CytoSoft? Elastic Modulus Plates,CytoSoft细胞弹性模量培养板
型号:CytoSoft
联系人:李胜亮
联系电话:18618101725
品牌:advanced biomatrix
CytoSoft® Elastic Modulus Plates
An Innovative Tool to Analyze the Effect of Matrix Stiffness/Rigidity on Regulating Cellular Behavior
Mechanotransduction refers to the processes through which cells sense and respond to mechanical stimuli by converting them to biochemical signals that elicit specific cellular responses. Focal adhesions are large macromolecular assemblies through which mechanical force and regulatory signals are transmitted between the extracellular matrix (ECM) and an interacting cell. Cells in the body interact with their local 3D microenvironments which can vary based on tissue location and disease states. Cells reside in various matrix stiffnesses throughout the body. The mechanical property that measures the stiffness of a material is termed Young's elastic modulus and is expressed in kilopascals of pressure. Tissue culture plasticware has an elastic modulus of 1X107kPa whereas tissues/organs have much lower values ranging from 0.2-64 kPa. In order to create more relevant in vitro cell models, researchers have started to grow on softer 3D hydrogels and substrates that more closely represent their native tissue rigidities. For example, changing to a stiffer substrate alters the differentiation potential of human mesenchymal stem cells to favor bone formation over cartilage and adipose tissues1.
Figure 1. Native matrix stiffnesses of various in vivo tissues.
| Cell Type | Observation | Reference |
|---|---|---|
| Mesenchymal Stem Cells | MSCs on soft substrates (1 kPa) had a higher expression of chondrogenic marker collagen-II and adipogenic marker lipoprotein lipase, less spreading, fewer stress fibers and lower proliferation rates than on hard substrates (15 kPa). | 1 |
| Fibroblasts | PDGF-induced chemotaxis and migration of pulmonary fibroblasts precultured on stiff substrates (25 kPa) were significantly higher than those of cells precultured on soft gels (2 kPa). | 2 |
| Endothelial Cells | Endothelial cell contraction-mediated neutrophil transmigration using an in vitro model of the vascular endothelium. Neutrophil transmigration increased with increasing substrate stiffness below the endothelium. | 3 |
| Neural Stem Cells | NSCs proliferated on substrates below 10 kPa and exhibited maximal proliferation on 3.5 kPa surfaces. Neuronal differentiation was favored on the softest surfaces with EY < 1 kPa. Oligodendrocyte differentiation was favored on stiffer scaffolds >7 kPa. Astrocyte differentiation was only observed on <1 and 3.5 kPa surfaces and represented less than 2% of the total cell population. | 4 |
| Tumor Cells | CCN1/CYR61 is highly regulated by stiffness in endothelial cells. Stiffness‐induced CCN1 activates β‐catenin nuclear translocation and signaling and that this contributes to upregulate N‐cadherin levels on the surface of the endothelium, increasing cancer cell metastasis. | 5 |
| Cancer Stem Cells | Fibrotic rigidities found in pancreatic cancer tissues promote elements of EMT, including increases in vimentin expression, decreases in E-cadherin expression, nuclear localization of β-catenin, YAP and TAZ, changes in cell shape towards a mesenchymal phenotype and induction of chemoresistance. | 6 |
| Mammary Epithelium | ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. | 7 |
| Hepatic Stellate Cells | Higher matrix stiffness leads to transdifferentiation of hepatic stellate cells into fibrogenic, proliferative, and contractile myofibroblasts. | 8 |
| Cardiomyocytes | Inverse correlation between the material stiffness and the amplitude of contraction of the cardiac tissue constructs by changing the modulus of the matrix using different compositions of PEG and fibrinogen. | 9 |
| Smooth Muscle Cells | Elastic modulus between 448 and 5804 Pa regulated SMC cytoskeletal assembly in 3D, with cells in stiff matrices having a slightly higher degree of F-actin bundling after prolonged culture. | 10 |
Table 1. Effects of matrix stiffness on cell behavior.
