Isolation of narrow subpopulations of cells using molecular computing cascades

使用分子计算级联分离狭窄的细胞亚群

• 批准号: 8302751
• 负责人: Milan N Stojanovic
• 金额: $ 26.66万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8302751
• 关键词: Address; Affinity; Antibodies; Autoimmune Diseases; Autoimmunity; Basic Science; Benchmarking; Biological Models; Biological Sciences; Biomedical Engineering; CD19 gene; CD28 gene; CD3 Antigens; Cell Line; Cell Separation; Cell surface; Cells; Communities; Complement 3d Receptors; Development; Disease; Dyes; Engineering; Evaluation; Failure; Flow Cytometry; Fluorescein; Funding; Gray unit of radiation dose; IL2RA gene; Immunoglobulin D; Immunology; Label; Lymphocyte; Lymphocyte Subset; Malignant Neoplasms; Medicine; Memory B-Lymphocyte; Methods; Molecular; Monitor; Natural Killer Cells; One-Step dentin bonding system; Organ; Pathogenesis; Peripheral Blood Mononuclear Cell; Population; Procedures; Protocols documentation; Reagent; Research; Sea; Series; Surface; T-Lymphocyte; TNFRSF6 gene; TNFSF5 gene; Techniques; Therapeutic; Tissue Therapy; Translating; base; interest; operation; programs; scale up; self organization

DESCRIPTION (provided by applicant): We describe here sets of molecules that compute the state of the cell surface. The sets can be programmed to self-organize on cell surfaces displaying antibody-based targeting moieties in a way that results in unique tags (labels) displayed on the surfaces of arbitrarily narrow subpopulations of cells. The decision to display or not the tag will depend on antibodies that are present, i.e., on the multiple cell surface markers that define uniquely the targeted cells via their presence or absence. The resulting tag can be used to isolate in a single step the targeted cells in the presence of other subpopulations. This single-step method for isolation of cells based on multiple surface markers will have unique advantages over existing methods because of its scalability, efficacy, and mildness. We will study the scope and limitations of new technique on an example of therapeutically interesting subpopulations of lymphocytes, comparing our technique with existing methods and multistep protocols: Aim 1: in which the molecules will compute the presence of multiple markers on a cell surface. We will start with evaluation of up to three markers on CD4+CD25+ and CD4+CD25+CD154+ T- cells. We will then demonstrate the ability to isolate groups of cells based on the intensity of a marker, separating two fractions of NK cells (CD56dim and CD56high), developing sets of molecules with thresholding (cut off value) function. Aim 2: in which molecules will evaluate both the presence and absence of markers. After initial optimization on a model system, we will demonstrate the ability to isolate narrow subpopulations of conventional memory B-cells (CD19+CD21+CD27+IgD-) and recently characterized unconventional memory B-cells (CD19+CD21-/lowCD27-IgD-CD95+). Our reagents can be combined with any moiety used for targeting cell-surface markers with only minimal optimization. Thus, through our results we will introduce the biomedical community to a highly modular approach to isolating narrow populations of cells using of-the- shelf reagents. The method will have wide applications in basic science research, personalized medicine, and biomedical engineering. PUBLIC HEALTH RELEVANCE: The problem we address appears often in life sciences, emerging therapeutic methods using cells, and biomedical engineering: from a sea of cells we need to isolate a narrow subpopulation that is uniquely distinguished from all other subpopulations through the presence and absence of multiple cell-surface markers. We describe in this proposal the development and optimization of a mild preparative isolation technique yielding viable arbitrarily narrow subpopulations of cells in a minimal number of steps. The technique is based on sets of molecules self-organizing according to sets of rules implemented through interactions with the cell surfaces, and labeling only cells that we target for isolation. The procedure will be widely applicable in studying pathogenesis of diseases (e.g., autoimmunity and cancer), for cellular therapies, and tissue and organ engineering, offering unique advantages over the existing methods for isolation of cells.

描述（由申请人提供）：我们在此描述了计算细胞表面状态的分子组。这些组可以被编程以在细胞表面上自组织，从而以导致在任意窄的细胞亚群的表面上显示独特标签（标记）的方式显示基于抗体的靶向部分。决定展示或 标签不依赖于存在的抗体，即，在多个细胞表面标记上，通过它们的存在或不存在唯一地限定靶细胞。所得标签可用于在其它亚群存在下在单个步骤中分离靶细胞。这种基于多个表面标志物分离细胞的单步方法由于其可扩展性、有效性和温和性而具有优于现有方法的独特优势。我们将研究新技术的范围和局限性的治疗感兴趣的淋巴细胞亚群的例子，比较我们的技术与现有的方法和多步骤协议：目的1：其中的分子将计算细胞表面上的多个标记的存在。我们将从评估CD 4 + CD 25+和CD 4 + CD 25 + CD 154 + T细胞上的多达三种标志物开始。然后，我们将展示基于标记物的强度分离细胞组的能力，分离NK细胞的两个部分（CD 56 dim和CD 56 high），开发具有阈值（截止值）功能的分子组。目标2：其中分子将评估标记物的存在和不存在。在对模型系统进行初始优化后，我们将证明分离常规记忆B细胞（CD 19 + CD 21 + CD 27 +IgD-）和最近表征的非常规记忆B细胞（CD 19 + CD 21-/低CD 27-IgD-CD 95+）的窄亚群的能力。 我们的试剂可以与任何用于靶向细胞表面标志物的部分组合，仅需最小的优化。因此，通过我们的研究结果，我们将向生物医学界介绍一种高度模块化的方法，使用现成的试剂分离狭窄的细胞群。该方法将在基础科学研究、个性化医疗和生物医学工程中有广泛的应用。 公共卫生关系：我们解决的问题经常出现在生命科学、使用细胞的新兴治疗方法和生物医学工程中：我们需要从细胞海洋中分离出一个狭窄的亚群，该亚群通过存在和不存在多种细胞表面标记物而与所有其他亚群独特地区分开来。我们在这个建议中描述了温和的制备分离技术的开发和优化，以最少的步骤产生可行的任意狭窄的细胞亚群。该技术基于一组分子，这些分子根据通过与细胞表面相互作用实现的一组规则进行自组织，并且只标记我们的目标细胞。 隔离该方法将广泛应用于研究疾病的发病机制（例如，自身免疫和癌症）、用于细胞治疗以及组织和器官工程，提供了优于现有细胞分离方法的独特优势。