Mizuno Mitsuru, Yori Kouichirou, Takeuchi Toshikazu, Yamamoto Takaaki, Ishikawa Natsumi, Kobayashi Megumi, Nishio Miwako, Sekiya Ichiro
Center for Stem Cell and Regenerative Medicine, Tokyo Medical and Dental University (TMDU), 1-5-45, Bunkyo-ku, Yushima, Tokyo 113-8519, Japan.
Department of HeartSheet Business, Terumo Corporation, 1500 Inokuchi, Nakaicho, Ashigarakami-gun, Kanagawa 259-0151, Japan.
Regen Ther. 2024 Jul 18;26:489-495. doi: 10.1016/j.reth.2024.07.002. eCollection 2024 Jun.
Cell-processing facilities face the risk of environmental bacteria contaminating biosafety cabinets during processing, and manual handling of autologous cell products can result in contamination. We propose a risk- and evidence-based cleaning method for cross-contamination, emphasizing proteins and DNA.
The transition and residual risks of the culture medium were assessed by measuring both wet and dried media using fluorescence intensity. Residual proteins and DNA in dried culture medium containing HT-1080 cells were analyzed following ultraviolet (UV) irradiation, wiping, and disinfectant treatment.
Wet conditions showed a higher transition to distilled water (DW), whereas dry conditions led to higher residual amounts on SUS304 plates. Various cleaning methods for residual culture medium were examined, including benzalkonium chloride with a corrosion inhibitor (BKC + I) and DW wiping, which demonstrated significantly lower residual protein and DNA compared to other methods. Furthermore, these cleaning methods were tested for residual medium containing cells, with BKC + I and DW wiping resulting in an undetectable number of cells. However, in some instances, proteins and DNA remained.
The study compared cleaning methods for proteins and DNA in cell products, revealing their advantages and disadvantages. Peracetic acid (PAA) proved effective for nucleic acids but not proteins, while UV irradiation was ineffective against both proteins and DNA. Wiping emerged as the most effective method, even though traceability remained challenging. However, wiping with ETH was not effective as it caused protein immobilization. Understanding the characteristics of these cleaning methods is crucial for developing effective contamination control strategies.
细胞处理设施在处理过程中面临环境细菌污染生物安全柜的风险，而人工处理自体细胞产品可能导致污染。我们提出了一种基于风险和证据的交叉污染清洁方法，重点关注蛋白质和DNA。
通过使用荧光强度测量湿培养基和干培养基来评估培养基的转移和残留风险。对含有HT-1080细胞的干培养基中的残留蛋白质和DNA进行紫外线（UV）照射、擦拭和消毒剂处理后进行分析。
湿条件下向蒸馏水（DW）的转移率更高，而干条件下在SUS304板上的残留量更高。研究了各种残留培养基的清洁方法，包括含缓蚀剂的苯扎氯铵（BKC + I）和DW擦拭，与其他方法相比，这两种方法的残留蛋白质和DNA显著更低。此外，对含细胞的残留培养基测试了这些清洁方法，BKC + I和DW擦拭使细胞数量检测不到。然而，在某些情况下，蛋白质和DNA仍然存在。
该研究比较了细胞产品中蛋白质和DNA的清洁方法，揭示了它们的优缺点。过氧乙酸（PAA）对核酸有效，但对蛋白质无效，而UV照射对蛋白质和DNA均无效。擦拭是最有效的方法，尽管可追溯性仍然具有挑战性。然而，用乙醇擦拭无效，因为它会导致蛋白质固定。了解这些清洁方法的特性对于制定有效的污染控制策略至关重要。
