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Project Question
Project Question
How does myogenic differentiation induced by cell-to-cell contact in 10% fetal bovine serum versus serum starvation using 2% horse serum impact the mRNA expression of selected genes during myogenesis?
Experimental flow
Experimental flow
Our project cultures C2C12 myoblasts obtained from the American Type Culture Collection (ATCC) until they reach 100% confluency (full coverage of the growth surface). Once achieved (day 0), we subject the cells to either a continuation of growth media (10% fetal bovine serum (FBS)) or an introduction to starvation media (2% horse serum (HS)). We sample the cells as they differentiate over a two week period. From these samples, we extract the RNA present and convert it into PCR readable cDNA. We then use specific primers to amplify and measure our genes of interest against the total cDNA present in the cell. This specific observation of target mRNA expression against total background RNA expression allows us to measure any changes in transcript quantities over the course of myogenesis between the two media conditions used during differentiation.
Figure 1: Literature scan of growth media and differentiation media used in studies of myogenesis with C2C12 myoblasts reveals an inconsistent approach to inducing myogenic differentiation via serum starvation. Growth media is used initially until researchers decide to induce myogenesis at a certain level of confluency (cell coverage on growth surface), at which point the media is switched for differentiation media.
FBS = fetal bovine serum, CS = calf serum, HS = horse serum.
Use of Serum in Cell Culture
Use of Serum in Cell Culture
Serum continues to be an essential part of cell culture media. It is an effective way to provide various nutrients, hormones, growth factors and attachment factors to aid in cell proliferation and survival for in vitro cell culture models.
As an animal-derived product, serum is highly variable between batches and may contain additional unidentified signalling molecules.
Usually, a fetal animal serum is used in growth media as it contains high growth factors. To enhance myotube formation and increase the rate of differentiation, most researchers decrease the serum concentration or switch to adult animal serum in an effort to decrease the growth factors.
Serum Starvation
Serum Starvation
The serum starvation method is commonly used to induce cell differentiation in vitro. Withdrawal of serum and associated growth factors drives proliferating cells to exit the cell cycle.
Serum starved C2C12 myoblasts will exit the cell cycle, undergo terminal differentiation, aggregate and fuse to form myotubes in vitro.
Differentiation of myoblasts can also be triggered via cell-to-cell contact when the cell culture reaches 100% confluency.
Throughout reading related literature in 4101, and past 4101 experiments, we have noticed that experiments using 2% horse serum have had earlier differentiation compared to using cell-to-cell contact or lower FBS concentrations.
Literature Scan Reveals Inconsistent Approach
Literature Scan Reveals Inconsistent Approach
Also indicated is a clear trend toward dropping serum conditions to 2% horse serum (HS) to induce differentiation. Very few researchers appear to maintain serum conditions and permit differentiation without using serum starvation.
Rationale
Rationale
Serum starvation is a common in vitro technique used amongst stem cell researchers. However, it is unclear whether varying the culture conditions to induce differentiation impacts the mRNA expression of genes related to myogenesis - which may confound results. By comparing cell-to-cell contact versus serum starvation induced myogenic differentiation, we can gain a better understanding of how culture conditions potentially impact the timing and abundance of mRNA expression for key genes. We hope this research will help optimize culture conditions for C2C12 cells and contribute to a more representative in vitro model of myogenesis.
Target Subgroups
Target Subgroups
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