Different concentrations of fetal bovine serum affect cytokine modulation in Lipopolysaccharide-activated apical papilla cells in vitro

Abstract Fetal bovine serum (FBS) is the most used supplement in culture media; however, it may interfere with in vitro assays via effects on cell proliferation and cytokine production. The ideal FBS concentration for assays using apical papilla cells (APCs) remains unknown. Therefore, this study aimed to evaluate the effects of FBS on APC activation, cell viability/proliferation, and cytokine production. Methodology Human APCs were cultured, plated, and maintained in media containing increasing concentrations of FBS for 24 h, 48 h, 72 h, 7 days, and 14 days in the presence of Lipopolysaccharide (LPS - 1 µg/mL). At each time point, the cells were subjected to the MTT assay. The cytokines transforming growth factor (TGF)-β1, osteoprotegerin (OPG), and interleukin (IL)-6, along with the chemokine CCL2, were quantified using the enzyme-linked immunosorbent assay at the 24-h time-point. Statistical analysis was performed using two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test (p<0.05). Results In general, APCs exhibited increasing metabolic activity in an FBS concentration-dependent fashion, regardless of the presence of LPS. In contrast, FBS interfered with the production of all the cytokines evaluated in this study, affecting the response induced by the presence of LPS. Conclusion FBS increased APC metabolism in a concentration-dependent manner and differentially affected the production of TGF-β1, OPG, IL-6, and CCL2 by APCs in vitro.


Introduction
Apical papilla cells (APCs) have recently received increased attention in the field of Dentistry due to their proliferation, differentiation, and immunomodulatory potential. These cells have been studied with a particular interest in regenerative endodontics, 1,2 and cytokine production has been a relevant research focus. 3,4 Fetal bovine serum (FBS) is the most widely used medium supplement for cell culture methodologies mainly due to its high levels of growth-stimulating factors. 5 However, FBS can interfere with phenomena such as cell proliferation and differentiation, modulation of molecules and cellular mechanisms, cell viability, and cytokine production. [6][7][8][9][10][11] The mere presence of FBS, even without stimulation, was found able to constitutively induce the release of cytokines such as interleukin (IL)-6 and 8, usually expected in immunologically activated cells. 11 These findings may reveal significant biases in cell culture methodologies; however, this behavior can vary significantly depending on cell population.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 9.0 (GraphPad Software, San Diego, CA, USA).
The Shapiro-Wilk test was used for normality testing, and the data were subjected to a two-way analysis of variance (ANOVA) followed by Tukey's post-test. The significance level was set at p<0.05.

Cell viability
The results of the MTT assay are shown in

Cytokine production
In general, medium supplementation with FBS resulted in an increase in TGF-β1 and IL-6 production by APCs ( Figures 3A and 3C). However, we observed TGF-β1 production by APCs in an FBS concentrationdependent manner ( Figure 3A), regardless of the presence of LPS. In contrast, when activated with LPS, APCs cultivated in serum-free medium decreased OPG ( Figure 3B) and CCL2 release ( Figure 3D) and increased the production of IL-6 ( Figure 3C). Data from CCL2 and OPG production by APCs showed lower amounts of protein released by FBS-supplemented media (1% and 10%) (Figures 3B and 3D). It is important to highlight that the statistical differences observed for LPS-activated APCs for OPG, IL-6, and CCL2 were abrogated by the presence of FBS, even at a lower concentration (1%) (Figures 3B, C, and D).

Discussion
APCs have been widely used in regenerative endodontic studies due to their proliferation, differentiation, and immunomodulatory potential. 1,2,24 APCs are considered the key cell population involved in revascularization procedures and were first described in 2004. 25 Figure 3A). Based on the absence of TGF-β1 in cell-free media (data not shown), our data suggest that the findings are not due to nonspecific reactions. TGF-β1 is a cytokine produced by several cell types with established roles in the regulation of other cytokines, including IL-6, OPG, and CCL2. [33][34][35] Production of TGF-β1 by MSC is known to occur when stimulus, such as cytokines, products from viruses and bacteria, and derivatives of enzyme reactions, lead to activation of the Nuclear Factor-κB pathway. 36 Recent literature has also demonstrated a higher production of TGF-β1 by MSCs when cultivated in a medium supplemented with amino acids. 37 The presence of these molecules in the composition of FBS may be a major contributor to the mechanism underlying TGF-β1 induction in APCs by FBS. Interestingly, LPS did not affect TGF-β1 production in any of the FBS concentrations tested ( Figure 3A).
OPG is a molecule that binds to RANKL (RANK ligand), a factor that promotes osteoclast formation, and prevents it from activating RANK [receptor activator of the (NF)-κB]. RANK is expressed on the cell surface of osteoclast precursors and its activation by RANKL induces osteoclast maturation. RANK/RANKL binding does not occur in the presence of OPG. 16,38 Our data showed high levels of constitutive OPG, which, in turn, was downregulated by LPS only in FBS-free medium ( Figure 3B). Interestingly, the addition of FBS CCL2 (monocyte chemoattractant protein-1, MCP-1) is a potent chemokine that activates signal transduction pathways, leading to monocyte chemotaxis. 40,41 In contrast to the increase in IL-6 levels, constitutive CCL2 production by APCs was downregulated by LPS activation in FBS-free medium.
As seen in the OPG data, this cytokine was present in lower levels in FBS-supplemented media than in FBSfree media. Additionally, the LPS activation response at 1% FBS was contrary to that observed in serumfree medium, and no response was observed for 10% FBS (Figure 4). Therefore, our CCL2 data also suggest that FBS interferes with APC chemokine production in vitro ( Figure 4).
Serum-induced TGF-β1 production by APCs may underlie the modulation of immunological challenges due to its role in modulating other cytokines. Therefore, this effect might explain IL-6 inhibition, 35 but not OPG and CCL2 reduction since TGF-β1 is described as being an OPG 33 and CCL2 34 inducer. Future studies should be conducted on other components and mechanisms involved in the modulation of cytokine release by FBS.
Taken together, our findings demonstrate that FBS supplementation may interfere with cell proliferation and cytokine production in vitro. By modulating TGF-β1, IL-6, OPG, and CCL2 release, FBS may create a microenvironment in vitro that differs from in vivo conditions (Figure 4). Therefore, we speculate efforts of researchers in developing substitutes, FBS remains the most commonly used supplement for cell culture. 30,42 The final decision regarding FBS usage and concentration should be made according to the target cell and molecules of each study.

Conclusion
In summary, this study showed that FBS supplementation may interfere with APC proliferation and cytokine production in vitro.