Debbie Knight

A Day in the Life: March 20, 2012 (in honor of the first day of Spring)

In research log on March 20, 2012 at 9:00 am

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab on a particular day. These entries I will call “A Day in the Life…”

In honor of the first day of Spring, I’m writing about cell culture. Specifically, thawing and growing cells after they have been in the deep freeze. Not so dissimilar to the grass turning a vibrant green and tulips stretching toward the sky after a long Winter’s nap.

Cultured cells can be stored for years in suspended animation simply by adding a cryopreservative (most labs use dimethyl sulfoxide or DMSO) and placing them in vat of liquid nitrogen (which freezes at a balmy 63 K (−210 °C; −346 °F) according to Wikipedia).

The cells I am working with today have been in liquid nitrogen for twelve years in our lab. And as long as we keep topping off the liquid nitrogen container (called a cryopreservation tank), they would keep indefinitely.

One of two cryopreservation tanks we have in the lab. Each tank has four racks each of which holds nine boxes which hold up to 81 vials of cells. So, one tank can hold up to 2,916 vials.

Because we have two cryopreservation tanks, each having four racks of nine boxes, we keep a record of where we put a vial of cells. Some labs use a computer-based system, but we find a handwritten logging system gives us more flexibility in the actual lab setting. This means we have to flip through a few pages in a 3-ring binder, but that’s okay.

This particular log dates back when the lab had only a few vials of cells and I simply didn’t know any better. I improved on this logging method when I worked in another lab — using a grid that shows the exact location of each vial in each box. While there was a computer database built into this system, it was still easier to just mark off the vial removed by hand.

Our cell log. We've found a handwritten logging system works better for us in the lab setting than a computer database. However, there are better ways than this to keep track of frozen vials of cells.

The cells I’m looking for are human brain cortex microvascular endothelial cells or BMVEC as we call them in our lab. We pronounce them “buh-muh-veck” rather than enunciating each individual letter — it’s easier to say.  So they are in rack #6 in box E. But didn’t I say that there were four racks per tank? Well, this means this rack is found in our second cryotank which contains racks 5 through 8.

I will be choosing the vials labelled “P5.” This indicates that the cells have been grown and moved (or passed) from a smaller growth flask into a larger one a total of five times. These cells can only divide so many times in their cultured lifetime.  BMVEC can only be passed about nine times before they will no longer grow.

Once the vial location has been found using the frozen cell log, I need to find them in the cryopreservation tank.

There are a couple of different cryopreservation tank systems. This one holds the cells under the liquid nitrogen surface rather than in the gas phase just above the liquid surface. You can see wisps of the gas phase coming out of the tank as I remove the insulated lid.

Here I'm pulling up one of four racks stored in the cryopreservation tank. Because the boxes of cells are stored in the liquid nitrogen, care must be taken as the excess liquid nitrogen pours off the rack and out of the boxes. Safety glasses and special insulated gloves should be worn at this point. At -346F, the liquid nitrogen can cause serious skin burns.

The frozen rack is set on a bench top or other surface that can withstand the extreme temperature.

Removing the box that contains the cells I will need. It should be noted that I am only wearing latex gloves. Ideally I should be wearing specially insulated gloves, but I find them too bulky to manipulate these boxes and vials. I work quickly because this stuff is really cold and can cause a serious freezer burn.

I have located the vial of cells I will be using today.

The cells need to be thawed relatively quickly (but gently) because they do not tolerate the cryopreservative very well. Using water that is room temperature and a styrofoam rack to help the vial float so water does not seep under the lid, the cells are thawed.

A frozen vial of cells is placed in a styrofoam rack and into room temperature water to thaw.

Vial of frozen cells thawing in a beaker of room temperature water. This only takes a minute or two.

Once thawed, the cells need to be quickly removed from the freezing medium which contains a cryopreservative. (Please pardon the poor focus in this photo)

The thawing process only takes a minute or two. The cells do not tolerate the cryopreservative so it needs to be removed in a timely manner. The cryopreservative is in the medium to coat the cells and prevent ice crystals from rupturing the cell’s membrane. But the cells are floating around in this cryopreservative, so how do we get them out of there? We first move them from the vial into a larger tube. More medium (without the cryopreservative) is added to the tube to dilute out the cryopreservative. Then the tube is placed in a centrifuge to spin the cells just hard enough so they accumulate at the bottom of the tube into what we call a “cell pellet.” The liquid on top of the cell pellet, or supernatant, is removed so that all that is left in the tube are the cells. At this point, some labs add more medium and centrifuge the cells again in a process called a “wash.” We don’t do this. We just add the culture medium and move it into the culture flask.

