Hashimoto Muneaki, Ido Yusuke, Tanaka Masato, Yokota Kazumichi, Kajimoto Kazuaki, Kataoka Masatoshi
Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2217-14, Hayashi-cho, Takamatsu, Kagawa 761-0301, Japan.
Parasitol Int. 2019 Apr;69:54-58. doi: 10.1016/j.parint.2018.11.005. Epub 2018 Nov 20.
It is very important to reduce the costs involved in malarial drug development by small-scale culture of Plasmodium falciparum, and automation of the assay system for drug efficacy against the parasites for high-throughput screening. In this study, we report that P. falciparum-infected erythrocytes can be stably cultured on μ-Slide Angiogenesis, which is used to investigate angiogenesis in tube formation assays, followed by automatic counting of the infection rate (parasitaemia). After 10 μL of parasite-infected erythrocytes were added to the inner well of μ-Slide Angiogenesis to prevent a multilayer of erythrocytes, 30 μL of silicon oil was overlaid on the culture medium to avoid evaporation of the medium, leading to stable small-scale parasite cultivation. The parasites were stained with a cell-permeant fluorescent nucleic acid stain (SYTO21) followed by cultivation. After taking bright field and fluorescent images using an inverted microscope, the infection rate could be calculated automatically by counting the number of erythrocytes and parasites using MetaMorph Offline software. The effect of anti-malarial drugs on parasite growth could be investigated on μ-Slide Angiogenesis, in which the parasite culture was added to the inner wells containing the drugs followed by their cultivation. Taken together, this method may be useful for image-based screening for anti-malarial drug candidates with automatic counting of parasite infection rates.
通过恶性疟原虫的小规模培养以及针对寄生虫的药物疗效检测系统的自动化，以进行高通量筛选，来降低疟疾药物研发成本非常重要。在本研究中，我们报告称，感染恶性疟原虫的红细胞可以在用于血管生成检测中研究血管生成的μ-Slide Angiogenesis上稳定培养，随后自动计数感染率（疟原虫血症）。将10μL感染寄生虫的红细胞添加到μ-Slide Angiogenesis的内孔中以防止红细胞形成多层，然后在培养基上覆盖30μL硅油以避免培养基蒸发，从而实现稳定的小规模寄生虫培养。寄生虫先用细胞渗透性荧光核酸染料（SYTO21）染色，然后进行培养。使用倒置显微镜拍摄明场和荧光图像后，可通过使用MetaMorph Offline软件计数红细胞和寄生虫的数量来自动计算感染率。可以在μ-Slide Angiogenesis上研究抗疟药物对寄生虫生长的影响，即将寄生虫培养物添加到含有药物的内孔中，然后进行培养。综上所述，这种方法可能有助于基于图像的抗疟候选药物筛选，并自动计数寄生虫感染率。
