Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system
Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system
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DOI：
10.1096/fasebj.11.8.9240969
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发表时间：
1997-07-01
期刊：
影响因子：
4.8
通讯作者：
Elson, EL
中科院分区：
文献类型：
--
作者：
Eschenhagen, T;Fink, C;Elson, EL
已经开发了一种在胶原基质中培养心肌细胞以产生一致收缩的三维模型心脏组织的方法，该模型心脏组织允许直接测量等长收缩力量。将鸡胚胎心肌细胞与胶原液混合，在两个尼龙搭扣涂层玻璃管之间进行凝胶。在培养过程中，心肌细胞形成自发跳动的心肌细胞填充基质(CMPM)，这些基质固定在尼龙搭扣覆盖的管的两端，通过这些基质它们可以连接到测力系统。免疫组织化学和电子显微镜显示α-肌动蛋白和α-原肌球蛋白阳性心肌细胞高度组织化的组织样结构，表现为典型的横纹、肌节肌丝、间盘、桥粒和紧密连接。在培养6-11天后，在器官浴中测量不起搏或非起搏的CMPM的作用力，最长可保持24小时。作用力随频率在0.8~2.0赫兹(正“阶梯”)、增加静息长度(Starling机制)和增加细胞外钙而增加。通过用携带重组β-半乳糖苷酶的腺病毒感染CMPM，证明了该系统作为基因操作试验台的有效性。转导效率从约5%(MOI0.1)提高到约50%(MOI100)。与单层培养相比，CMPM显示出更多完整心脏组织的生理特性。这种方法比转基因动物的产生更简单、更快，应该可以在高度受控的条件下研究体外培养心肌细胞的遗传或药物操作的功能后果。
A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3-dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro-coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte-populated matrices (CMPMs) anchored at opposite ends to the Velcro-covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue-like structure of alpha-actin and alpha-tropomyosin-positive cardiac myocytes exhibiting typical cross-striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements off paced or unpaced CMPMs were performed in organ baths after 6-11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive ''staircase''), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant beta-galactosidase-carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.