counting + freezing down, plus the durability of the hog gut matrix

Oh, just a cow head in formaldehyde in the lab. No biggie.

Looking for viable cells in a primary cell culture is like looking for a planet in the universe that can sustain life. In fact, looking at a cell culture under the microscope and scanning for living cells might as well be looking through a telescope at the stars. So many mysteries. Where is there possibly life?

On Friday, I scanned and scanned... aaaaaaand scanned my culture flasks under the imaging microscope, and found ONLY ONE possible viable cell. This was tragic. Guy Ben-Ary trained me in using the very posh digital microscope, and also informed me that cells are social creatures - they like to be able to reach out and touch other cells, make a colony. He said without other like cells around, they will die. There were plenty of red blood cells, in various stages of life and death: bright ones still alive, blue ones in a state of asphyxiation (dying) and dead black ones. Then, one larger round one, a viable cell! Only, it was a poor, lonely cell, looking miserable. Its centre was blackening and it was possibly going to just die. I was crestfallen.

The success is that my cultures have no contamination. This is a big deal. Especially since I'm a noob in the lab. And, especially since I was digging in a messy (non-sterile) bone for these cells. So, I should be happy about that.

Today when I looked again, I saw things that looked potentially exciting. Alas, they were air bubbles. That's what Ionat told me, anyway. Microscopic air bubbles. I then went through a process of removing all of the media (bone soup or DMEM, whatever each flask had) and replacing it with new. The old media (junk media) was stored in new, sterile flasks and returned to the incubator, just in case at some point something unexpected decides to grow. The fresh media in the old flasks was likewise then returned to the incubator on top of the flask with its old media. I looked again through the microscope, hunting for life. Nothing. I will dig out another bone from the butcher tomorrow and try again with new cultures. I might also get to have some bone cancer cells from the freezer if Stuart can find them. Now, THOSE will grow.

Today I began decellularization and freezing down cells. After speaking with a surgeon this weekend about what tissues might have the strongest extracellular matrix (a.k.a. collagen), I now know that pig intestine is my best bet for decellularization. He tells me that pig tissue is by far the strongest, not to mention the closest to human. Also, on top of that, veins, arteries and bowels have the strongest tissue matrix. This is extremely interesting, given that I was doing my preliminary research with hog gut through a biomimetic inquiry into the process of tissue engineering. Somehow I intuitively knew that hog gut was the best material to work with. But, as with so many things in that project, intuition was always the best guide. This is what I consider listening to the haptic intelligence of my body: it tells me things on a subtle level that I can't explain at the time, but that later prove to be scientifically accurate. Anyway, I secured a good amount of hog gut from the butcher and away we went.

The cells that I froze down were half of the C2C12s that I began in my first day at the lab. Cells just keep multiplying, so freezing them down is the only way to keep them to use them, but also stop them from continuing to proliferate.

Here are my new protocols that were put into play today in the lab. Yes, I counted cells. How did I keep track? With a handheld clicker, while I stared through the microscope.

Hog gut stretched over sterile vials. Stage I.

PROTOCOL for Decellularization using trypsin

1. Wash tissue in ultrapure H2O overnight (in beaker/container) on a rocker @ 4˚C
2. Incubate tissue in 0.05% trypsin with EDTA for 30-60 mins @37˚C
3. Wash the tissue briefly with ultrapure H2O to remove the trypsin
4. Neutralize the trypsin by incubating the tissue in culture media (e.g. DMEM) containing 10% FBS at RT, 2x30 min (can leave O/N at 4˚C)
5. Wash the tissue in 1% Triton-X-100 made up in ultrapure H2O for 1-2 days, changing the wash buffer 2x per day
6. Wash the tissue in ultrapure H2O for another day

Notes:

• Standard trypsin-EDTA used to dissociate cells from tissue culture plastic: http://products.invitrogen.com/ivgn/product/25300062
• Timing is flexible - originally the TX-100 wash was done for 5 days and rinsed in H2O for several days at a time, but didn't seem to make much difference



The unused intestine gets frozen in a tube for later.

• To check how well the decellularization has worked, check sections with DAPI

PROTOCOL for Counting cells

1. May need to dilute cell suspension
2. Prepare the haemocytometer:
• Make sure the slide and cover slip is clean. Use EtOH if necessary.
• Moisten edges of cover slip and apply to grid surface of slide.
3. Touch drop of cell suspension (recently stirred) onto edge of cover slip at the surface of the slide.
4. Capillarity will draw a volume of cell suspension between slide and cover slip. Do not allow volume to spill into grooves flanking the grid on the slide. The cover slip must be totally covered inside, however.
5. Inspect under microscope. If cells are really clumpy, you will need to break up the clumps and restart. Clumps of 3-10 cells are OK as long as you can count them easily. If not, dilute suspension. 
6. Count the cells in the grid of 25 small squares (each of which is itself divided into 16 smaller squares). 
7. Count cells that touch only two border lines as being in the grid. Ignore cells on the border of the other two lines.
8. Make an average count (use both grids) and note dilution factor. 
9. The volume of suspension contained between the slide and cover slip within the 25 square grid is 1x10[-4]ml (according to the dimensions of each square and the distance between slide and cover slip). 
10. Multiply the average number of cells by 1x10[4] and this will give you a cell number/ml of suspension. Take into account any dilution. 
• For example, if you count 150 cells in the grid specified, then your cell count is 150x10[4] = 1.5x10[6] cells per ml.

My C2C12s ready for the sleep of deep freeze.

PROTOCOL for Freezing down cells

1. Obtain cell count using trypsinisation.
2. Spin or dilute cells to give a count of 1.5 million/ml medium.
3. Label vials with cell type, passage number, date, initials, number of cells.
4. Add 100µl DMSO to each vial.
5. Add equal volume of neat FCS (or NCS depending on cell type) and mix well.
6. Add 0.8ml cell suspension per vial and mix well.
7. IMMEDIATELY start the freezing process which should be relatively slow by placing vials on dry ice covered with tissue lagging for 1hr then transferring to liquid nitrogen or -80˚ freezer or placing in heavily lagged container directly into -80˚ freezer.

Oh, by Friday I will have microscopic images.