Optimization and validation of FePro cell labeling method.
Optimization and validation of FePro cell labeling method.
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DOI：
10.1371/journal.pone.0005873
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发表时间：
2009-06-11
期刊：
影响因子：
3.7
通讯作者：
Arbab AS
中科院分区：
文献类型：
--
作者：
Janic B;Rad AM;Jordan EK;Iskander AS;Ali MM;Varma NR;Frank JA;Arbab AS
目前使用铁氧化物（Fe）-鱼精蛋白（Pro）硫酸盐（FePro）磁性标记细胞的方法是基于在无血清培养基中产生FePro复合物，然后将其与细胞孵育过夜以进行有效标记。然而，这种标记技术需要长时间（>12-16小时）孵育，并使用相对高剂量的Pro（5-6 μg/ml），从而产生大的细胞外FePro复合物。这些复合物可能难以用简单的细胞洗涤来清洁，并且可能在T2* 加权MRI上产生不期望的低信号强度。本研究的目的是通过使用低剂量的Pro并在产生任何FePro络合物之前将Fe和Pro直接添加到细胞中来修改当前的标记方法。分别以人肿瘤胶质瘤（U251）和人单核细胞白血病细胞（THP-1）系作为贴壁和悬浮细胞类型的模型系统，进行剂量依赖性（Fe 25 - 100 μg/ml和Pro 0.75 - 3 μg/ml）和时间依赖性（2 - 48 h）标记实验。评估这些细胞的标记效率和细胞活力。普鲁士蓝染色显示95%以上的细胞被标记。当用100 μg/ml Fe和3 μg/ml Pro标记时，U251细胞中的细胞内铁浓度在24小时达到30-35 pg-铁/细胞。然而，在所述FePro浓度下，4小时后观察到相当的标记。类似地，当用100 μg/ml Fe和3 μg/ml Pro标记时，THP-1细胞在48小时达到100 pg-铁/细胞。同样，对于所描述的FePro浓度，4小时后观察到了相当的标记。FePro标记并不显著影响细胞活力。在简单的细胞洗涤后几乎没有观察到细胞外FePro复合物。为了验证和确定修订技术的有效性，标记了人T细胞、人造血干细胞（hHSC）、人骨髓基质细胞（hMSC）和小鼠神经元干细胞（mNSC C17.2）。使用100 μg/ml Fe和3 μg/ml Pro标记4小时，导致这些细胞的非常有效的标记，而不损害它们的活力和功能能力。使用100 µg/ml Fe和3 µg/ml Pro的短孵育时间的新技术可有效标记细胞用于细胞MRI。
Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12–16 hours) incubation time and uses relatively high dose of Pro (5–6 µg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 µg/ml and Pro 0.75 to 3 µg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached ∼30–35 pg-iron/cell at 24 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved ∼10 pg-iron/cell at 48 h when labeled with 100 µg/ml of Fe and 3 µg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17.2) were labeled. Labeling for 4 hours using 100 µg/ml of Fe and 3 µg/ml of Pro resulted in very efficient labeling of these cells, without impairing their viability and functional capability. The new technique with short incubation time using 100 µg/ml of Fe and 3 µg/ml of Pro is effective in labeling cells for cellular MRI.
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