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approximately 25 kDa. In the body, TGF-β exists in an and between cells and the matrix, promoting cell growth,
inactive pre-cursor form, consisting of a secreted pre-cursor differentiation, and functional expression. 60,61 In contrast,
protein complex with latent TGF-β-binding proteins. The synthetic matrix hydrogels are chemically synthesized,
protein is activated through enzymatic cleavage or other allowing for high tunability and precise control over their
activation mechanisms when required by cells, producing composition, mechanical properties, and degradation
biologically active TGF-β. In a humanized skin organoid characteristics. In addition, synthetic matrices can be
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model, aggregating hPSCs into multicellular clusters, the chemically modified or functionalized to precisely regulate
addition of a matrix, BMPs, and LSB (a TGF-β inhibitor) cell behavior and organoid development. 62
in the culture dish inhibits TGF-β signaling and facilitates 3.1. Components of natural hydrogels
ectoderm induction from pluripotent stem cells, leading to
the formation of initial epithelial cysts. Similarly, in the 3.1.1. Decellularized ECM (dECM)
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formation of expanded neuroepithelium organoids (ENOs), The ECM is a complex network secreted by cells,
a gradient of decreasing TGF-β concentrations is employed. primarily composed of proteins, glycosaminoglycans
Adding LSB during cortical neural induction suppresses (GAGs), and bioactive factors, such as TGF-β1 and FGF.
TGF-β signaling, resulting in organoid morphology closely It plays a crucial role in the cellular microenvironment,
resembling cortical organoids. In contrast, organoids not contributing to cell survival and modulating cellular
treated with TGF-β inhibitors exhibit morphology similar behavior. ECM provides physical support to cells
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to ENOs. Thus, generating ENOs from different hESC/iPSC and conveys biological information while facilitating
lines is associated with varying levels of TGF-β inhibition. intercellular signaling. By decellularizing ECM to create
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The relevant pathways are presented in Table 1. dECM, potential immune responses can be avoided
3. Application of different types of while retaining the functional properties of the ECM,
which support organoid cultivation and functionality.
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hydrogels in organoid culture dECM provides a biomimetic microenvironment that
Compared to traditional 2D cell culture methods, enhances cell adhesion, differentiation, and proliferation.
organoid culture requires a specialized 3D culture system Decellularization also allows matrix customization,
to facilitate cell growth and adhesion. Conventionally, making it more suitable for various organoid cultures and
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66
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organoid culture has primarily depended on extracellular clinical applications. Kim et al. utilized renal dECM
matrices derived from animal or tumor sources, hydrogels for the in vitro culture of kidney organoids,
which has resulted in poor reproducibility in organoid resulting in more extensive vascular networks than
preparation and potential tumorigenic risks. However, traditional Matrigel. Moreover, dECM provides enhanced
with the advancements in tissue engineering, hydrogel support and guidance for cell growth and differentiation,
scaffolds based on natural and synthetic polymers have facilitating the development of more mature and
become crucial components of organoid culture due to functionally complete kidney organoids. 66
their unique advantages. Natural matrix hydrogels are
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typically derived from biological materials and possess 3.1.2. Alginate
excellent biocompatibility, providing cells with a growth Alginate, a natural polymer derived from seaweed,
environment similar to their natural state. These hydrogels is known for its excellent non-immunogenicity and
contain bioactive components, such as proteins and gelation properties, making it a popular choice in various
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polysaccharides, that can enhance interactions among cells medical biomaterials. During organoid cultivation,
Table 1. Organoid-related signaling pathways
Signaling Receptor type Ligand type Key signal Main physiological functions References
pathway molecules
Wnt/ Frizzled and LRP5/6 Wnt protein β-catenin, etc. Cell proliferation, differentiation, and 43
β-catenin embryonic development
Notch Notch receptor (single Jagged 1/2/ Notch Maintaining stem cell gene expression 48
transmembrane protein) Delta 1/3/4
BMP BMP receptor type I/II BMP ligand Smad, MAPK, etc. Cell growth, differentiation and 53
development, organogenesis, and stem
cell regulation
TGF-β TGF-β receptor type I/II/III TGF-β ligand R-Smad, Co-Smad Cell proliferation, differentiation, 55
(Smad4) apoptosis, and stromatogenesis
Abbreviations: BMP: Bone morphogenetic protein, TGF: Transforming growth factor.

Volume 1 Issue 2 (2025) 6 doi: 10.36922/or.8262