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Engineered skeletal muscle tissue networks with controllable architecture (Biomaterials, 2008)

This publication by W. Bian et al was summarized by Tom Ben Arye, with edits by Natalie Rubio. The full, original paper can be accessed here.

Overview

The article describes a method to generate a perforated muscle tissue in an artificial environment with an area of 0.5-2 cm2 and a thickness of 0.1-0.4mm with highly specialized and aligned skeletal muscle fibers.

The motivation behind this construct is to increase the tissues surface area, providing oxygen and nutrient supply to the cell and to provide directional cues for myofiber alignment.

The System

In this method cells are put inside a liquid mixture of a hydrogel. Before the gel polymerizes, it is poured on top of a PDMS mold that contains PDMS posts (Fig. 1B) to generate the porous structure of the gel after PDMS mold removal (Fig. 1C). PDMS mold is created using standard replica molding from an SU-8 master (Fig. 1A)

The gel is anchored to velcro, which can be used to easily handle the tissue, provide it with structure and exert uniaxial tension which serve as an alignment cue.

culturedmeat

Figure 1: Engineered tissue formation. (A) PDMS mold formation. (B) pouring cell/gel solution on the mold. (C) After gel polymerization, PDMS mold is removed, leaving the porous hydrogel tissue.

Generally, after hydrogels are formed, they compact. Gel compaction increases the cell density and promote cell alignment, however it may cause problems while generating the architecture of this tissue, deforming and even ripping the gel. The authors quantified the compactness of the gel, with different gel content, different PDMS posts height and geometry to optimize the preservation of the tissue geometry.

They found that hexagonal, staggered posts, which is made of pure fibrin, offered predictable tissue geometry.

With this method the tissue’s thickness, porosity, and cell alignment can be independently changed by tuning the thickness of the mold, the size of the posts and the distance/size between the subunits of the design.

The hydrogel contains either fibrincollagen or a mixture of both and is supplemented with matrigel. Fibrin and Collagen support high viability and spreading of skeletal muscle stem cells. Matrigel is added to support faster cell spreading. These options are expensive and are not suitable for commercial food industries.

Cell Viability, Distribution and Differentiation

Cell type: C2C12 (mouse muscle cells)

Tissue porosity increased cell viability. After 3 days cell viability in the depth of 40µm was 94.3% compared to 90.6% in a non-porous control, meaning a 40% decrease in cell death.

Cell alignment was also assessed and found to be aligned and directed by the posts and geometry and tissue architecture. In general, muscle cell alignment is considerably important for cultured meat in the aspect of tissue texture, although not relevant here since this work is only relevant for minced meat.

Using this method, cells became highly differentiated and formed contracting myofibers (movie), another crucial factor for cultured meat, as this factor is important for protein production and overall taste of meat.

 

Summary

The authors offer a new method to generate engineered muscle tissue using microfabrication technology. By designing pores inside the muscle tissue they were able to generate larger tissues which are highly differentiated with higher cell viability with high cell density. From the cultured meat aspect, this technology cannot be used to generate complex 3D tissues like a steak, however it can be used for minced meat products, and with a few modifications it can make Mark Post’s muscle cell bundles generation (0:52-1:13) more efficient and automatic.