目的: 建立用于定量检测脐带间充质干细胞悬液中细菌内毒素的方法,并验证其可行性。方法: 参考2020年版《中华人民共和国药典》(简称《中国药典》)第四部通则1143细菌内毒素光度测定法的标准,从标准曲线建立及可靠性验证、供试品干扰初筛及干扰性检测2个方面,初步建立脐带间充质干细胞悬液中细菌内毒素含量的检测方法,并用此方法对3批脐带间充质干细胞悬液的细菌内毒素进行了定量检测。结果: 在标准曲线建立可靠性验证中,得到线性回归方程:lgT=2.778 0-0.281 4lgC,r=-0.999 8,|r|>0.980,阴性对照的T＞3 600 s(标准曲线最低点平均反应的T为2 690 s),反应重复管的RSD均≤10%,表明标准曲线建立成功;干扰初筛试验结果显示,干细胞悬液在20、40、80稀释倍数下,对鲎试剂检测均无干扰作用,且40倍稀释时的回收率接近100%,表明在无干扰的情况下,40倍为最佳干扰稀释倍数;3批供试品的试验结果显示,40倍稀释下,3批供试品的内毒素含量均小于限值0.5 EU·mL-1,且RSD均＜10%,阳性回收率在86.8%~101.2%,符合2020年版《中国药典》的规定。结论: 本方法能快速定量检测脐带间充质干细胞悬液中内毒素含量,抗干扰能力强,能较好地满足细胞悬液中细菌内毒素含量测定。
Objective: To establish a quantitative detection method of bacterial endotoxin in umbilical cord mesenchymal stem cell suspension and verify its feasibility. Methods: According to the standards for photometric determination of bacterial endotoxin in General notice 1143 of Chinese Pharmacopoeia part Ⅳ (2020 edition). A preliminary detection method of bacterial endotoxin content in umbilical cord mesenchymal stem cell suspension was established from two aspects: standard curve establishment, reliability verification, sample interference screening and interference detection. Bacterial endotoxins in three batches of umbilical cord mesenchymal stem cell suspensions were quantitatively detected by this method. Results: In the establishment of standard curve and reliability verification test, the linear regression equation was lgT=2.778 0-0.281 4lgC, r=-0.999 8,|r|>0.980, negative control time>3 600 s (standard curve low average response of time was 2 690 s), repeated reaction tube RSD were 10% or less, the standard curve was established successfully. The results of interference screening test showed that the stem cell suspension had no interference on limulus test at the dilution ratio of 20, 40 and 80, and the recovery rate was close to 100% at the dilution ratio of 40, indicating that 40 times was the best interference dilution ratio without interference. The results of the three batches of samples showed that the endotoxin content of the three batches of samples were less than 0.5 EU·mL-1 under 40 times dilution, and the RSD was less than 10%. The positive recovery was 86.8%-101.2%, which complied with The Chinese Pharmacopoeia 2020 edition. Conclusion: This method can quickly and quantitatively detect the content of endotoxin in umbilical cord mesenchymal stem cell suspension, and has strong anti-interference ability, which can meet the determination of bacterial endotoxin in cell suspension.
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