understanding material feminism

Feminist materialism... this post is not perfectly pieced together, somewhat meandering, but if I try to make it perfect right now, I'll never publish it.

These are some of my notes and thoughts as I try to understand exactly what material feminism is now. The material feminism I'm interested in is new feminist materialism or new materialism from a feminist perspective, though the materialist feminism that precedes it and which relates to Marxism/labour is certainly relevant to my art practice and my thinking around the work I do in the laboratory. I don't only wish to understand it as it's been philosophized so far--I wish to contribute to the meaning of it by adding my own philosophical standpoint and relevant praxis.

New feminist materialism as I understand it, embraces the materiality of the body and bodies (nonhuman), rejects the cerebral privileging of discourse around or "about" the body in favour of embodied knowledge (being IN it) and rejects postmodern social constructivist assertions that The Material is constituted entirely from language built up around it.

Of particular interest to me is that feminist materialism turns an eye to SCIENCE and its examination of bodies/material/life/stuff/matter, not as simply an androcentric realm that defines science reality according to its own self-serving semiotics, but as a place we can look to in order to radically redefine matter as we know it, animate or inanimate. We must do this by looking at ways where the discursive and the material intersect (not discard discourse entirely in favour of materiality).

I ask (rhetorically), how do words manifest something real? They don't go beyond the realm of ideas unless a material ground exists from which to conjure things. I'm currently working on an abstract I've entitled, The Witch in the Lab Coat. This is for a presentation I plan to give that compares witchcraft (as synonymous with art practice/ manipulating material) with laboratory practice, grounded in new material feminist theory. I embrace the occult and manipulation in its purest meaning and reject the dichotomy between science and spirituality. This draws on postmodern feminism in tearing down the notion of dichotomies but pushes it further to establish a new theoretical place and space that is currently still somewhat undefined. Dialogue around materiality from a feminist perspective looks not only at human agency, but also nonhuman agency, the agency of nature, the agency of unseen things (invisibilia) and asks that we rethink what we think we know about matter and materials and their capacity to affect/act upon us and upon the world. It draws from quantum mechanics, biology, neuroscience, even empathy and creativity. I'm interested in inventing a realization about new methods of knowledge-making and in collaborating with matter to invent new materials. Personally, I draw from my own wealth of experience in adult education and learning theory, hands-on practice and teaching, craft/art, as well as poetry (speech acts), prose, myth and narrative production, witchcraft and postmodern feminism. Oh, and let's not forget motherhood - an unconventional practice of motherhood, whereby I have never made it the centre of my identity, have bucked norms and nuclear family expectations, queered it, and done a damn good job with it while pursuing my own goals. These are all one in my being, intricately interlaced to form a complete mode of material feminist existence.

One way of assessing an intersection between dialogue and material would be to ask the (rhetorical) question: is materialization a perlocutionary effect? I would answer: in part. Perlocution, I would argue, represents the agency of one human through words, the expression of will or presentation of a compelling utterance, that urges another agent to act. So, then, in addition to asking another person to do something for us, we could extend this to the basic notion behind spellcraft: utterances meant to persuade human OR nonhuman material to act in accordance with human intention in order to create a consequence (bad or good). Faith can move mountains, but only if the mountains agree to go along.

Feminist materialism presents an opportunity to imagine a new standard of ethics: one where the material consequences of actions, practices or activities inspire not just ethical considerations but also ethical practices. Working in biotechnology (or any technological field) with human/ nonhuman/ human-derived/ animal-derived/ disembodied and semi-living agents for the purposes of creating something 'other' (even if that 'other' is an augmented human) demands a deep reconsideration of ethical practice. The age-old question is raised: Should we do it just because we can?

