Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

Abstract

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. “biobanking” of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

The structural and functional integrities of porcine myocardium are mostly preserved by cryopreservation

“…Here, we preserved the porcine myocardium by simply immersing chunks of muscle tissue (∼0.5 cm 3 ) in liquid nitrogen without any pretreatment, seemingly a slow and crude way of preserving tissue, although it is a widely used technique for freezing purified proteins. Similarly, clinical specimens are often preserved in this way and it has been shown that ultrastructural features of such samples can be well conserved when thawed and fixed in glutaraldehyde ( Galhuber et al, 2021 ), consistent with our findings. Strikingly, when flash-freezing (by impact with liquid helium–cooled copper block) is used to ultrarapidly freeze (<10 ms) striated muscle tissue, followed by freeze substitution to chemically preserve frozen components, molecular structural preservation is superb within 5–10 µm of the surface ( Craig et al, 1992 ; Luther et al, 2011 ) but deteriorates rapidly at deeper levels (>10 µm), with severe ice crystal damage apparent (as distortion of the filament lattice; Padron et al, 1988 ).…”

Analytical methods used for the characterisation of specific features of biological tissues related with obesity: A review

“…The SEM results could provide an important information about the structural composition of adipose tissue layers in the human body, as well as the main microstructural features; however, the sample preparation for SEM measurements should be performed with a special care. Recently it was demonstrated that a simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy [132]. In addition, transmission electron microscopy (TEM) was also applied to study the role of adipose tissue function in obesity [133].…”

Adipocyte p53 coordinates the response to intermittent fasting by regulating adipose tissue immune cell landscape

“…Electron microscopy of eWAT samples (n = 3 for each HFD-IF and HFD-IF-KO groups) was performed according to a previously published protocol 100 . In short, eWAT was dissected and the bulk tissue immediately flash frozen in liquid nitrogen without cryo-preserving agents.…”