A system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy.
A system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy.
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DOI：
10.1002/nbm.1940030502
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发表时间：
1990-10-01
影响因子：
2.9
通讯作者：
Sillerud, L O
中科院分区：
文献类型：
--
作者：
Freyer, J P;Fink, N H;Sillerud, L O
我们开发了一种在核磁共振波谱分析过程中灌注多细胞球体搅拌悬浮液的系统。培养基温度、pH、氧张力以及葡萄糖和乳酸浓度的测量表明，灌注过程中球体周围的宏观环境条件与标准旋转培养瓶中的环境条件相匹配。在 NMR 灌注室中培养长达 48 小时的球体在体积和细胞数量生长、中心坏死范围、细胞克隆性和增殖状态方面与在标准条件下培养的球体几乎相同。为了避免解释 NMR 谱时出现问题，我们使用了含有 10% 正常无机磷酸盐浓度的介质；比较生长和核磁共振研究表明该培养基对报告的结果没有影响。 31P NMR 光谱分析表明，在 8 小时的分析过程中，平均 pH、三磷酸核苷酸 (NTP) 与无机磷酸盐 (Pi) 的比率、NTP 总量和总能量基本保持恒定。在分析过程中停止球体培养物的搅拌会导致核苷酸磷酸池在 30 分钟内耗尽，同时 Pi 积累并转变为更酸性的细胞内 pH 值。如果在 30 分钟内恢复搅拌，这种效果可以逆转。停止灌注同时保持搅拌会导致 31P 光谱恶化，直到 120 分钟不再残留高能磷酸盐且 pH 值降至约 6。再灌注 30 分钟后，这种效应也部分可逆，需要 10 小时才能恢复到正常 31P 光谱。球体模型系统与 31P NMR 光谱分析的结合将为研究肿瘤细胞能量代谢和活力调节的基本问题提供强大的工具。
We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.