Automated drug screening with contractile muscle tissue engineered from dystrophic myoblasts

Abstract

Identification of factors that improve muscle function in boys with Duchenne muscular dystrophy (DMD) could lead to an improved quality of life. To establish a functional in vitro assay for muscle strength, mdx murine myoblasts, the genetic homologue of DMD, were tissue engineered in 96-microwell plates into 3-dimensional muscle constructs with parallel arrays of striated muscle fibers. When electrically stimulated, they generated tetanic forces measured with an automated motion tracking system. Thirty-one compounds of interest as potential treatments for patients with DMD were tested at 3 to 6 concentrations. Eleven of the compounds (insulin-like growth factor-1, creatine, beta-hydroxy-beta-methylbutyrate, trichostatin A, lisinopril, and 6 from the glucocorticoid family) significantly increased tetanic force relative to placebo-treated controls. The glucocorticoids methylprednisolone, deflazacort, and prednisone increased tetanic forces at low doses (EC(50) of 6, 19, and 56 nM, respectively), indicating a direct muscle mechanism by which they may be benefitting DMD patients. The tetanic force assay also identified beneficial compound interactions (arginine plus deflazacort and prednisone plus creatine) as well as deleterious interactions (prednisone plus creatine inhibited by pentoxifylline) of combinatorial therapies taken by some DMD patients. Since mdx muscle in vivo and DMD patients respond in a similar manner to many of these compounds, the in vitro assay will be a useful tool for the rapid identification of new potential treatments for muscle weakness in DMD and other muscle disorders.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

“…Eschenhagen and coworkers have advanced the field by developing aligned tissue models suitable for some multiwell-plate analytics and that are capable of measuring forces of contraction (22). Together, these and other systems (23,24) have already proven their utility as models for cell transplantation (25) and drug candidate evaluation (12,26,27). We have recently used microfabrication to miniaturize such aligned tissue models into microscale tissues (11).…”

High-Content Drug Screening with Engineered Musculoskeletal Tissues

“…Thirty-one compounds of interest as potential treatments for patients with DMD were tested with MFAS at three to six concentrations. 75 Eleven of the compounds significantly increased mdx mBAM tetanic force relative to placebo-treated controls. The assay also identified beneficial compound interactions as well as deleterious interactions of combinatorial therapies taken by some DMD patients.…”

“…4B) for tissue engineering mBAMs that enables the use of standard robotic liquid-handling systems. 75 The liquid handler mixes a cell suspension with soluble extracellular matrix in each well, a process that takes approximately 7-8 min per 96-well plate. Each construct is engineered with 50,000-100,000 cells=mBAM and, after differentiation, contains several hundred aligned striated muscle fibers.…”

“…Adapted from Refs. 70,75 sterility. 75 MAD is encased in an insulated box so that temperature, CO 2 level, and humidity can be maintained in a similar manner to a standard tissue culture incubator.…”

“…70,75 sterility. 75 MAD is encased in an insulated box so that temperature, CO 2 level, and humidity can be maintained in a similar manner to a standard tissue culture incubator. Once loaded onto the MAD on a heated stage, the device automatically transports the microwell plate to the stimulating electrodes and electrically stimulates each well for 1-2 s with preselected parameters for tetanic force generation, and 40-60 images of the mpost deflections are captured.…”