Fetal bovine serum—a cell culture dilemma

Ethical and possible reproducibility issues arise when using fetal bovine serum in cell culture media

Effect of Fetal Bovine Serum on the Sperm Quality of Depik (Rasbora tawarensis) after Short-term Cryopreservation

Background: Sperm cells are susceptible to oxidative stress during cryopreservation. Therefore, an antioxidant is necessary to protect them from damages. Fetal bovine serum (FBS) is one of potent antioxidants for fish sperm cryopreservation. Hence, the aims of this study are to examine the effect of FBS on sperm quality after a short period and to determine its optimum concentration on depik (Rasbora tawarensis). Methods: Depik fish were obtained from the Fish hatchery of Lukup Badak, Aceh Tengah District, Indonesia. Sperms collected from the fish were diluted in Ringer extenders containing FBS concentration of 10% (P1), 20% (P2), 30% (P3), 40% (P4), 50% (P5) and 60% (P6), filled into 2 ml cryotubes and equilibrated prior immersed into liquid nitrogen for 15 days. The parameters observed were sperm motility, consistency, pH, fertilization and hatching rates and DNA fragmentation post-thawing. Result: The ANOVA test indicates that the application of FBS in Ringer had a significant effect on sperm motility, fertilization and hatching rates (P less than 0.05). The highest motility (58.33%) was recorded at FBS 60% and significantly different from those at other concentrations. The laddering analysis showed that applying FBS protected the integrity of depik sperms DNA. It is concluded that the optimum concentration of FBS on depik sperm led to a short-term cryopreservation of 60%.

The Development and Evaluation of Brain and Heart Cell Lines from a Marine Fish for Use in Xenobiotic-Induced Cytotoxicity Testing

Two cell lines derived from the brain and heart of a Pacific white snook specimen ( Centropomus viridis) were developed and evaluated in terms of their responsiveness to glyphosate-induced cytotoxicity. The cells were grown in Leibovitz-15 (L-15) medium supplemented with 10% fetal bovine serum (FBS) and were passaged 36 times. Growth was tested at different concentrations of FBS (5, 10 and 20%) at 27°C. The cell lines were cryopreserved at different passages and were successfully thawed, with a survival rate greater than 80% without detectable contamination. At passage 36, the cells were used to assess the deleterious effects of glyphosate, and cell proliferation was measured by direct counting and with the MTT assay. Similar LC50 values were obtained with both methods. Although the principles behind these two assessment methods differ, our results show that both are suitable for evaluating glyphosate toxicity. In addition, heart- and brain-derived cells showed similar sensitivity, suggesting that the same mode of action might be responsible for the toxicity of glyphosate at the cellular level. The newly developed Pacific white snook brain and heart cell lines could be useful to investigate cellular and molecular mechanisms of toxicity, satisfying the need to reduce the use of animals in experiments. Glyphosate-related toxicological data obtained in the present study will allow us to continue investigating the effects of this herbicide directly on brain and heart fish cells since similar studies have only been carried out on either live organisms or on human cell lines such as neuroblastoma, which are immortalised by oncogenes or similar.

