Vascular Transcriptomics: Investigating Endothelial Activation and Vascular Dysfunction Using Blood Outgrowth Endothelial Cells, Organ-Chips, and RNA Sequencing.
Vascular Transcriptomics: Investigating Endothelial Activation and Vascular Dysfunction Using Blood Outgrowth Endothelial Cells, Organ-Chips, and RNA Sequencing.
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DOI：
10.1002/cpz1.582
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发表时间：
2022-10
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影响因子：
--
通讯作者：
Jain, Abhishek
中科院分区：
文献类型：
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作者：
Mathur, Tanmay;Kumar, Ankit;Flanagan, Jonathan M;Jain, Abhishek
血管器官芯片或血管芯片技术已经显著影响了我们对微生理血管系统建模的能力。这些仿生平台作为药物筛选模型引起了科学家和制药商的极大兴趣。然而，这些模型仍然缺乏患者来源的内皮细胞形式的患者特异性脉管系统的包含。血液生长内皮细胞是患者血液来源的内皮祖细胞，其作为自体内皮细胞替代物已引起血管生物学界的兴趣，并且也已与血管芯片模型结合。下一代测序技术如RNA测序可以进一步释放个性化血管芯片在识别内皮功能障碍的患者特异性标志方面的潜力。在这里，我们提出了一个详细的协议（1）分离血液生长内皮细胞（BOEC）从病人的血液样本，（2）培养他们在微流控血管芯片，（3）分离和制备RNA从个人血管芯片测序，和（4）进行差异基因表达和生物信息学分析血管功能障碍和内皮激活途径。该方法特别关注血管稳态和病理学中涉及的通路和基因的识别，然而用户可以根据其系统要求轻松地更改分析。基本方案1：基础方案2：微流控血管芯片中的BOEC培养基础方案3：自体血管芯片中的RNA分离基础方案4：内皮激活途径的差异基因表达和生物信息学分析
Vascular organ-chips or vessel-chip technology has significantly impacted our ability to model microphysiological vasculature. These biomimetic platforms have garnered significant interest from scientists and pharmaceuticals as drug screening models. However, these models still lack the inclusion of a patient-specific vasculature in the form of patient derived endothelial cells. Blood outgrowth endothelial cells are patient blood derived endothelial progenitors that have gained interest from the vascular biology community as an autologous endothelial cell alternative and have also been incorporated with the vessel-chip model. Next generation sequencing techniques like RNA sequencing can further unlock the potential of personalized vessel-chips in discerning patient-specific hallmarks of endothelial dysfunction. Here we present a detailed protocol for (1) isolating blood outgrowth endothelial cells (BOECs) from patient blood samples, (2) culturing them in microfluidic vessel-chips, (3) isolating and preparing RNA from individual vessel-chips for sequencing, and (4) performing differential gene expression and bioinformatic analysis of vascular dysfunction and endothelial activation pathways. This method focuses specifically on identification of pathways and genes involved in vascular homeostasis and pathology however the users can easily change the analysis based on their systems requirements. Basic Protocol 1: Blood outgrowth endothelial cell isolation from blood Basic Protocol 2: BOEC culture in microfluidic vessel-chips Basic Protocol 3: RNA isolation from autologous vessel-chips Basic Protocol 4: Differential gene expression and bioinformatic analyses of endothelial activation pathways