Development of a 96-well platform for human skeletal muscle tissue drug screening

Afshar Bakooshli, Mohammad* 1 ; Abraha, Haben Y.* 1 ; Davoudi, Sadegh 1 ; Afshar Bakooshli, Mohsen 1 ; Gilbert, Penney M 1, 2

 1.  Institute of Biomaterials and Biomedical Engineering, University of Toronto; 2.  Department of Biochemistry, University of Toronto

Background:

3D models of human skeletal muscle (hSKM) tissue have been engineered and are responsive to pharmacological stimulation, but scalable processes to produce these tissues are needed. Traditional 2D muscle cell cultures do not lend themselves to measurement of contraction force and thus can only provide 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 muscle wasting treatments 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 viable, it must be able to produce hSKMs in bulk and allow simple quantification of hSKM strength.

Objective:

We aim to develop a human muscle microtissue (hMMT) engineering platform that is demonstrably suitable for the study of skeletal muscle and for drug candidate testing. Specifically, the platform must be easily fabricated, encouraging adoption; the platform must generate uniform, 3D hMMTs in bulk; hMMTs must mimic endogenous skeletal muscle architecture and drug response; and the platform must provide a non-invasive measurement of skeletal muscle strength.

Results:

We report the development of a human skeletal muscle microtissue (hMMT) platform, capable of generating hSKM tissues in bulk. The platform consists of a custom polydimethylsiloxane (PDMS) 96-well plate that holds hMMTs via two micropost anchor points. Fabrication of the platform is simple and only requires PDMS, an oven, and the polyurethane platform mold. hMMTs contract in response to chemical and electrical stimulation, and this contraction induces intracellular calcium flux and measurable deflection of the micropost tissue anchors. We have characterized the relationship between micropost deflection and contractile strength, used MATLAB to automate post-deflection measurement, and designed an apparatus that enables non-invasive electrical stimulation of individual hMMTs. Together this allows non-invasive, automated measurement of hMMT contraction strength. hMMT characterization showed that muscle fiber diameter increased over 14 days of tissue culture, as did expression of the maturation marker myosin heavy chain. We also found that hMMT drug response reflected in vivo drug response. Treating hMMTs with insulin-like growth factor 1 did not affect fiber diameter, while administration of the glucocorticoid dexamethasone, and the toxic cholesterol drug cerivastatin, significantly reduced fiber diameter.