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.

中文翻译：

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使用微流体集成光学成像技术在活淋巴结组织中扩散细胞因子

免疫系统中的通讯和药物功效在很大程度上依赖于蛋白质（例如细胞因子）通过组织基质的扩散。分析组织中扩散的可用方法需要显微注射或使组织中的蛋白质饱和，这可能会由于损伤或细胞快速反应而改变局部运输特性。在这里，我们开发了一种新颖的、用户友好的方法——微流体集成光学成像 (micro-IOI)——来量化活体组织样本中生物活性蛋白的有效扩散系数。微流控平台用于将荧光标记的细胞因子的皮克传递到小鼠淋巴结切片内的微尺度区域，并通过宽场荧光显微镜监测扩散。Micro-IOI 已根据理论和现有方法进行了验证。自由扩散系数分别在 Stokes-Einstein 预测的葡聚糖和细胞因子的 8% 和 24% 以内。此外，模型矩阵中葡聚糖和蛋白质的扩散系数在光漂白后荧光恢复 (FRAP) 报告结果的 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的有效扩散 - 肿瘤坏死因子 α (TNF-α)、干扰素 γ (IFN-γ) 和白细胞介素 2 (IL-2) ，来自人和小鼠 - 通过活的淋巴结组织。这是第一种直接测量活组织切片中细胞因子转运的方法，未来应该促进对细胞间通讯动力学的更深入了解，并实现靶向免疫治疗设计。