常用组织材料脱细胞试剂残留量与细胞毒性相关性研究

Correlation between the residual amount of decellularization reagents in common tissue materials and cytotoxicity

目的研究常用组织材料脱细胞试剂十二烷基硫酸钠(SDS)、聚乙二醇辛基苯基醚(Triton X-100)和硫代甜菜碱10(SB-10)的残留量与细胞毒性的相关性,并找出控制试剂残留的安全阈值。方法参照GB/T 16886.5中的细胞毒性检测方法,分别检测不同质量浓度的SDS、Triton X-100、SB-10对L-929细胞和MC3T3-E1细胞的细胞毒性。细化上述3种脱细胞试剂的质量浓度区间,确定单种试剂残留无细胞毒性的阈值。将3种试剂两两混合或3种共混,通过细胞毒性实验确定试剂混合后无细胞毒性的阈值。结果 SDS、Triton X-100和SB-10对L-929细胞产生细胞毒性的临界浓度分别为4×10^(-3)、2×10^(-2)和6×10^(-1) mg/ml,对MC3T3-E1细胞产生细胞毒性的临界浓度分别为6×10^(-3)、4×10^(-2)和2 mg/ml。3种试剂细胞毒性大小趋势为SDS>Triton X-100>SB-10。将临界浓度的3种试剂两两混合或3种共混,对L-929细胞和MC3T3-E1细胞均无细胞毒性。结论采用上述3种脱细胞试剂制备脱细胞支架时应采用多种试剂清洗工序,高效清洗组织使试剂残留量低于临界值,以确保脱细胞支架的生物安全性。

Objective To study the correlation between the residual amounts of commonly used tissue decellularization reagents like sodium dodecyl sulfate(SDS),Triton X-100,sulfobetaine 10(SB-10)and cytotoxicity,and to find the safety threshold for controlling the residual of the reagent.Methods SDS,Triton X-100,SB-10 solutions of different concentrations were prepared,and the cytotoxicity of solutions of different concentrations was detected by L-929 cells and MC3T3-E1 cells according to the method of GB/T 16886.5.The mass concentration range of the above three decellularization reagents was refined,and the threshold for the residual non-cytotoxicity of a single reagent was determined.Mix the three reagents in pair or three blends,and determine the threshold of non-cytotoxicity after the reagents are mixed by cytotoxicity test.Results SDS,Triton X-100 and SB-10 caused L-929 cells to produce cytotoxic concentration values of 4×10^(-3),2×10^(-2) and 6×10^(-1) mg/ml,and MC3T3-E1 cells 6×10^(-3),4×10^(-2) and 2 mg/ml.The toxicity trend of the three reagents is SDS>Triton X-100>SB-10.When three reagents at critical concentrations are mixed in pairs or three,they have no cytotoxicity to L-929 cells and MC3T3-E1 cells.Conclusions When using the above three decellularization reagents to prepare decellularized scaffolds,a variety of reagent cleaning procedures should be used to efficiently clean the tissues so that the residual amount of reagents is below the critical value to ensure the biological safety of the decellularized scaffolds.