Precision-cut bladder slices: an enabling technology for urologic research

精密切割膀胱切片：泌尿学研究的支持技术

基本信息

批准号：
10785220
负责人：
Rosalyn M Adam
金额：
$ 33.73万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-20 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10785220
关键词：
Acceleration Animal Model Benign Biochemical Biological Assay Biological Models Biomimetics Bladder Bladder Dysfunction Bladder Tissue Calcium Signaling Cell Communication Cells Cholinergic Agonists Coupled Cryopreservation Development Devices Disease Dissociation Distal Environment Evaluation Extracellular Matrix Functional disorder Generations Genetically Engineered Mouse Health Heart Histologic Human Image In Vitro Intestines Laboratories Lower urinary tract Lung Measurement Microscopy Molecular Molecular Analysis Mus Muscle Contraction Muscle function Neurogenic Bladder Organ Organoids Pharmacotherapy Phenotype Physiological Physiology Preparation Proteomics Recovery Relaxation Research Signal Transduction Slice Smooth Muscle Specimen Stretching Structure System Technology Testing Tissue Slice Technology Tissues Traction Translational Research Urologic Diseases Validation Wild Type Mouse bioprinting cell type chromatin immunoprecipitation experimental study fluid flow human disease human tissue insight novel novel therapeutic intervention novel therapeutics organ on a chip pharmacologic preservation pressure reconstitution response technology development three dimensional cell culture tool translational study urologic

项目摘要

Mechanistic evaluation of smooth muscle is essential to a rigorous understanding of lower urinary tract function in both health and disease, and to facilitate translational urologic disease research. Existing strategies for smooth muscle evaluation include whole bladder preparations or bladder strips from animal models as well as single cells from human tissues. However, with these approaches the ability to perform integrated analysis of contraction, stiffness and underlying signaling within a single experiment in specimens that capture the native tissue environment is limited. Precision-cut tissue slice technology has been applied to a number of hollow organs yielding novel insights into cell and tissue physiology and pathophysiology. Research from the Krishnan laboratory has demonstrated that precision-cut lung slices (PCLS) can be coupled with tissue traction microscopy (TTM) to enable quantitative assessment of physiologically relevant parameters in distal airways, including tissue contraction and stiffness. In this application we propose to develop the precision-cut bladder slice (PCBS) as a novel platform to interrogate contraction, relaxation and stiffness of the bladder in vitro, while preserving the native cellular microenvironment. The overall objective of this proposal is to demonstrate the utility of PCBS technology for evaluation of bladder contraction and stiffness in healthy murine and healthy and diseased human bladder tissues. We hypothesize that development of precision-cut bladder slices will enable quantitative assessment of smooth muscle function and signaling in vitro while preserving the native cellular environment. We will test this hypothesis with the following Specific Aims: Aim 1: Evaluate precision-cut bladder slices for measurement of smooth muscle contraction. Aim 2: Evaluate precision-cut bladder slices for assessment of bladder wall stiffness. We will use tissue traction microscopy to assess evoked contraction and relaxation in mouse and human bladder tissues. In addition, we will perform biochemical and immunohistochemical analysis to determine the extent to which PCBS retain viability, phenotypic and functional responses, including after cryopreservation. We will also apply a novel tissue stretching device to explore stiffness in PCBS from diseased versus healthy human bladders, enabling us to evaluate smooth muscle activity within the microenvironment of the extracellular matrix. At the end of the project period, we expect to have created a novel platform enabling measurement of physiologically relevant endpoints in bladder tissue, including contraction, relaxation and stiffness, and to have determined the extent to which PCBS can be modulated with pharmacological agents to enable mechanistic studies. Successful completion of these studies is expected to establish a new platform that can facilitate the validation of new therapeutic interventions for bladder dysfunction.

平滑肌的机制评估对于严格了解健康和疾病中下尿路功能以及促进转化泌尿系统疾病研究至关重要。平滑肌评估的现有策略包括来自动物模型的整个膀胱制剂或膀胱条以及来自人体组织的单细胞。然而，通过这些方法，在捕获天然组织环境的样本中的单个实验中对收缩、硬度和潜在信号进行综合分析的能力是有限的。精密切割组织切片技术已应用于许多中空器官，为细胞和组织生理学和病理生理学提供了新的见解。 Krishnan 实验室的研究表明，精密切割肺切片 (PCLS) 可以与组织牵引显微镜 (TTM) 结合使用，从而能够定量评估远端气道的生理相关参数，包括组织收缩和硬度。在此应用中，我们建议开发精密切割膀胱切片（PCBS）作为一种新颖的平台，用于在体外检查膀胱的收缩、松弛和僵硬，同时保留天然细胞微环境。该提案的总体目标是展示 PCBS 技术在评估健康小鼠以及健康和患病人类膀胱组织的膀胱收缩和硬度方面的实用性。我们假设精确切割的膀胱切片的开发将能够在体外定量评估平滑肌功能和信号传导，同时保留天然细胞环境。我们将通过以下具体目标来检验这一假设：目标 1：评估精确切割的膀胱切片以测量平滑肌收缩。目标 2：评估精确切割的膀胱切片以评估膀胱壁硬度。我们将使用组织牵引显微镜来评估小鼠和人类膀胱组织的诱发收缩和松弛。此外，我们将进行生化和免疫组织化学分析，以确定 PCBS 保留活力、表型和功能反应的程度，包括在冷冻保存后。我们还将应用一种新型组织拉伸装置来探索患病人类膀胱与健康人类膀胱的 PCBS 硬度，使我们能够评估细胞外基质微环境内的平滑肌活动。在项目期结束时，我们期望创建一个新的平台，能够测量膀胱组织的生理相关终点，包括收缩、松弛和僵硬，并确定 PCBS 可以用药物调节的程度，以实现机理研究。这些研究的成功完成预计将建立一个新平台，促进膀胱功能障碍新治疗干预措施的验证。