mini-MEndR: A 96-well mixed species culture assay to co-evaluate human and mouse Pax7⁺cell-mediated skeletal muscle repair

Abstract

Functional evaluation of novel molecules that promote stem cell mediated endogenous repair often require multiplexed in vivo transplant studies that are low throughput and hinder the rate of discovery. Here, we optimized and miniaturized a previously developed muscle endogenous repair (MEndR) in vitro assay that captures significant events of in vivo muscle endogenous repair to offer greater throughput for functional validation studies. The mini-MEndR assay consists of miniaturized cellulose scaffolds designed to fit in 96-well plates, the pores of which are infiltrated with myoblasts encapsulated in a fibrin-based hydrogel to form engineered skeletal muscle tissues. Pre-adsorbing thrombin to the cellulose scaffolds facilitates in situ tissue polymerization, a critical modification that enables new users to rapidly acquire assay expertise. Following the generation of the 3D myotube template, muscle stem cells (MuSCs), prospectively isolated from mouse skeletal muscle tissue, are engrafted onto the engineered template. A regenerative milieu is then introduced by injuring the muscle tissue with a myotoxin. We evaluated two different commercially available human primary myoblast lines and were able to successfully generate miniaturized 3D muscle templates, as well as recapitulate the in vivo outcomes of a known modulator of MuSC mediated repair but only in the presence of both the stem cells and the regenerative milieu. Thus, the mini-MEndR culture assay captures the ability of different molecular treatments to modulate donor MuSC skeletal muscle production and niche repopulation. The miniaturized predictive assay offers a simple, scaled platform with which to investigate MuSC endogenous repair molecular modulators, and thus is a promising strategy to accelerate the muscle endogenous repair discovery pipeline.

A cryopreservation strategy for myoblast storage in paper-based scaffolds for inter-laboratory studies of skeletal muscle health

“…Four paper scaffold candidates were selected from a larger pool of candidate cellulose-based papers based on thickness and similarity of the material to the original paper scaffold used in for the MEndR [27] and mini-MEndR [32] protocols (Mini-Minit Products, R10, Scarborough, Canada). Each of the four paper candidates were characterized using scanning electron microscopy (SEM) to assess their microstructural properties and for their ability to support myoblast differentiation, and compared to the original paper scaffold.…”

“…To evaluate tissue homogeneity, tissues were imaged at either 4X or 10X magnification, with the settings described in Table 4. SAA + coverage was quantified as previously described; [27,32] briefly, confocal image stacks were maximum Z-projected and thresholded either manually or using FIJI's triangle algorithm, where the percentage of pixels brighter than the threshold was taken as a metric of image coverage by SAA + myotubes. To assess myofiber morphology, tissues were imaged at 40X magnification, with the objective correction collar being adjusted to optimize focus (Table 4).…”

“…All subsequent steps were performed as described in our previously optimized protocol. [32] Briefly, on Day 5 of differentiation, integrin α-7 + GFP + muscle stem cells MACS-enriched from enzymatically digested Tg:Pax7-nEGFP (i.e. Pax7-nGFP) transgenic mouse [39] hindlimb skeletal muscle tissue were re-suspended in SAT10 media replete of FGF2.…”

“…To do this, we performed a complete MEndR repair assay. [27,32] Briefly, we generated myotube templates derived from myoblasts either unfrozen or cryopreserved in mini-MEndR (Figure 3A) and engrafted GFP + murine muscle stem cells on Day 6 (Figure 6A). The following day, we injured the myotube template with cardiotoxin (CTX), a snake venom toxin, leading to a reduction in myotube template fiber area coverage by ~50% relative to uninjured (CTX-) controls as optimized by our previous protocol [32] (Figure 6A-B).…”

“…For example, we previously noted that 3-5 seeding attempts were required to master the manufacturing of mini-MEndR myotube templates by new users already proficient in myoblast 2D cell culture. [32] Therefore, we set out to establish a protocol to cryopreserve myotube templates after manufacturing to enable their distribution to the research community with a view to improving platform adoption. Specifically, we built upon previous work from Bashir and colleagues, who demonstrated that myoblasts encapsulated in a hydrogel mixture seeded onto 3D-printed polyethylene glycol dimethacrylate (PEGDMA) molds could be cryopreserved and successfully maintain structure and function upon thawing.…”