For example, culturing cancer cells. When I spoke to my father about my recent work, he caringly advised me, "Don't get any on you!" Contagion is a huge force in making magic, the concept of transmission from one being to another or one thing to another. Fear of contagion has to be one of the most prolific fears throughout humankind. We are afraid of 'catching' things that can't biologically be caught, of them somehow rubbing off on us and affecting us with their own special imagined agency: death, bad luck, madness, evil, loss, demons, guilt. Guilty by association. This unique brand of possession, of violation by a nonhuman agent no doubt has its roots in actual disease contagion or pestilence. Culturally, we are obsessed with preventing contagion by any means, avoiding disease even if the means of avoidance may cause other problems. In my work, if I align myself with the imagined or understood goals of cancer cells, can I say I'm collaborating with them? Will this make me guilty by association? Do I become a monster, a murderer and a force for evil in the material world? Could I infect someone or infect myself? Is playing with living cells like playing God and therefore immoral? Where do all of my materials really come from? How is that acquisition of materials affecting the world? Ethically, it's difficult to reconcile. I don't mind playing God. However, I do mind the waste produced by standard laboratory practice--all the plastic pipettes, glass pipettes, plastic flasks, sterile wrapping, nitrile gloves, petri dishes, plastic bottles, etc in massive quantity that are thrown away into the landfill after being autoclaved. Each time I feed a single petri dish of cells, I use one sterile glass pipette to suck out the waste media, one sterile and packaged plastic pipette to aspirate fresh media from its container and expel it into the cell dish/flask. I go through a few pairs of nitrile gloves. When I trypsinize cells, and split them into new flasks, I use three times more pipettes and flasks. They are all single use, disposable, in order to maintain sterility, prevent contagion, control biohazard. The ecofeminist in me cringes. The materials? Animal rights activists would have a cow. I'm no longer a vegetarian so I can't get hypocritical about that. It gets more complex from here. 

So, what if I get cancer cells on me, which my father cautions me not to do? How much protection do my lab coat and gloves afford me? Is cancer contagious? Will anybody suffer loss through my interaction with these microorganisms (the loss of me)? You cannot 'catch' cancer--it falls into that intangible realm of things that people fear catching. However, "tumorigenic human cells are... potential hazards as a result of self-inoculation", as in, an accidental needle prick, which happened once in laboratory history and someone formed a tumour in their own body. So, while getting some of the cells on me wouldn't necessarily be a problem, getting some in me might be. Should I go splashing them around where others might be exposed to them, there might potentially be a problem as well, but I'm trained in how to handle these potent creatures as safely as possible, in a contained manner. They aren't airborne particles. Ethical? Checkmark there.  

The economics of material feminism, as I imagine them, are enmeshed in social capital. Touch = community = communication = currency. Haptic economy, a currency of connection. This reflects ideas of Marxist materialism where social activity is synonymous with economy. My brand of new material feminism takes this notion and highlights the importance of touch and haptics in the bonding required to form social networks, how this equates to information exchange and transmission and builds social capital. Social capital is that intangible, nontaxable exchange of resources vis a vis friends, family, colleagues, associates (your network) that assigns fluid value to services and goods, where materials matter to individuals in varying degrees, meaning there can be no fixed value or worth. This radically shifts ideas around profit, from surplus to promoting greater quality of life. Of course, I'm talking about a barter system, where trade is direct.

Enter textiles. Interconnectedness. Intersections. Social Fabric. Mesh. Matrix. Materia. Quilting Bees and other collective works in cloth. Digitextiles. Digits. Hands. Handmade. Computer-assisted. Jacquard.

To be continued in a later post.

READ for reference to early material feminism:

1. http://www.feministes-radicales.org/wp-content/uploads/2012/03/Stevi-Jackson-Why-a-Materialist-Feminism-is-still-possible-Copie.pdf

order + chaos

My pretties today:

These are the low grade osteosarcoma cells I've been nurturing for a month now. This image shows how slowly they grow, since the flask I first plated them in is not even confluent yet after almost 30 days. This small cluster is representative of SAOS-2 growth over a month, which is super slow, but also very organized. I haven't actually seen cells organize themselves so well before and it's interesting because one of the challenges of growing muscle tissue is that the cells don't grow in any organized fashion (as in, all in the same direction). This small cluster isn't the best example I have of the cell organization of SAOS-2s. The below photo, that I took with my iPhone through the microscope lens, is a much better example of their tendency to self-organize.
*By the way, that text bit is my handwriting that I made into a font, here. 

