Fetal bovine serum—a cell culture dilemma

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

Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same

Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Modification of Biocorrosion and Cellular Response of Magnesium Alloy WE43 by Multiaxial Deformation

The study shows that multiaxial deformation (MAD) treatment leads to grain refinement in magnesium alloy WE43. Compared to the initial state, the MAD-processed alloy exhibited smoother biocorrosion dynamics in a fetal bovine serum and in a complete cell growth medium. Examination by microCT demonstrated retardation of the decline in the alloy volume and the Hounsfield unit values. An attendant reduction in the rate of accumulation of the biodegradation products in the immersion medium, a less pronounced alkalization, and inhibited sedimentation of biodegradation products on the surface of the alloy were observed after MAD. These effects were accompanied with an increase in the osteogenic mesenchymal stromal cell viability on the alloy surface and in a medium containing their extracts. It is expected that the more orderly dynamics of biodegradation of the WE43 alloy after MAD and the stimulation of cell colonization will effectively promote stable osteosynthesis, making repeat implant extraction surgeries unnecessary.

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%.

Degradation Analysis of Thin Mg-xAg Wires Using X-Ray Near-Field Holotomography

Magnesium-silver alloys are of high interest for the use as temporary bone implants due to their antibacterial properties in addition to biocompatibility and biodegradability. Thin wires in particular can be used for scaffolding, but the determination of their degradation rate and homogeneity using traditional methods is difficult. Therefore, we have employed 3D imaging using X-ray near-field holotomography with sub-micrometer resolution to study the degradation of thin (250 &mu;m diameter) Mg-2Ag and Mg-6Ag wires. The wires were studied in two states, recrystallized and solution annealed to assess the influence of Ag content and precipitates on the degradation. Imaging was employed after degradation in Dulbecco&rsquo;s modified Eagle&rsquo;s medium and 10% fetal bovine serum after 1 to 7 days. At 3 days of immersion the degradation rates of both alloys in both states were similar, but at 7 days higher silver content and solution annealing lead to decreased degradation rates. The opposite was observed for the pitting factor. Overall, the standard deviation of the determined parameters was high, owing to the relatively small field of view during imaging and high degradation inhomogeneity of the samples. Nevertheless, Mg-6Ag in the solution annealed state emerges as a potential material for thin wire manufacturing for implants.

Nucleofection of Adipose Mesenchymal Stem/Stromal Cells: Improved Transfection Efficiency for GMP Grade Applications

Nucleofection (NF) is a safe, non-viral transfection method, compatible with Good Manufacturing Practice guidelines. Such a technique is useful to improve therapeutic effectiveness of adipose tissue mesenchymal stem cells (ASC) in clinical settings, but improvement of NF efficiency is mandatory. Supernatant rich in growth factors (SRGF) is a clinical-grade medium additive for ASC expansion. We showed a dramatically increased NF efficiency and post-transfection viability in ASC expanded in presence of SRGF (vs. fetal bovine serum). SRGF expanded ASC were characterized by increased vesicle endocytosis but lower phagocytosis properties. SRGF increased n-6/n-3 ratio, reduced membrane lipid raft occurrence, and lowered intracellular actin content in ASC. A statistical correlation between NF efficiency and lipid raft availability on cell membranes was shown, even though a direct relationship could not be demonstrated: attempts to selectively modulate lipid rafts levels were, in fact, limited by technical constraints. In conclusion, we reported for the first time that tuning clinical-grade compatible cell culture conditions can significantly improve ASC transfection efficiency by a non-viral and safe approach. A deep mechanistic characterization is extremely complex, but we can hypothesize that integrated changes in membrane structure and intracellular actin content could contribute to explain SRGF impact on ASC NF efficiency.

Development of an in vitro tissue culture system for hammer coral (Fimbriaphyllia ancora) ovaries

In vitro gonad culture systems have proven useful to investigate intrinsic mechanisms of sexual reproduction in animals. Here we describe development of an in vitro culture method for coral ovaries. Mesenterial tissues containing both ovaries and mesenterial filaments were microscopically isolated from the scleractinian coral, Fimbriaphyllia ancora, and culture conditions were optimized. M199 diluted 10× (10% M199, pH 8.1) and supplemented with 25 mM HEPES and the antibiotics, ampicillin, penicillin and streptomycin, supported oocyte survival and maintained the structural integrity of ovaries during short-term culture (~ 6 days). Addition of a commercial antibiotic–antimycotic solution (Anti–Anti) and fetal bovine serum adversely affected ovary maintenance and caused tissue disintegration. Characterization of cultured ovaries showed that there is no difference in cell proliferation of ovarian somatic cells between culture Days 1 and 6. Moreover, the presence of oogonia and expression of a major yolk protein, vitellogenin, were confirmed in ovaries cultured for 6 days. This system will be useful for studying effects of a wide range of substances on coral oogenesis.