Bioassay

Dynamic chromogenic method for detection of bacterial endotoxin content in umbilical cord mesenchymal stem cell suspension

Expand
  • Zhejiang Free Trade Zone Hopkins Biotechnology Co., Ltd., Zhoushan 316000, China

Received date: 2022-08-21

  Online published: 2024-06-26

Abstract

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.

Cite this article

YUAN Chao-lu, ZHANG Si-shi, CHEN Yi-ru, LI Jia-hui, TANG Jia . Dynamic chromogenic method for detection of bacterial endotoxin content in umbilical cord mesenchymal stem cell suspension[J]. Chinese Journal of Pharmaceutical Analysis, 2022 , 42(12) : 2110 -2115 . DOI: 10.16155/j.0254-1793.2022.12.06

References

[1] PHINNEY DG, PROCKOP DJ. Concise review: mesenchymal stem/multipotent stromal cells: the state of trans differentiation and modes of tissue repair—current views[J].Stem Cells, 2007, 25(11):2896
[2] REINE EO, JACQUELINE B, STOLTZ JF, et al. Umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal stem cell-based therapies[J].Tissue Eng Part B Rev, 2014, 20(5):523
[3] 刘梦婷,饶巍,韩兵,等.人脐带间充质干细胞的体外免疫调节特性[J].中国组织工程研究,2020,24(7):1063
LIU MT, RAO W, HAN B, et al. Immunomodulatory characteristics of human umbilical cord mesenchymal stem cells in vitro[J].Chin J Tiss Eng Res, 2020, 24(7): 1063
[4] 项楠,汪国生,厉小梅.我国干细胞临床研究现状分析、政策回顾及展望[J].中华细胞与干细胞杂志(电子版),2020,10(5):303
XIANG N, WANG GS, LI XM. Status quo, policy review and prospect of stem cell clinical trials in china[J].Chin J Cell Stem Cell (Elect Editn), 2020, 10(5): 303
[5] 蒋伟民,黄元华,马燕琳.人胚胎干细胞建株与维持培养条件的优化[J].生命科学研究,2014,18(4):349
JIANG WN, HUANG YH, MA YL. The optimization of generating and culturing human embryonic stem cells[J].Life Sci Res, 2014, 18(4): 349
[6] 周琪,任小波,杨旭,等.面向未来的新一轮医疗技术革命—干细胞与再生医学研究战略性先导科技专项进展[J].中国科学院院刊,2015,30(2): 262
ZHOU Q, RENG XB, YANG X, et al. Confronting coming medical renovation—progress on stem cell and regenerative medicine research[J].Bull Chin Acad Sci, 2015, 30(2): 262
[7] 祝贺,郝捷,周琪,等.临床级干细胞库及干细胞制剂[J].生命科学, 2016, 28(8): 895
ZHU H, HAO J, ZHOU Q, et al. Clinical-grade stem cell bank and stem cell products[J].Life Sci, 2016, 28(8): 895
[8] 张军霞,郭姣梅,周继春,等.动态显色法检测 4 种清热解毒类中药注射液中细菌内毒素含量[J].中国药房,2018,29(13):1806
ZHANG JX, GUO JM, ZHOU JC, et al. Content determination of bacterial endotoxin in 4 kinds of heat-clearing and detoxifying TCM injections by kinetic chromogenic assay[J].China Pharm, 2018, 29(13): 1806
[9] 李树馀,梁进.微量动态显色法定量检测3种大输液的细菌内毒素[J].中国药师,2014,17(6): 1052
LI SY, LIANG J.Application of micro-kinetic chromogenic method in endotoxin quantitative detection of three kinds of infusions[J].China Pharm, 2014, 17(6): 1052
[10] 杨晨曦.细菌内毒素试验凝胶定性法与动态浊度定量法的比较[J].临床血液学杂质,2010,23(6): 366.
YANG CX.Comparison of gel qualitative method and dynamic turbidity quantitative method for bacterial endotoxin test[J].J Clin Hematol, 2010, 23(6): 366
[11] 中华人民共和国药典2020年版. 三部[S].2020:179
ChP 2020. Vol Ⅲ[S].2020:179
[12] 中华人民共和国药典2020年版.四部[S].2020:178
ChP 2020. Vol Ⅳ[S].2020:178
[13] 林金海,陈晓佳,丁友玲.注射用盐酸溴己新原料中细菌内毒素的检测[J].上海医药, 2020, 41(7): 75
LIN JH, CHEN XJ, DING YL. Study on the bacterial endotoxin test in the raw material of bromhexine hydrochloride for injection[J].Shanghai Med Pharm J, 2020, 41(7): 75
[14] 马莉,张凇,王安航,等.细菌内毒素定量检测方法的常见问题剖析[J].中国医院药学杂志,2013,33(9): 733
MA L, ZHANG S, WANG AH, et al. Analysis of common problems in quantitative detection of bacterial endotoxin[J].Chin J Hosp Pharm, 2013, 33(9): 733
[15] 田祥学.细菌内毒素检查法及其在药品检验中的应用[J].中国医药指南, 2015, 13(23): 278
TIAN XX. Bacterial endotoxin test and its application in drug inspection[J].Guid Chin Med, 2015, 13(23): 278
[16] 王巨才,王雪.动态显色法定量检测磺达肝癸钠注射液中细菌内毒素[J].药物分析杂志,2021,41(1): 180
WANG JC, WANG X. Determination of bacterial endotoxin in fondaparinux sodium injection by kinetic chromogenic assay[J].Chin J Pharm Anal, 2021, 41(1): 180
[17] 裴宇盛,蔡彤,高华.细菌内毒素检查新方法进展[J].药物分析杂志, 2014, 34(3): 392
PEI YS, CAI T, GAO H. Progress on new methods of bacterial endotoxin test[J].Chin J Pharm Anal, 2014, 34(3):392
Outlines

/