NanoShuttleTM-PL

What is NanoShuttleTM-PL and what is its mechanism of action?
NanoShuttleTM-PL is a nanoparticle assembly consisting of gold, iron oxide, and poly-L-lysine. The poly-L-lysine will non-specifically bind to cell membranes via electrostatic interactions. NanoShuttleTM-PL will be retained by the cells for roughly 8 days, after which they are released into the extracellular space.1 If in 3D, NanoShuttleTM-PL will be released into the extracellular matrix, and the 3D culture will retain its magnetic nature.

Is the NanoShuttleTM-PL cytotoxic?
We have found no effect of NanoShuttleTM-PL on metabolism, proliferation, or inflammatory stress in lung2 and aortic valve cells3. Additionally, we have found cultures proliferate more in 3D than 2D.1,4

Can I perform biological experiments (immunohistochemistry, Western blotting, qRT-PCR, biochemical assays) on 3D structures with NanoShuttleTM-PL?
NanoShuttleTM-PL should have no effect on any experimental conditions or fluorescent signal, and 3D cultures created with NanoShuttleTM-PL should be treated like any other 3D culture or microtissue. Given the brown color of NanoShuttleTM-PL, we do advise against using DAB-based immunohistochemical staining, as the similarity in color can lead to misinterpretation of results.

Will the NanoShuttleTM-PL affect imaging under a microscope?
NanoShuttleTM-PL should have no effect on imaging, either with brightfield, fluorescent, confocal, or scanning electron microscopy. Magnetic 3D cultures should be imaged as any other 3D culture or microtissue would be imaged.

Can NanoShuttleTM-PL affect cell counting?
No, the nanoparticles are too small to have any effect

How do I store NanoShuttleTM-PL and for how long?
NanoShuttleTM-PL should be stored at 4°C, and can be used for at least a year.

The NanoShuttleTM-PL appears clear or separated, what do I do?
The reason for this appearance is that the NanoShuttleTM-PL has settled at the bottom of the vial. Mix the NanoShuttleTM-PL 10X with pipette action to ensure even distribution of NanoShuttleTM-PL before use.

Are there any specific disposal procedures for NanoShuttleTM-PL?
No, treat NanoShuttleTM-PL as you would any other biological waste.

The NanoShuttleTM-PL looks like a delicious chocolate milkshake, would drinking it be a bad idea?
Yes, while NanoShuttleTM-PL is non-toxic, we strongly advise against intaking it, although you could see it pass through your system using MRI.

Magnetizing Cells with NanoShuttleTM-PL

How do I magnetize cells?
Cells are magnetized by adding NanoShuttleTM-PL directly to a flask of cells that is 70-80% confluent, and incubating overnight. Typically, we suggest adding NanoShuttleTM-PL at a concentration of 1 μL/10,000 cells.

Can I magnetize cells in suspension?
Yes, see our protocol on magnetizing suspension cells. Briefly, suspension cells are magnetized by mixing them with NanoShuttleTM-PL for ~30 min.

When I detach adherent cells using trypsin, will the NanoShuttleTM-PL come off?
No, cells that are trypsinized with routine protocols will not lose the NanoShuttleTM-PL during detachment.

I treated too many cells with NanoShuttleTM-PL, can I replate the cells?
Yes, you can replate the cells to continue culture. The cells will behave exactly as they would without NanoShuttleTM-PL, and the NanoShuttleTM-PL will eventually detach off.

Can I freeze cells with NanoShuttleTM-PL?
Yes, NanoShuttleTM-PL should have no effect on cryopreservation. You can freeze cells with NanoShuttleTM-PL, and if you thaw those cells later on, they will retain their magnetism.

Culturing in 3D

Does this system require any specialized equipment or media?
No, there is no specialized equipment or media in this system outside of the NanoShuttleTM-PL and magnets.

What multiwell plates can I use? Do they need to be low-binding?
We offer kits for 35 mm petri dishes, 6-well, 24-well, 96-well, and 384-well plates. While low-binding plates are not necessarily required, we strongly suggest their use. In magnetic levitation, using low-binding plates limits the number of cells to adhere quickly to the bottom of the plate. In magnetic 3D bioprinting, low-binding plates are necessary, as spheroids are formed on the bottom of the well plate, and its adhesion to the bottom would yield a 2D environment at the interface.

How fast will 3D cultures appear after applying a magnetic force?
This will vary with cell type, cell number, and media volume, but typically cells will begin to aggregate within minutes and start to form larger structures within hours.

Can I aspirate media out of wells without losing the culture?
You can hold the cultures down by placing the magnet underneath the well plate while aspirating liquids. It should be noted that the magnetic forces are weak, and while most cultures can be held down, some may not as a result of the culture outgrowing the magnetic field. Be careful when aspirating liquids.

My cells appear very aggregated when centrifuged and remain aggregated when I try to resuspend them, what should I do?
Use vigorous pipette action to break apart clumps or large aggregates to create an even suspension of cells.

Can I use the magnets to stretch the 3D culture?
While theoretically yes, the magnets in this system are very weak, only strong enough to levitate or print cells. Either miniscule distances (<5 mm) or stronger magnets are required, but the magnetic field strength needed to pull the 3D culture approaches a level harmful to cells. Thus, using the magnets to pull the 3D culture is not advised.

Should my 3D cultures appear loose or dense?
Different cell types will yield different morphologies in 3D. The main factors will be the number of cells and time of culture. See Fig. 8 of our Nature Protocols publication to see what different cells would look like in 3D. Also check out our blog to see how we levitated HepG2 hepatocytes and how the culture grows over time.

BiO Assay – Viability Screening

Will bubbles affect my analysis?
For automated analysis, yes, bubbles will affect analysis, which depends on the contrast between the dark 3D structures and the light media. If bubbles appear, they will normally go away with time, but if they are still present when you are ready to take images, take time during printing to pop bubbles.

Can the n3Dock image cells without NanoshuttleTM-PL?
No, NanoshuttleTM-PL is not only needed to magnetize the cells and print them, but also to provide contrast for imaging. The iPodTM has a resolution of 200 μm5, so it cannot image cells.

Can the iPodTM be left in the incubator for the length of an experiment?
While there maybe adverse effects by leaving the iPodTM long-term due to humidity, from our experience, by using the iPodTM for just the experiment and maintaining it properly, the iPodTM will last a long time.

References

1. Souza, G. R. et al. Three-dimensional tissue culture based on magnetic cell levitation. Nat. Nanotechnol. 5, 291–6 (2010).
2. Tseng, H. et al. Assembly of a three-dimensional multitype bronchiole coculture model using magnetic levitation. Tissue Eng. Part C. Methods 19, 665–75 (2013).
3. Tseng, H. et al. A three-dimensional co-culture model of the aortic valve using magnetic levitation. Acta Biomater. 10, 173–82 (2014).
4. Castro-Chavez, F., Vickers, K. C., Lee, J. S., Tung, C.-H. & Morrisett, J. D. Effect of lyso-phosphatidylcholine and Schnurri-3 on osteogenic transdifferentiation of vascular smooth muscle cells to calcifying vascular cells in 3D culture. Biochim. Biophys. Acta 1830, 3828–34 (2013).
5. Timm, D. M. et al. A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis. Sci. Rep. 3, 3000 (2013).