cancer in a contact lens, creative visualization + freezing potential

In the lab with my pets, examining their growth. Tristan Matheson photo.

 

 To give a sense of the rate of growth of osteosarcoma cells in vitro, consider this... 

• On February 25, I thawed and plated 2 cryovials of U-2 OS osteosarcomas, frozen in liquid nitrogen since 2010. Each cryovial went into a 75cm2 vented culture flask, meaning I had two flasks. I also thawed 2 cryovials of SAOS, each vial to one flask, meaning two more flasks, for a total of four flasks (you can see the flasks in the image above).

• Today, March 6, approximately nine days later, I did the following: 
• passaged one overly confluent flask of U-2 OS cells into 3 flasks and returned them to the incubator to continue working with them in 3 days (when they'll all be confluent again)
• passaged two overly confluent flasks of U-2 OS cells into 10 cryovials and put them in the -80˚F freezer to begin building my own cell bank, and to replace the 2 that I'd taken from the frozen stock belonging to the Sacher lab (leaving me 8 vials in my bank)
• passaged one overly confluent flask of cells into two 100mm petri dishes onto hydrogel contact lenses (one per dish, for myopic cells ahaha)
• changed the media in two sad and lonely SAOS flasks of cells
• changed the media in the one flask of waste media I'd kept, which now has some adherent cells on the bottom of the flask, so the earlier suspended cells I'd noticed in mitosis did in fact eventually adhere, meaning U-2 OS can seemingly replicate in suspension and thrive (odd). 

Ultimately, if I'd passaged and frozen everything I had earlier today, I'd have been able to produce roughly 23 cryovials of cells, meaning approximately 6x what I started with a week and a half ago. This is an enormous rate of growth and actually, had I passaged the cells when they should have been passaged, two days ago (because they were already confluent then), I'd have at least 2x that amount if not more, leading me to estimate that osteosarcomas grow and spread at an approximate rate of 10x their volume per week or so (rounding down). This is a very, very loose approximation. I'm sure that somewhere in a scientific journal is the calculated rate of growth for osteosarcomas. But, I think I've made my point here: this is an aggressive microorganism, even immediately after being woken from a five-year cryo-sleep. My particular cultures are what are referred to a 'high grade' osteosarcoma, meaning very likely to spread all over, with tons of cells in mitosis at any given time. In tissue culture terms, U-2 OS need to be passaged about every two days, whereas most noncancer cells (the ones I've worked with, anyway) only need to be fed fresh media every 2-3 days and passaged once a week at the very most. 

A photo of a digital microscopic image onscreen of one of my osteosarcoma cultures. Tristan Matheson photo.

The culture dishes I placed a contact lens in are one of my initial new experiments with growing the osteosarcomas on a 3D form. The contact lenses are prescriptive (for nearsightedness), which may be relevant only in terms of interesting narrative. I'm not sure how or what it will grow, but I do know that cells can be grown in hydrogel, recalling my very first tissue culture experiments with Oron Catts and Andrew Pelling at the Pelling Lab in 2013. During that experiment, Oron had us rip open baby diapers and shake out the hydrogel pellets, stir them with culture media and HeLa cells before the gels gelled. I'm a contact lens wearer and have experienced firsthand how easily protein (cells) grow on them, causing all manner of irritation. We use protein remover on our contact lenses each night for a reason. Our eyeballs are the only place on our entire bodies where live cells are exposed to the outside environment, barring a flesh wound. We might say that each contact lens wearer is an unwitting tissue culturist of sorts, growing a small bit of eye tissue (fibroblasts or laminae) on each lens every day. Contact lens websites might refer to the tissue as, "eye-produced buildup". Should my experiment prove to be wildly successful as I envision it (no pun intended), I could end up with a tiny tumour/bone matrix tissue cup, with the contact lens as its initial, underlying architecture. I'll update on that progress when I have some to report. In theory, I might even be able to primary source human fibroblast cells from culturing a dirty contact lens, but the risk of contamination would be pretty high. Extra antibiotics would be needed, which means extra funds to buy them.

I've learned a few new things about osteosarcoma as well: children with osteosarcoma are usually tall for their age as osteosarcoma is tied to rapid bone growth. It's more common in males than females and more common in African Americans than Caucasians. This means my osteosarcoma cultures from young white girls are not the most common kind. Osteosarcoma itself is quite rare. It can be caused by radiation therapy for a different kind of cancer, and it's not known if it can be caused by x-rays or not, but sometimes osteosarcoma appears in the jaw bones.

Protocol for freezing down cells*

1. Trypsinize cells according to cell passaging protocol (published in an earlier post)
2. Neutralize cell suspension with 10mL culture media per flask (instead of 40mL) and store all contents together (if using same cell type/passage no.) in a 50mL falcon tube
3. Spin down cells in falcon tube in centrifuge @1000x for 3 mins
4. While cells are spinning down, prepare freezing media in a 15mL falcon tube:
• 9mL neat FBS
• 1mL Dimethyl sulfoxide (DMSO** "cryoprotectant" should always be at a 1:10 ratio in the media or at 10%)
• gently aspirate media in falcon tube to mix
5. Also, prepare cryovials by unscrewing caps and mark cell type, passsage number, date and name on cryovials
6. After removing 50mL falcon tube from centrifuge, observe cell pellet at bottom--aspirate all media from falcon tube so that only the pellet remains
7. Add 10mL freezing media to the 50mL falcon tube and resuspend cell pellet in the media
8. Add 1mL cell suspension to each cryovial and screw cap on tightly--immediately place in Mr Frosty freeze container (lag container)
9. Place Mr Frosty in -80˚ freezer
10. Remove cryovials from Mr Frosty after 24 hours (can be left in container up to a month) and place in liquid nitrogen for long-term storage

*This is a loose protocol, where cells have not been counted first and so there is no way to really know how many tens of thousands of cells will go into each cryovial. But with U-2 OS, you don't need a lot anyway.