Surface engineering of living myoblasts via selective periodate oxidation

Abstract

Cell surface molecules are vital for normal cell activity. To study the functions of these molecules or manipulate cell behavior, the ability to decorate cell surfaces with bioactive molecules of our choosing is a potentially powerful technique. Here, we describe the molecular engineering of living L6 myoblast monolayers via selective periodate oxidation of sialic acid residues and the application of this surface modification in the artificial aggregation of cells. The aldehyde groups generated by this reaction were used to selectively ligate a model molecule, biotin hydrazide, to the cell surfaces. Flow cytometry analysis after staining with fluorescently conjugated avidin revealed a concentration-dependent increase in fluorescence compared to untreated cells with a maximal shift of 345.1 +/- 27.4-fold and an EC(50) of 17.4 +/- 1.1 microM. This mild oxidation reaction did not affect cell number, viability, or morphology. We then compared this chemical technique with the metabolic incorporation of reactive cell surface ketone groups using N-levulinoylmannosamine (ManLev). In this cell line, only a 22.3-fold fluorescence shift was observed compared to untreated cells when myoblasts were incubated with a high concentration of ManLev for 48 hours. Periodate oxidation was then used to modify myoblast surfaces to induce cell aggregation. Crosslinking biotinylated myoblasts, which do not spontaneously aggregate in culture, with avidin resulted in the rapid formation of millimeter-sized, multicellular structures. These data indicate that sodium periodate treatment is an effective, noncytotoxic method for the in vitro molecular engineering of living cell surfaces with the potential for cell biology and tissue engineering applications.

Controlled embryoid body formation via surface modification and avidin–biotin cross-linking

“…We have previously demonstrated effective surface modification and biotinylation. After periodate treatment, cell viability was found to be at least 98% over a 24 h period assessed via trypan blue exclusion (De Bank et al 2003). ES cell viability was also unaffected by periodate treatment and processing (data not shown).…”

“…The method involves mild oxidation of surface sialic acid residues with sodium periodate to generate nonnative reactive aldehyde groups which are then biotinylated with biotin hydrazide (De Bank et al 2003). Biotin-avidin binding is then exploited to rapidly cross-link engineered ES cells in a controlled density-dependent manner upon avidin supplementation.…”

Fluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates

“…To functionalize ATDC5 cell surfaces for OSB binding, we followed a protocol previously described by De Bank et al [29] Cells were grown to ~60 to 80% confluency, trypsinized and counted with a haemocytometer. Expression of aldehyde groups on the cell surfaces resulted from subsequent incubation with 5 ml cold (4°C) sodium periodate (NaIO 4 ) solution in a dark environment for ~5-10 min.…”

“…Because of the disparity in density between OSBs and cells, forced conjugation was anticipated to assure robust integration and uniform distribution of the fluorescent beads into the dense cell aggregate environment. A method based on the interaction between streptavidin and biotin (SB) was chosen [29,32]. Taking advantage of the strong adsorption properties of streptavidin to a poly(dimethylsiloxane) (PDMS) layer [33], bead surfaces were coated uniformly with streptavidin molecules (Figure 2).…”

“…Taking advantage of the strong adsorption properties of streptavidin to a poly(dimethylsiloxane) (PDMS) layer [33], bead surfaces were coated uniformly with streptavidin molecules (Figure 2). Cell surfaces were prepared for interaction with the coated microbeads by periodate treatment followed by biotin conjugation [29]. This approach resulted in a complete inclusion of the fluorescent beads by conjugated cells during the initial phase of aggregation ( Figure S1) and homogeneous distribution of OSBs in the aggregate ( Figure 1D).…”