Three-dimensional cell culturing by magnetic levitation

Abstract

Recently, biomedical research has moved toward cell culture in three dimensions to better recapitulate native cellular environments. This protocol describes one method for 3D culture, the magnetic levitation method (MLM), in which cells bind with a magnetic nanoparticle assembly overnight to render them magnetic. When resuspended in medium, an external magnetic field levitates and concentrates cells at the air-liquid interface, where they aggregate to form larger 3D cultures. The resulting cultures are dense, can synthesize extracellular matrix (ECM) and can be analyzed similarly to the other culture systems using techniques such as immunohistochemical analysis (IHC), western blotting and other biochemical assays. This protocol details the MLM and other associated techniques (cell culture, imaging and IHC) adapted for the MLM. The MLM requires 45 min of working time over 2 d to create 3D cultures that can be cultured in the long term (>7 d).

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Figure 1: Human pulmonary fibroblasts before and after incubation with magnetic nanoparticles.
Figure 2: Magnetically levitated 3D cultures of A549 cells.
Figure 3: Magnetic levitation in 96-well plates.
Figure 4: Magnetically levitated 3D cultures levitating in medium in a 24-well plate.
Figure 5: Replacing medium with magnetically levitated 3D cultures.
Figure 6: Transferring 3D cultures from a 24-well plate to a 96-well plate using the Teflon pen.
Figure 7: Magnetically levitated 3D cultures of HepG2s.
Figure 8: Micrographs of magnetically levitated 3D cultures of various cell types.
Figure 9: Immunohistochemical staining patterns of 3D cultures of A549s for mucin-5AC (Abcam, cat. no. ab3649, 1:100 dilution), cytokeratin-19, (Abcam, cat. no. ab15463, 1:100 dilution), E-cadherin (Invitrogen, cat. no. 13-1700, 1:200 dilution), and N-cadherin (Invitrogen, cat. no. 33-3900, 1:100 dilution) after 2 d of culture.

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Acknowledgements

This work was supported by a US National Science Foundation (NSF) Small Business Innovation Research Award Phase I (0945954) and Phase II (1127551) from the NSF IIP Division of Industrial Innovation and Partnerships, and by an award from the State of Texas Emerging Technology Fund.

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Affiliations

Authors

Contributions

W.L.H. and D.M.T. contributed equally to this protocol by running the majority of experiments, gathering the bulk of the data presented in this protocol and preparing the manuscript. J.A.G. both helped in gathering data and images for this protocol. H.T. helped to write the manuscript. T.C.K. and G.R.S. invented and optimized the technique described in this protocol.

Corresponding author

Correspondence to Glauco R Souza.

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Competing interests

The University of Texas M. D. Anderson Cancer Center (UTMDACC) and Rice University, along with their researchers, have filed patents on the technology and intellectual property reported here. If licensing or commercialization occurs, the researchers are entitled to standard royalties. G.R.S. and T.C.K. have equity in Nano3D Biosciences, Inc. UTMDACC and Rice University manage the terms of these arrangements in accordance with their established institutional conflict-of-interest policies.

Supplementary information

Supplementary Figure 1

Magnetic levitation in 24-well plates. First, take a 24-well plate (A) and add 300-400 μL of media with cells to each well (B). Next, cover the plate with a 24-well white lid insert (C,D), 24-well magnetic drive (E,F), and lid (G,H). The plate lid can then be annotated and the plate can be transferred into an incubator (I). (PDF 1821 kb)

Supplementary Figure 2

Imaging magnetically levitated 3D cultures in a 96-well plate. First, bring the plate into a sterile environment (A), and remove the lid (B), then magnet (lid insert if necessary) (C). Next, replace the lid atop the 96-well plate (D) and move the plate out of the sterile environment (E) onto a microscope stage to image (F). (PDF 1491 kb)

Supplementary Figure 3

Transferring magnetically levitated 3D cultures from a well plate to a coverslip for imaging. Take a coverslip and place atop the 96-well magnetic drive with the magnets facing upwards (A,B). Next, pick up a 3D culture from a well plate with a Teflon pen (C). With the culture attached to the pen (D), remove the magnet from the pen (E). The 3D culture should still stay on the pen (F). Finally, place the culture on the coverslip by moving it close to the magnetic drive over the coverslip (G). (PDF 3614 kb)

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Haisler, W., Timm, D., Gage, J. et al. Three-dimensional cell culturing by magnetic levitation. Nat Protoc 8, 1940–1949 (2013). https://doi.org/10.1038/nprot.2013.125

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