CytoSoft® elastic modulus plates are used to culture cells on substrates with various defined rigidities covering a broad physiological range (0.2kPA- 64kPa). On the bottom of each well, there is a thin layer of specially formulated biocompatible silicone, whose elastic modulus (rigidity) is carefully measured. The surfaces of the gels in CytoSoft® products are functionalized to form covalent bonds with amines on proteins. This chemical functionalization is stable and the reaction does not require a catalyst, facilitating the coating of the gel surfaces with matrix proteins and plating cells. For example, coating with an ECM protein, such as PureCol® (5006), is recommended before plating cells.
The silicone substrates of CytoSoft® plates are optically clear and have a low auto-florescence. The layer of silicone in each well is firmly bonded to the bottom of the well. Unlike hydrogels (such as polyacrylamide gels), silicone gels are not susceptible to hydrolysis, do not dry nor swell, are resilient and resistant to tearing or cracking, and their elastic moduli (rigidities) remain nearly unchanged during extended storage periods. CytoSoft® products accommodate the harvesting of cells using enzymes such as trypsin and collagenase. There is no biochemical breakdown of the substrate during or after enzyme treatment, and there are no residuals of the substrate in the sample retrieved from a CytoSoft® plate. For researchers who are unsure on which stiffness to use, we offer a Discovery Kit (5190) that has various elastic moduli of approximately 0.2, 0.5, 2, 8, 16, 32 and 64 kPa in 7 individual 6-well plates
CytoSoft® References
- Kidambi, et al. Astrogliosis in a dish: substrate stiffness induces astrogliosis in primary rat astrocytes. Royal Socoety of Chemistry, 2016, 6, 34447-34457.
- Tanner, et al. Decoupling cellular response to topography and stiffness in three dimensions. BioRxiv, December 10, 2017.
- Henke, E. et al. LOX-catalyzed collagen stabilization is a proximal cause for intrinsic resistance to chemotherapy. Oncogene, 2018, volume 37, pages 4921–4940.
Figure 2. Fluorescent cell imaging of F-actin in HeLa cells using CytoSoft® imaging plates (8 kPA). Cell migration and adhesion can be analyzed by monitoring the expression of F-actin filamentous cytoskeleton protein.
Figure 3. Primary human dermal fibroblast matrix stiffness optimization. A elastic modulus of 8 kPA is an optimal matrix stiffness for dermal fibroblasts showing a reduction in F-actin stress fibers and increased cell adhesion (Vinculin) when compared with 0.2 or 64 kPa matrix stiffnesses.
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References
- Song Li, et al. The Effect of Matrix Stiffness on the Differentiation of Mesenchymal Stem Cells in Response to TGF-β. Biomaterials. 2011 Jun; 32(16): 3921–3930.
- Hasegawa Y, et al. Matrix stiffness regulates migration of human lung fibroblasts. Physiol Rep. 2017 May;5(9).
- Helim Aranda-Espinoza, et al. Endothelial cell substrate stiffness influences neutrophil transmigration via myosin light chain kinase-dependent cell contraction. Blood. 2011 Aug 11; 118(6): 1632–1640.
- Molly S. Shoichet, et al. The effect of substrate stiffness on adult neural stem cell behavior. Volume 30, Issue 36, December 2009, Pages 6867-6878.
- Sara Zanivan, et al. Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium. EMBO J. 2017 Aug 15; 36(16): 2373–2389.
- Río Hernández, et al. Matrix stiffness induces epithelial–mesenchymal transition and promotes chemoresistance in pancreatic cancer cells. Oncogenesis volume 6, page e352 (2017).
- David J. Mooney, et al. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nature Materials volume 13, pages 970–978 (2014).
- Wells RG, et al. The role of matrix stiffness in hepatic stellate cell activation and liver fibrosis. J Clin Gastroenterol. 2005 Apr;39(4 Suppl 2):S158-61.
- DrorSeliktar, et al. Matrix stiffness affects spontaneous contraction of cardiomyocytes cultured within a PEGylated fibrinogen biomaterial. Acta Biomaterialia. Volume 3, Issue 1, January 2007, Pages 33-41.
- Andrew J.Putnam, et al. The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system. Biomaterials, volume 29, Issue 17, June 2008, Pages 2597-2607.
