Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro
在体外试验中采用剪切流变学测量载有细胞水凝胶的相对硬度
发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2101 浏览次数: 11776
评审: Ralph BottcherVikash VermaSaskia F. Erttmann
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参见作者原研究论文
The authors used this protocol in:
Oct 2015
Abstract
Hydrogel systems composed of purified extracellular matrix (ECM) components (such as collagen, fibrin, Matrigel, and methylcellulose) are a mainstay of cell and molecular biology research. They are used extensively in many applications including tissue regeneration platforms, studying organ development, and pathological disease models such as cancer. Both the biochemical and biomechanical properties influence cellular and tissue compatibility, and these properties are altered in pathological disease progression (Cox and Erler, 2011; Bonnans et al., 2014). The use of cell-embedded hydrogels in disease models such as cancer, allow the interrogation of cell-induced changes in the biomechanics of the microenvironment (Madsen et al., 2015). Here we report a simple method to measure these cell-induced changes in vitro using a controlled strain rotational rheometer.
Keywords: Shear rheology (剪切流变学)
Matrix stiffness (基质硬度)
Cancer-associated fibroblasts (癌症相关成纤维细胞)
Hydrogels (水凝胶)
Background
Fibrosis and solid tumours are both accompanied by pathological remodelling of their native tissue (Cox and Erler, 2011; Bonnans et al., 2014). In both pathological conditions, the local tissue environment experiences physico-chemical as well as biological changes, resulting in increased tissue stiffness (elastic modulus) (Humphrey et al., 2014). The strengthened tissue/matrix regulates mechano-signaling that leads to altered cell behaviour, cell morphology, differentiation state, proliferation, migration and stemness. In preclinical animal models of cancer, these changes can drive malignant progression and metastatic spread (Bonnans et al., 2014). Not surprisingly, targeting matrix stiffening has received substantial attention in recent years, and several clinical trials have been initiated (Kai et al., 2016).
The elasticity and mechanical properties of a matrix component can readily be examined using atomic force microscopy (AFM), which is a technique that provides nanometre resolution and concurrent measurement of the applied force with picoNewton resolution (Kasas and Dietler, 2008). However, AFM is not applicable to understand the elastic properties of larger 3D matrices. The mechanical properties of bulk 3D matrices can more accurately be examined using shear rheology (Picout and Ross-Murphy, 2003). Rheology is the study of how materials deform when forces are applied to them. Thus applying shear stress to a 3D matrix can determine the elastic modulus (stiffness) of a bulk 3D matrix. In this protocol we describe a method to measure cell-induced changes on matrix stiffness of hydrogels embedded with cancer-associated fibroblasts by shear rheology.
Materials and Reagents
- NuncTM cell-culture treated multidishes, 24-well (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 )
- 100 μl sterile pipet tip
- 1,000 μl sterile pipet tip
- 1.5 ml sterile microcentrifuge tubes
- 8 mm disposable biopsy punch (KAI, catalog number: BP-80F )
- Syringe filter, minisart, 0.20 µm (VWR, catalog number: 514-7011 )
- Cells: immortalized human cancer-associated fibroblasts (CAFs) (Gaggioli et al., 2007)
- Collagen type I, high concentration, rat tail (Corning, catalog number: 354249 )
- Matrigel® basement membrane matrix, *LDEV-Free (Corning, catalog number: 354234 )
- Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
- Sterile PBS, pH 7.2 (Thermo Fisher Scientific, catalog number: 20012068 )
- Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
- DMEM (Thermo Fisher Scientific, catalog number: 41966-052 )
- Insulin-transferrin-selenium (Thermo Fisher Scientific, GibcoTM, catalog number: 41400045 )
- Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
- Y-27632 (Sigma-Aldrich, catalog number: Y0503 )
- MEM α, nucleosides (Thermo Fisher Scientific, GibcoTM, catalog number: 11900-073 )
- Sodium bicarbonate, NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
- 1 M HEPES buffer (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
- 5x collagen buffer (see Recipes)
- Growth medium (see Recipes)
- 1 ml collagen type I/Matrigel hydrogel (+/- cancer-associated fibroblasts) (see Recipes)
Equipment
- Timer
- Centrifuge
- Pipette
- Cell incubator at 37 °C, 5% CO2
- Discovery Series Hybrid rheometer (TA Instruments, model: DHR-2 )
- 8 mm geometry, Figure 1a (TA Instruments)
- 8 stepped mm Peltier plate, Figure 1a (TA Instruments)
- Stainless Steel Spatula, One End Flat, One End Bent, 6 in. in length (UNITED SCIENTIFIC SUPPLIES, model: SSFB06 )
- Hemocytometer
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Cox, T. R. and Madsen, C. D. (2017). Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro. Bio-protocol 7(1): e2101. DOI: 10.21769/BioProtoc.2101.
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分类
癌症生物学 > 通用技术 > 生物力学试验
细胞生物学 > 细胞分离和培养 > 细胞生长
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