DIY CO2 Incubator - The Proof is in the Pudding
After building everything and testing the code its time to see if this incubator actually supports life. I suggest letting the incubator stabilize for a few hours before putting cells in. Its also important to put a small reservoir of water in the incubator to keep the environment humid in order to minimize evaporation from the culture dishes.
Mouse Fibroblast Proliferation
To test out the ability for this incubator to support the proliferation of mammalian cells I started with a very common mouse fibroblast cell line known as NIH3T3 (ATCC). The cells were originally derived in the 1960s and are used worldwide in cell biology research. What you see below are images of these cells proliferating over the course of a few days. Those who work with NIH3T3 cells will notice that the growth rate is indistinguishable from the growth rate of these cells in a commercial incubator. I also want to note the absence of any obvious signs of infection.
NIH3T3 cells proliferating over the course of 3 days in standard culture medium (DMEM, 10% FBS, 1% PenStrep). Scale bar = 200 μm and applies to all.
Human Neural Stem Cell Proliferation and Differentiation
I decided to then try out a more sensitive culture of cells. In this case, I was able to get my hands on some human neural stem cells. These were derived from a skin biopsy of Guy Ben-Ary, artist and member of SymbtioticA. Following the Yamanaka protocol, his cells were modified to become induced pluripotent stem cells (iPSCs) and then driven towards neuronal progenitors. Some images of their growth over the course of several days in the DIY Incubator are below.
Human NSCs proliferating in maintenance medium (Invitrogen Neural Induction Media mixed with 50% NeuroBasel Media and 50% Advanced DMEM/F12) over 6 days. After adding retinoic acid (RA) to differentiate the cells, they begin to acquire a neuronal morphology by Day 12. This does not happen if RA is not added. Scale bar = 200 μm and applies to all.
Finally, I employed the common C2C12 mouse muscle myoblast cell line (ATCC) to see if I could perform a fairly standard differentiation protocol. These cells were originally derived in the 1970’s and are used routinely in many labs. They grow quickly and will also fuse and differentiate into muscle fibres. With this cell line it is possible to not only examine their proliferation but also the more complex process of myotube differentiation. What we see in the images below are C2C12 cells proliferating over a few days. Following standard protocols I allowed them to grow until fully confluent and then switched to a low serum differentiation medium. Keeping these particular cells in low serum promotes cell fusion and the differentiation of these cells into of myotubes. In vivo, this process is typically stimulated in response to stress (damage, exercise, etc) in which muscle satellite cells are activated, proliferate as myoblasts, which then fuse to form new muscle fibre. What we see is that over the course of a weeks in the DIY Incubator, I can reproduce the results from the same cells grown in standard commercial incubators. No infections occurred and the timing of cell proliferation, fusion and differentiation appeared no different than when cultured in a commercial incubator.
C2C12 Mouse Myoblasts growing in the DIY Incubator (scale bar = 200 μm and applies to all). Single cells are visible on Day 1, by Day 5 the culture has grown to confluence in standard culture medium (DMEM, 10% FBS, 1% PenStrep). On day 5 differentiation media (DMEM, 2% Horse Serum, 1% PenStrep) is added and myotubes are visible by Day 7.