Wartenberg M, Acker H
Max-Planck Institut für molekulare Physiologie, Dortmund, Germany.
Micron. 1995;26(5):395-404. doi: 10.1016/0968-4328(95)00009-7.
Fluorescent dyes were used in conjunction with confocal microscopy to record the vitality status of cells in multicellular glioma spheroids. Multicellular spheroids are in vitro models for micrometastases or intravascular microregions of large tumors. With progressing growth three distinct concentric annular shells develop. A rim of proliferating cells in the periphery is followed towards the center by layers of quiescent cells and at a defined spheroid diameter cell death occurs in the central core. Fluorescein diacetate (FDA) and Calcein/AM were used as vital stains and Lucifer Yellow/VS (LYVS) was used as a marker for dead cells. For loading multicellular spheroids with the esterase substrate dyes we used a two step cold incubation technique to avoid dye accumulation in the most peripheral cell layers. Homogenously stained tissue allowed to describe the fluorescence attenuation in depth as a monoexponential decay. An attenuation coefficient C was calculated from calibration experiments to be 12.5 x 10(-3) in vital stained tissue and 17.9 x 10(-3) in lethal stained tissue. Using the respective attenuation coefficient the raw data were corrected for light absorption and scattering in depth. In radial recordings of the vitality status of multicellular glioma spheroids using CLSM-technique we showed that spheroids up to a diameter of 250 microns were homogenously stained with Calcein/AM and FDA. Spheroids larger than 250 microns consist of vital stained cells and unstained cells. They do not show dead cell staining until they reach a diameter of about 400 microns. The thickness of the rim of vital stained cells decreased with increasing diameter of the spheroids to 64 +/- 7 microns in spheroids of a diameter of 550 +/- 25 microns. Thereafter the thickness of the Calcein/AM or FDA stained rim augmented again, reaching 93 +/- 9 microns in spheroids of 700 microns in diameter. The first signs of dead cell staining in the central core occurred at a diameter of 400 +/- 25 microns. The radius of the core increased in an exponential way. The cell layer which was stained neither by vital nor by lethal dyes showed a thickness of 150 microns in spheroids of 550 +/- 25 microns in diameter. Our staining technique and the radial recording of mean field fluorescence signals in living multicellular spheroids will be a valuable tool for experimental cancer research providing a non invasive quantification of cell vitality in living multicellular spheroids.
荧光染料与共聚焦显微镜结合使用，以记录多细胞胶质瘤球体中细胞的活力状态。多细胞球体是微转移或大肿瘤血管内微区域的体外模型。随着生长的进行，会形成三个不同的同心环形壳。外围是一层增殖细胞，接着向中心依次是静止细胞层，当球体直径达到一定程度时，中心核心会出现细胞死亡。荧光素二乙酸酯（FDA）和钙黄绿素/AM用作活细胞染料，而路西法黄/VS（LYVS）用作死细胞标记物。为了用酯酶底物染料加载多细胞球体，我们采用了两步冷孵育技术，以避免染料在最外围细胞层中积累。均匀染色的组织可将深度荧光衰减描述为单指数衰减。通过校准实验计算得出，活细胞染色组织中的衰减系数C为12.5×10⁻³，死细胞染色组织中的衰减系数C为17.9×10⁻³。使用各自的衰减系数对原始数据进行深度光吸收和散射校正。在使用共聚焦激光扫描显微镜（CLSM）技术对多细胞胶质瘤球体活力状态进行径向记录时，我们发现直径达250微米的球体被钙黄绿素/AM和FDA均匀染色。直径大于250微米的球体由活细胞染色的细胞和未染色的细胞组成。直到直径达到约400微米时，它们才会出现死细胞染色。活细胞染色边缘的厚度随着球体直径的增加而减小，在直径为550±25微米的球体中降至64±7微米。此后，钙黄绿素/AM或FDA染色边缘的厚度再次增加，在直径为700微米的球体中达到93±9微米。中心核心出现死细胞染色的最初迹象是在直径为400±25微米时。核心半径呈指数增长。在直径为550±25微米的球体中，既未被活细胞染料也未被死细胞染料染色的细胞层厚度为150微米。我们的染色技术以及对活的多细胞球体中平均场荧光信号的径向记录，将成为实验性癌症研究的一个有价值工具，可对活的多细胞球体中的细胞活力进行非侵入性定量分析。
