Rapid Magnetic 3D Printing of Cellular Structures with MCF-7 Cell Inks

Abstract

A contactless label-free method using a diamagnetophoretic ink to rapidly print three-dimensional (3D) scaffold-free multicellular structures is described. The inks consist of MCF-7 cells that are suspended in a culture medium to which a paramagnetic salt, diethylenetriaminepentaacetic acid gadolinium (III) dihydrogen salt hydrate (Gd-DTPA), is added. When a magnetic field is applied, the host fluid containing the paramagnetic salt is attracted towards regions of high magnetic field gradient, displacing the ink towards regions with a low gradient. Using this method, 3D structures are printed on ultra-low attachment (ULA) surfaces. On a tissue culture treated (TCT) surface, a 3D printed spheroid coexists with a two-dimensional (2D) cell monolayer, where the composite is termed as a 2.5D structure. The 3D structures can be magnetically printed within 6 hours in a medium containing 25 mM Gd-DTPA. The influence of the paramagnetic salt on MCF-7 cell viability, cell morphology, and ability of cells to adhere to each other to stabilize the printed structures on both ULA and TCT surfaces is investigated. Gene expressions of hypoxia-inducible factor 1-alpha (HIF1α) and vascular endothelial growth factor (VEGF) allow comparison of the relative stresses for the printed 3D and 2.5D cell geometries with those for 3D spheroids formed without magnetic assistance. This magnetic printing method can be potentially scaled to a higher throughput to rapidly print cells into 3D heterogeneous cell structures with variable geometries with repeatable dimensions for applications such as tissue engineering and tumour formation for drug discovery.

Figure 1

Effect of Gd-DTPA on cell proliferation. Approximately, 1000 MCF-7 cells are incubated in 0, 1, 10, 25, 50, 75, 100, and 125 mM Gd-DTPA. Cell proliferation is measured by MTT assay at 3, 24, 48, and 72 hours. The viable cells are (a) quantified by a standard curve (for n=3 analyzed by standard error) and (b) control normalized percent viability using Gd-DTPA free medium (0 mM) using SEM and a two-way ANOVA with Bonferroni posttests to evaluate the relative differences in viability for each concentration of Gd-DTPA. A p < 0.05 is considered to be statistically significant. As the Gd-DTPA concentration increases, cell proliferation is reduced. However, at 24 hours, the effects of Gd-DTPA to cell viability are similar to that of Gd-DTPA free medium. Significant decreases in cell viability are observed for MCF-7 cells in 25 mM and above Gd-DTPA at 48 and 72 hours. Therefore, exposure to Gd-DTPA should be limited to a maximum of 24 hours in order to limit harmful effects of Gd-DTPA on cell proliferation.

Figure 2

Effect of Gd-DTPA on cell morphology. MCF-7 cell morphologies in 0, 1, 10, 25, 50, 75, 100, and 125 mM Gd-DTPA (n=12) within 24 hours when incubated on (a) a ULA plate and (b) TCT plate. Within 6 hours, there are no apparent effects on cell morphology. At concentrations above 25 mM Gd-DTPA at 6 hours, the cell morphologies begins to differ from structures produced with 0 mM Gd-DTPA (control samples) for both ULA and TCT surfaces. In (a), as the concentration of Gd-DTPA ≥ 50 mM, the ability of cells to adhere together diminishes. Cell-cell adhesion is key for formation of a 3D structure. Similarly, in (b) concentrations of ≥ 50 mM limit intercellular attachment within 1-3 hours of exposure to Gd-DTPA. However, at 6 hours, intercellular adhesion overcomes the limiting influence of Gd-DTPA on cell-cell attachment. Therefore, to reduce harmful effects on cell morphology, exposure to Gd-DTPA should be limited to 25 mM for at most 6 hours. Scale bar = 50 μm.

Figure 3

Effect of Gd-DTPA on diamagnetic cell printing. Formation of 3D and multidimentional cell structures (2.5D) through diamagnetophoresis on (a) a ULA surface and (b) a TCT surface. Approximately 1000 cells (n=5 analyzed by SD) are incubated in both cases. For (a, i), 0 and 1 mM Gd-DTPA are insufficient to coalesce cells into a 3D structure. At 24 hours, accumulation of numerous globular cluster aggregates is observed. Only 25 and 10 mM Gd-DTPA are able to print cells through diamagnetophoresis. (a, ii) As the concentration of Gd-DTPA decreases from 25 mM, the formation of globular cell clusters increases and their ability to form a single spherical 3D structure is reduced. Only 25 mM is able to produce a single spherical cluster that remained intact until 24 hours. For (b, i), concentrations of 0 and 1 mM are again insufficient to coalesce cells into a 3D structure. The diameters of the cellular structures are equivalent those of their wells since these cells have formed 2D monolayers. Only 10 and 25 mM Gd-DTPA were able to produce a 3D structure; however, for (b, ii) 25 mM Gd-DTPA produced a denser 3D structure. Therefore, 25 mM Gd-DTPA is an appropriate concentration for forming 3D cell structures using diamagnetophoresis. Scale bar = 50 μm.

Figure 4

Incubation period of cells in the presence of external magnetic field. Cell aggregates following washes with a nonparamagnetic medium after diamagnetic 3D cell printing of (a) 5000 MCF-7 cells on a ULA surface (n=3) and (b) 3000 MCF-7 cells on a TCT surface (n=3) in 25 mM Gd-DTPA for 1, 3, 6, and 24 hours. Incubations periods indicate durations for exposure to the paramagnetic medium and the externally applied magnetic field after which the medium is replaced by 0 mM Gd-DTPA removed to prevent overexposure of Gd-DTPA and the magnetic field. At 1 and 3 hours of incubation, the cells are successfully concentrated to the zones of low magnetic field strength, which are determined by the arrangements of the magnets. However, following medium changes to remove Gd-DTPA, the 3D structures do not maintain their aggregated structures. Only for 6 hours of exposure do the cells remain as a 3D structure following medium changes. Therefore, a minimum of 6 hours is sufficient for producing a single cell structure for cell suspensions in both ULA and TCT surfaces. Scale bar = 200 μm.

Figure 5

Growth and viability of cell structures formed by diamagnetophoresis. Box-and-whisker plots for area measurements of 3D cell structures (n=3) are formed on (a) a flat ULA surface (3D) and (b) a TCT surface (2.5D), as well as 3D structures using (c) round ULA plates that allow the formation of self-assembled spheroids and (d) flat ULA plates to allow the formation of numerous spontaneously formed spheroids per well. Central 3D cell structures were (i) imaged and (ii) measured at 6 hours (following medium changes to remove Gd-DTPA), 24, 48, and 72 hours. Upper and lower whiskers are placed at the 95^th and 5^th percentile, respectively. Points beyond the whisker ranges are plotted as single dots. At 6 hours, there is a relatively large variation between the forms of the 3D structures. However, at 6 hours, the level of variation between (a) 3D spheroids printed with diamagnetophoresis on a flat ULA surface is much lower than for (c) 3D spheroids printed on a round ULA surface. At 24 hours, the projected areas of both samples are equivalent. Scale bar = 200 μm.

Figure 6

Control normalized fold change gene expression to G A P D H. Expression of HIF1α is not significant for 3D and 2.5D cell structures in comparison to the normalized expression in 2D monolayers. Expression of VEGF is significant only for 2.5D cell structures.