Standardization of myotube formation using micropatterns

Experiments were carried out by the Muscle and Pathology team from Grenoble Institute of Neurosciences (I. Marty, Y. Even). Protocol and images were generously provided by C. Villard (Institut Néel, Grenoble) and Y. Even who ran the experiments

Background

This comparative study aimed at establishing an optimized protocol for myotube assay differentiation.

A major bottleneck for current myogenesis assays is the heterogeneity in myotube morphology and differentiation, and thus complexity in terms of image analysis and quantification of neo-formed structures.

The objective was to test whether restricting the surface of adhesion of myoblast cells would favor the formation of fully-functional standardized myotubes.

Experimental Description

In order to investigate the impact of adhesion geometry (fibronectin coating), 3 conditions were tested.

L6 myoblasts (Rat) were seeded (10e6 cells /chip) on:

• fully adhesive FN coated glass (“standard” condition),
• micropatterned FN islands (Arena discs of 500µm in diameter)
• micropatterned FN lines (20µm width, 500µm length)

 

Results: Observation of differentiated myotubes 3 days and 7 days post seeding (10X confocal microscope)

at Day 3
at Day 7
Fully adhesive surface (FN)	Arena 500µm FN discs	20µm x 500µm FN lines

L6 myoblasts (Rat) were stained for nucleus (Hoechst, blue), ER (Calnexin/clnx, red)

and a marker of differentiated myotubes (Myosin Heavy Chain Ab, green)

Conclusions

Isolated and individualized differentiated myotubes were obtained as early as 3 days post-seeding on micropatterns of finite shape and size.

Micropatterned myotubes were functional - as revealed by the myosin heavy chain staining- and  presented a typical elongated morphology.

The geometry of the substrate is a key parameter in order to obtain a standardized and homogenous array of fully-differentiated myotubes. They form as individualized structures easy to localize on the substrate. Image segmentation and analysis is simplified and more precise on mature myotubes (Day 7).

Overview of standardized differentiated myotubes (L6 rat) aligned on 20 µm width adhesive micropatterns.

The image is a mosaic of 18 fields (x10). There is approximately one myotube per pattern. Labeling is the same as above.