Department of Chemistry, University of Virginia, McCormick Rd., PO Box 400319, Charlottesville, VA 22904, USA.
Department of Chemistry, University of Virginia, McCormick Rd., PO Box 400319, Charlottesville, VA 22904, USA.
Anal Chim Acta. 2018 Feb 13;1000:205-213. doi: 10.1016/j.aca.2017.11.048. Epub 2017 Nov 23.
Communication and drug efficacy in the immune system rely heavily on diffusion of proteins such as cytokines through the tissue matrix. Available methods to analyze diffusion in tissue require microinjection or saturating the tissue in protein, which may alter local transport properties due to damage or rapid cellular responses. Here, we developed a novel, user-friendly method - Microfluidic Integrated Optical Imaging (micro-IOI) - to quantify the effective diffusion coefficient of bioactive proteins in live tissue samples ex vivo. A microfluidic platform was used to deliver picograms of fluorescently labelled cytokines to microscale regions within slices of murine lymph node, and diffusion was monitored by widefield fluorescence microscopy. Micro-IOI was validated against theory and existing methods. Free diffusion coefficients were within 8% and 24% of Stokes-Einstein predictions for dextrans and cytokines, respectively. Furthermore, diffusion coefficients for dextrans and proteins in a model matrix were within 1.5-fold of reported results from fluorescence recovery after photobleaching (FRAP). We used micro-IOI to quantify the effective diffusion of three cytokines from different structural classes and two different expression systems - tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and interleukin-2 (IL-2), from human and mouse - through live lymph node tissue. This is the first method to directly measure cytokine transport in live tissue slices, and in the future, it should promote a deeper understanding of the dynamics of cell-cell communication and enable targeted immunotherapy design.
在免疫系统中，蛋白质（如细胞因子）的扩散对于通讯和药物疗效至关重要。目前用于分析组织扩散的方法需要进行微注射或使组织饱和蛋白质，这可能会因损伤或快速的细胞反应而改变局部传输特性。在这里，我们开发了一种新颖的、用户友好的方法——微流控集成光学成像（micro-IOI），用于量化活体组织样本中生物活性蛋白质的有效扩散系数。使用微流控平台将皮克级荧光标记细胞因子递送至鼠淋巴结切片的微尺度区域内，并通过宽场荧光显微镜监测扩散。micro-IOI 经过了理论和现有方法的验证。对于葡聚糖和细胞因子，自由扩散系数分别在理论预测的斯托克斯-爱因斯坦（Stokes-Einstein）值的 8%和 24%以内。此外，在模型基质中，葡聚糖和蛋白质的扩散系数与荧光恢复后光漂白（FRAP）的报道结果相差 1.5 倍以内。我们使用 micro-IOI 来量化来自不同结构类别和两种不同表达系统的三种细胞因子（肿瘤坏死因子-α（TNF-α）、干扰素-γ（IFN-γ）和白细胞介素-2（IL-2））在活体淋巴结组织中的有效扩散。这是直接测量活体组织切片中细胞因子传输的第一种方法，它将有助于深入了解细胞间通讯的动态，并能够实现靶向免疫疗法的设计。
