Klauke Norbert, Smith Godfrey L, Cooper Jon
Department of Electronics, University of Glasgow, Glasgow, United Kingdom.
Biophys J. 2003 Sep;85(3):1766-74. doi: 10.1016/S0006-3495(03)74606-2.
Microchannels (40- microm wide, 10- microm high, 10-mm long, 70- microm pitch) were patterned in the silicone elastomer, polydimethylsiloxane on a microscope coverslip base. Integrated within each microchamber were individually addressable stimulation electrodes (40- microm wide, 20- microm long, 100-nm thick) and a common central pseudo-reference electrode (60- microm wide, 500- microm long, 100-nm thick). Isolated rabbit ventricular myocytes were introduced into the chamber by micropipetting and subsequently capped with a layer of mineral oil, thus creating limited volumes of saline around individual myocytes that could be varied from 5 nL to 100 pL. Excitation contraction coupling was studied by monitoring myocyte shortening and intracellular Ca(2+) transients (using Fluo-3 fluorescence). The amplitude of stimulated myocyte shortening and Ca(2+) transients remained constant for 90 min in the larger volume (5 nL) configuration, although the shortening (but not the Ca(2+) transient) amplitude gradually decreased to 20% of control within 60 min in the low volume (100 pL) arrangement. These studies indicate a lower limit for the extracellular volume required to stimulate isolated adult cardiac myocytes. Whereas this arrangement could be used to create a screening assay for drugs, individual microchannels (100 pL) can also be used to study the effects of limited extracellular volume on the contractility of single cardiac myocytes.
在显微镜盖玻片基底上的聚二甲基硅氧烷硅橡胶弹性体中制作了微通道（宽40微米、高10微米、长10毫米、间距70微米）。每个微腔中集成了可单独寻址的刺激电极（宽40微米、长20微米、厚100纳米）和一个公共的中央伪参考电极（宽60微米、长500微米、厚100纳米）。通过微量移液将分离的兔心室肌细胞引入腔室，随后覆盖一层矿物油，从而在单个肌细胞周围形成有限体积的盐水，其体积可在5纳升至100皮升之间变化。通过监测肌细胞缩短和细胞内Ca(2+)瞬变（使用Fluo-3荧光）来研究兴奋收缩偶联。在较大体积（5纳升）配置中，刺激的肌细胞缩短和Ca(2+)瞬变的幅度在90分钟内保持恒定，尽管在低体积（100皮升）配置中，缩短幅度（但Ca(2+)瞬变幅度未变）在60分钟内逐渐降至对照的20%。这些研究表明了刺激分离的成年心肌细胞所需的细胞外体积下限。虽然这种配置可用于创建药物筛选试验，但单个微通道（100皮升）也可用于研究有限细胞外体积对单个心肌细胞收缩性的影响。
