别再让你培养的细胞睡在硬板床上了!!众所周知,体内细胞所处周边环境的软硬度(softness,stiffness)可谓千差万别,凭直觉想想脂肪细胞和骨细胞吧。可是我们体外培养细胞时却选择使用了几乎相同的材质,硬邦邦的培养皿、板或培养瓶。你可能说:“这没啥呀,大家不都是这样做的吗?我的细胞也长的挺好呀!”别再固执的认为细胞生长在“硬板床”上对其没有什么影响了,大量的研究表明细胞所处环境的硬度对其生物学行为影响可谓相当的大。下面选择几篇CNS级别文章的摘要,感兴趣的话可以下载读一读,即刻更新一下我们陈旧的观点吧。Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell. 2009 Nov 25;139(5):891-906.SUMMARYTumors are characterized by extracellular matrix (ECM) remodeling and stiffening. The importance of ECM remodeling to cancer is appreciated; the relevance of stiffening is less clear. We found that breast tumorigenesis is accompanied by collagen crosslinking, ECM stiffening, and increased focal adhesions. Induction of collagen crosslinking stiffened the ECM, promoted focal adhesions, enhanced PI3 kinase (PI3K) activity, and induced the invasion of an oncogene-initiated epithelium. Inhibition of integrin signaling repressed the invasion of a premalignant epithelium into a stiffened, crosslinked ECM and forced integrin clustering promoted focal adhesions, enhanced PI3K signaling, and induced the invasion of a premalignant epithelium. Consistently, reduction of lysyl oxidase-mediated collagen crosslinking prevented MMTV-Neu-induced fibrosis, decreased focal adhesions and PI3K activity, impeded malignancy, and lowered tumor incidence. These data show how collagen crosslinking can modulate tissue fibrosis and stiffness to force focal adhesions, growth factor signaling and breast malignancy. Matrix elasticity directs stem cell lineage specification.Cell. 2006 Aug 25;126(4):677-89.SUMMARYMicroenvironments appear important in stem cell lineage specification but can be difficult toadequately characterize or control with soft tissues. Naive mesenchymal stem cells (MSCs)are shown here to specify lineage and commit tophenotypes with extreme sensitivity to tissuelevel elasticity. Soft matrices that mimic brainare neurogenic, stiffer matrices that mimic muscle are myogenic, and comparatively rigidmatrices that mimic collagenous bone proveosteogenic. During the initial week in culture,reprogramming of these lineages is possiblewith addition of soluble induction factors, butafter several weeks in culture, the cells committo the lineage specified by matrix elasticity,consistent with the elasticity-insensitive commitment of differentiated cell types. Inhibitionof nonmuscle myosin II blocks all elasticitydirected lineage specification–without stronglyperturbing many other aspects of cell functionand shape. The results have significant implications for understanding physical effects of thein vivo microenvironment and also for therapeutic uses of stem cells. Role of YAP/TAZ in mechanotransduction. Nature. 2011 Jun 8;474(7350):179-83.SUMMARYCells perceive their microenvironment not only through soluble signals but also through physical and mechanical cues,such as extracellular matrix (ECM) stiffness or confined adhesiveness. By mechanotransduction systems, cells translatethese stimuli into biochemical signals controlling multiple aspects of cell behaviour, including growth, differentiationand cancer malignant progression, but how rigidity mechanosensing is ultimately linked to activity of nucleartranscription factors remains poorly understood. Here we report the identification of the Yorkie-homologues YAP(Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif, also known as WWTR1) asnuclear relays of mechanical signals exerted by ECM rigidity and cell shape. This regulation requires Rho GTPaseactivity and tension of the actomyosin cytoskeleton, but is independent of the Hippo/LATS cascade. Crucially, YAP/TAZ are functionally required for differentiation of mesenchymal stem cells induced by ECM stiffness and for survival ofendothelial cells regulated by cell geometry; conversely, expression of activated YAP overrules physical constraints indictating cell behaviour. These findings identify YAP/TAZ as sensors and mediators of mechanical cues instructed by thecellular microenvironment. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture.Science. 2010 Aug 27;329(5995):1078-81.SUMMARYStem cells that naturally reside in adult tissues, such as muscle stem cells (MuSCs), exhibit robustregenerative capacity in vivo that is rapidly lost in culture. Using a bioengineeredsubstrate to recapitulatekey biophysical and biochemical niche features in conjunction with a highly automated single-celltracking algorithm, we show that substrate elasticity is a potent regulator of MuSC fate in culture. UnlikeMuSCs on rigid plastic dishes (~106kilopascals), MuSCs cultured on soft hydrogel substrates that mimicthe elasticity of muscle (12 kilopascals) self-renew in vitro and contribute extensively to muscleregeneration when subsequently transplanted into miceandassayedhistologically and quantitatively bynoninvasive bioluminescence imaging. Our studies provide novel evidence that by recapitulatingphysiological tissue rigidity, propagation of adult muscle stem cells is possible, enabling future cell-basedtherapies for muscle-wasting diseases. 需要更多关于细胞培养表面弹性对不同类型细胞的生物学行为影响的研究文献?下面是针对不同组织细胞的另外一些例子:

