Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Anal Bioanal Chem. 2010 Aug;397(7):2939-47. doi: 10.1007/s00216-010-3897-z. Epub 2010 Jun 12.
A three-layer microfluidic device was developed that combined perfusion of cultured cells with on-line chemical analysis for near real-time monitoring of cellular secretions. Two layers were reversibly sealed to form a cell chamber that allowed cells grown on coverslips to be loaded directly into the chip. The outlet of the chamber was in fluidic contact with a third layer that was permanently bonded. Perfusate from the cell chamber flowed into this third layer where a fluorescence enzyme assay for non-esterified fatty acid (NEFA) was performed on-line. The device was used to monitor efflux of NEFAs from approximately 6,200 cultured adipocytes with 83 s temporal resolution. Perfusion of murine 3T3-L1 cultured adipocytes resulted in an average basal concentration of 24.2 +/- 2.4 microM NEFA (SEM, n = 6) detected in the effluent corresponding to 3.31 x 10(-5) nmol cell(-1) min(-1). Upon pharmacological treatment with a beta-adrenergic agonist to stimulate lipolysis, a 6.9 +/- 0.7-fold (SEM, n = 6) sustained increase in NEFA secretion was observed. This multilayer device provides a versatile platform that could be adapted for use with other cell types to study corresponding cellular secretions in near real-time.
开发了一种三层微流控装置，将培养细胞的灌注与在线化学分析相结合，实现对细胞分泌物的近实时监测。两层可反复密封，形成一个细胞腔，允许载玻片上生长的细胞直接装入芯片。腔室的出口与第三层保持流体接触，该层永久性地结合在一起。细胞腔室的灌注液流入这第三层，在那里进行非酯化脂肪酸 (NEFA) 的荧光酶分析在线进行。该装置用于监测约 6200 个培养脂肪细胞中 NEFA 的流出，时间分辨率为 83 秒。对鼠 3T3-L1 培养脂肪细胞进行灌注，在流出物中检测到平均基础浓度为 24.2 +/- 2.4 microM NEFA（SEM，n = 6），相当于 3.31 x 10(-5) nmol 细胞(-1) min(-1)。用β-肾上腺素能激动剂进行药理学处理以刺激脂肪分解时，观察到 NEFA 分泌持续增加 6.9 +/- 0.7 倍（SEM，n = 6）。这种多层装置提供了一个通用平台，可以适应其他细胞类型，用于近实时研究相应的细胞分泌物。
