Development of a human skeletal micro muscle platform with pacing capabilities

Abstract

Three dimensional engineered culture systems are powerful tools to rapidly expand our knowledge of human biology and identify novel therapeutic targets for disease. Bioengineered skeletal muscle has been recently shown to recapitulate many features of native muscle biology. However, current skeletal muscle bioengineering approaches require large numbers of cells, reagents and labour, limiting their potential for high-throughput studies. Herein, we use a miniaturized 96-well micro-muscle platform to facilitate semiautomated tissue formation, culture and analysis of human skeletal micro muscles (hµMs). Utilising an iterative screening approach we define a serum-free differentiation protocol that drives rapid, directed differentiation of human myoblast to skeletal myofibres. The resulting hµMs comprised organised bundles of striated and functional myofibres, which respond appropriately to electrical stimulation. Additionally, we developed an optogenetic approach to chronically stimulate hµM to recapitulate known features of exercise training including myofibre hypertrophy and increased expression of metabolic proteins. Taken together, our miniaturized approach provides a new platform to enable high-throughput studies of human skeletal muscle biology and exercise physiology.

A 96-well culture platform enables longitudinal analyses of engineered human skeletal muscle microtissue strength

“…3d-f and Supplementary Movie S5). Force magnitudes were in line with prior reports of human skeletal muscle microphysiological systems at similar culture timepoints 24,35 . While a noticeable variation in the magnitude of the contractile forces generated by hMMTs was observed between each muscle donor at reported culture time points, a significant increase in contractile forces www.nature.com/scientificreports www.nature.com/scientificreports/ with culture time (Days 7-14) was observed consistently within each muscle donor (Fig.…”

“…In vitro models of human skeletal muscle are generally incapable of studying muscle contraction, and often those that can are limited to doing so as an experimental endpoint owing to the need to remove tissues from the culture device so as to implement a force transducer for measurements 16,24 . MyoTACTIC micro-posts, as with a recently reported cell culture insert approach 35 , were designed to sustain hMMT long-term culture and to allow for non-invasive contractile force measurements of hMMTs in an easy and robust manner ( Fig. 3a).…”

“…As a non-invasive alternative, others have engineered elegant culture devices that employ flexible posts or pillars to support tissue maturation and measure active force as it relates to post-deflection following electrical or chemical stimulation 17,18,27,28,[30][31][32][34][35][36][37][38][39] . However, some of these technologies have footprints that are not amenable to high-throughput tests.…”

Can we mimic skeletal muscles for novel drug discovery?

“…Incorporating macrophages, in particular, was critical to successful in vitro and in vivo regeneration of adult rat-engineered muscle, highlighting the increased physiological relevance of multicellular models [11]. Miniaturization of engineered human muscle size [14] without the loss of maturation, structure, or functional phenotype will be required for increased experimental throughput.…”

Modelling functional human neuromuscular junctions in a differentially-perturbable microfluidic environment, validated through recombinant monosynaptic pseudotyped ΔG-rabies virus tracing

“…Indeed, a number of studies have demonstrated the applicability of various bioengineering methods for creating muscle-neuron co-cultures, including functional NMJs in vitro. [15][16][17][18][19][20][21] In the present study, we model functional human NMJs by compartmentalized culture of iPS cell-derived MN pseudo-organoids and myotubes in a differentially perturbable multi-nodal microfluidic chip and demonstrate that NMJ function can be validated through monosynaptic retrograde tracing using a designer pseudotyped ΔG-rabies virus (RV).…”