Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Newly designed synchrotron x-ray fluorescence nano-probes permit visualization of the elemental distribution of a sample. The use of cryogenic approaches is mandatory to slow down x-ray radiation damages. Therefore, a rapid cryofixation is essential for obtaining frozen hydrated cells to optimize the preservation of the cell structure and its chemical integrity.

The specificity of the cellular preparations for synchrotron studies lead to the optimization of this protocol for acquiring frozen hydrated cell monolayers. Visual demonstration is important for understanding how to manipulate the cell monolayer samples to achieve reproducibly cryofixed cells embedded within a thin layer of ice. To prepare a silicon nitride membrane support, open the capsule containing the membrane support and gently squeeze the capsule to lightly loosen the support.

Use thin tweezers to grasp one corner of the silicon frame taking care not to touch the silicon nitride membrane in the center of the frame and place the frame into an uncovered Petri dish in a laminar flow cabinet. Then UV sterilize the membrane for 25 to 30 minutes. At the end of the sterilization, add 10 microliters of attachment factor such as poly-L-lysine.

Incubate for 25 minutes at 37 degrees Celsius and at 5%carbon dioxide with humidity. Then fill the wells of a sterile 48-well plate with 200 to 250 microliters of 0.22 micrometer filtered ultra pure and ultra trace water per well. Use fine tweezers to carefully submerge the membrane vertically with three successive 10-second rinses in one well of water.

After rinsing, place the membrane vertically in an empty well of a sterile 96-well plate under the laminar flow hood and leave it overnight until dry. For seeding of the cells of interest, place the coated membrane with the flat side facing up in one well of a sterile four-well plate. Add five times 10 to the four cells in 10 microliters of an appropriate culture medium to the surface of the silicon nitride membrane without touching the membrane.

The drop should cover the entire membrane. Then culture the cells at 37 degrees Celsius and 5%carbon dioxide with humidity. When the cells have started to attach to the substrate, slowly add one milliliter of complete culture medium down the wall of each well covering each membrane.

Then culture the cells at 37 degrees Celsius and 5%carbon dioxide with humidity. For cryoimmobilization of the cellular preparation, first turn on an automatic plunge freezer and fill the appropriate number of wells in a 12-well plastic plate with 37 degrees Celsius ammonium acetate buffer. Immediately before rinsing and plunge freezing the membrane, remove the sample from the incubator and use the black clamp ring of a pair of quick release tweezers to unlock the tweezers.

Use the unlocked tweezers to grasp the membrane support and immerse the membrane vertically within the ammonium acetate buffer solution for about five seconds. To remove any excess buffer, press the bottom of the membrane support onto filter paper and blot each side of the frame onto the filter paper without allowing the membrane to touch the paper. When the excess buffer has been removed, open the environmental chamber door, quickly slide the tweezers into the forceps interlock, and close the door of the chamber.

Press blot A plunge. The tweezers holding the silicon nitride membrane will be quickly plunged into the cryogen. After plunge freezing, remove the lid of the transfer container with pre-cooled forceps and press transfer.

The silicon nitride membrane will be moved slightly above the cryogen. In a quick single movement, slide and slightly tilt the tweezers out of the forceps interlock to bring the tweezers directly into an empty slot of the cryobox in the transfer container filled with nitrogen liquid. Then release the black clamp ring to free the membrane.

For freeze drying of the plunge-frozen cells, use the rocker switch located on the rear panel of the freeze drying instrument to power on the instrument. The display will show press enter when ready to load sample. Mount the silicon nitride membrane brass holder on top of the sample transfer holder provided by the supplier in the polystyrene storage container keeping the level of liquid nitrogen to about one to two millimeters below the top edge of the first brass piece.

Place the membrane with the cell sample side facing up in the brass holder numbered cavity and pre-cool the transfer rod in a liquid nitrogen-filled polystyrene foam box. Use the rod to lock in the full assembly, press enter to break the vacuum to release the lid of the freeze dryer chamber, immediately transfer the sample in the freeze dryer chamber, and close the freeze dryer chamber lid. Then immediately press start to continue with the freeze drying cycle.

At the end of the freeze drying cycle, press stop to vent the chamber and remove the full assembly to access the freeze-dried samples. Here a typical optical video microscope view of frozen hydrated breast cancer cell line cells subcultured onto a poly-L-lysine coated silicon nitride membrane support as demonstrated is shown. X-ray fluorescence elemental mapping of the frozen hydrated cells reveals the distributions of physiological elements such as potassium, sulfur and zinc.

In this representative example, the tightly bound sulfur element was evenly distributed within the cell in a similar pattern to that observed for potassium and represents a good estimate of the cellular mass profile. The zinc distribution, however, exhibited higher signal within the nucleus than in the cytosol with a clearly defined separation of the signal between the cytosol and the nucleus. In this typical Brightfield microscopy view of freeze-dried primary mouse hippocampal neurons directly cultured on a silicon nitride membrane as demonstrated, the cells exhibit a typical neural morphology that is unaffected by their culture or preservation method.

X-ray fluorescence imaging of plunge-frozen cells also reveals typical elemental distributions as exhibited by these representative freeze-dried cells at a 50 to 100 nanometer spatial resolution. Plotting the silicon nitride window is critical for obtaining the thinnest possible ice layer of cells after plunge freezing. Preserving the chemical integrity of the cells at the subcellular level allows the metallome of the cells to be explored with synchrotron nano-probes as well as with other microanalytical techniques.

As not all synchrotron nano-probe analysis can be performed at cryogenic temperature, the frozen hydrated cells can be further freeze-dried to facilitate their analysis at room temperature. Remember to use appropriate personal protective equipment when working with liquid nitrogen and to always use ethane gas in a fume hood away from flames.