Characterizing Intracellular Ice Formation of Lymphoblasts Using Low-Temperature Raman Spectroscopy

Abstract

Raman microspectroscopy was used to quantify freezing response of cells to various cooling rates and solution compositions. The distribution pattern of cytochrome c in individual cells was used as a measure of cell viability in the frozen state and this metric agreed well with the population-averaged viability and trypan blue staining experiments. Raman imaging of cells demonstrated that intracellular ice formation (IIF) was common and did not necessarily result in cell death. The amount of intracellular ice as well as ice crystal size played a role in determining whether or not ice inside the cell was a lethal event. Intracellular ice crystals were colocated to the sections of cell membrane in close proximity to extracellular ice. Increasing the distance between extracellular ice and cell membrane decreased the incidence of IIF. Reducing the effective stiffness of the cell membrane by disrupting the actin cytoskeleton using cytochalasin D increased the amount of IIF. Strong intracellular osmotic gradients were observed when IIF was present. These observations support the hypothesis that interactions between the cell membrane and extracellular ice result in IIF. Raman spectromicroscopy provides a powerful tool for observing IIF and understanding its role in cell death during freezing, and enables the development, to our knowledge, of new and improved cell preservation protocols.

Rapid Freezing Enables Aminoglycosides To Eradicate Bacterial Persisters via Enhancing Mechanosensitive Channel MscL-Mediated Antibiotic Uptake

“…6C). On the other hand, cell membranes (including the cytoplasmic membrane and organelle membranes in eukaryotic cells) are generally thought to be the primary site of freezing injury according to cryobiological studies (48,50,53,54,71). In line with this notion, our combined analyses by NPN staining, PI staining, protein leakage assay, and electron microscopy collectively and unambiguously revealed the injurious effects of freezing on the membranes of E. coli cells (Fig.…”

“…Conceivably, freezing leads to membrane damage and destabilization ( Fig. 7) (48,50,53,54), which, in turn, changes the membrane tension and thus activates MscL. Alternatively, freezing can directly result in osmotic shock (48,52,71), which, in turn, could also activate MscL (73,74).…”

“…Freezing-induced aminoglycoside potentiation is linked to freezing injury of the cell membrane. Prompted by cryobiological studies revealing that the cell membrane (mostly in eukaryotic cells) is the primary site of freezing injury (48,50,53,54), we examined whether freezing causes damage to E. coli cell membranes by a combination of membrane integrity staining, protein leakage assay, membrane permeability assay, and membrane morphology analysis. To this end, first, we found that the hydrophobic fluorescent probe 1-N-phenylnaphthylamine (NPN), which fluoresces weakly in aqueous environments but becomes strongly fluorescent when interacting with the hydrophobic tail of membrane phospholipids (55), exhibited a much higher fluorescence intensity upon binding to the frozen E. coli cells than to untreated cells (column 2 versus column 1 and column 4 versus column 3 in Fig.…”

Characterizing modes of action and interaction for multicomponent osmolyte solutions on Jurkat cells

“…These compounds are used in nature to stabilize biological systems subjected to environmental extremes (P. Yancey, Clark, Hand, Bowlus, & Somero, 1982). In previous studies, we demonstrated that various osmolytes can act in concert to improve cell viability during cryopreservation (Pi, Yu, Petersen, & Hubel, 2018;Pollock, Yu et al, 2016;Pollock et al, 2017;Pollock, Budenske, McKenna, Dosa, & Hubel, 2016;Yu, Yap, Pollock, & Hubel, 2017). We have shown that certain combinations of sugars, sugar alcohols and amino acids not only maintained post-thaw recovery but also resulted in cells that had improved function compared with those that were cryopreserved with DMSO.…”

“…Boundary and area of ice crystal are generated through MATLAB. AIC: area of ice-to-cell [Color figure can be viewed at wileyonlinelibrary.com] Moens (2009),Yu et al (2017)…”