BACKGROUND Cells are continuously exposed to changes in their environment. Endocrine systems, in particular, communicate by rhythms and feedback loops. In this study, we developed an automated system to produce such conditions for cultured cells in a precisely timed manner. We utilized a programmable pair of syringe pumps for inflow and a peristaltic pump for outflow to create rhythmic pulses at 5-min intervals in solutions that mimic the endogenous patterns of insulin produced by pancreatic islets as a test case. RESULTS This perifusion system was first tested by measuring trypan blue absorbance, which was intermittently added and washed out at 3:3 and 2:3 min (in:out). Absorbance corresponded with patterns of trypan blue delivery. We then created patterns of forced oscillations in islets by intermittently switching between solutions containing 28 millimolar (mM) glucose (producing high levels of intracellular calcium ([Ca2+]i) and insulin secretion) and 28 mM glucose + calcium-channel blocker nifedipine (producing low levels of [Ca2+]i and insulin secretion). Forced perifusion effects were monitored by fura-2 AM fluorescence measurements of [Ca2+]i. Islets showed uniform oscillations in [Ca2+]i at time intervals consistent with the perifusion pattern, mimicking endogenous pulsatility. CONCLUSIONS This study highlights a valuable method to modify the environment of the cell culture over a period of hours to days.

中文翻译：

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一种用于随着时间改变细胞培养条件的自动灌注系统。

背景技术细胞连续地暴露于其环境的变化。内分泌系统尤其通过节律和反馈回路进行交流。在这项研究中，我们开发了一种自动化系统，以精确定时的方式为培养的细胞产生这种条件。我们使用可编程的一对注射泵进行流入，并使用蠕动泵进行流出，以在5分钟间隔内在模拟胰岛产生的胰岛素内源性模式的溶液中产生有节奏的脉动，作为测试案例。结果首先通过测量台盼蓝吸光度测试了该灌注系统，该台盼蓝吸光度是在3：3和2：3 min（in：out）间断添加并洗出的。吸光度与台盼蓝传递的模式相对应。然后，我们通过在包含28毫摩尔（mM）葡萄糖（产生高水平的细胞内钙（[Ca2 +] i）和胰岛素分泌）和28 mM葡萄糖+钙通道阻滞剂硝苯地平（产生低水平的[Ca2 +] i和胰岛素分泌）。通过[Ca2 +] i的fura-2 AM荧光测量监测强迫的灌注效果。胰岛显示[Ca2 +] i的均匀振荡，时间间隔与周围灌注模式一致，模仿内源性搏动。结论这项研究强调了一种有价值的方法，可以在数小时到数天的时间内改变细胞培养的环境。