Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets

Cell–cell interactions drive immune activation, tissue repair, and stem cell fate, yet there are few methods that can create large numbers of pre-defined cell pairs to study cell crosstalk. Droplet microfluidics allows high-throughput compartmentalization of multiple cells, but random loading results in < 1 % of droplets containing the desired combinations. Here, we present Pair Isolation by Coalescence and Sorting (PICS), a microfluidic platform that can generate specific cell pairs through droplet merging and sorting (‘merge-sorting’). PICS detects target combinations using fluorescence and triggers simultaneous electrocoalescence and dielectrophoretic sorting. Using fluorescent dye–loaded droplets, we achieved 98.6 % purity of merged and sorted droplets. In experiments using cells stained with three distinct dyes, > 90 % of desired cell pairs were recovered – compared to fewer than 1 % when using random Poisson loading. To demonstrate the utility of PICS for extended co-culture..., Droplet microfluidic images and videos were collected using an AE31 inverted microscope and a Phantom Miro M110 high-speed camera. Droplet/gel collection images were collected on a Leica DMi8 inverted microscope using the brightfield and Leica GFP (11525314), Y3 (11525311), and Cy5-naEx (11536078) filter cubes to detect FITC/Calcein Green, Cell Trace Yellow, and Cell Trace Far-red, respectively. Length scale information was determined from the automatically generated scale bar from the Leica DMi8. Individual microscope image channels were then imported to ImageJ, where fluorescent intensity, droplet/gel size, cell pairing, and viability data were analyzed using the software plugins. The analyzed data were then input to GraphPad Prism to generate data visualizations. Device geometry was drafted in Autodesk AutoCAD. Additional experimental methods description can be found in the associated manuscript. , # Data from: Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets Dataset DOI: [10.5061/dryad.wstqjq2zr](10.5061/dryad.wstqjq2zr) ## Description of the data and file structure This dataset was used to generate the results and figures included in \"Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets\" (Joslin et al., 2025). The dataset consists of microscopy images, included as TIFF (.tif) or JPEG (.jpg) files, and AutoCAD drawing files (.dwg). More details on data collection can be found in the Methods section of this dataset and the manuscript. ### Files, folders, and variables #### File: data_from_Joslin_et_al_2025.zip **Description:** Zip file for complete data for \"Deterministic Cell Pairing with Simultaneous Microfluidic Merging and Sorting of Droplets\" (Joslin et al., 2025). Contains the folder *data_from_Joslin_et_al_2025*, which contains subfolders organized in the following structure: * data_from_J...,细胞间相互作用驱动免疫激活、组织修复与干细胞命运决定，但目前鲜有方法能够制备大量预定义的细胞对以研究细胞串扰。液滴微流控（droplet microfluidics）技术可实现多种细胞的高通量分隔，但随机加载仅能让不到1%的液滴包含目标组合。本文介绍了基于聚合并分选的细胞配对（Pair Isolation by Coalescence and Sorting, PICS），这是一种通过液滴融合与分选（简称“融选”）生成特异性细胞对的微流控平台。PICS利用荧光检测目标组合，并同步触发电融合（electrocoalescence）与介电泳（dielectrophoretic）分选。通过负载荧光染料的液滴实验，我们实现了98.6%的融选后液滴纯度。在使用三种不同染料染色的细胞实验中，目标细胞对的回收率超过90%——而随机泊松加载法的回收率仅不足1%。为验证PICS在长期共培养中的应用潜力…… 液滴微流控图像与视频通过AE31倒置显微镜及Phantom Miro M110高速相机采集。液滴/凝胶成像通过Leica DMi8倒置显微镜完成，分别使用明场及Leica GFP（11525314）、Y3（11525311）与Cy5-naEx（11536078）滤光块，以分别检测FITC/钙黄绿素绿、细胞追踪黄与细胞追踪远红荧光。长度标尺信息由Leica DMi8自动生成的标尺确定。随后将单通道显微镜图像导入ImageJ软件，通过内置插件分析荧光强度、液滴/凝胶尺寸、细胞配对情况及细胞活性数据。分析所得数据随后导入GraphPad Prism以生成数据可视化结果。器件几何结构通过Autodesk AutoCAD绘制。更多实验方法细节可参见相关研究论文。 , # 数据来源：《通过液滴同步微流控融合与分选实现确定性细胞配对》 数据集DOI: 10.5061/dryad.wstqjq2zr ## 数据与文件结构说明 本数据集用于支撑《通过液滴同步微流控融合与分选实现确定性细胞配对》（Joslin等，2025）中的结果与图表。数据集包含以TIFF（.tif）或JPEG（.jpg）格式存储的显微图像，以及AutoCAD绘图文件（.dwg）。更多数据采集细节可参见本数据集的方法章节及相关论文。 ### 文件、文件夹与变量 #### 文件：data_from_Joslin_et_al_2025.zip **描述：** 包含《通过液滴同步微流控融合与分选实现确定性细胞配对》（Joslin等，2025）完整实验数据的压缩包。其内包含名为*data_from_Joslin_et_al_2025*的文件夹，该文件夹下的子文件夹按以下结构组织： * data_from_J...,