cell passage + decalcification ftw

More activities this week: cell passage

Protocol for cell passage (for most cell types*):  ----> I'll unpack this process further below

• Culture should be around 60% confluent, no more than 80%
• Aspirate (remove with pipet) all present cell medium
• Add PBS to vessel to rinse cell layer of any remaining medium and dispose (3x)
• Tripsinization (from http://www.qiagen.com/knowledge-and-support/spotlight/protocols-and-applications-guide/animal-cell-culture/):

1. Add enough warmed 1x trypsin–EDTA solution (see table 1x trypsin–EDTA solution) to cover the monolayer, and rock the flask/dish 4–5 times to coat the monolayer.
2. Place the flask/dish in a CO₂ incubator at 37°C for 1–2 min.
3. Remove flask/dish from incubator and firmly tap the side of the flask/dish with palm of hand to assist detachment.
4. If cells have not dislodged, return the flask/dish to the incubator for a few more minutes.
5. IMPORTANT: Do not leave cells in 1x trypsin–EDTA solution for extended periods of time. Do not force the cells to detach before they are ready to do so, or clumping may occur.
6. Overly confluent cultures, senescent cells, and some cell lines may be difficult to trypsinize. While increasing the time of trypsin exposure may help to dislodge resistant cells, some cell types are very sensitive to trypsin and extended exposure may result in cell death. In addition, some cell lines will resist this treatment and will produce cell clumps.
7. Once dislodged, resuspend the cells in growth medium containing serum.
8. Gently pipet the cells up and down in a syringe with a needle attached to disrupt cell clumps.
9. If pipetted too vigorously, the cells will become damaged. Ensure that pipetting does not create foam.

• Separate the liquid evenly into new vessels.
• Label vessels with cell type, date, researcher name, and passage number.
• Place vessels in incubator and leave them a couple days to recover.

Using all of the above protocols, I created two new cell cultures today: C2C12s. I'm calling them Mighty Mouse cell cultures, because C2C12s are mouse muscle tissue cells. These two cultures have been named after two of my Cell Parent donors, Peter Steggall and Sonny Assu (photos will come your way once I am able to print them - Peter, I hope to print yours and ship it off to you before I leave Australia, and Sonny, I'll print and deliver yours to you once I'm back in Canada). These cell cultures will also receive quantum mechanical psychoenergetic transfer from an energy worker/ artist (and former student of mine), Amber Friedman of Blue Lotus Wholistic Healing in Powell River, BC. This should make them even mightier Mighty Mouse muscle cells! Amber will be building crystal grids to transfer strength-building energy. I'll have more information from her in the form of a report after the work is complete. This work complements the work of the professor I'm working with in the lab right now, Ionat Zurr. Ionat is working with the C2C12s on a special miniature laboratory device that she ordered from Sweden - it is essentially, as she describes it, like a gym for the cells - it's a small surgical steel device that expands and contracts the cells once they've differentiated into their elongated form. This cellular exercise will make them stronger and she hopes, will cause them to align in the same direction with each other in order to reproduce muscle tissue that is similar to what grows in vivo (in a body).
I'll also be working with another energy worker/hypnotherapist from Montreal as well, but not on these particular Mighty Mouse cultures. I'm looking forward to seeing what results.

Now to unpack the cell passage process a little bit: cells grow in a monolayer on the bottom of the vessel (pitre dish, etc). They are firmly attached to the vessel and will continue to grow until they cover the entire bottom - this is called confluence. At this time, they will either stop growing or die. So, we don't let them cover the bottom entirely, but instead do the cell passage process to divide them up into more vessels, to keep growing. Or, they go into the freezer to suspend growth until they are required for more experiments. As you can see in the first part of the cell passage protocol, we do the passage when they are about 60% confluent. They begin to differentiate at about 80% confluence, meaning they change into another cell stage. We need to passage them before then.

PBS is phosphate-buffered saline, a solution that cells like because it's got the right amount of salt. This is used to give them a cleanse prior to separating them from the bottom of the dish. They must be cleansed of all cell medium (which is the blood serum + antibiotics that keep cells alive in their culture dishes) because the cell medium, called DMEM, will neutralize the Trypsin and it won't work. Trypsin is like a soap that essentially begins to break down the cell wall, so it is used to separate them, but only for a minute so that they don't become mortally wounded. Just think: every time you wash your hands, you are removing a cell layer. After a minute, the cells are floating around in the solution freely and can be pipetted into new vials and once more have space enough to grow. After they are in their new cell condos, they can be fed again with the cell medium (DMEM), which neutralizes the Trypsin and helps them recover from the stress. They are then returned to the incubator, which keeps them at body temperature, and begin their growth process anew.

The Mighty Mouse cultures at home in the incubator.

When Amber is ready to do her energy work on the Mighty Mouse cultures this coming Friday morning, they will be cozy in the incubator, well fed and ready to receive good energy. Then I'll look at them again under the microscope and see if I can determine what difference she might have made.

Also, in the next few days or week, I will be doing bone decalcification/decelluarization with Dr Mary Lee. This is extremely exciting. She will help me remove all of the calcium crystals from the bone extracellular matrix and then we will fill it with wax, slice it with the bone saw, dye it beautiful colours and then photograph it under a  microscope. Oh, and one final interesting thing to add: bone cells don't become confluent! They can grow while floating in suspension and do not adhere to the wall of a dish. Why? Because they are magical.