TissueReferenceEmbryonic(胚胎)1. Culturing of mouse and human cells on soft substrates promote the expression of stem cell markers.243602052. Differential regulation of morphology and stemness of mouse embryonic stem cells by substrate stiffness and topography.245296273. Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions.211794494. Effect of substrate stiffness on early mouse embryo development.228600095. Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling.225531656. Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions.211794497. Matrix elasticity directs stem cell lineage specification.16923388Hepatocytes(肝细胞)1. Relative rigidity of cell-substrate effects on hepatic and hepatocellular carcinoma cell migration. 235655952. Hepatic stellate cells require a stiff environment for myofibroblastic differentiation. 215277253. Increased stiffness of the rat liver precedes matrix deposition: implications for fibrosis. 179322314. Functional modulation of ES-derived hepatocyte lineage cells via substrate compliance alteration. 182661085. Engineering hepatocellular morphogenesis and function via ligand-presenting hydrogels with graded mechanical compliance. 15744840Neural(神经)1. Migration of glial cells differentiated from neurosphere-forming neural stem/progenitor cells depends on the stiffness of the chemically cross-linked collagen gel substrate. 240419352. Photocured biodegradable polymer substrates of varying stiffness and microgroove dimensions for promoting nerve cell guidance and differentiation. 228570113. Effects of substrate stiffness and cell density on primary hippocampal cultures. 205473724. The effects of substrate elastic modulus on neural precursor cell behavior. 234299625. The influence of substrate stiffness on the behavior and functions of Schwann cells in culture. 22738780Heart(心脏)1. Substrate stiffness modulates gene expression and phenotype in neonatal cardiomyocytes in vitro. 225195492. The constant beat: cardiomyocytes adapt their forces by equal contraction upon environmental stiffening. 235195953. Cardiomyocytes from late embryos and neonates do optimal work and striate best on substrates with tissue-levelelasticity: metrics and mathematics. 22752667 Bone, cartilage skeletal muscle(骨骼、软骨、骨骼肌)1. Osteocyte differentiation is regulated by extracellular matrix stiffness and intercellular separation. 239949432. Effect of substrate stiffness on the osteogenic differentiation of bone marrow stem cells and bone-derived cells. 234475013. Response of sheep chondrocytes to changes in substrate stiffness from 2 to 20 Pa: effect of cell passaging. 233237694. Substrate stiffness and oxygen as regulators of stem cell differentiation during skeletal tissue regeneration: a mechanobiological model. 22911707 General and mechanistic(常规及机械组织细胞)1. Determination of local and global elastic moduli of valve interstitial cells cultured on soft substrates. 237465972. Computational model predicts cell orientation in response to a range of mechanical stimuli. 237088753. Investigating the role of substrate stiffness in the persistence of valvular interstitial cell activation. 225817284. Mechanochemical model of cell migration on substrates of varying stiffness. 223041165. Influence of substrate stiffness on circulating progenitor cell fate. 22169135 Eye(眼组织)1. Substrate elasticity as biomechanical modulator of tissue homeostatic parameters in corneal keratinocytes. 23664838Endotheial and blood(上皮和血液)1. Effect of substrate stiffness and PDGF on the behavior of vascular smooth muscle cells: implications for atherosclerosis. 206486292. Endothelial barrier disruption and recovery is controlled by substrate stiffness. 232960343. Influence of membrane cholesterol and substrate elasticity on endothelial cell spreading behavior. 232396124. OxLDL and substrate stiffness promote neutrophil transmigration by enhanced endothelial cell contractility and ICAM-1. 225602865. Endothelial cell responses to micropillar substrates of varying dimensions and stiffness. 22389314 Mesenchymal(间充质细胞)1. The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-β. 213979422. Differential regulation of stiffness, topography, and dimension of substrates in rat mesenchymal stem cells. 238634543. Physical and chemical microenvironmental cues orthogonally control the degree and duration of fibrosis-associated epithelial-to-mesenchymal transitions. 230185984. Mesenchymal stem cell durotaxis depends on substrate stiffness gradient strength. 23390141

