Three-dimensional tissue culture based on magnetic cell levitation

Abstract

Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.

Figure 1. Magnetic iron oxide-containing hydrogels

a, Human glioblastoma cells (lower arrow) treated with magnetic iron oxide (MIO)-containing hydrogel held at the air-medium interface by a magnet. The image was captured at 48 h of culture and depicts a ~1 mm spheroid. Scale bar, 5mm. b, Vial of a MIO-containing hydrogel (arrow) in water. c, Scheme of electrostatic interaction of nanoparticles (spheres) with phage (elongated structures). Gold (yellow sphere) and MIO (brown sphere) nanoparticles are depicted (not drawn to scale). d, MRI image (T2*-weighted) of purified hydrogel in solution: MIO-containing hydrogel (top panel), average T2* = 16 ms and MIO-free hydrogel control (bottom panel), average T2* = 46.2 ms. Scale bar, 2 mm.

Figure 2. 3D cell culture with magnetic-based levitation

The top row shows the general cell levitation strategy and the bottom row shows the corresponding optical micrograph of neural stem cells at each stage. a, Hydrogel is dispersed over cells and the mixture is incubated. The dark blotches are fragments of hydrogel. b, Washing steps remove non-interacting hydrogel fragments. Fractions of phage, gold, and MIO nanoparticles enter cells or remain membrane-bound. c, Application of an external magnet causes cells to rise to the air-medium interface. Image shows culture 15 min after levitation. d, After 12 h of levitation, characteristic multicellular structures form (single structure is shown in the schematic). Scale bar, which applies to bottom row, 30 µm.

Figure 3. Comparison of 3D cell growth with standard 2D tissue culture

a, Phase contrast (top row) and fluorescence (mCherry; bottom row) images of levitated human glioblastoma cells monitored over 8 days. Cells coalesced within hours and formed spheroids by 24 h. Scale bar, 200 µm. b, Number of cells as a function of time for levitated cell culture (a; blue squares) and representative 2D culture (red triangles). Line fits indicate an exponential trend for levitated cells (blue line) and linear trend for surface attached (red line). c, Immunofluorescence detection of N-cadherin (red) and DAPI nuclear staining (blue) in a mouse xenograft, 3D magnetic levitation for 48 h, and 2D standard culture with human glioblastoma cells. Scale bar, 10 µm.

Figure 4. Shape control of magnetically levitated culture

3D culture derived from a magnet with a 12 mm (a–f) and 6 mm (g–l) outer radius. a, g, Magnetic field profiles were obtained by direct integration of the Biot-Savart law using Mathematica (Wolfram Research Inc.; Champaign, IL) and the surface current K⃗ = M⃗ × n̂ (M⃗, magnetization and n̂, unit vector normal to magnet surface). b, h, Hall probe measurements along a diameter perpendicular to the symmetry axis at the air-medium interface. Lines are spline fits. c–e, i–k, Human glioblastoma cells at the onset of levitation (c, i), 30 h (d, j), and following magnet removal at 30 h (e, k). f, l, Spheroid images after growth for one week. Scale bar, 400 µm.

Figure 5. Confrontation assay of magnetically levitated multicellular spheroids

a, Brightfield and fluorescence images of human glioblastoma cells (green; GFP-expressing cells) and normal human astrocytes (red; mCherry-labeled) cultured separately and then magnetically guided together (time=0). b, Confrontation between human glioblastoma cells and normal astrocytes monitored for 10.5 d. Invasion of the spheroid composed of normal human astrocytes by human glioblastoma cells serves as a standard assay of glioma invasiveness. Scale bar, 200 µm.