A Novel Microfluidic HIV-1 Co-Culture Assay to Quantify Latent Reservoirs

一种新颖的微流体 HIV-1 共培养测定法来量化潜在储库

基本信息

批准号：
8768888
负责人：
Utkan Demirci
金额：
$ 22.62万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8768888
关键词：
3-Dimensional Activated Lymphocyte Allogenic Anti-Retroviral Agents Automation Biological Assay Biological Markers Biomedical Engineering Biophysics Blast Cell CD4 Positive T Lymphocytes CD8B1 gene Cell Communication Cell Culture Techniques Cells Chromosomes Coculture Techniques Code Complex Coupled Culture Media DNA Detection Development Disease remission Frequencies Genetic Materials Goals Grant Growth HIV HIV-1 Human Individual Infection Laboratories Laboratory Study Measurement Measures Methods Microfluidics Modeling Organ Patients Performance Peripheral Blood Mononuclear Cell Principal Investigator Proteins Protocols documentation Proviruses Reaction Reagent Research Rest Retrovirology Sampling Stem cell transplant System Technology Test Result Testing Therapeutic Time Tissues Toxin Variant Viral Viral Genome Virus Virus-Cell Membrane Interaction antiretroviral therapy base cell type chemotherapy cohort design experience improved in vivo innovation miniaturize novel public health relevance research study response viral DNA viral RNA virology

3-Dimensional Activated Lymphocyte Allogenic Anti-Retroviral Agents Automation Biological Assay Biological Markers Biomedical Engineering Biophysics Blast Cell CD4 Positive T Lymphocytes CD8B1 gene Cell Communication Cell Culture Techniques Cells Chromosomes Coculture Techniques Code Complex Coupled Culture Media DNA Detection Development Disease remission Frequencies Genetic Materials Goals Grant Growth HIV HIV-1 Human Individual Infection Laboratories Laboratory Study Measurement Measures Methods Microfluidics Modeling Organ Patients Performance Peripheral Blood Mononuclear Cell Principal Investigator Proteins Protocols documentation Proviruses Reaction Reagent Research Rest Retrovirology Sampling Stem cell transplant System Technology Test Result Testing Therapeutic Time Tissues Toxin Variant Viral Viral Genome Virus Virus-Cell Membrane Interaction antiretroviral therapy base cell type chemotherapy cohort design experience improved in vivo innovation miniaturize novel public health relevance research study response viral DNA viral RNA virology

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项目摘要

DESCRIPTION (provided by applicant): HIV-1 reservoirs continue to exist in latent form despite long-term suppression of circulating virus with antiretroviral therapy, and a main challenge in achieving HIV-1 antiretroviral (ART)-free remission is the persistence of these latent viral reservoirs. Quantifying the replication competent viral reservoir is critical to understanding how HIV-1 persists and to measuring how this reservoir changes in response to therapeutic strategies. Quantitative co-culture assay have been developed to measure the replication competent reservoir by activating CD4+ T cells from patients in the presence of feeder cells for viral outgrowth and measurements of HIV-1 proteins or genetic material. However, traditional quantitative co-culture assays are time-consuming, labor-intensive and require parallel reactions in large-well format culture. Novel methods to miniaturize the co- culture platform and to increase sample throughput have the potential significantly to improve the study of HIV latency and could be used to streamline and standardize testing of novel reservoir eradication strategies. We propose to adapt and validate a novel, miniaturized chip-based microfluidic co-culture assay and compare its performance with the traditional co-culture assay. We hypothesize that miniaturizing these assays will allow for higher throughput and be less labor intensive than traditional platforms. The proposed method will also allow for tight control of the growth microenvironment which will help to improve sensitivity and standardize test results between laboratories. Our aims are: (1) adapt a chip-based microfluidic cell culture/expansion protocol for viral co-culture to quantify integrated, replication-competent provirus as infectious units per million cells, and, (2) validate the performance of the novel microfluidic co-culture assay compared with traditional laboratory viral outgrowth assays. This two-year development grant will utilize innovative approaches and adaptations of existing microfluidic technologies to develop an assay to characterize HIV- reservoirs in patients on antiretroviral therapy. Our proposal involves principal investigators with different but complimentary research backgrounds and experiences, including translational virology and bioengineering/biophysics. Our proposed assay has the potential to be used for a variety of applications involving the isolation and growth of infected human cell subsets and tissues, and we ultimately plan to use the assay to quantify replication competent reservoirs in various cell types and from patients undergoing allogeneic stem cell transplantation and cytoreductive chemotherapy.

描述（由申请人提供）：尽管长期抑制了使用抗逆转录病毒疗法的循环病毒，但HIV-1储层仍以潜在形式存在，并且在实现HIV-1抗逆转录病毒（ART）抗逆转录病毒（ART）的主要挑战中是这些潜在病毒储存的持续性。量化复制能力的病毒储存库对于了解HIV-1如何持续以及测量该储层如何响应治疗策略的变化至关重要。已经开发了定量共培养分析，以通过在有进料细胞的情况下激活患者的CD4+ T细胞来测量复制储存库，以进行病毒生长和HIV-1蛋白或遗传材料的测量。然而，传统的定量共培养测定是时必的，劳动力密集的，需要大孔格式文化中的平行反应。微型化合物平台并增加样品吞吐量的新方法具有显着改善艾滋病毒潜伏期研究的潜力，可以用来简化和标准化新型储层消除策略的测试。我们建议适应和验证一种新颖的，微型芯片的微流体共培养分析，并将其性能与传统的共文化测定法进行比较。我们假设将这些测定法的小型化将允许更高的吞吐量，并且比传统平台更少。提出的方法还将允许严格控制生长微环境，这将有助于提高敏感性并在实验室之间标准化测试结果。我们的目的是：（1）适应病毒共培养的基于芯片的微流体细胞培养/膨胀方案，以量化一百万个细胞的综合综合，复制能力病毒作为传染病单元，以及（（2）与传统的微富集培养物的性能相比，与传统的实验室病毒式分析相比，验证新型的微流体共文化分析的性能。这项为期两年的开发赠款将利用现有微流体技术的创新方法和适应性，以开发一种抗逆转录病毒治疗患者的HIV-储藏的测定法。我们的建议涉及具有不同但免费研究背景和经验的主要研究人员，包括翻译病毒学和生物工程/生物物理学。我们提出的测定有可能用于各种应用，涉及感染的人类细胞子集和组织的隔离和生长，我们最终计划使用该测定法来量化各种细胞类型中的复制依从性储层，并与接受同种异体干细胞移植和细胞降解性化学疗法的患者中的患者。