My two last trial runs ended up here:
First came the squares. Before even attempting imaging, Dan – a PhD student in the Pelling Lab who has been guiding me on this project – told me the squares wouldn’t work for the imaging. Another problem was that cells had seemingly fallen from the square onto the petri dish: seeding time of an hour did not seem to be enough.
My next try was to make circles that would fit snuggly in the bottom of a 35mm petri dish. Came and went many sizes of circles in Sketch Up and later on the bed of the 3D printer.
I settle for the size that fit the tightest in my 35mm dish. My goal was to prevent the cells from falling off. During printing, the wood filament gave me some problems. It seems to be prone to dry extrusion, especially when there are too many items in a single build. I’ve adapted my practices around this problem and print less items at a time. After managing the prints and struggling with the printer, I ended up with 3 34.5mm wooden circles that I attached at the bottom of the petri dishes with thermal grease. One of the circles went in a little bit crooked, but I only had the 3. Dan had shown me the previous Friday how to calculate the amount of cells re-suspended in 1ml of media. Up until then, I had been re-suspending them in 5ml of media and, after counting them, plugging them in a simple crossed multiplication to obtain the volume of cell-media substance necessary for cultivation. From now on, I re-suspend my cells in 1ml of media and create, in another vial, a diluted solution. 900 microliters of media join 100 microliters of my cell-media substance and it is from this I extract an aliquot of cells to count. The diluted substance allows for much faster cell counting as the 1ml substance can be quite concentrated when the petri dish is very confluent. After counting my cells, I took respectively 1/4, 1/8 and 1/16 of my cells for each dish. In cell numbers, it was 968 750, 484 375 and 242 187.5 cells per dish. By trying out different concentrations, we can track the amount of cells resulting in the best growth rate.
We were Monday August 24th and, after arriving at the lab at 2pm, I left around 3 hours later. I came back 3 times during the week to change the media. On Friday the 28th, I came to maintain my C2C12s still growing in petri dishes.
On Saturday August 29th, I had to throw this trial in the garbage. As Dan puts it: “That’s science.” The circles at the bottom of the petri dish fit snuggly. Maybe a bit TOO snuggly in fact. This resulted on a slight curve in the wood from being pressed fitted in the petri dish. Whether or not cells have successfully grown onto the wood is still a mystery: the curve in the wood made it impossible to image. We did start the imaging protocol for two of the dishes.
The first step was to proceed to staining. We used Hoechst stain. The whole reason we need to do imaging for this project is to monitor the status of the cells: the wood is not see-through therefore we can’t look at it with the phase contrast microscope in the cell culture room. This Hoechst stain is a blue fluorescent used to stain DNA that can be used to image live or dead cells(Thermofisher; Chazotte, 2010; Bruckner). It binds best to thymine and adenine though it can attach itself to any nucleic acids (Chazotte, 2010). The Hoechst becomes fluorescent under UV light. 1 microliters of stain is needed per milliliters of media in the dish. For 2 ml I therefore dropped 2 microliters of stain in each dish. They went back in the incubator for 30 minutes: this gives the time for the stain to bind to the DNA.
The next step was to remove the media and stain and wash the dish thoroughly with PBS (saline water): this way any unbound staining is removed (Bruckner). Next, the cells need to be fixed with paraformaldehyde (PFA). Fixing is a step required to prevent any movement of the cell-structures during imaging by increasing rigidity (Bruckner; Redig, 2013). The PFA forms bonds between proteins and other tissue structures in the cells which makes for good preservation (Redig, 2013). Following 10 minutes at room temperature or in the incubator, PFA needs to be well washed with PBS.
The following step was to put a drop of mounting medium on the wooden circle at the bottom of the dish and place a small glass slide on top. This is where the rounded edges of the snug circles became problematic: the glass slide didn’t have enough room to lay flat on top of the wood. This rendered imaging impossible and it is at this moment that the dishes ended up, once again, in the biological waste.
