Department of Plastic Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Mol Cell Biochem. 2011 May;351(1-2):65-75. doi: 10.1007/s11010-011-0712-x. Epub 2011 Jan 19.
5-bromo-2-deoxyurudine (BrdU) can be used as a methodological tool for in vivo investigations following in vitro prelabeling of isolated stem cells for subsequent cell tracking within the recipient host. The objective of this study was to determine how useful BrdU may be as a labeling modality for adipose derived stem cells (ASC) by examining BrdU toxicity, BrdU intracellular stability, and potential effects on ASC differentiation. Porcine and human ASC (pASC and hASC, respectively) were labeled with BrdU at 5 or 10 μM for 2, 6, 24, and 48 h. BrdU toxicity and stability over time in monolayer cultures, in 3-D collagen scaffolds implanted to a porcine model and after thawing from long-term storage were evaluated by MTT assays and immunohistochemistry. ASC differentiation was evaluated by Oil Red O staining. BrdU was not cytotoxic at all tested concentrations and incubation times. BrdU color intensity within each cell and the number of ASC labeled with BrdU decreased as a function of both incubation time and BrdU concentrations. Labeling intensities decreased over time and were undetectable after 6 passages for pASC and 4 passages for hASC. In 3-D scaffolds, BrdU-labeled ASC were identifiable after 90 days of in vitro cultures and for 30 days in a porcine model. BrdU did not prevent preadipocyte differentiation and BrdU labeling was still detectable after subsequent thawing after long-term storage of ASC. BrdU is an excellent candidate reagent to label and track ASC that will allow distinction between BrdU-labeled donor cells and host cells. The data provides a foundation for conducting future tissue engineering projects using BrdU-labeled ASC.
5-溴-2-脱氧尿苷（BrdU）可作为一种方法学工具，用于体外标记分离的干细胞后，对其在受体宿主中的细胞进行跟踪。本研究的目的是通过研究 BrdU 的毒性、细胞内稳定性以及对脂肪来源干细胞（ASC）分化的潜在影响，确定 BrdU 作为 ASC 标记方法的有用性。猪 ASC（pASC）和人 ASC（hASC）分别用 5 或 10 μM 的 BrdU 标记 2、6、24 和 48 小时。通过 MTT 分析和免疫组织化学法评估 BrdU 在单层培养物中的毒性和稳定性、在植入猪模型的 3-D 胶原支架中的稳定性以及从长期储存中解冻后的稳定性。通过油红 O 染色评估 ASC 分化。在所有测试浓度和孵育时间下，BrdU 均无细胞毒性。随着孵育时间和 BrdU 浓度的增加，每个细胞内 BrdU 的颜色强度和被 BrdU 标记的 ASC 数量减少。标记强度随时间降低，在 pASC 中孵育 6 代和 hASC 中孵育 4 代后，强度完全不可检测。在 3-D 支架中，在体外培养 90 天后和植入猪模型 30 天后，仍可识别 BrdU 标记的 ASC。BrdU 不会阻止前体脂肪细胞分化，并且在 ASC 长期储存后进行后续解冻后仍可检测到 BrdU 标记。BrdU 是标记和跟踪 ASC 的优秀候选试剂，可区分 BrdU 标记的供体细胞和宿主细胞。该数据为使用 BrdU 标记的 ASC 进行未来组织工程项目提供了基础。
