/ml,hMSC-1为1ng/ml,hMSC-2为100ng/ml;hMSC-3增殖不受内毒素存在的影响。hMSC-1的蛋白质组学分析显示,与细胞周期相关的各种蛋白质,凋亡,内毒素刺激改变了宿主对感染的防御,而SOD2表达在培养期间显着并且一致地上调。后者也通过ELISA证实。此外,重组SOD2以类似于内毒素的方式增加hMSC-1细胞的增殖能力。这些结果表明,内毒素通过上调SOD2以改善细胞存活来保护MSC免受氧化应激。
结论:由于内毒素可以影响各种细胞功能,应设定体外MSC培养物的内毒素限值。基于对MSC增殖的影响,观察到的最低不良反应水平被确定为0.1ng/ml。
BACKGROUND: Multipotent mesenchymal stem cells (MSCs) are widespread in adult organisms and are implicated in tissue maintenance and repair, regulation of hematopoiesis, and immunologic responses. Human (h)MSCs have applications in tissue engineering, cell-based therapy, and medical devices but it is unclear how they respond to unfavorable conditions such as hypoxia or inflammation after in vivo transplantation. Although endotoxin testing is a requirement for evaluating the quality and safety of transplanted MSCs, there have been no reports on the dose response to endotoxins to establish limits for in vitro MSC culture systems. The present study aimed to accurately quantify the risk of endotoxin contamination in cell culture systems in order to establish the acceptable endotoxin limit for hMSC proliferation.
METHODS: Three types of bone marrow-derived hMSC (hMSC-1: 21 years, M/B; hMSC-2: 36 years, M/B; hMSC-3: 43 years, M/C) and adipose-derived stem cells (ADSCs; StemPro Human) were cultured in medium from commercial kits containing various concentrations of endotoxin (0.1-1000 ng/ml). The proliferative capacity of cells was estimated by cell counts using a hemocytometer. To clarify the molecular mechanism underlying the effect of endotoxin on hMSCs proliferation, cellular proteins were extracted from cultured cells and subjected to liquid chromatograph-tandem mass spectrometry shotgun proteomics analysis. The expression of Cu/Zn-type superoxide dismutase (SOD1) and Fe/Mn-type superoxide dismutase (SOD2) induced in hMSCs by endotoxin stimulation were evaluated by enzyme-linked immunosorbent assay (ELISA), and the effect of SOD2 on hMSC proliferation was also estimated.
RESULTS: Although there was no change in cell morphology during the culture period, proliferative capacity increased with endotoxin concentration to over 0.1 ng/ml for ADSCs, 1 ng/ml for hMSC-1, and 100 ng/ml for hMSC-2; hMSC-3 proliferation was unaffected by the presence of endotoxin. A proteomic analysis of hMSC-1 revealed that various proteins related to the cell cycle, apoptosis, and host defense against infection were altered by endotoxin stimulation, whereas SOD2 expression was significantly and consistently upregulated during the culture period. The latter was also confirmed by ELISA. Moreover, recombinant SOD2 increased proliferative capacity in hMSC-1 cells in a manner similar to endotoxin. These results suggest that endotoxin protects MSCs from oxidative stress via upregulation of SOD2 to improve cell survival.
CONCLUSIONS: Since endotoxins can affect various cellular functions, an endotoxin limit should be set for in vitro MSC cultures. The lowest observed adverse effect level was determined to be 0.1 ng/ml based on the effect on MSC proliferation.