practice + protocol

I've got four flasks of U-2 OS osteosarcomas growing extremely aggressively, and two flasks of SAOS osteosarcomas trying very hard to establish themselves. When I thawed the cells, I had no idea how long they'd been in liquid nitrogen. The SAOS cryovials had no date on them, only a passage number. The U-2 OS cryovials had been frozen since 2010.
Below are my basic tissue culture protocols, which might not be very interesting for experienced tissue culturists but might be valuable info for someone who either would like to try tissue culture or for someone who is interested in exactly what I do in the lab every two days while I'm mucking around with cancer:

Protocol for mixing osteosarcoma nutrient media (used for both SAOS and U-2 OS) -- in other words, HOW TO FEED BONE CANCER CELLS in vitro:

1. In warm water bath, thaw and warm (usually takes approx 20 mins): 
• 50 mL fetal bovine serum (FBS) in aliquot tube
• 5 mL penicillin/streptomycin (P/S) in aliquot tube
• 500 mL McCoy's 5a medium in unopened container
2. While this is warming, UV sterilize laminar flow hood @20 mins
3. Turn off UV (if necessary) and raise sash to appropriate height for working. Turn on light
4. Spray down hood with 70% ethanol (EtOH), wipe dry with paper towel and prepare with sterile 10mL or 20mL plastic pipettes and 50mL falcon tubes
5. Spray down all tubes and containers removed from warm water bath (with EtOH) before entering them into the flow hood

Under the hood:

1. Aspirate 55mL McCoy's 5a medium from container and store in sterile falcon tube (recap tube)
2. Add entire quantity of warmed P/S and FBS to McCoy's 5a medium container for a total liquid of 500mL. Recap container and shake gently to mix contents
• This gives a proper 10% FBS and 1% P/S mixture, standard for cell culture
• McCoy's 5a is particular to osteosarcoma cell culturing

Outside of hood:

1. Refrigerate falcon tube of remaining McCoy's 5a medium for later use, after sealing lid with a thin strip of parafilm. Make sure to write name, date and contents on the tube
2. Write name, date and new contents of mixed media on container and refrigerate after use 
• Media has to be warmed to body temperate (37˚C) before each use with live cells

Cleanup:

1. Spray down hood with EtOH and wipe dry before closing sash and turning off light

*I've seen different protocols online for mixing osteosarcoma media - some don't use McCoy's 5a medium but just use standard DMEM that is used in almost every cell culture. However, I'm going with the mixture stated on a cell line supplier website, which calls for McCoy's 5a (despite the fact that I did not get my cells from them, but from a random source lab that supplied cryovials to the lab I'm working in now).

Here is my protocol for thawing cells (from liquid nitrogen, but similar process for thawing from a -80˚ freezer):

1. Warm nutrient media in warm water bath
2. UV sterilize (20 mins), spray down and prepare hood with plastic pipettes, pasteur pipettes, flasks, falcon tubes, phosphate buffered solution (PBS), warmed media. Turn on suction hose
3. Put on protective cryo gloves, open liquid nitrogen vault, pull up rack and quickly remove desired cryovials from racks/boxes
4. Replace boxes/racks in vault and return lid, lock
5. Immediately warm cryovials for 1-2 minutes in warm water bath, until thawed
6. Spray vials with EtOH before entering hood 
7. Immediately aspirate contents of cryovial and place in sterile falcon tube. Fill tube with PBS and recap
8. Spin down falcon tube of thawed cells and PBS in centrifuge @1500x for 4 minutes
9. Observe the cell disk at the bottom of the falcon tube, spray down tube and return to hood
10. Aspirate PBS from tube, leaving the cell disk at the bottom
11. Refill tube with fresh PBS, recap and shake tube to re-suspend cells
12. Spin down tube of resuspended cells @1500x for 4 minutes
13. In hood, fill culture flasks (2 per cryovial) with 10mL warmed media
14. Spray down tube and return to hood
15. Aspirate PBS from tube, again leaving the cell disk at the bottom
16. Add 10mL warmed media to tube, recap and shake to resuspend cells in media
17. Aspirate cell suspension and put in flasks (to make 20mL total liquid media/ cell suspension in each flask)
18. Write name, date, cell type, passage number on flask 
19. Incubate flasks immediately in 5% CO2 at 37˚C (standard cell culture incubator)
20. Do not remove flasks from incubator for 2-3 days, in order to allow cells to settle and adhere to bottom of flask (establish the culture) *osteosarcomas grow rapidly and consume media a little more quickly than regular cells, so changing media after two days is advised
21. Change media every 2-3 days and passage cells once confluent (80-85% monolayer of cells covering bottom of flask)

*Frozen cells are suspended in a combination of nutrient media and DMSO, which is essentially antifreeze and toxic to cells when in a thawed state. Hence, the very rapid action to rinse the cells with PBS 2x before re-suspending them in nutrient media for culturing.

