Development of a novel microperfusion chamber for determination of cell membrane transport properties
Development of a novel microperfusion chamber for determination of cell membrane transport properties
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DOI：
10.1016/s0006-3495(96)79246-9
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发表时间：
1996-07-01
影响因子：
3.4
通讯作者：
Critser, JK
中科院分区：
文献类型：
--
作者：
Gao, DY;Benson, CT;Critser, JK
一种新型的微灌流室的开发，以测量动力学细胞体积的变化，在各种细胞外条件下，并定量测定细胞膜转运特性。该装置消除了建模的模糊性和固有的限制，在使用的微扩散室和微量移液管灌注技术，这两者都已经过验证，并密切相关的光学技术，使用光学显微镜和图像分析。由此产生的简单性应被证明是特别有价值的耦合运输的水和渗透溶质通过细胞膜的研究。利用该微灌流室测定了小鼠卵母细胞对水和二甲基亚砜（DMSO）的渗透系数以及金黄地鼠胰岛细胞对水的渗透系数。在这些实验中，将单个细胞保持在室中，并在22 ℃下用高渗培养基灌注，有或没有DMSO（1.5M）。将细胞体积变化录像并通过图像分析定量。根据实验数据和跨膜耦合传质的不可逆热力学理论，确定了卵母细胞的透水系数为0.47 μ m。. min（-1）。atm（-1），在不存在DMSO和0.65 μ m .最小值（-1）。atm（-1）下进行。卵母细胞膜的DMSO渗透系数和膜对DMSO的反射系数分别为0.23和0.85 μ m/s。这些值与使用微量移液管灌注和微扩散室技术测定的值一致。金黄地鼠胰岛细胞的水渗透系数测定为0.27 μ m。最小值（-1）。atm（-1），这与先前使用电子尺寸测量（库尔特计数器）技术确定的值非常一致。使用微灌注室具有以下主要优点：1）该方法允许通过用制备的培养基灌注单个细胞或细胞群来容易地改变细胞外条件（可以用不同的培养基再灌注细胞以研究相同细胞对不同渗透条件的响应）。2)细胞和灌注介质的短混合时间允许精确控制细胞外渗透压摩尔浓度，并确保相应数学公式（建模）的准确性。3)该技术在研究细胞渗透反应和测定细胞膜转运特性方面具有广泛的适用性。
A novel microperfusion chamber was developed to measure kinetic cell volume changes under various extracellular conditions and to quantitatively determine cell membrane transport properties. This device eliminates modeling ambiguities and limitations inherent in the use of the microdiffusion chamber and the micropipette perfusion technique, both of which have been previously validated and are closely related optical technologies using light microscopy and image analysis. The resultant simplicity should prove to be especially valuable for study of the coupled transport of water and permeating solutes through cell membranes. Using the microperfusion chamber, water and dimethylsulfoxide (DMSO) permeability coefficients of mouse oocytes as well as the water permeability coefficient of golden hamster pancreatic islet cells were determined. In these experiments, the individual cells were held in the chamber and perfused at 22 degrees C with hyperosmotic media, with or without DMSO (1.5 M). The cell volume change was videotaped and quantified by image analysis. Based on the experimental data and irreversible thermodynamics theory for the coupled mass transfer across the cell membrane, the water permeability coefficient of the oocytes was determined to be 0.47 mu m . . min(-1) . atm(-1) in the absence of DMSO and 0.65 mu m . min(-1) . atm(-1) in the presence of DMSO. The DMSO permeability coefficient of the oocyte membrane and associated membrane reflection coefficient to DMSO were determined to be 0.23 and 0.85 mu m/s, respectively. These values are consistent with those determined using the micropipette perfusion and microdiffusion chamber techniques. The water permeability coefficient of the golden hamster pancreatic islet cells was determined to be 0.27 mu m . min(-1). atm(-1), which agrees well with a value previously determined using an electronic sizing (Coulter counter) technique. The use of the microperfusion chamber has the following major advantages: 1) This method allows the extracellular condition(s) to be readily changed by perfusing a single cell or group of cells with a prepared medium (cells can be reperfused with a different medium to study the response of the same cell to different osmotic conditions). 2) The short mixing time of cells and perfusion medium allows for accurate control of the extracellular osmolality and ensures accuracy of the corresponding mathematical formulation (modeling). 3) This technique has wide applicability in studying the cell osmotic response and in determining cell membrane transport properties.