Establishment And Standardization Of Rapid Detection Methods Of The Contaminated Microorganisms (Bacteria,Fungi And Mycoplasma) In Cell Preparations

With the development of cell preparations in cell therapy and clinical research,it is particularly important to establish a strict quality control system for cell preparations and ensure that the cell preparations are safe for clinical use.Cell preparations are highly susceptible to microbial contamination during cultivation,can be tested using the compendial sterility test protocols for biological products,are time consuming,have significant lag in results,and cannot meet the requirements for release QC of cell preparations.Therefore,it is urgent to establish rapid and sensitive detection methods for microbial contamination that are suitable for cell preparations.The specific research content is as follows:（1）Establishment of a serum-free culture system for MSC:a serum-free culture system for MSC was established,and the cultured MSC were characterized.MSc morphology was uniformly spindle shaped and cell viability was>90%;The expression rate of cell surface marker positive antigen molecules（CD90,CD105）was≥95%and that of negative antigen molecules（CD45,CD34）was≤2%;With the ability to differentiate into osteoblasts and adipocytes.（2）Cell preparation detection method for ATP biofluorimetry of bacterial and fungal contamination establishment:an ATP based biofluorescent assay suitable for the detection of contaminating bacteria and fungi in cell preparations was developed using MSC.The optimized reaction system(using Hepes buffer at p H 7.6,0.4mg·m L^-1luciferase concentration,0.7 mg·m L^-1luciferin concentration,8 mmol·L^-1Mg²⁺concentration,1 mmol·L^-1DTT concentration,24℃ temperature,0.5% Triton X-100 extraction reagent for ATP extraction,and 0.015%CTAB extraction solution)was determined by analyzing various factors affecting the biofluorescent detection of ATP;A standardized operation flow for the ATP detection method was established.The simulated contamination detection results showed that:the detection limit was 60 cfu·m L^-1for representative strain C.albicans and 150 cfu·m L^-1for E.coli.Reference to the Pharmacopoeia’s alternative method of microbiological testing,the established ATP biofluorescent assay was methodologically validated,and the results showed that the exclusivity of the ATP biofluorescent assay was good and the assay was reproducible.（3）Establishment of a mycoplasma fluorescence test for mycoplasma contamination of cell preparations:a fluorescent test for mycoplasma that is suitable for the detection of mycoplasma contamination in cell preparations has been established,various factors affecting the detection have been explored,and a standardized operation flow for the detection method has been established.The validity of the established mycoplasma fluorescence assay for the detection of mycoplasma contamination was demonstrated by comparative verification with the mycoplasma staining method and the mycoplasma PCR method.In this study,using MSC cultured in serum-free medium,an ATP biofluorimetric method to detect bacterial and fungal contamination in cell preparations and a mycoplasma fluorometric method to detect mycoplasma Contamination in cell preparations were successfully established,and the established assays were validated and preliminary standardized in this study,forming a standard proposal for related detection techniques and providing a new choice for rapid detection of microbial contamination in cell preparations.