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Development of a surface tension mediated technique for dry stabilization of mammalian cells.

ABSTRACT: Dry state preservation at ambient temperatures (lyopreservation) is a biomimetic alternative to low temperature stabilization (cryopreservation) of biological materials. Lyopreservation is hypothesized to rely upon the creation of a glassy environment, which is commonly observed in desiccation-tolerant organisms. Non-uniformities in dried samples have been indicated as one of the reasons for instability in storage outcome. The current study presents a simple, fast, and uniform surface tension based technique that can be implemented for lyopreservation of mammalian cells. The technique involves withdrawing cells attached to rigid substrates to be submerged in a solution of lyoprotectant and then withdrawing the samples at a specific rate to an inert environment. This creates a uniform thin film of desiccated lyoprotectant due to sudden change of surface tension. The residual moisture contents at different locations in the desiccated film was quantified using a spatially resolved Raman microspectroscopy technique. Post-desiccation cellular viability and growth are quantified using fluorescent microscopy and dye exclusion assays. Cellular injury following desiccation is evaluated by bioenergetic quantification of metabolic functions using extracellular flux analysis and by a Raman microspectroscopic analysis of change in membrane structure. The technique developed here addresses an important bottleneck of lyoprocessing which requires the fast and uniform desiccation of cellular samples.

SUBMITTER: Solocinski J

PROVIDER: S-EPMC5837090 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Development of a surface tension mediated technique for dry stabilization of mammalian cells.

Solocinski Jason J Osgood Quinn A QA Rosiek Eric E Underwood Lukas L Zikanov Oleg O Chakraborty Nilay N

PloS one 20180305 3

Dry state preservation at ambient temperatures (lyopreservation) is a biomimetic alternative to low temperature stabilization (cryopreservation) of biological materials. Lyopreservation is hypothesized to rely upon the creation of a glassy environment, which is commonly observed in desiccation-tolerant organisms. Non-uniformities in dried samples have been indicated as one of the reasons for instability in storage outcome. The current study presents a simple, fast, and uniform surface tension ba ...[more]

PMID: 29505556

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Project description:Extraction of membrane proteins from biological membranes has traditionally involved detergents. In the past decade, a new technique has been developed, which uses styrene maleic acid (SMA) copolymers to extract membrane proteins into nanodiscs without the requirement of detergents. SMA nanodiscs are compatible with analytical techniques, such as small-angle scattering, NMR spectroscopy, and DLS, and are therefore an attractive medium for membrane protein characterization. While mass spectrometry has also been reported as a technique compatible with copolymer extraction, most studies have focused on lipidomics, which involves solvent extraction of lipids from nanodiscs prior to mass-spectrometry analysis. In this study, mass spectrometry proteomics was used to investigate whether there are qualitative or quantitative differences in the mammalian plasma membrane proteins extracted with SMA compared to a detergent control. For this, cell surface proteins of 3T3L1 fibroblasts were biotinylated and extracted using either SMA or detergent. Following affinity pull-down of biotinylated proteins with NeutrAvidin beads, samples were analyzed by nanoLC-MS. Here, we report for the first time, a global proteomics protocol for detection of a mammalian cell "SMALPome", membrane proteins incorporated into SMA nanodiscs. Removal of SMA from samples prior to processing of samples for mass spectrometry was a crucial step in the protocol. The reported surface SMALPome of 3T3L1 fibroblasts consists of 205 integral membrane proteins. It is apparent that the detergent extraction method used is, in general, quantitatively more efficient at extracting proteins from the plasma membrane than SMA extraction. However, samples prepared following detergent extraction contained a greater proportion of proteins that were considered to be "non-specific" than in samples prepared from SMA extracts. Tantalizingly, it was also observed that proteins detected uniquely or highly preferentially in pull-downs from SMA extracts were primarily multi-spanning membrane proteins. These observations hint at qualitative differences between SMA and detergent extraction that are worthy of further investigation.

Dataset Information

Development of a surface tension mediated technique for dry stabilization of mammalian cells.

Publications

Development of a surface tension mediated technique for dry stabilization of mammalian cells.

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