DISC-3D: dual-hydrogel system enhances optical imaging and enables correlative mass spectrometry imaging of invading multicellular tumor spheroids.

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging.

Exercise tolerance through severe and extreme intensity domains.

The power-duration relationship accurately predicts exercise tolerance for constant power exercise performed in the severe intensity domain. However, the accuracy of the prediction of time to task failure (Tlim ) is currently unclear for work rates (WR) above severe intensities; that is, within the extreme intensity domain (Tlim  < 2 min). We hypothesized that Tlim would be shorter for WRs within the extreme intensity domain than predicted from the linear 1/time relationship of the severe intensity domain which would suggest mechanisms limiting exercise are different between intensity domains. Six men completed 7 knee-extension tests. Tlim of extreme intensity exercise (60%, 70%, 80%, and 90% 1RM; Tlim  < 2 min) were compared to the predicted Tlim from the slope of the S1-S3 (Tlim  ≥ 2-15 min) regression. Twitch force (Qtw ) and maximal voluntary contraction (MVC) were measured on the right vastus lateralis before and after each test. Tlim at 70-90% 1RM were shorter than the Tlim predicted by the severe domain 1/time model (P < 0.05); however, Tlim at 60% 1RM was not different than the predicted severe Tlim , suggesting the mechanisms limiting extreme exercise manifest ≥60% 1RM. A significant linear relationship for 60-90% 1RM was observed which suggested a curvature constant unique to the extreme domain ( Wext' ) that was smaller than the W ' of the severe domain (1.5 ± 0.6 vs. 5.9 ± 1.5 kJ, P < 0.001). Qtw and MVC were significantly decreased following severe exercise, however, Qtw and MVC were not significantly decreased following 80% and 90% 1RM, giving evidence that mechanisms causing task failure were recovered by the time post-exercise measurements were made (~90 sec).