Detailed protocol for evaluation of dynamic perifusion of human islets to assess β-cell function
Detailed protocol for evaluation of dynamic perifusion of human islets to assess β-cell function
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DOI：
10.4161/isl.3.5.15938
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发表时间：
2011-09-01
期刊：
影响因子：
2.2
通讯作者：
Al-Abdullah, Ismail H.
中科院分区：
文献类型：
--
作者：
Bentsi-Barnes, Kwamina;Doyle, Maire E.;Al-Abdullah, Ismail H.
胰岛中P细胞质量的确定性测量是β细胞功能的测量，即，胰岛响应周围葡萄糖水平的微小变化以受控方式释放胰岛素的能力。含有葡萄糖和促分泌素的溶液通过胰岛的连续流动或动态灌注是体外调节胰岛素释放的最准确评估。在这里，我们详细描述了一种低成本，迷你灌注系统，可以适用于任何实验室，以评估胰岛功能，通过检查动态胰岛素释放响应于葡萄糖浓度升高和添加促分泌素。将纯度>80%且存活率>90%的人胰岛用低葡萄糖（1 mM）灌注，随后用高葡萄糖（16.8mM +/- KCl，25 mM）攻击。观察到对葡萄糖浓度升高的典型双相反应，峰值时胰岛素浓度平均增加8倍（高于基础）。类似地，用卡巴胆碱或毒蜥外泌肽-4（Byetta）与葡萄糖（6 mM）灌注导致胰岛素分泌高于基础水平的1.32倍和1.35倍增加。胰岛可维持在灌流装置中，并持续对葡萄糖反应长达3 h。以最低的财务成本和技术专长，该设备可以在任何生物实验室中设置，以在不到6小时的时间内评估许多细胞类型的调节激素释放。这将允许其他实验室在体外以更好地接近体内胰岛功能的方式测量胰岛素对他们的药物或修饰剂的反应。
The definitive measure of P-cell quality in an islet is the measurement of beta-cell function, i.e., the ability of the islets to release insulin in a controlled manner in response to minute changes in ambient glucose levels. Continuous flow or dynamic perifusion of the solution containing glucose and secretagogues through the islets is the most accurate assessment of regulated insulin release in vitro. Here, we describe in detail a low cost, mini-perifusion system that can be adapted to any laboratory to assess islet function by examining dynamic insulin release in response to elevated glucose concentrations and addition of secretagogues. Human islets with purity >80% and viability >90% were perifused with low glucose (1 mM) and subsequently challenged with high glucose (16.8 mM +/- KCl, 25 mM). A prototypical biphasic response to elevated glucose concentrations was observed with an average 8-fold (above basal) increase in insulin concentration at peak values. Similarly, perifusion with carbachol or exendin-4 (Byetta) with glucose (6 mM) resulted in 1.32- and 1.35-fold increase in insulin secretion above basal. Islets could be maintained in the perifusion apparatus and continued to respond to glucose for up to 3 h. At minimal financial cost and technical expertise, this apparatus can be set-up in any biological laboratory to evaluate regulated hormone release from many cell types in less than 6 h. This will allow other laboratories to measure insulin responses to their drug or modifier of interest in vitro, in a manner that better approximates islet function in vivo.