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the molecular mechanisms of intracellular mechano- 5.2. Non-uniform construction standards
signaling are significantly disrupted. 253,254 For example, The successful transition of MSK organoids from the
YAP/transcriptional coactivator with postsynaptic density laboratory research stage to the field of wide application
protein 95/Drosophila disc large tumor suppressor/zonula must first overcome the key challenge of standardization.
occludens-1-binding motif transcriptional co-activators is The current lack of standardization in the preparation of
unable to efficiently translocate into the nucleus, and the organoids has become a major bottleneck constraining
Wnt/β-catenin signaling pathway activity is abnormal, their development. This lack of standardization is reflected
which leads to the expression profiles of key downstream at multiple levels. In terms of experimental reproducibility,
genes regulating bone formation and the dominant factors it is often difficult to corroborate the results of different
of myogenic differentiation to deviate from physiological research teams or even different batches of experiments
status. This dysregulation at the molecular level will trigger by the same team. Regarding data comparability, various
a profound shift in cell fate. For example, the mineralization research methods make it difficult to compare and analyze
capacity of osteoblasts and the anabolic activity of the data horizontally. In terms of large-scale production,
chondrocytes will decrease. The ultimate manifestation the lack of standardization poses a significant challenge to
is cellular dedifferentiation or reduced function, which stable production. Together, these factors seriously limit
completely fails to reflect their true state of activation in the potential of organoid technology for translational
the mechanical microenvironment in vivo. applications.

However, current organoid culture for MSK systems is Achieving organoid standardization is an extremely
mostly limited to static environments or primary devices complex, systematic project, as it covers the complete
that can only provide simple unidirectional cyclic strain, process chain from cell acquisition to final data analysis,
making it difficult to effectively reproduce the complex and each of which contains variability factors that may lead
variable mechanical environment in vivo. This serious lack to biased results. The primary source of variation is the
of mechanical simulation has led to significant deviations heterogeneity of cell sources. Stem cells from different
from the in vivo situation in terms of structural integrity, donor sources will exhibit inherent differences in genetic
functional maturity, and physiological responsiveness and epigenetic characteristics due to differences in age,
of organoids. Several studies have provided empirical gender, genetic background, and health status of individual
evidence for the critical role of mechanical stimulation donors. 257,258 These differences will directly affect the
in organoid culture. Schädli et al. inoculated hMSCs differentiation potential of stem cells, their proliferative
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in PLGA scaffolds and subjected the scaffolds to cyclic properties, and the phenotypic characteristics of the final
compression through a bioreactor. It was found that organoid formed. To unify the heterogeneity of tumor cells,
hMSCs cultured under dynamic conditions of cyclic Ji et al. collected 65 types of liver cancer tissues from
259
compression secreted more collagen, while the scaffold different patients and constructed a liver cancer organoid
mineral density increased significantly. These findings biobank (LICOB). Employing this LICOB for multiple
establish the necessity of cyclic mechanical simulation in omics analyses and high-throughput drug screening
inducing the maturation of functional bone organoids. In can comprehensively collect histological and molecular
the study of the muscle mechanical response mechanism, characteristics of various types of liver cancer, avoiding the
Bieling et al. resolved the force feedback regulation limitations of a single cell type.
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mechanism of the actin network. It was reported that Secondly, Matrigel and fetal bovine serum, the critical
when the self-assembled actin fiber network encounters components of the organoid culture, have a significant
external mechanical resistance, it results in a significant “black box” effect problem. These two substances, as
increase in the density and structural rigidity of filaments indispensable culture matrices for maintaining the normal
within the network. This strengthening effect, directly proliferation and functional status of stem cells, contain
driven by mechanical stimuli, is essentially a microscopic a variety of proteins and cytokines that are essential for
pre-adaptation of the muscle in response to mechanical cell behavior. 260,261 They cannot be completely replaced
loading. This discovery elucidates at the subcellular level by synthetic means. However, the marked batch-to-batch
how mechanical stress regulates cytoskeletal dynamics variability of these naturally occurring substances, as well
and ultimately influences the maturation of muscle as the risk of immunogenicity, can significantly interfere
structure and function at the tissue level. By accurately with cell behavioral patterns and the stability of organoid
modeling the complex mechanical environment in vivo developmental processes. The core of solving this problem
and reintroducing the core element of force into the is to decipher the secrets of Matrigel and fetal bovine serum
organoids of the MSK system, can we promote the active ingredients, and formulate an economically effective
organoids of the MSK system to truly become organoids synthetic substitute. Sekine et al. developed a completely
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capable of moving? synthetic, chemically well-defined animal-free culture

Volume 1 Issue 3 (2025) 20 doi: 10.36922/OR025280024