New Anti-Cancer Drug Compounds to Treat FLT-3 Mutated Leukemia

Around 30 % of the acute myeloid leukemia (AML) patient present a FLT3 mutations, that can be divided in 2 groups: punctual mutations represent 5 % and internal tandem duplicates represent around 25 % (FLT3-ITD). AML with FLT3-ITD mutations lead to significantly poor prognosis often requiring allogenic stem cell transplant. Different drugs are used to treat AML such as midostaurin, azacytidine, and daunorubicin. In this study, MOLM-13 cells (adult acute myeloid leukemia), expressing the FLT3-ITD mutation, were used to test different drugs to decrease their cell proliferation and increase their apoptosis level, compared to midostaurin (an inhibitor of FLT3), for 24 hours treatment. MOLM-13 cells were cultured in RPMI1640, in presence of 2, 10 and 25 % of fetal bovine serum (FBS). 25 % FBS was tested to be as close as possible with the level of serum present in the blood. Adipose stromal cells (ASC) were used as a reference control, in absence of serum. Compounds A (targeting the FLT3 pathway) was used at various concentrations (0.05 to 100 µM), over 24h of treatment, in combination or not with compound B (targeting the DNA). Compound A has a high affinity for IRAK4, downstream kinase of TLR pathway, and has the potential to block FLT3 pathway. TLR pathway activation in leukemia, through Myd88 and IRAK4, is involved in the pro-inflammation response but it is also inducing cell proliferation and cell survival. Compound B is an inhibitor of the poly (ADP-ribose) polymerase (PARP), which are enzymes that are involved in DNA transcription, cell cycle regulation and DNA repair. When ASC were treated with the compound A, the caspase 3/7 activity was not different from the control, but the number of cells was lower at 50 and 100 µM, indicating that the compound A was delaying the cell growth. The number of MOLM-13 cells decreased, in a dose response manner with the compound A (1 to 100 µM). The level of caspase 3/7 activity was elevated, in a dose response manner, which is consistent with the decreased number of cells. MOLM-13 cells resisted more to the anti-cancer effect of the compound A at 25 % FBS, indicating that it could work on patients. Midostaurin, from 0.05 to 100 µM, was reducing the number of cells and increasing the caspase 3/7 activity in the cells, in a dose response manner, at 2, 10 and 25 % FBS. However, data suggest that at the lowest doses, midostaurin was more efficient than compound A, but the compound A was more efficient at higher dose than midostaurin. Compound B alone, at 10 µM, was decreasing the number of MOLM-13 cells and increase the apoptotic level for the 2, 10 and 25 % FBS. A synergistic effect was demonstrated with compound B (10 µM) mixed with low concentration of the compound A (from 1 to 50 µM), at 25 % FBS. This latest result suggest that it will be possible to lower the posology of the compound A when it will be given to the patients, in combination with the compound B. In conclusion, the data showed the compound A and compound B decreased the level of proliferation and increased the level of apoptosis in MOLM-13 cells, cultured with 2 to 25 % of FBS fetal bovine serum, even if MOLM-13 cells were more resistant to the deleterious drugs effect at 25% FBS. Their combination had a synergic effect in increasing the caspase 3/7 activity and decreasing the cell proliferation. Further studies will be necessary to understand more the molecular mechanism affected by the compound A and/or B in the FLT-3 pathway. The compounds A and B demonstrate potential in treatment of AML expressing FLT-ITD. Disclosures Oliva: Emmaus Lifesciences, Inc.: Current Employment. Villanueva: Emmaus Lifesciences, Inc.: Current Employment. Ochiai: Emmaus Lifesciences, Inc.: Ended employment in the past 24 months. Niihara: Emmaus Lifesciences, Inc.: Current Employment.

Chitosan-TiO2 nanotubes scaffolds for proliferation and early differentiation of MG63 by functionalization with fetal bovine serum

Scaffolds have been used as alternative biomaterials to overcome physiological bone disorders. Production of scaffolds has been challenging to fulfil the following criteria: biodegradability, mechanical sustainability, and biocompatibility. For cellular interaction, protein adsorbed on scaffold surface is important for osteoblastic activities. This study aimed to functionalize chitosan-TiO2 nanotubes scaffolds with fetal bovine serum and investigate in vitro efficacy of such scaffolds with fetal bovine serum. Chitosan-TiO2 nanotubes scaffolds were prepared via direct blending and lyophilization. They were then functionalized with fetal bovine serum via adsorption for 4, 8, 12 and 24 h. The in vitro efficacy of the functionalized scaffolds was evaluated using MG63 cells. The adsorption of fetal bovine serum onto the scaffolds was complex where saturation of adsorption was hardly attained. The in vitro efficacy of scaffolds with adsorbed fetal bovine serum was higher than that of those without fetal bovine serum by promoting better osteoblastic functions. Notably, the scaffolds functionalized for 4 h enhanced cell adhesion and proliferation on 7 day suggesting good regulation of osteoblastic binding and proliferation. ALP protein was expressed on 26 day in all functionalized scaffolds. Chitosan-TiO2 nanotubes scaffolds with adsorbed fetal bovine serum can be a potential regenerative material for bone regeneration.