Cell culture techniques under static two-dimensional (2D) cell culture conditions have advanced our understanding of cellular and molecular mechanisms that underlie normal and pathological conditions of various tissues. However, this platform has limits. In a 2D cell culture system, cells adhere to an inert, flat surface and are forced into a physiologically distorted state. Furthermore, cells are deprived of surrounding cellular contact that limits cell-cell signaling through secreted factors, gap junctions, and extracellular matrix components. The disconnect between cells can lead to deviations in normal tissue-specific structure and behavior such as loss of differentiated status, altered migration, and abnormal growth.

Organoids and spheroids are three-dimensional (3D) aggregates of cells in which the cells’ normal shape and physical contacts are maintained, enabling the formation of complex structures with realistic biochemical and biomechanical environments. This added dimensionality can lead to improvements in cell survival, proliferation and differentiation. Spheroids are typically composed of cancer cells, but could be seeded by other cell types that are cultured under scaffold-free, non-adherent conditions, with a relatively shorter survival period due to hypoxia and cell death. Organoids, also called “mini-organs,” use a scaffolding matrix to support the self-organization of organ-specific stem cells or progenitor cells that differentiate and lead to tissue architecture and function that resemble normal development in vivo. There are limitations to the complexity throughput and reproducibility in the production of these 3D structures. Therefore, efforts to improve methodological techniques would aid in generating reliable and consistent spheroid and organoid models for use in research.

The microgravity environment fosters the formation of organoids and spheroids. Culturing stem cells and progenitor cells in microgravity stimulates proliferation as well as preserves ‘stemness,’ helping to maintain population numbers in culture. In addition, microgravity forces cells to interact and anchor to each other which promotes the development of tridimensional cultures, producing larger and wider cell clusters with higher order structures. Microgravity therefore would be a platform to optimize conditions for large-scale production of organoids and spheroids for research, regenerative medicine applications, and pre-clinical testing of drug candidates. Many studies have looked at organoid and spheroid formation and behavior using different tissue types under simulated gravity. The positive results produced on Earth demonstrate the viability and potential of utilizing 3D cultures in space to study a range of biological processes and pathologies. 

The presence of cancer stem cells in tumors leads to poor outcomes in patients and has therefore been a focus for cancer treatment. When cancer stem cells are cultured in microgravity, the cells adopt two fates: (1) differentiated cancer cells that adhere to the bottom of the dish and (2) suspended, multicellular 3D spheroids (MCS). These MCSs retain features of the primary tumor, and therefore can be used as an intermediate model (between 2D cultures and in vivo tumors) to understand the behavior of different cancer cell types, as well as test potential therapies.  Interestingly, microgravity itself appears to be able to disrupt cancer spread and induce cell death through disruptions in mechanical sensing between cells. Microgravity conditions could therefore offer a platform to discover novel targets for drug discovery aimed at curing cancer.

Organoid technology has been utilized in microgravity to study neural development and function. These studies use brain organoids, which are small clusters of self-assembled brain cells generated from pluripotent stem cells that form functional neural networks and 3D structures resembling the embryonic brain. Brain organoids grew larger following spaceflight, which was predicted based on previous observations that progenitors proliferate faster in microgravity. The larger organoid may, however, display functional differences among the cellular components as well as rearrangements in the network architecture that could have negative effects on brain development. They are exploring whether changes in the epigenome may be driving these changes. This study could have significant implications for conditions such as autism and psychiatric disorders that arise due to changes in neural function and connectivity. Future experiments are aimed at exploring how microgravity affects neural properties in brain organoids grown using young vs. old progenitor cells and whether the microgravity platform can be used to study and address accelerated aging and neurodegeneration.