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Summary: Multicellular spheroids are useful models for drug testing or studying tumor biology, but their production requires specialized approaches. Here, we present a protocol to produce viable spheroids by slow rotation around a horizontal axis using standard culture tubes. We describe steps for both seed and starter culture, and maintenance and expansion of spheroids. We detail assessment of spheroid size, count, viability, and immunohistochemistry. This protocol reduces gravitational forces that lead to cell clumping and is amenable to high-throughput use. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Abstract The advancement of microgravity simulators is helping many researchers better understanding the impact of the mechanically unloaded space environment on cellular function and disfunction. However, performing microgravity experiments on Earth, using simulators such as the Random Positioning Machine, introduces some unique practical challenges, including air bubble formation and leakage of growth medium from tissue culture flask and plates, all of which limit research progress. Here, we developed an easy-to-use hybrid biological platform designed with the precision of 3D printing technologies combined with PDMS microfluidic fabrication processes to facilitate reliable and reproducible microgravity cellular experiments. The system has been characterized for applications in the contest of brain cancer research by exposing glioblastoma and endothelial cells to 24 h of simulated microgravity condition to investigate the triggered mechanosensing pathways involved in cellular adaptation to the new environment. The platform demonstrated compatibility with different biological assays, i.e., proliferation, viability, morphology, protein expression and imaging of molecular structures, showing advantages over the conventional usage of culture flask. Our results indicated that both cell types are susceptible when the gravitational vector is disrupted, confirming the impact that microgravity has on both cancer and healthy cells functionality. In particular, we observed deactivation of Yap-1 molecule in glioblastoma cells and the remodeling of VE-Cadherin junctional protein in endothelial cells. The study provides support for the application of the proposed biological platform for advancing space mechanobiology research, also highlighting perspectives and strategies for developing next generation of brain cancer molecular therapies, including targeted drug delivery strategies.

With the advancement in reusable rocket propulsion technology, space tourist trips into outer space are now becoming a possibility at a cost-effective rate. As such, astronauts will face a host of health-related challenges, particularly on long-duration space missions where maintaining a balanced healthy microbiome is going to be vital for human survival in space exploration as well as mission success. The human microbiome involves a whole list of micro-organisms that reside in and on the human host, and plays an integral role in keeping the human host healthy. However, imbalances in the microbiome have been directly linked to many human diseases. Research findings have clearly shown that the outer space environment can directly affect the normal microbiome of astronauts when the astronaut is exposed to the microgravity environment. In this study, we show that the simulation of microgravity on earth can mimic the outer space microgravity environment. Staphylococus aureus (S. aureus) was chosen for this study as it is an opportunistic pathogen, which is part of the normal human skin microflora and the nasal passages. This study’s results show that S. aureus proliferation was significantly increased under a microgravity environment compared to Earth’s gravity conditions, which complements previous work performed on bacteria in the outer space environment in the International Space Station (ISS). This demonstrates that this technology can be utilised here on Earth to mimic the outer space environment and to study challenging health-related questions. This in return saves us the cost on conducting experiments in the ISS and can help advance knowledge at a faster rate and produce countermeasures to mitigate the negative side effects of the hostile outer space environment on humans.

Matrix metalloproteinases (MMPs) play central roles in vertebrate tissue development, remodeling, and repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate proteolytic activity by binding tightly to the MMP active site. While each of the four TIMPs can inhibit most MMPs, binding data reveal tremendous heterogeneity in affinities of different TIMP/MMP pairs, and the structural features that differentiate stronger from weaker complexes are poorly understood. Here we report the crystal structure of the comparatively weakly bound human MMP-10/TIMP-2 complex at 2.1 Å resolution. Comparison with previously reported structures of MMP-3/TIMP-1, MT1-MMP/TIMP-2, MMP-13/TIMP-2, and MMP-10/TIMP-1 complexes offers insights into the structural basis of binding selectivity. Our analyses identify a group of highly conserved contacts at the heart of MMP/TIMP complexes that define the conserved mechanism of inhibition, as well as a second category of diverse adventitious contacts at the periphery of the interfaces. The AB loop of the TIMP N-terminal domain and the contact loops of the TIMP C-terminal domain form highly variable peripheral contacts that can be considered as separate exosite interactions. In some complexes these exosite contacts are extensive, while in other complexes the AB loop or C-terminal domain contacts are greatly reduced and appear to contribute little to complex stability. Our data suggest that exosite interactions can enhance MMP/TIMP binding, although in the relatively weakly bound MMP-10/TIMP-2 complex they are not well optimized to do so. Formation of highly variable exosite interactions may provide a general mechanism by which TIMPs are fine-tuned for distinct regulatory roles in biology.

Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG(20K)-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG(20K)-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.