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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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.
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).
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).
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).