其实,要根据所培养细胞所处的体内环境环境选择相应软硬度的基质,尽最大可能保持细胞固有的特性并不难:Matrigen Life Technologies根据不同细胞类型设计生产了硬度各异的Softwell透明水凝胶,并已经将这些水凝胶铺设在我们常用细胞培养板、皿等用材的表面,用户可以根据所培养细胞的体内环境选择相应硬度的水凝胶培养板或培养皿,十分方便。 Matrigen Life Technologies专一致力于重塑细胞培养之景象:你只需简单的改变培养用品即可让你的细胞培养条件更接近于体内真实状况。Matrigen Life Technologies生产的Softwell水凝胶产品主要包括如下三种类型:

水凝胶功能Easy coat系列Collagen I系列Non-Activated系列水凝胶已活化,可根据自身实验需要自行包被EMC蛋白,如胶原蛋白、纤连蛋白、层粘连蛋白等水凝胶上已包被好I型胶原蛋白(取自鼠尾或绵羊皮),为即拆即用型,使用便捷水凝胶未活化,其表面结合活性极低。用于悬浮细胞培养或自行活化

Matrigen Life Technologies生产的Softwell水凝胶产品适用于各组织细胞的培养:

弹性指数(kPa)Soft(柔软)Intermediate(中度)Stiff(硬)0.20.51248122550适用于培养脂肪、肝、肺、胚胎、脂肪、神经部位的细胞适用于培养肌肉、心脏部位的细胞适用于培养骨骼部位的细胞

Matrigen Life Technologies生产的Softwell水凝胶产品有多种样式的培养器皿

培养器皿样式Softwell®系列Softwell GTM系列PetrisoftTM系列SoftviewTM系列聚苯乙烯材质的水凝胶培养板,规格有6孔、12孔、24孔、96孔黑色聚苯乙烯框架、硼硅玻璃孔径的水凝胶培养板,规格有6孔、12孔、24孔、96孔、384孔聚苯乙烯材质的水凝胶培养皿,规格有35mm、100mm、150mm35mm培养皿,里面放置覆盖水凝胶的高透度玻璃(直径为10mm和20mm)

别担心使用铺设了水凝胶的培养皿(或板)对你的实验结果会造成不良影响,softwell系统与各种实验相兼容,不会干扰实验结果;也不会因为有了这层薄薄的水凝胶而给你的实验带来麻烦:softwell不影响核酸和蛋白提取,不影响显微镜下观测拍照。

(聊城荣恒医疗器械有限公司)