Cells Growth in Different Media
The goal of this set of experiments was to study the growth rate of cells in 3 different media; 1. Complete Media 2. Exosome Free Media 3. Serum Free Media. We studied the effect of media on both ADSCs and FBs. Cells growth was monitored by a Matlab code that Henry has made for us. 8 images of every flasks were taken and the number of cells in the images were counted by the code and then averaged over 8 images for each data point.
Fig 1; Matlab code for counting the number of cells based on images taken.
In order to do this experiment, cells were thawed and split into 3 flasks with complete, exo free and serum free media. Interestingly, ADSCs from day one did not seem happy with serum free and they were dead after 3 days. FBs also died over time, even though at day one, they seemed happy with serum free. The conclusion is ADSCs and FBs will not be happy with serum free media immediately after being thawed.
Fig 2; ADSCs and FBs over 5 days in serum free media
We decided to test a hypothesis that maybe we should first condition cells with complete media after thawing and change the media to serum free after the first passage. In order to test that, when the complete media flask reached a confluence layer, we split cells again into 3 different medias. Interestingly, this time, cells in serum free seemed to survived. However, when cells in serum free were passaged, after trypsinization, no pellet was observed by spinning down the cells with 150 g for 5 minutes and cells were most likely dead. This suggests that we are not able to culture cells in serum free media for a long time, and for our experiments cells have to be conditioned with complete media and the media needs to be switched to serum free right before the experiments.
Fig 3; Fibroblasts in 3 different media over 5 days.
Fig 4; ADSCs in 3 different media over 5 days.
From the minimal information for studies extracellular vesicles 2018:
“All culture medium composition and preparation details should be provided in methods. This should be customary for cell culture studies, and is doubly important here since supplements like glucose, antibiotics, and growth factors can affect EV production and/or composition. Of special note are medium components that are likely to contain EVs, such as serum. EVs are ideally obtained from culture medium conditioned by cells in the absence of fetal calf serum (FCS or FBS), serum from other species, or other complex products such as platelet lysate, pituitary extract, bile salts, and more, to avoid coisolation of exogenous EVs. When use of these supplements is unavoidable, experiments should include a non- conditioned medium control to assess the contribution of the medium itself. However, depending on down- stream use, it may not be necessary or desirable to deplete EVs. In the case of depletion, since nutrient or EV deprivation of cells that are normally cultured in serum- or lysate-containing medium can change cellular behavior and the nature and composi-tion of released EVs, it is important to specify culture history (how and when the switch to serum- free medium occurred, including acclimatization steps). Alternatively, cells can be exposed during the EV release period to medium that has been pre-depleted of EVs. Here, too, effects on cells and EVs may be expected, and the methods and outcome of depletion vary greatly and should be reported.”
Using the Matlab code, following plots were made for both ADSCs and FBs comparing the effect of media on cells growth over time. As it was expected, Complete media did a better job compared to exosome free, and exosome free better than serum free media.
Fig 5; The effect of media on growth of FBs.
Fig 6; The effect of media on growth of ADSCs.