Marsden K M, Kershaw T R, Sinden J D, Lantos P L
Department of Psychology, Institute of Psychiatry, London, U.K.
Brain Res. 1991 Dec 24;568(1-2):76-84. doi: 10.1016/0006-8993(91)91381-a.
The visualisation of transplanted cell lines is essential to determine both their viability and possible functional properties. Fluorescent latex microspheres were used to label cultured human neuroblastoma IMR-32 cells prior to transplantation. IMR-32 cells were first rendered amitotic by treatment with mitomycin C and bromodeoxyuridine and subsequently incubated with fluorescent microspheres for 3 days. Cell suspensions were prepared from these cultures and transplanted into the cortex and hippocampus of male Sprague-Dawley rats bearing ibotenate lesions of forebrain cholinergic projections. The animals were perfused at 4, 8 and 12 weeks post-transplantation and tissue was prepared for electron and light microscopy. IMR-32 cells containing fluorescent microspheres were clearly visualised in cryostat sections at all time points. Greater survival was seen in the hippocampus, with evidence of migration of cells from the site of implantation. Macrophages were seen at the electron and light microscope level, and were distinct from the discrete fluorescent labelled IMR-32 cells. Ultrastructurally, transplanted IMR-32 cells resembled cells in vitro, with microspheres clearly distinguished within the cytoplasm. Fluorescent microspheres provide a simple and direct labelling technique suitable for long-term transplant experiments using characterised cell lines.
对移植细胞系进行可视化对于确定其活力和可能的功能特性至关重要。在移植前，使用荧光乳胶微球标记培养的人神经母细胞瘤IMR - 32细胞。首先用丝裂霉素C和溴脱氧尿苷处理IMR - 32细胞使其停止有丝分裂，随后将其与荧光微球孵育3天。从这些培养物中制备细胞悬液，并将其移植到患有前脑胆碱能投射鹅膏蕈氨酸损伤的雄性Sprague - Dawley大鼠的皮质和海马中。在移植后4周、8周和12周对动物进行灌注，并制备用于电子显微镜和光学显微镜观察的组织。在所有时间点的低温恒温器切片中都能清楚地看到含有荧光微球的IMR - 32细胞。在海马中观察到更高的细胞存活率，并有细胞从植入部位迁移的证据。在电子显微镜和光学显微镜水平都观察到了巨噬细胞，它们与离散的荧光标记的IMR - 32细胞不同。在超微结构上，移植的IMR - 32细胞类似于体外细胞，微球在细胞质中清晰可辨。荧光微球提供了一种简单直接的标记技术，适用于使用特征明确的细胞系进行长期移植实验。
