Download Case Study: Can A549 Cells Maintain Functionality and Signaling in

Case Study: Can A549 Cells Maintain Functionality and Signaling in Serum-Free
Media?
Introduction
Fetal bovine serum (FBS) has been used in cell culture for decades. FBS is thought to provide basic
elements required for cell culture, including growth factors, attachment factors, iron transporters,
vitamins, amino acids, lipids, carbohydrates, hormones, and trace elements. However, these elements
can change from lot-to-lot, potentially impacting cell growth and performance in an uncontrolled
manner and reducing experimental control. To manage the lack of control and performance
inconsistency, researchers sacrifice convenience, time and materials when screening multiple lots to
secure a large inventory of a satisfactory lot for future use.
A549 cells are derived from adenocarcinoma human alveolar basal epithelial cells. A549 cells are used
as a model to study human cell biology, including signaling pathways. It is common to transfect reporter
constructs into cell lines such as A549 cells, to track transcriptional activation. NF-kB is a transcription
factor that plays a key role in regulating the immune response to infection. One of the several pathways
NF-kB can be activated is through signaling of inflammatory cytokine TNF-α by binding its receptor
cognate TNF-R on the cell surface.
There are factors in serum such as TGF-b that can interfere in immune signaling; consequently, there is a
need to study cell signaling in a serum-free environment.
The Challenge
Perform cell signaling research in a reproducible and defined serum-free environment.
Cells need lipids for membrane formation (cellular membrane, Golgi apparatus, endoplasmic reticulum).
Lipids are required for effective cell signaling on the membrane and removal of serum removes a source
of lipids. Additionally the current serum free media options do not provide a lipid alternative. In order
to study signaling lipids should be present for optimal responses.
In this case study, we investigated whether the stably transfected A549 cell line with an NF-kB reporter
can be activated by a known activator, TNF-α in a reproducible and defined serum-free environment
containing a novel lipid formulation.
Method
Cell-Ess is a chemically-defined media supplement that can be used in place of FBS to ensure high lot-tolot consistency while using the same base media. The benefit of using this approach is that the base
media has a nutrient mix that if changed may alter the signaling of the cell based on nutritional changes.
Cell-Ess can replace FBS in culturing immortalized cells.
Stably transfected A549 cells were adapted to Cell-Ess by weaning
cells away from FBS by following a sequential adaptation (Figure 1).
Once NF-kB luc A549 had grown for several passages in a
completely serum-free environment consisting of 10% Cell Ess, then
it was used to test the signaling of NF-kB in response to TNF-α.
TNF-α was added at equal amount to NF-kB luc A549 and cultured
in either FBS or Cell-Ess. Luciferase was measured as relative light
units (RLU) for each group.
To ensure the activation of NF-kB was specific to TNF-α, a kinase
inhibitor was added that would block the TNF-α signal. Ro 106-9920
is a kinase inhibitor used in these experiments. Ro 106-9920 was
added to the cultures with TNF-α, and then the luciferase activity
was measured.
Results
A549 cells grown in serum-free Cell-Ess conditions had similar cell
Figure 1. Sequential adaptation of
signaling activity as A549 cells grown in FBS. TNF-α induced NF-kB
HEK293 cells from FBS to Cell-Ess.
activation was equivalent in DMEM with either 10% FBS or 10%
Cell-Ess. NF-kB luciferase activity was also specific to TNF-α addition in the presence of TNF-α inhibitor
Ro 106-9920, as indicated by reduced activity observed in cells grown in the presence of either FBS or
Cell-Ess. (Figure 2).
(-) TNF-α
(-) Ro 106-9920
(+) TNF-α
(-) Ro 106-9920
(+) TNF-α
(+) Ro 106-9920
Figure 2. Comparison of NF-kB luc A549 in DMEM with FBS (red) versus Cell-Ess (blue). Luciferase was
measured in relative light units (RLU).
Conclusions
In this case study, we demonstrated that controlled cell signaling studies can be performed in a serumfree environment. Cell-Ess was able to replace FBS in culturing A549, providing a serum-free media
platform to study cell signaling and many other networks. Furthermore, we demonstrated that a stably
transfected cell line retained the NF-kB-luc construct and is inducible.
The level of NF-kB activation in either 10% FBS or 10% Cell-Ess suggests that the protein-protein
interaction of TNF-α and its receptor TNF-R on the cell surface was unaltered in serum-free conditions.
Further, the integrity of the intra-cellular protein-cascade, including kinase activation of NF-kB, was also
maintained. The activation was shown to be specific NF-kB activity by an inhibitor Ro 106-9920
targeting NF-kB, which knocked down TNF-α mediated luciferase induction.Cell-Ess provided a serumfree media solution while maintaining the similar protein-protein interaction and NF-kB activation of
A549 cells.
Phone: (844) Ess Prod (377-7763)
Email: [email protected]