Reviewed and edited by New Harvest Journal Club’s Tom Ben Arye and Matt Sharp, respectively.
Abstract: One of the obstacles to the potential clinical utility of bioengineered skeletal muscle is its limited force generation capacity. Since engineered muscle, unlike most native muscle tissue, is composed of relatively short myofibers, we hypothesized that its force production and transmission would be profoundly influenced by cell-matrix interactions. To test this hypothesis, we systematically varied the matrix protein type (collagen I/fibrin/Matrigel) and concentration in engineered, hydrogel-based neonatal rat skeletal muscle bundles and assessed the resulting tissue structure, generation of contractile force, and intracellular Ca2+ handling. After two weeks of culture, the muscle bundles consisted of highly aligned and cross-striated myofibers and exhibited standard force-length and force-frequency relationships achieving tetanus at 40 Hz. The use of 2 mg/ml fibrin (control) yielded isometric tetanus amplitude of 1.4±0.3 mN as compared to 0.9±0.4 mN measured in collagen I-based bundles. Higher fibrin and Matrigel concentrations synergistically yielded further increase in active force generation to 2.8±0.5 mN without significantly affecting passive mechanical properties, tetanus-to-twitch ratio, and twitch kinetics. Optimized matrix composition yielded significant cellular hypertrophy (protein/DNA ratio=11.4±4.1 vs. 6.5±1.9 μg/μg in control) and a prolonged Ca2+ transient half-width (Ca50=232.8±33.3 vs. 101.7±19.8 ms). The use of growth-factor-reduced Matrigel instead of standard Matrigel did not alter the obtained results suggesting enhanced cell-matrix interactions rather than growth factor supplementation as an underlying cause for the measured increase in contractile force. In summary, biomaterial-based manipulation of cell-matrix interactions represents an important target for improving contractile force generation in engineered skeletal muscle.
Overview: This was an Extracellular Matrix (ECM) optimization study. The authors changed the matrix composition (fibrin and matrigel concentrations) in order to find the optimal matrix for skeletal muscle growth and performance. They tried several combinations of fibrin (0.2%,0.4%,0.6%) with matrigel (10%,20%,40%) to form a hydrogel. They also compared it to a control which contained 0.14% collagen I and 10% matrigel. They assessed the tissue properties, such as structure, ability of the muscle to contract and Ca+2 intracellular handling.
From the results it seems as though they did not find an optimum concentration in the range. They found that in the range of the study the more matrigel and the more fibrin they added, the better the tissue grew. However the difference between 20% matrigel and 40% matrigel was not significant in any parameter.
This experiment was done with only one type of cell (neonatal rat skeletal myoblasts). A co-culture or tri-culture may have given contradicting results which are more relevant for growing a tissue. However, cell population was not pure as they did not use a cell line, but dissected and isolated the cells from rats.
Cells were not evenly distributed in the hydrogel and were mostly found surrounding the tissue. This may be related to low diffusion into the center of the hydrogel.
This article shows the importance of ECM study. From the cultured meat aspect, matrix composition should be focused on texture and taste. Optimization parameters should be increased cell growth and proliferation, rather than optimizing the ability of the cells to apply contractile forces (functionalize the tissue).
In addition, matrigel, which is secreted from cancerous mouse cells, may not be approved for consumption. It is imperative for ECM studies to be done with cheap, edible proteins.
Key Points:
- Optimizing the matrix enhanced myotube-matrix interaction which allowed for stronger contraction and increased muscle development.
- Optimizing the matrix also affected intracellular conditions, probably due to signaling cause by integrin to ECM interaction
- Collagen I based muscle bundles ruptured often, while fibrin ones do not.
- Cells are not evenly distributed in the hydrogel and were mostly found on the edges.
- Optimizing matrix composition did not affect myotubes depth into the hydrogel, percent of cross-striated myotubes or cell maturation.
- Optimizing matrix composition significantly changed/increased cell number (survival+proliferation) tissue’s contractile force duration and kinetics, tissue shrinkage over time, myotube diameter, protein content of the bundle cells, protein-to-dna ratio (amount of protein normalized to the number of cells), and lower stiffness for fibrin vs collagen I.
- Main change was seen when moving from 10% to 40% matrigel.
Notable Statistics:
- Optimizing the ECM content allowed a 3-fold increase in force amplitude between collagen I-based and optimized fibrin-based bundles.
Glossary:
Extracellular matrix (ECM) – the gel in which cells live in. consist of water, and large structures of sugars and proteins.
Myofiber – muscle fiber
Striated muscle – aligned muscle fibers. can contracts and relaxes in short, intense bursts. Can be found in skeletal and cardiac muscles.
Tetanus – prolonged contraction of skeletal muscle fibers.
Hypertrophy – growth of organ size not due to cell divisions.
Collagen – a structural protein which muscle cells cannot directly attach to.
Fibrin – a structural protein which muscle cells can attach to. More convenient to work with compared to Collagen and has better physical properties.
Matrigel – a product by BD. a gelatinous protein mixture that resembles native ECM. Secreted by a type of cancerous mouse cells.
Integrin – proteins in the cell membrane which promote cell attachment to the ECM. Attachment of the integrin also send signals/information into the cell from the surrounding ECM.
Ca+2 (Calcium) intracellular handling – muscle cell contraction is regulated by intracellular calcium concentration.
