RESUMO
Fibroblasts (mouse, NIH/3T3) are combined with MDA-MB-231 cells to accelerate the formation and improve the reproducibility of 3D cellular structures printed with magnetic assistance. Fibroblasts and MDA-MB-231 cells are cocultured to produce 12.5 : 87.5, 25 : 75, and 50 : 50 total population mixtures. These mixtures are suspended in a cell medium containing a paramagnetic salt, Gd-DTPA, which increases the magnetic susceptibility of the medium with respect to the cells. A 3D monotypic MDA-MB-231 cellular structure is printed within 24 hours with magnetic assistance, whereas it takes 48 hours to form a similar structure through gravitational settling alone. The maximum projected areas and circularities, and cellular ATP levels of the printed structures are measured for 336 hours. Increasing the relative amounts of the fibroblasts mixed with the MDA-MB-231 cells decreases the time taken to form the structures and improves their reproducibility. Structures produced through gravitational settling have larger maximum projected areas and cellular ATP, but are deemed less reproducible. The distribution of individual cell lines in the cocultured 3D cellular structures shows that printing with magnetic assistance yields 3D cellular structures that resemble in vivo tumors more closely than those formed through gravitational settling. The results validate our hypothesis that (1) fibroblasts act as a "glue" that supports the formation of 3D cellular structures, and (2) the structures are produced more rapidly and with greater reproducibility with magnetically assisted printing than through gravitational settling alone. Printing of 3D cellular structures with magnetic assistance has applications relevant to drug discovery, lab-on-chip devices, and tissue engineering.
RESUMO
Controlled cell assembly is essential for fabricating in vitro 3D models that mimic the physiology of in vivo cellular architectures. Whereas tissue engineering techniques often rely on intrusive magnetic nanoparticles placed in cells and hydrogel encapsulation of cells to produce multilayered cellular constructs, we describe a high-throughput, label-free, and scaffold-free magnetic field-guided technique that assembles cells into a layered aggregate. An inhomogeneous magnetic field influences the diamagnetic cells suspended in a paramagnetic culture medium. Driven by the magnetic susceptibility difference and the field gradient, the cells are displaced toward the region of lowest field strength. Two cell lines are used to demonstrate the sequential assembly of layer-on-layer aggregates in microwells within 6 h. The effect of magnet size on the assembly dynamics is characterized and a microwell size criterion for the highest cell aggregation provided. Label-free magnetic-field-assisted assembly is relevant for on-demand scalable biofabrication of complex layered structures. Potential applications include drug discovery, developmental biology, lab-on-chip devices, and cancer research.
Assuntos
Campos Magnéticos , Engenharia Tecidual , Agregação Celular , HidrogéisRESUMO
AIM: The goal of our research is to develop an ultrasensitive diagnostic platform called 'NanoMonitor' to enable rapid label-free analysis of a highly promising class of biomarkers called glycans (oligosaccharide chains attached to proteins) with high sensitivity and selectivity. The glycosylation of fetuin - a serum protein - and extracts from a human pancreatic cancer line was analyzed to demonstrate the capabilities of the NanoMonitor. MATERIAL & METHODS: The NanoMonitor device consists of a silicon chip with an array of gold electrodes forming multiple sensor sites and works on the principle of electrochemical impedance spectroscopy. Each sensor site is overlaid with a nanoporous alumina membrane that forms a high density of nanowells on top of each electrode. Lectins (proteins that bind to and recognize specific glycan structures) are conjugated to the surface of the electrode. When specific glycans from a test sample bind to lectins at the base of each nanowell, a perturbation of electrical double-layer occurs, which results in a change in the impedance. Using the lectins Sambucs nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA), subtle variations to the glycan chains of fetuin were investigated. Protein extracts from BXPC-3, a cultured human pancreatic cancer cell line were also analyzed for binding to SNA and MAA lectins. The performance of the NanoMonitor was compared to a conventional laboratory technique: lectin-based enzyme linked immunosorbent assay (ELISA). RESULTS & DISCUSSION: The NanoMonitor was used to identify glycoform variants of fetuin and global differences in glycosylation of protein extracts from cultured human pancreatic cancerous versus normal cells. While results from NanoMonitor correlate very well with results from lectin-based ELISA, the NanoMonitor is rapid, completely label free, requires just 10 microl of sample, is approximately five orders of magnitude more sensitive and highly selective over a broad dynamic range of glycoprotein concentrations. CONCLUSION: Based on its performance metrics, the NanoMonitor has excellent potential for development as a point-of-care handheld electronic biosensor device for routine detection of glycan biomarkers from clinical samples.