This lovely moon-like image shows a larger cluster of cells than the one above--much larger. The dark, fuzzy spot on the right side is an out-of-focus air bubble on the surface of the media (the cells form their layer on the bottom of the flask). You can clearly see how the cells have organized themselves to grow fairly smoothly, all in the same direction. They are so astoundingly beautiful to me. The rounded cells are in mitosis. They do seem to be picking up the pace of cell division and growth a little more now that they have a colony formed. I wish that there was a way I could push them to grow faster, because I prefer the SAOS-2s to the U-2 OSs. Why? The U-2 OS cells grow extremely chaotically (no organization at all, it seems) and are rather monstrous. They are growing in clumps on the weavings, and don't seem to want to flatten and adhere properly to the fibres. I suspect these SAOS cells would do a much better job, but I won't be able to find out until they've become confluent (abundant) enough for me to trypsinize them and try it out. I've been waiting. One must practice excruciating patience in working with live cells. Below is an image of the state of the weavings now with the U-2 OS cells growing on them.

It's extremely difficult, with the digital imaging microscope I'm currently using, to get a clear focus on the cells on weavings, because it's 3-dimensional and there are many depths of field. This image shows the weaving right after I re-seeded it with another flush of cells, so the floating ones are the out of focus ones, while the growing colonies are better in focus at the edges of the fibres. This growth is drastically different from the 3T3 connective tissue growth on my weavings at SymbioticA. The connective tissue actually did what is in its nature to do: connect things. These cancer cells shown above are unruly as hell. I can't imagine how they form a matrix! Time will tell.

My son (like probably all of humanity) has ascribed the human moral value of evil to these osteosarcoma cells. He says they're creepy. He describes that they are selfish and lazy, that they do not work within a system but their only purpose is to reproduce. I can't see them this way when working with them in vitro. I can't imagine them as evil or any other human value--I can only see them as vibrant microorganisms. They can't harm anyone in the dish and they can't disrupt a system because there isn't one to disrupt. How interesting then, between the two types, the one has seemingly established its own order while one is the very embodiment of chaos. Think of cancer as an organized force, and then think of it as a meandering chaos with no conscious will (it is both, depending on the type). Is cancer actually cancer outside of a human body or does it depend on a human context, with a system to disrupt, to be what we consider 'cancer'. If there is no imposed order to follow, is it still deviant?

PS - I threw out the contact lenses today. Nothing would grow on them. Dan warned me that cells may not like them, because they aren't firm enough. I guess cells need the opposing pressure of our eyeballs to the contact lenses to find an environment they enjoy.

fibersarcoma + naming new realities

Beginning of a 3D print of frame looms for tissue culture: the ground layer.

I've begun to assemble a glossary of new terms related to my creative biotextile work, including the spontaneous contributions of others, towards the development of a new language around the intersection of art + biology. My old friend, artist/comedian, Troy Haines, invented 'fibersarcoma' in response to my work with weaving and osteosarcoma (not to be confused with - or maybe to be confused with - fibrosarcoma). It's a collective effort -- I'm not the only artist engaged in this process of establishing new terminologies to explain philosophical and technical overlaps in science and culture. In fact, it was my colleague Tristan Matheson who suggested I do this, as he is developing his own glossary with regards to his work with cancer culture from an ethnographic standpoint. Artists and philosophers/ writers have long invented and established new terms to explain their expanding cultural realities. In contemporary culture, we have an ever-broadening reach of artistic and cultural practice as interdisciplinary values encourage experimentation between fields. Part of my work as a BioArtist has been to learn the language of biology/science so that I can thoroughly understand the materials I work with and the technical applications enough to manipulate them to my own ends. This involves not only learning a new language AND practice, but also becoming fascinated with how the various languages (artspeak, biospeak, communications theory, educational theory, material philosophies, etc) influence cross-disciplinary work being done, as much as the work can influence languages. This represents the beginning of accessibility to previously closed or tightly restricted, exclusive realms of knowledge--similar to the "Word of God" no longer being spoken in Latin in churches. <--my analogy using religion in comparison to science is no accident here, either.

Pure caffeine used in the Pelling Lab to 'excite' cells.