I came back Monday the 1st of September to set up another trial. The first step was to print some circles that would fit flat at the bottom of the dish. Following the printing of two test sizes (33mm and 34mm), I settled on the 34mm which fits flat at the bottom of the dish but can still be removed by turning the dish upside down. I had another few problems with 3D printing wood in the afternoon. The filament did more dry extrusion and another lab member had to interrupt the printing for me. I had to proceed to printing in small batches, loading and unloading the wood filament between tries to ensure it wouldn’t get clogged. This slowed me down a little bit and I still wasn’t satisfied with the printing process. Once my circles were printed though, I proceeded to repeat the protocol set with Dan. I set up 3 dishes with a wood cover at the bottom. Unfortunately, my cells weren’t as confluent as I would have liked them to be. Respectively, they came to 293 750 (1/4), 146 875 (1/8) and 73 437.5 (1/16) cells each; and I added 2 ml of media per dish.
The media was changed early Tuesday afternoon, after a morning spent experimenting with the 3D printing. I changed speed of printing, temperature and layer height after reading recommendations online. This seems to have given me much less trouble printing. I also went down to printing one item at a time instead of two and that seems to have helped as well. Compared to my last experiences, it seems giving myself more time to play around with the settings gave me the greatest satisfaction. I printed another circle and another square. Dan had mentioned he had imagined the wood to be thicker. Instead of the 1mm I had been printing at, I printed 2mm thick. I had also experience some wrapping with the thinner layers and it seems going thicker solved that problem! I decided to try out another square just because the microscope slides are square. I measured the 35mm petri dish and ended up with a 24x24mm square that fits snugly in there. I did my circle at 33mm. Another change I brought to this trial was to put more cells. When I first met with Andrew, he had suggested putting all of the cells from one dish on the wood. Since my trials from Monday were with much less than that, I decided to grab half of my cells per dish. I also didn’t have to cultivate my cells which meant I could grab more for trials. At 450 microliter per dish, it came to over a million and half cells per dish!
I will keep changing the media each day and will try imaging once again at the end of the week!
During my time at the lab, I feel myself being hybridized more and more. The laboratory is not only a physical space, it is much more than a set of rooms in the basement of a building on campus. It is a milieu. As a “physical or social setting in which people live or in which something happens or develops” (Merriam-Webster), the lab is a place of human interaction with each other, with other living beings and with technical beings (Simondon, 1958). Simondon underlines the relationship between technical beings and human beings in the lab : « The machine with superior technicality is an open machine, and the ensemble of open machines assumes man as permanent organizer and as a living interpreter of the inter-relationships of machines. […] He is among the machines that work with him. » (Simondon, 1958, p. 4). It is only as we act with them that these machines and tools allow us to reach our goals. It is as we open ourselves to new techniques, not only of usage but of being with; and with consideration for constitutive elements and participating individualities, that the whole itself emerges as a dynamic milieu of anti-disciplinary and curiosity-based research for the physical manipulation of biology (Pelling, 2015). And in the meantime, scientific processes still depend on this life that, interestingly, has come to itself depend on us
Bruckner, M. Z. Basic Cellular Staining. Microbial Life Educational Resources. Retrieved from http://serc.carleton.edu/microbelife/research_methods/microscopy/cellstain.html.
Chazotte, B. (2010). Labeling Nuclear DNA with Hoechst 33342. Cold Spring Harbor Protocols. Retrieved from http://cshprotocols.cshlp.org/content/2011/1/pdb.prot5557.full.
Merriam-Webster. Milieu. Retrieved from http://www.merriam-webster.com/dictionary/milieu.
Pelling, A (2015). “Re-purposing Life in an Anti-disciplinary and Curiosity Driven Context.” Leonardo, 48, 274-275.
Redig, J. (2013). How to fix adherent cells for microscopy and imaging. BitesizeBio. Retrieved from http://bitesizebio.com/13460/how-to-fix-adherent-cells-for-microscopy-and-imaging/.
Simondon, G. (1958). On the Mode of Existence of Technical Objects. Paris: Aubier, Editions Montaigne. 122 p.
Thermofisher. Hoeschst 33342 Solution. Retrieved from https://www.thermofisher.com/order/catalog/product/62249.