| Mechanical Tissue Compression and Whole-mount Imaging at Single Cell Resolution for Developing Murine Epididymal Tubules
|
|
Author:
Date:
2020-08-05
[Abstract] Cells inside the body are subjected to various mechanical stress, such as stretch or compression provided by surrounding cells, shear stresses by blood or lymph flows, and normal stresses by luminal liquids. Force loading to the biological tissues is a fundamental method to better understand cellular responses to such mechanical stimuli. There have been many studies on compression or stretch experiments that target culture cells attached to a flexible extensible material including polydimethylsiloxane (PDMS); however, the know-how of those targeting to tissues is still incomplete. Here we present the protocol for mechanical tissue compression and image-based analysis by focusing on developing murine epididymis as an example. We show a series of steps including tissue dissection from ...
[摘要] [摘要 ] 细胞我n侧主体经受各种机械应力，例如通过管腔液体拉伸或通过血液或淋巴周围细胞的剪切应力提供的压缩流，并且正常的应力。力加载到生物组织上是一种基本方法，可以更好地了解细胞对此类机械刺激的反应。关于压缩或拉伸实验，已有许多研究针对以附着于包括聚二甲基硅氧烷（PDMS）在内的柔性可扩展材料的培养细胞为目标的研究。然而，知道的-如何与靶向组织仍然是不完整的。 在这里，我们以开发小鼠附睾为例，介绍用于机械组织压缩和基于图像的分析的协议。我们显示了一系列步骤，包括从鼠胚胎中解剖组织，使用手动设备进行基于水凝胶的压缩方法以及无损容积组织成像。该协议对于定量和探索组织水平的生物机械反应系统很有用。
[背景 ] 细胞可以对机械刺激作出反应通过细胞内的生物化学信号传导途径。已知这种细胞机械反应在诸如胚胎发育，再生，组织稳态和癌症转移的各种生物过程中起着基本作用（Mammoto 等，2013； Humphrey 等，2014； Vining和Mooney，2017）。最近有关向细胞外部施加力的实验表明细胞如何对给定的机械刺激作出反应。例如，已显示出拉伸或压缩附着在诸如聚二甲基硅氧烷（PDMS）之类的柔性有机硅基材上的细胞单层会触发机械敏感离子通道蛋白Piezo1，从而引起各种细胞行为，例如细胞分裂和挤压，从而导致体内稳态的细胞数量增加。组织（Eisenhoffer ...
|
|
|
| Porous Scaffold Seeding and Chondrogenic Differentiation of BMSC-seeded Scaffolds
|
|
Author:
Date:
2015-12-20
[Abstract] Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects (Bornes et al., 2014). BMSCs can be seeded within porous biomaterial scaffolds that support three-dimensional cell organization, chondrogenic differentiation and extracellular matrix deposition for the creation of engineered cartilage. This protocol describes our defined methods for isolation and expansion of human and ovine BMSCs, seeding of BMSCs within porous scaffolds and in vitro chondrogenic differentiation (Adesida et al., 2012; Bornes et al., 2015).
[摘要] 骨髓来源的间充质干细胞（BMSCs）是治疗关节软骨缺陷的有希望的细胞来源（Bornes等人，2014）。 BMSCs可以种植在支持三维细胞组织，软骨形成分化和细胞外基质沉积的多孔生物材料支架中，用于创建工程化软骨。 该方案描述了我们定义的用于分离和扩增人和绵羊BMSCs，在多孔支架内接种BMSCs和在体外软骨形成分化的方法（Adesida等人，2012; Bornes et al。，2015）。
|
|
|
| Binding to Secreted Bone Matrix in vitro
|
|
Author:
Date:
2014-02-20
[Abstract] This method examines the bone matrix binding capacity of proteins. Using osteogenic differentiation medium, multipotent stromal cells (MSC) are induced to differentiate into osteocytes in vitro and to secrete bone matrix. The latter is confirmed using Alizarin red S staining, which detects the presence of calcific deposits (hydroxyapatite). These calcific deposits are used to test the bone binding properties of proteins. The binding to the calcific deposits is assessed by Western blot analysis.
[摘要] 这种方法检查蛋白质的骨基质结合能力。 使用成骨分化培养基，诱导多潜能基质细胞（MSC）在体外分化成骨细胞和分泌骨基质。 后者使用茜素红S染色证实，其检测钙化沉积物（羟基磷灰石）的存在。 这些钙沉积物用于测试蛋白质的骨结合性质。 通过蛋白质印迹分析评估与钙化沉积物的结合。
|
|
|