 
The Internet and coding culture (thanks, coders!) have done much to promote this sense of open, collaborative development and exchange, simply in the push for open source programming, which has bled into other facets of our worldly interactions and knowledge industries.

Here I am holding a 3D-printed hollow form used as a mold for casting HeLa cells in agar. Dan Modulevsky printed this for our experiments with growing cells in sculptural forms. I greased the entire inside of the cube with vacuum grease, which is, as it was explained to me, hydrophobic and therefore will not contaminate a cell culture (because it won't mix with liquid).

The agar is melted and dissolved in fetal bovine serum (FBS), and then the HeLa cells and media are added. We had to work quickly, before the agar hardened again.

This shows the HeLa/agar/culture media mix in the cube mold before it has completely hardened.

Ladies and gentlemen, here we have a cervical cancer culture cube (C4)! This is a beautiful image that Dan sent me of the cube form after being removed from the mold - my idea for using the vacuum grease (based on my experience with art mold-making + cement pouring, and using Vaseline) worked very well--we weren't sure how else to get the cube out of the mold. Dan's maintenance of the C4 has shown us that the HeLas can grow and thrive just fine in the agar for a time. We're not sure about longer term yet. Dan also injected some 3T3s (connective tissue cells) into the cube to see what would happen. So far, they are all growing together and the colour of the cube is gone as the media has been consumed by the cells.

Another term I'm including in my glossary is one I invented last year: haptic epistemology. I wrote about this a bit in my other two related research blogs, here and here. This is a term to explain knowledge generation through hands-on making, particularly as it relates to materiality and craft-based practices (I include both textiles and tissue engineering in the category of "craft-based practice"). Craft: process-oriented work with a technical focus, deep aesthetic consideration and functional (or semi-functional) outcomes. Also, this doesn't preclude philosophical outcomes or conceptual development... those are variable (e.g. material feminism). Easy enough definition?

The final term I'll elucidate today is a word I invented for a blog post I wrote a few posts ago (on this blog): omniphilic. Omniphilic is used specifically to refer to a cancer cell culture (for now)—meaning, a microorganism that thrives indiscriminately in all manner of host bodies regardless of age, gender, species, etc.    

Meta-material: In this case, it's a micrograph of my last tissue culture on weaving, which I've imported into a program called Pointcarre, and which I've used to reduce the image to 6 or 8 greys. I've assigned weave structures to each grey: a different density of satin weave per colour. This will become a physical jacquard weaving that I'll be working on at Hexagram at the end of the month. My miniature weaving and tissue/material growth will become a large-scale weaving about a weaving and the haptic intelligence of cells to 'weave' themselves into structured material. I might have to include meta-material in my glossary, even though metamaterial has been used as a term in a different context already. For my weaving, the contact with the body and its use as a covering for skin is also important in its meshy meta-ness.

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.

