Three‐dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system

Abstract

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 of 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.

Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue

“…Delivery of cells in tissue-like structures that preserve cellular attachments could increase cell delivery efficiency and reduce cell death (4). Most heart tissue engineered in vitro to date has focused on creating tissues by seeding neonatal rat or chick cardiomyocytes into polymer or extracellular matrix scaffolds and gels (5)(6)(7)(8)(9)(10)(11). Creation of 3D tissues that are composed only of cells and the matrix they secrete (12)(13)(14)(15), which we refer to as ''scaffoldfree'' tissue engineering, addresses limitations associated with polymer and exogenous matrix-based tissues (e.g., unfavorable host response to biomaterials).…”

Engineering Cardiovascular Regeneration

“…Since immature cardiomyocytes beat spontaneously and have the capacity to form a 3D network of communicating cells, the first steps towards forming engineered heart tissue (EHT) can be quite easily achieved (review in [92]). The incorporation of cells into a hydrogel is one of the oldest ideas in tissue engineering, which was introduced 35 years ago [93] and was applied to generate the first engineered cardiac tissues [94]. The most commonly used hydrogels are based on collagen I [87], Matrigel [95], fibrin [88, 89••, 96], and mixtures thereof.…”

“…Various systems have been developed to fix the remodeling tissue and provide the mechanical strain necessary for cell orientation between the fixation points. The setup ranges from Velcro-coated glass tubes [94], rings [95], Flexcell chambers [97], and mesoscopic micropost arrays [98] to newer systems in which the fixation posts serve as means to measure contractile force [99,100] (overview in [48]). For cardiac repair, the hydrogel systems can be upscaled, either by employing larger casting molds [101] or assembling of several smaller elements [102].…”

Rapid Prototyping of Engineered Heart Tissues through Miniaturization and Phenotype-Automation

“…We demonstrated the phenotypic difference in tissue mechanics between wildtype cells and mutant cells with truncated 1 integrin [40]. We also extended the applications of engineered tissue models by creating cardiac tissue constructs [41]. Recently, we introduced the concept of growing mini-tissues in 96-well plates for high-throughput drug screening [42].…”