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Metabolic Glycoengineering of Cell-Derived Matrices and Cell Surfaces: A Combination of Key Principles and Step-by-Step Procedures.

ACS biomaterials science & engineering (2019-01-14)
Marcus Gutmann, Julian Bechold, Jürgen Seibel, Lorenz Meinel, Tessa Lühmann
ABSTRACT

Metabolic glycoengineering allows insertion of non-natural monosaccharides into glycan structures during biosynthesis thereby enabling extracellular matrices (ECMs), cell surfaces, or tissues for decoration with functional cues with ultimate spatial control while deploying aqueous and toxicologically benign coupling chemistries. In this work, we discuss relevant methods in the design of metabolic glycoengineered systems, ranging from synthetic procedures to decoration of cell surfaces and ECM components by bioorthogonal chemistries for widespread biomedical applications. As representative example, we chose a tetra-acetylated azide-bearing monosaccharide as model compound to be metabolically incorporated into glycans of the glycocalyx and ECM components generated by NIH 3T3 cells. Detailed guidance in fabrication and functionalization of azide-bearing glycan structures via bioorthogonal click chemistries in glycoengineered extracellular matrices is provided. In addition, a biocompatible design space of the copper(I)-catalyzed azide-alkyne cycloaddition due to the toxicity of the copper catalyst is detailed enabling effective and safe modification of living cell systems. Thereby, this set of methods provides the blueprint enabling the design and characterization of metabolically glycoengineered systems for novel applications in drug delivery and tissue engineering.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Gelatin from bovine skin, Type B, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Acetic acid, glacial, ReagentPlus®, ≥99%
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2-Mercaptoethanol, ≥99.0%
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Propidium iodide, ≥94.0% (HPLC)
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Sodium azide, ReagentPlus®, ≥99.5%
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TWEEN® 20, viscous liquid, suitable for cell culture
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Dulbecco′s Modified Eagle′s Medium - high glucose, With 4500 mg/L glucose, L-glutamine, and sodium bicarbonate, without sodium pyruvate, liquid, sterile-filtered, suitable for cell culture
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4′,6-Diamidino-2-phenylindole dihydrochloride, BioReagent, suitable for fluorescence, ≥95.0% (HPLC)
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DL-Dithiothreitol, ≥99.0% (RT)
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Copper(II) sulfate, anhydrous, powder, ≥99.99% trace metals basis
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Ponceau S, BioReagent, suitable for electrophoresis
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