One thing that should be noted, the culture flasks that BMVEC grow in need to be pre-coated with a solution that helps the cells “stick” to the culture flask’s surface. In this case, we use a solution of fibronectin, a substance produced by another cell type called a fibroblast.

Gently mixing the cells before moving them to a new tube.

Transferring the cells from the vial to a tube.

Putting the tube into the centrifuge "bucket" to pellet the cells.

The centrifuge must be balanced with another tube of similar weight, including the same volume of fluid. It is critical to balance a centrifuge to prevent damage to the rotor.

The cells are pelleted at 1200 rpms (400 times gravity) for ten minutes in a centrifuge. The spin is fast enough to pellet the cells but not so fast as to break them open. Note: the rpms may be different depending on the centrifuge size.

Checking for a cell pellet at the bottom of the tube. While it may not be apparent in this photo, there is a small pellet of cells (whitish color) at the bottom of the tube (the end with a slight point).

About 10 milliliters of culture medium is added to the cell pellet, gently mixed to break up the pellet, and moved to a pre-coated culture flask. This is what we call a T75 flask -- it has a surface growth area of 75 centimeters squared.

Once the cells and their growth medium is placed in the culture flask, the flask is placed in a cell incubator. This incubator hold the cells at body temperature (37C or 98.6F). The atmosphere is made of 95% air that’s in the room and 5% carbon dioxide — the carbon dioxide helps the culture medium maintain a neutral pH.

The culture flask is placed in the incubator so the cells can grow so they pack in the flask.

The cell culture incubator. It keeps the cells at body temperature (37C or 98.6F). It also maintains a carbon dioxide level of five percent inside the incubator to help the culture medium stay at neutral pH.

These cells will take a few days to completely cover the growing surface (we call this “confluence”). The growing surface is the bottom of the flask which is covered by the culture medium. Once completely packed in, these cells will stop growing (until they are passed into a larger flask). This is different than other cell lines that are taken from cancerous tumors — those tend to keep growing despite crowded conditions because their growth is unchecked (they are cancer after all).

We can look at the cells using a special microscope called an inverted phase contrast microscope. The optics are such that cells that are attached and spread out on the growing surface look dark and the cells that are lightly tethered to the growing surface or floating in the culture medium look white (we call them refractile).  I have included a few photos of what the cells look like right after passing them and over 72 hours.

We use an inverted phase-contrast microscope to look at the cells in the culture flask. Light passes up through the flask and the light is captured and redirected to the eye pieces. In most light microscopes the light passes from the top and collected under the stage for observation.

Cells appear as bright white spheres immediately after they are passed into a culture flask. They have yet to settle out of the medium. Time = 0 hours

Cells two hours after passing. They no longer look white but are a deep gray. They also are still somewhat spherical because they have only just begun to "stick" and spread on the growing surface of the culture flask. They will spread out a little further on the culture surface over the next few hours. Time = 2 hours

The cells 24 hours after passing. They are now completely attached and have gone through a round or two of cell division -- there are more cells here than there were in the previous photo. Time = 24 hours

The cells after two days. They have undergone more cell division -- there are more. The two cells circled in the lower left-hand corner have just completed a round of cell division and will spread out. Time = 48 hours

Cells after three days in culture. They have gone through many cell divisions and have filled in all the empty spaces. We call this a "confluent monolayer" and these cells will be moved from this flask to a larger one (see later post). There are still some cells actively dividing in this photo. Time = 72 hours

And for some photos of BMVEC going through cell division…

The cell that has a whitish "glow" around it is in the early stages of cell division. Here the chromosomes can be seen lining up (dark squiggles with halo around them inside the cell).

The cell in the upper left corner (with a white glow) has just divided its chromosomes (dark matter) into two compartments and a cell membrane has begun to form across the middle to form two cells.

The white-haloed cells in the center have completed cell division, the chromosomes are the dark objects inside, and are in the process of separating from each other.

And there you have it: how to thaw and culture cells. Of course, I didn’t cover how to do all this so that the cultures do not become contaminated with bacteria or fungus. Aseptic technique is something that needs to be taught either in person or in a very well-produced video.

All photos were taken by me with cell phone in hand — not so easy to juggle cell culture and a cell phone while trying to keep the culture sterile, let me tell you!  But hopefully this gives you an idea how cells are cultured in the lab.

Happy first day of Spring, everyone!


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