FSL Constructs: A Simple Method for Modifying Cell/Virion Surfaces with a Range of Biological Markers Without Affecting their Viability

Abstract

The ability to modify/visualize biological surfaces, and then study the modified cell/virion in a range of in vitro and in vivo environments is essential to gaining further insight into the function of specific molecules or the entire entity. Studies of biological surface modification are generally limited to genetic engineering of the organism or the covalent attachment of chemical moieties to the cell surface 1,2 . However these traditional techniques expose the cell to chemical reactants, or they require significant manipulation to achieve the desired outcome, making them cumbersome, and they may also inadvertently affect the viability/functionality of the modified cell. A simple method to harmlessly modify the surface of cells is required.Recently a new technology, KODE Technology has introduced a range of novel constructs consisting of three components: a functional head group (F), a spacer (S) and a lipid tail (L) and are known as Function-Spacer-Lipid or FSL constructs3. The spacer (S) is selected to provide a construct that is dispersible in water, yet will spontaneously and stably incorporate into a membrane.FSL construct functional moieties (F) so far include a range of saccharides including blood group-related determinants, sialic acids, hyaluronan polysaccharides, fluorophores, biotin, radiolabels, and a range of peptides [3][4][5][6][7][8][9][10][11][12] . FSL constructs have been used in modifying embryos, spermatozoa, zebrafish, epithelial/endometrial cells, red blood cells, and virions to create quality controls systems and diagnostic panels, to modify cell adhesion/ interaction/ separation/ immobilization, and for in vitro and in vivo imaging of cells/virions [3][4][5][6][7][8][9][10][11][12] .The process of modifying cells/virions is generic and extremely simple. The most common procedure is incubation of cells (in lipid free media) with a solution for FSL constructs for 1-2 hours at 37°C [4][5][6][7][8][9][10] . During the incubation the FSL constructs spontaneously incorporate into the membrane, and the process is complete. Washing is optional. Cells modified by FSL constructs are known as kodecytes [6][7][8][9] , while virions are kodevirions 10 . FSL constructs as direct infusions and kodecytes/kodevirions have been used in experimental animal models 7,8,10 . All kodecytes/kodevirions appear to retain their normal vitality and functionality while gaining the new function of the F moiety 7,8,10,11 . The combination of dispersibility in biocompatible media, spontaneous incorporation into cell membranes, and apparent low toxicity, makes FSL constructs valuable research tools for the study of cells and virions. Video LinkThe video component of this article can be found at https://www.jove.com/video/3289/ ProtocolThe following protocol describes the generic procedure for the insertion of FSL constructs (Figure 1) into biological membranes. For simplicity the protocol will only refer to cells (kodecytes), which are at a 100% concentration. However, the term cells is interchangeable with v...

PEGylation of Vesicular Stomatitis Virus Extends Virus Persistence in Blood Circulation of Passively Immunized Mice

“…A range of concentrations of PEG (0 to 20 mg) was added to 5 ϫ 10 8 TCID 50 s of VSV with a final volume of 500 l in 100 mM potassium PBS (pH 7.4) for each preparation. PEG conjugation reactions were performed as described previously (40,45,53). Briefly, all conjugation reactions were performed at 25°C with gentle agitation.…”

Incorporation of fluorescein conjugated function-spacer-lipid constructs into the red blood cell membrane facilitates detection of labeled cells for the duration of ex-vivo storage

“…100µL of FSL-FLRO4 (50 µg/mL in PBS) was added to an equal volume of RBC and mixed by vortexing [7]. Cells were incubated at 37 o C for one hour, followed by three washes with PBS and centrifugation (515 g, 5 mins at room temperature).…”

“…derivative of dioleoylphosphatidylethanolamine ( Figure 1) [7]. Phosphate buffered saline (PBS; GIBCO, Life Technologies, Victoria, Australia) was used to reconstitute lyophilised FSL-FLRO4 (stock concentration 2 mg/mL), prepare FSL-FLRO4 working concentration (50 µg/mL) and as a wash buffer following RBC labelling.…”

“…FSL constructs are not only useful in providing visualisation and tracking cell survival, but can be used to modify cells to generate a desired phenotype (e.g. blood group antigens) without altering the host cell function [7,8]. This is a valuable technology that has been utilised to model transfusion reactions and predict in-vivo RBC survival in animals [9], and has the potential to be applied in human clinical studies, though this avenue has not yet been explored.…”

“…These constructs are comprised of three components: functional head (F), which can represent a variety of biological functional groups; a spacer (S) selected to provide spacing between F and the membrane; and, a lipid (L) tail for spontaneous incorporation into cell membrane via anchoring into the lipid bi-layer [7] (Figure 1). FSL constructs are not only useful in providing visualisation and tracking cell survival, but can be used to modify cells to generate a desired phenotype (e.g.…”