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Those are fairly standard protocols that anyone who has any experience with tissue culture knows. I haven't invented anything new there - I'm just transcribing my methods exactly as I practice them in the lab. Other labs and researchers will do things slightly differently, with more or less precision and greater or lesser degrees of concern for aseptic technique (sterility).

So, I thawed four cryovials: 2x U-2 OS and 2x SAOS. The U-2 OS, which I know were frozen in 2010, were only passage 5, which means they'd only become confluent and had to be split 5x before they were frozen (young in the scheme of a cell life cycle). They did very well after being thawed and cultured. The SAOS, on the other hand, were passage 18 (kind of a teenager, relatively speaking) and had no date on the vial, meaning I haven't the slightest clue how long they'd been frozen for. Obviously, the longer a vial is frozen, the more potential damage to the cells and the more difficult it becomes to revive them. Sometimes they can't be revived if it's been too long. My SAOS cells are struggling. There are very few viable cells in the flasks - almost none when I first checked after 2 days. However, a week later, they've begun to just barely start to establish themselves, though they are still lonely in there. I'll give them some more time. The U-2 OS are so aggressive that they were confluent after two days and had to be passaged.

Here's my cell passaging protocol (also standard with only minor variations):

1. UV sterilize, spray down and prepare hood with pipettes, Trypsin-EDTA, warmed media, PBS, 75cm2 vented culture flasks and waste container (if necessary)
2. Warm culture media and aliquot tube of Trypsin-EDTA
3. Aspirate all exhausted media from established culture flasks (with suction hose and glass pasteur pipettes, OR with plastic pipettes into a waste container inside the hood)
4. Add small amount of PBS (approx 10mL) to flasks, recap and gently swish liquid over cell monolayer
5. Aspirate PBS and repeat PBS wash process again
6. Aspirate PBS and add 2mL Trypsin to each 75cm2 flask, gently rocking flask after recapping to ensure it covers cell monolayer 
7. Incubate flasks 1-2 minutes or until cell monolayer separates and cells are suspended in the Trypsin (inspect under microscope to be sure cells have completely detached and are free floating). Tap flasks gently 5x to detach stubborn cells, if necessary (this may make cells clump, particularly if they are prone to clumping--such as with osteosarcomas, I've noticed)
8. Return flasks to hood and immediately add 40mL fresh media to flasks to neutralize the enzymatic action of the Trypsin
9. Aspirate 20mL cell suspension from each full flask and add it to new, sterile flasks (this leaves the two older flasks with 20mL of suspension and gives the two new flasks 20mL of suspension as well)
10. In the case of osteosarcomas, it's a time- and money-saver to actually split the cells 1:4 (instead of 1:2) into new flasks since they proliferate so much more quickly than other cell types--in that case, aspirate 10mL from older flask at a time, to add to a new flask and do this 3x to end up with 4 flasks with 10mL cell suspension each--then add another 10mL fresh media to each flask to create 4 flasks @ 20mL cell suspension each
• Also, splitting cells can include an additional step of obtaining a cell count to determine how many cells to seed in each new flask - a high cell count may require a larger division of cells (1:4) - I don't typically do this extra step because I'm an artist, not a scientist and I don't need extreme accuracy at this point--however, seeding too few cells in a flask might make the culture not viable, as cells typically need the company of other cells in close proximity in order to thrive well (awwwwww)
11. Recap and incubate flasks immediately, leaving alone for 2-3 days (depending on cell type)
12. Return Trypsin to freezer after sealing aliquot tube with parafilm, return media to fridge (PBS doesn't need to be refrigerated or warmed, FYI)
13. Clean up hood

I should report that interestingly, the U-2 OS cultures have begun to *immediately* form tumour tissue - that is, multiple cell layers are piling all on top of each other and looking nasty, before the monolayer has even fully formed. This is deviant behavior for cells, for sure and it may have been encouraged with my tapping action during cell passaging that caused the osteosarcoma cells to clump hard. Also, the U-2 OS cultures APPEAR to have cells floating around as well, in mitosis in suspension. This is also deviant behavior, and it might mean that the cells don't need to be adhered to anything in order to replicate themselves. Versatile mofos. I've put my waste media from one flask into a new flask by itself to see if those floating cells will establish a new culture or not. IF they do, this probably means that indeed the cancer cells can replicate and thrive in suspension.