Yamashita Kyohei, Yamada Koji, Suzuki Kengo, Tokunaga Eiji
Department of Physics, Faculty of Science, Tokyo University of Science, Tokyo, Japan.
euglena Co., Ltd., Tsurumi-ku, Yokohama-shi, Kanagawa, Japan.
PeerJ. 2019 Apr 4;7:e6636. doi: 10.7717/peerj.6636. eCollection 2019.
Noninvasive and safe cell viability assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined eight typical natural food pigments to find pigment (MP) or anthocyanin pigment (AP) works as a good viability indicator of dye exclusion test (DET) for which is an edible photosynthetic green microalga. This is the first report using natural food pigments as cell viability assay. stained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imaging (, ) microscopy of single live cells and principal component analysis (PCA). To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth of , but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, viability of cells in culture can be monitored with them over a long period.
在再生医学、基因工程、单细胞分析和微生物食品培养等许多领域，都需要无创且安全的细胞活力检测方法。在这种情况下，一种安全且廉价的方法是优选的，它对细胞和环境的负荷较小，无需昂贵且占空间的设备以及技术人员来操作。我们研究了八种典型的天然食用色素，以寻找能作为染料排斥试验（DET）良好活力指标的色素（MP）或花青素色素（AP），该试验针对的是可食用的光合绿色微藻。这是首次使用天然食用色素进行细胞活力检测的报告。用MP或AP染色的细胞可以通过明场显微镜进行肉眼判断。这通过对单个活细胞进行无扫描、无创吸收光谱成像（，）显微镜观察和主成分分析（PCA）得到了光谱证实。为了确认这两种天然色素对死细胞的染色能力并检测其对细胞的负荷，将它们与传统用于DET的合成染料台盼蓝（TB）和亚甲基蓝（MP）进行了比较。结果表明，MP和AP对经微波处理的死细胞的染色能力与TB和MB相当，并且在苯扎氯铵中对死细胞的染色速度更快、更均匀。培养物中的生长曲线和死细胞比例表明，合成染料会抑制的生长，但天然色素不会。然而，随着细胞密度的增加，AP会增加染色细胞的比例，添加葡萄糖可防止这种情况发生。MP比AP能在更短时间内染色死细胞，而AP在强光长期照射下比MP颜色更稳定。由于这些色素的低毒性，可以用它们长期监测培养细胞的活力。
