Program of Molecular Medicine, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
Program of Translational Medicine, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.
Cells. 2023 Apr 17;12(8):1174. doi: 10.3390/cells12081174.
It has been suggested that in vitro studies of the rescue effect of CFTR modulator drugs in nasal epithelial cultures derived from people with cystic fibrosis have the potential to predict clinical responses to the same drugs. Hence, there is an interest in evaluating different methods for measuring in vitro modulator responses in patient-derived nasal cultures. Commonly, the functional response to CFTR modulator combinations in these cultures is assessed by bioelectric measurements, using the Ussing chamber. While this method is highly informative, it is time-consuming. A fluorescence-based, multi-transwell method for assaying regulated apical chloride conductance (Fl-ACC) promises to provide a complementary approach to theratyping in patient-derived nasal cultures. In the present work, we compared Ussing chamber measurements and fluorescence-based measurements of CFTR-mediated apical conductance in matching, fully differentiated nasal cultures derived from CF patients, homozygous for F508del ( = 31) or W1282X ( = 3), or heterozygous for Class III mutations G551D or G178R ( = 5). These cultures were obtained through a bioresource called the Cystic Fibrosis Canada-Sick Kids Program in Individual CF Therapy (CFIT). We found that the Fl-ACC method was effective in detecting positive responses to interventions for all genotypes. There was a correlation between patient-specific drug responses measured in cultures harbouring F508del, as measured using the Ussing chamber technique and the fluorescence-based assay (Fl-ACC). Finally, the fluorescence-based assay has the potential for greater sensitivity for detecting responses to pharmacological rescue strategies targeting W1282X.
有人提出，在体外研究 CFTR 调节剂药物对囊性纤维化患者鼻上皮培养物的挽救作用，有可能预测这些药物的临床反应。因此，人们有兴趣评估评估患者来源的鼻培养物中体外调节剂反应的不同方法。通常，使用 Ussing 室通过生物电测量来评估这些培养物中 CFTR 调节剂组合的功能反应。虽然这种方法非常有信息量，但很耗时。一种基于荧光的、用于测定调节顶端氯离子电导的多跨膜方法（Fl-ACC）有望为患者来源的鼻培养物中的定型提供一种补充方法。在本工作中，我们比较了源自 CF 患者的完全分化的鼻培养物的 Ussing 室测量和基于荧光的 CFTR 介导的顶端电导率测量，这些患者为 F508del 纯合子（=31）或 W1282X（=3），或 G551D 或 G178R （=5）的 III 类突变杂合子。这些培养物是通过一个名为囊性纤维化加拿大- Sick Kids 个体化 CF 治疗计划（CFIT）的生物资源获得的。我们发现，Fl-ACC 方法在检测针对所有基因型的干预措施的阳性反应方面是有效的。在含有 F508del 的培养物中，使用 Ussing 室技术和基于荧光的测定法（Fl-ACC）测量的患者特异性药物反应之间存在相关性。最后，基于荧光的测定法有可能更灵敏地检测针对 W1282X 的药理学挽救策略的反应。
