Development of a 96-well Platform for Human Skeletal Muscle Tissue Drug Screening

Abraha, Haben Y.* 1; Afshar, Mohammad E.* 1; Afshar, Mohsen 1; Gilbert, Penney M 1

1. Institute of Biomaterials and Biomedical Engineering, University of Toronto
* denotes co-first authorship

Background:

Three dimensional (3D) models of human skeletal muscle (hSKM) tissue have been engineered successfully and shown to be responsive to pharmacological stimulation, but scalable processes to produce these tissues for drug screening are needed. Initial efforts to model hSKM were limited to two dimensional (2D) muscle cell cultures. However, 2D cultures do not lend themselves to measurement of contraction force, and so are generally restricted to providing indirect measures of muscle strength. Recently, scientists have been able to engineer 3D hSKM tissues which contract upon electrical stimulation. Significantly, the effects of drugs on 3D hSKM contraction reflected their effects in vivo. One of the central benefits of these in vitro hSKM models is their utility in phenotypic drug screens. Candidate treatments for myopathic diseases (e.g. spinal muscle atrophy) can be assessed for positive muscle strength effects, while other drugs can be tested for off-target adverse effects on the skeletal muscle. However, current hSKM engineering methods are limited in scale – only two hSKM tissues can be made per mold, and measurement of contraction force is laborious. For an hSKM drug screening platform to be truly viable, it must be able to produce hSKMs in bulk and allow simple quantification of hSKM strength.

Hypothesis:

We expect that developing a custom 96-well plate capable of 3D hSKM bulk production, and simple strength quantification, will result in a platform suitable for drug screening applications. We specifically hypothesize that as seen in vivo, anabolic androgenic steroid (AAS) treatment and glucocorticoid treatment will cause hSKM strength to increase and decrease respectively. We further hypothesize that treatment with cerivastatin will cause tissue weakening, consistent with its reported myolytic effects in vivo.

Results:

Here we report the development of a human skeletal muscle microtissue (hMMT) platform, capable of producing generating hSKM tissues in bulk. The platform consists of a custom polydimethylsiloxane (PDMS) 96-well plate that holds engineered tissues through two micropost anchor points. We show that hMMTs contract in response to acetylcholine stimulation, and that this contraction induces a measurable deflection of the micropost tissue anchors. We have characterized the relationship between micropost deflection and contractile strength, and have automated post-deflection measurement using a custom Matlab program. Through confocal image analysis, we have demonstrated a time-dependent increase in muscle fiber size over 14 days of tissue culture. Finally, western blotting results suggest that increased length of tissue culture is correlated with increased levels of the maturation marker myosin heavy chain.