Cell adhesion molecules such as cadherins and integrins play significant roles in spheroid formation 29. Spheroid formation does not require a special culture medium and can be divided into three steps: loose cellular aggregation caused by interactions between integrin and the ECM, accumulation of cadherin, and the formation of compact spheroids via cadherin-cadherin interactions 28. We describe their advantages and disadvantages and summarize future research directions. In this mini-review, we explore the use of spheroids for the regeneration of bone, cartilage, liver tissues, nerves, and skin, dealing separately with scaffold-free spheroids and scaffolded spheroids. Several previous works have shown that spheroids can be used for various TE applications, alone or with a biomedical scaffold fabricated using conventional or novel processes such as electrospinning, 3D printing, and hydrogel contraction. However, the scaffold materials may trigger immune responses and cause infections 24 - 27. Spheroids have thus been incorporated into various biomedical scaffolds (made of collagen, elastin, or ECM), yielding complex 3D structures. Some tissues (such as cardiac tissues) require extensive cell-ECM interactions to maximize tissue-specific functions 23. Nonetheless, spheroids have been widely used for the regeneration of skin, blood vessels, nerves, and liver tissues. In efforts to counter these limitations, cryoplastic sectioning, light-sheet fluorescence microscopy, and optical clearing methods have been employed 21, 22.įurthermore, it is difficult to accurately shape spheroids and obtain complex 3D cell constructs. Moreover, spheroids scatter light and feature inhomogeneous dye diffusion 21, rendering imaging very challenging. To overcome these limitations, efforts have been made to artificially supply oxygen via fiber injection 17 - 20. Necrosis usually develops in spheroids of larger diameters, e.g., 400 to 600 µm, and reduces cell viability and proliferation 16. Therefore, the interiors of spheroids lack nutrients and oxygen and retain CO 2, ultimately triggering necrosis, whereas the cells in the outer layer successfully transfer nutrients and remove waste 14, 15. Cells rely exclusively on diffusion to transport nutrients or eliminate waste. However, spheroids are associated with critical shortcomings attributable to their geometrical structure. Spheroids more closely resemble the geometrical and physiological in vivo microenvironment than do monolayers, enhancing their clinical and regenerative utility. Finally, as spheroids are cell aggregates, they protect cells from unwanted external stimuli or drug inflows by ensuring the maintenance of a dense ECM network. Furthermore, spheroids exhibit higher-level tissue-specific gene expression compared to monocultures, thus affecting the regulation of cell signaling and cytokine expression, because they more closely mimic in vivo microstructures 13.

The geometrical similarity between spheroids and cells of native tissue may allow spheroids to mimic cellular functions (including viability and differentiation) better than do monolayer cultures 11, 12. The ECM interacts with growth factors and facilitates contact between cells and ECM components 10. Spheroids feature extensive cellular interactions, mimicking the in vivo extracellular matrix (ECM) that mechanically supports cells and regulates most cellular functions, including migration, survival, proliferation, and differentiation. Spheroids thus find many applications in tissue engineering (TE) 9. 2D monocultures also do not engage in in vivo cellular behavior 5 and are much more drug-sensitive than are spheroids 6, 7, reflecting differences in physical and physiological properties, the expression and spatial organization of surface receptors, cancer gene expression levels, cell stage, drug accessibility, and the local pH 8. However, 2D cultures do not mimic the in vivo 3D microenvironment cell-cell and cell-matrix interactions are limited 4. 2D cell cultures have found many applications in in vitro studies, disease modeling, analyses of cellular responses, drug screening, and regenerative medicine 2, 3. Before spheroids were introduced by Moscona and Moscona 1, conventional two-dimensional (2D) cell cultures created in Petri dishes or culture flasks were widely used these were convenient, inexpensive, and very reproducible. A spheroid is a three-dimensional (3D), self-assembling cell aggregate 1.

Hanging scaffold nude