Zheng C H, Levenston M E
Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305-4038,
Eur Cell Mater. 2015 Apr 19;29:224-36; discussion 236. doi: 10.22203/ecm.v029a17.
The 1,9-dimethylmethylene blue (DMMB) assay is widely used to quantify sulfated glycosaminoglycan (sGAG) contents of engineered tissues, culture media, tissue samples and bodily fluids, but the assay is subject to interference from polyanions such as hyaluronic acid (HA), DNA and RNA. We examined whether specific combinations of dye pH and absorbance wavelength could minimize non-sGAG artifacts without compromising DMMB assay sensitivity. HA and DNA solutions generated substantial signal at pH 3 but not at pH 1.5. Reducing dye pH did not significantly alter sGAG measurements for normal cartilage and meniscus tissues, but eliminated anomalously high apparent sGAG contents for enzymatically isolated chondrocytes, adipose-derived stem cell (ADSC)-agarose constructs and ADSC pellets. In a cartilage tissue-engineering case study, pH 3 dye indicated high apparent sGAG readings throughout culture in both basal and chondrogenic media, with a marked decline between day 14 and 21 for chondrogenic constructs. The pH 1.5 dye, however, indicated minimal sGAG accumulation in basal medium and stable sGAG content throughout culture in chondrogenic medium. As it is often difficult to know a priori whether all groups in a study will have sGAG contents high enough to overwhelm artifacts, we recommend modifying the standard DMMB assay to reduce the risk of spurious findings in tissue engineering and clinical research. Specifically, we recommend shifting to a pH 1.5 DMMB dye and basing quantification on the absorbance difference between 525 nm (µ peak) and 595 nm (β peak) to compensate for the moderate loss of sensitivity associated with reducing the dye pH.
1,9 - 二甲基亚甲基蓝（DMMB）测定法被广泛用于定量工程组织、培养基、组织样本和体液中的硫酸化糖胺聚糖（sGAG）含量，但该测定法会受到透明质酸（HA）、DNA和RNA等多聚阴离子的干扰。我们研究了染料pH值和吸光度波长的特定组合是否能在不影响DMMB测定法灵敏度的情况下，将非sGAG假象降至最低。HA和DNA溶液在pH 3时产生大量信号，但在pH 1.5时则不会。降低染料pH值对正常软骨和半月板组织的sGAG测量值没有显著影响，但消除了酶解分离的软骨细胞、脂肪来源干细胞（ADSC）-琼脂糖构建体和ADSC微球中异常高的表观sGAG含量。在一个软骨组织工程案例研究中，pH 3染料显示在基础培养基和成软骨培养基中整个培养过程的表观sGAG读数都很高，而成软骨构建体在第14天至21天之间有明显下降。然而，pH 1.5染料显示基础培养基中sGAG积累极少，而成软骨培养基中整个培养过程sGAG含量稳定。由于通常很难事先知道研究中的所有组的sGAG含量是否高到足以掩盖假象，我们建议修改标准DMMB测定法，以降低组织工程和临床研究中出现虚假结果的风险。具体而言，我们建议改用pH 1.5的DMMB染料，并基于525 nm（µ峰）和595 nm（β峰）之间的吸光度差异进行定量，以补偿因降低染料pH值而导致的灵敏度适度损失。
