Engineering fully functional, integrated skeletal muscle

There is an urgent and growing need for non-animal testing of biologically active agent?s across UK industry, government agencies and healthcare research. Currently, new research from tissue & cell engineering is generating increasingly complex forms of simple 3-Dimensional model tissues (potentially ?human?), which can be developed in the laboratory. These models can now provide us the opportunity to monitor how whole tissue responds to challenges (e.g. toxic chemicals) by replacing conventional animal testing. The key to this is first developing simple individual tissue models and then combining different models to generate normal tissue complexity. We have developed in our laboratories simple 3 dimensional individual models of muscle and nerve using collagen or fibrin as a support material; these are both naturally occurring proteins. We now propose to combine these models and develop a 3D muscle with nerve endings, grown in the laboratory, to test the effect of various drugs and chemicals for potential human use replacing traditional testing on animals. Our remit will be to assess whether this complex model can be developed and test if it will be able to replace animal testing in the future. The final aim is better testing with less use of animals.

Interrogation of the neuromuscular interface is essential for both insights into, and therapies for, neuromuscular disease. However, animal systems are used extensively because of the difficulty in sourcing relevant human materials, despite the models not being totally ideal. The aim of this project is therefore to develop a 3D tissue engineered neuromuscular junction ? this will give a more biologically relevant system but will also, in the long term, reduce the number of animals used in neuromuscular experimentation. In the short term, the number of experiments that can be completed per animal will be increased leading to a reduction in the number of animals used. Our previous work has succeeded in developing and characterising both early skeletal muscle and neural model tissues individually in culture. This project aims to investigate integrating these two models, as an in vitro neuro-muscular test-bed. We are proposing using two skeletal muscle model systems ? a developmental, fibrin-based system and a regenerative, collagen-based system. In both of these systems, muscle cells are incorporated into a matrix that has a ?pseudo? tendon at each pole of the construct. The formed constructs can be attached to force transducers to measure the amount of tension generated over time. It is important that in any neuromuscular system the neural component is well covered and there is extensive expertise available as successfully culturing such cells is a non-trivial task. The applicants in this grant include a well established motor neuron laboratory with a long track record of such cultures.

Both of the skeletal muscle systems have been shown to generate tension over time and our preliminary data indicates that motor neurons can be successfully cultured on the regenerative/collagen system. We are seeking to extend these preliminary findings and co-culture both skeletal muscle systems with motor neurons to promote construct maturation and then to allow monitoring of functional output. The effect of the presence of motor neurons on such outputs will be studied. Various pharmacological agents will be added to the cultures to ensure that any neuromuscular junction is behaving physiologically. Finally, pathological motor neurons or muscle cells will be added to normal cells in constructs and several of the described parameters measured to see if the system can recreate the response of a pathological system.