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differentiation protocols to generate either myogenic to culture muscle organoids, significantly improving the
precursor cells or quiescent satellite-like cells, both of survival rate and differentiation efficiency of the organoids
which subsequently mature into functional, contractile through sustained mechanical stimulation. To better
myotubes. Primary myoblasts serve as another study the function and mechanism of muscle organoids,
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fundamental building block, possessing an intrinsic researchers have also employed gene-editing and cell-
capacity to fuse and form multinucleated myotubes, the labeling technologies. A recent study by Li et al. developed
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basic functional units of skeletal muscle. Under 3D skeletal muscle organoids using microdroplet-engineering
culture conditions, myoblasts can self-assemble into technology. Derived directly from primary tissues without
skeletal muscle organoids, simulating the structure and requiring primary cell culture, microdroplet-engineered
function of natural skeletal muscle. Moreover, myoblasts skeletal muscle organoids significantly reduce the
can dedifferentiate into cells similar to satellite cells under generation time of skeletal muscle organoids. In addition,
3D culture conditions, known as idSCs, which can support while employing CRISPR/Cas9 technology to investigate
multiple rounds of muscle regeneration, similar to natural the roles of specific genes in muscle development and
satellite cells, after transplantation. Satellite cells are the diseases has become well-established, studies utilizing
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stem cells in skeletal muscle, responsible for muscle growth this technology to construct muscle organoids remain at a
and regeneration. However, it remains unclear how to nascent stage.
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effectively increase the number of satellite cells in vitro
while maintaining their stem cell characteristics, especially 3.4. Composite organoids/multi-tissue organoids
their ability to repopulate the niche. In addition, both MSCs The MSK system does not exist in isolation; it is also
and idSCs can be induced toward myogenic lineages under under the control and regulation of the vascular and
appropriate conditions, further expanding the available cell nervous systems. The vascular system supplies muscles
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sources for organoid construction. This diverse cellular and bones with oxygen and nutrients, ensuring their
toolkit enables the generation of increasingly sophisticated normal metabolism and functional maintenance. The
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skeletal muscle organoids with significant potential for nervous system precisely controls muscle contraction and
applications. The construction of skeletal muscle organoids relaxation through nerve impulses, coordinates movement,
also requires suitable ECM materials. These primarily and senses muscle tension and joint position to prevent
include Matrigel, collagen, and fibrin. 165-167 Synthetic injury. The vascular, nervous, and MSK systems work
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hydrogel materials mainly consist of poly(lactic-co-glycolic together to ensure the body’s mobility and overall health.
acid) (PLGA), PEG, and polyacrylamide. These ECM Extensive research has been conducted on the
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materials offer a diverse range of options for culturing construction of vascularized bone organoids. Duan et al.
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skeletal muscle organoids, catering to various research and proposed a new strategy to produce pre-vascularized
application needs. bone organoids with self-organizing vascularization
The cultivation techniques for muscle organoids have and enhanced osteogenic properties on a large scale by
seen significant advancements in recent years, particularly combining MSCs, human umbilical vein endothelial
in the application of 3D culture systems and bioreactors. cells (HUVECs), and osteogenic microparticles. Besides,
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These technologies provide conditions closer to the Li et al. successfully constructed vascularized bone
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in vivo environment for constructing muscle organoids organoids by leveraging the ability of dental pulp stem cells
and promoting the alignment, fusion, and formation to differentiate into endothelial lineages in conjunction
of muscle fiber-like structures. 3D culture systems, by with BMSCs. This vascularized bone organoid not only
simulating the mechanical tension within muscle tissue, increased mineralization deposition and reduced cell
offer an environment closer to that of the body for the necrosis but also formed hollow structures, demonstrating
construction of muscle organoids. For instance, Price et good vascularization effects. In addition, Jusoh et al.
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al. developed a 3D organoid culture method to produce a designed a microfluidic device composed of four parallel
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large number of adult skeletal muscle satellite cells in vitro. channels (vasculature, bone, medium, and lung
This method not only increased the quantity of satellite fibroblasts), using fibrin and HA nanoparticles as the
cells but also maintained their stem cell characteristics, ECM to mimic the structure of real bone tissue. Although
which is crucial for the growth and regeneration of skeletal cartilage is devoid of vascularization, blood vessels play
muscle. Bioreactor technology has also played a significant a critical role in constructing cartilage organoids. Chen
role in the cultivation of muscle organoids. By introducing et al. leveraged the natural vascularization gradient
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mechanical stretching stimuli in the bioreactor, researchers within osteochondral tissues using single-BMSCs-derived
can simulate the physiological conditions of muscle tissue cartilage organoids, achieving gradient heterogeneous
in the body, promoting the alignment and fusion of muscle osteochondral regeneration. Vascularization of skeletal
fibers. Similarly, Chromiak et al. applied an orbital shaker muscle organoids can be achieved either through the
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Volume 1 Issue 3 (2025) 10 doi: 10.36922/OR025280024