post-title

Let's talk a moment about my new relationship with human bone cancer in vitro. Our relationship has just begun but I've been researching the two types, who I've personified as Sonya and Osanna, and so know a fair bit about them/it already. I see them as female, if microorganisms can have a gender (can they? should they?). These particular cell lines are derived from female donors, so I've assigned them that gender for now because it serves my feminist discussion thus far--however, bone is universal and osteosarcoma can happen to any gender and probably any species, even. I could suggest that it's ultimately trans/inter/omniphilic.
I've read that human osteosarcoma is one of the most aggressive forms of cancer a child can suffer, afflicting mainly adolescents during stages of rapid bone formation. I also understand that it forms a malignant bone tissue mass, or osteoid, which in healthy circumstances is the precursor to the crystallized bone we all depend on to solidly frame up the rest of our squishy biomateria. Osteoid will mineralize, and so bone cancer will in part become a calcified monster, a crystalline nightmare.
But I'm learning to appreciate it from my safely distant observer position (on the other, 'outer' side of the flask). I'm learning to love it (if love can be defined as a profound appreciation, which is in fact how I define it) because I'm developing an intimate relationship with it. The intimacy does have boundaries--it isn't technically inside me, but it's inside my thoughts and inside my emotional processes. It's inside my daily routines, as I have to feed it and care for it like a mother. I'm investing a ton of money into its 'well being'. I want to see it grow and thrive and become beautiful, in the way only a mother could appreciate. I watch it closely, monitoring its changes, recording its activities, registering its surprises, making adjustments according to its needs. I have plans for its future. I take my job of protecting it from danger seriously.
Does it love me back? This question is seemingly ludicrous but also an important one to digest: am I in a one-sided relationship with this specimen or do my observations of it somehow change it, affect it in any way? Certainly, practically speaking, my decisions around its maintenance have an effect on it. But does it register an outside observer/ provider? Does it respond energetically to my presence, my body's magnetic field in close proximity to its cellular electromagnetic functioning? Does it feel pulled towards me the way I feel pulled by the moon--almost unconsciously? Do the vibrations of my words, as I look through the microscope and utter soft approvals and appreciation to it out loud, have an impact on the quantum level? What might that look like, if so?
Known research, such as the Double Slit Experiment (the relational interpretation of the experiment), posits that observation does indeed change the nature of a subject/ material--in the case of the experiment, from wave to particle and vice versa but we can employ this philosophy elsewhere. What would Jane Bennett say? She is critical of a singular pontiff power of perception, in the subject/object dichotomy where the only 'actant' is the observer of a subject or object and where an object has no agency to a(e)ffect anything in return (though a subject might). Bennett has compellingly written about the possible inherent 'thing-power' of the inanimate object, to act on the observer and be acted upon. What about the thing-power of a cancer cell? It is animate, but is it the same kind of animate as any other living thing? What kind of animate is it? Simply plasmic animation? We know it can be devastating to us. Can it perceive, too? How does it perceive? How would it express? At this point in time, aside from the cancer culture thriving or failing to thrive, I have no way to know how it feels or if it feels.
The prospect of feeling a profound appreciation for cancerous growth is perhaps incomprehensible, seemingly perverse. My mother and grandmother, as well as my grandfather have suffered (and long recovered) from cancers--thyroid, colon, uterine. They know firsthand the fear and anguishing lack of control that ensues and I have witnessed and internalized some of this. For humans, cancer is a culture of theft, stealing our very lives. It is every dirty, despised thing known in human nature, anthropomorphized on the micro level. What happens to the person who embodies it then, even if briefly? How much of the cancer do they become, how much shame do they feel when they contain the representation of that deplorable thing, when it becomes visible in their hair loss, their thinness? How much do they struggle to not identify with it?
How much is my body, MY own body? Aside from a contested, problematic legal definition of bodily ownership and autonomy, let's consider our interactions with all microorganisms: 3lbs of bacteria in the gut, influencing our hormonal secretions/synthesis and ultimately our emotional reactions and perceptions of the world, not to mention gender and systemic health (life). This is a massive paradigm shift. Suddenly, how much control do we ever have, anyway? How much of who we are IS actually who we imagine or enact that we are, and how much of who we are IS someone/thing else, another entity, a microentity or entire colonies of microentities? Can we appreciate them? Can the earth appreciate us even though we are collectively destroying it? What if the earth hated us and would do anything to be rid of us, take an antibiotic to kill us off in droves or blast us with so much radiation from the sun that we wither, even though we might love it, need it? What do we do with our irreconcilable differences?

I've just begun my relationship with osteosarcoma and already I'm talking about irreconcilable differences. Perhaps it is better to adhere to scientific protocol and remain completely impartial towards my specimens after all? From an ethical standpoint, that is just as dangerous.

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.  

3T3 cells in vitro

Below is a video of a 24-hour time lapse of live cell imaging at the Pelling Lab. My trusty lab partner, Tristan Matheson, helped me capture two similar videos--the one shown here was the best result. This video is exciting because it captures mitosis (two cells dividing) as well as displays cell motility (the ways cells move across the bottom of a petri dish). This is a beginning experiment, with the hopes that I can use the same technique to effectively capture video of osteosarcoma cell motility and other processes on my weavings, once they are well developed tissue samples. 

*Please note: the video quality isn't conducive to fullscreen viewing, unfortunately - but for a better version of the video that can be viewed fullscreen, you can see it on Vimeo here.