Concentration Of Fetal Bovine Serum Research Articles

Establishment a cell line of splenic fibroblasts from golden pompano and its application in heavy metal toxicology and pathogen infection

A cell line derived from golden pompano Trachinotus ovatus splenic fibroblasts (TOSF) was established, its application in toxicology and virus susceptibility was demonstrated in this study. The TOSF cells grew fastest with the L-15 medium containing 10% fetal bovine serum concentration at 28 °C. The chromosomal number of normal TOSF cells was 2N = 48. TOSF cells showed high sensitivity to grouper iridovirus (SGIV) and nervous necrosis virus (NNV). Cadmium and copper had toxic effects on TOSF cells, which could cause an oxidative stress response in TOSF cells and activate the antioxidant system to regulate the reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione reductase (GSH) level in the cells. Cadmium and copper had an impact on the replication of SGIV and NNV in the TOSF cells, and copper could limit the proliferation of Aeromonas hydrophila in TOSF cells. Generally, the TOSF cell line can apply for heavy metal toxicity mechanisms study and environment monitoring, as well as the pathogen infection mechanism research.

The influence of fetal bovine serum concentration on stemness and neuronal differentiation markers in stem cells from human exfoliated deciduous teeth

Dental Stem Cells (DSCs) from discarded teeth are a non-invasive and ethically favorable source with the potential for neurogenesis due to their ectodermal origin. Stem cells from human exfoliated deciduous teeth (SHED) are particularly promising due to their high differentiation potential and relative immaturity compared to other Mesenchymal Stromal Cells (MSCs). Markers like CD56 and CD271 are critical in identifying MSC subpopulations for therapeutic applications because of their roles in neurodevelopment and maintaining stemness. This study investigates how fetal bovine serum (FBS) concentrations affect the expression of CD56 and CD271 in SHED, influencing their stemness and neuronal differentiation potential. SHEDs were isolated from various donors, cultured, and characterized for MSC traits using markers such as CD14, CD19, CD29, CD34, CD45, CD73, CD90, CD105, CD56, and CD271. Culturing SHED in different FBS conditions (standard 15 %, reduced 1 % and 5 %, and FBS-free) showed that lower FBS concentrations increase CD271 and CD56 expression while maintaining the standard MSC immunophenotype. Importantly, the enhanced expression of these markers can be induced even after SHEDs have been expanded in standard FBS concentrations. These findings suggest that FBS concentration can optimize SHED culture conditions, enhancing their suitability for regenerative medicine and tissue engineering applications.

Gradient-induced instability in tumour spheroids unveils the impact of microenvironmental nutrient changes

Tumours often display invasive behaviours that induce fingering, branching and fragmentation processes. The phenomenon, known as diffusional instability, is driven by differential cell proliferation, migration, and death due to the presence of metabolite and catabolite concentration gradients. An understanding of the intricate dynamics of this spatially heterogeneous process plays a key role in the investigation of tumour growth and invasion. In this study, we developed an in vitro tumour invasion assay to investigate cell invasiveness in tumour spheroids under a chemotactic stimulus. Our method, employing tumour spheroids seeded in a 3D collagen gel within a microfluidic chemotaxis chamber, focuses on the role of diffusive gradients. Using Time-Lapse Microscopy, the dynamic evolution of tumour spheroids was monitored in real-time, providing a comprehensive view of the morphological changes and cell migration patterns under different chemotactic conditions. Specifically, we explored the impact of fetal bovine serum (FBS) gradients on the behaviour of CT26 mouse colon carcinoma cells and compared the effects of varying FBS concentrations to two isotropic control conditions. Furthermore, a finite element in silico model was developed to quantify the diffusive flow of nutrients in the chemotaxis chamber and obtain a detailed understanding of tumour dynamics. Our findings reveal that the presence of a chemotactic gradient significantly influences tumour invasiveness, with higher concentrations of nutrients associated with increased cancer growth and cell migration.

Rapid and Accurate Ecotoxicological Assessment of Heavy Metals Using Cyprinus carpio Cells.

Heavy metals have serious negative effects on various aquatic organisms, and therefore rapid and accurate ecotoxicological assessments of heavy metals are necessary. Fish-derived cells sensitive to heavy metals have been used as valuable tools for ecotoxicological assessments. However, this method requires a minimum toxicity treatment time of 96 h, which limits its use when rapid ecotoxicological assessments are required or ecotoxicological assessments of a large number of toxicants are performed. In this study, these limitations were overcome by adjusting parameters including the concentration of fetal bovine serum (FBS) in the medium and the treatment time of the toxicant. Specifically, we found that the maximum time for fish cells to remain unstarved was 6 h when using a medium containing 1% FBS. We applied both parameters to the ecotoxicological assessment (using a medium containing 1% FBS for the toxicity assessment and treating the toxicant for only 6 h). Surprisingly, these adjusted parameters allowed us to obtain faster and more accurate data than the traditional assessment. This improvement was due to the new assessment conditions that minimized the possibility that the growth-inducing effects of nutrients present in excess in the medium could interfere with the cellular response to the toxicant. The accuracy of this assessment was not limited to measuring the toxicity of heavy metals. In conclusion, we have established an ecotoxicity assessment that can generate rapid and accurate data on heavy metals. This new platform will become the cornerstone of rapid and accurate ecotoxicity assessments of heavy metals.

Alteration of mature neuronal marker of β-III tubulin expression in differentiated SH-SY5Y cells by refinement of foetal bovine serum concentration

BackgroundThe process of differentiating neuroblastoma cells (SH-SY5Y) is crucial for obtaining mature neuronal markers. However, there is variability in the concentrations of foetal bovine serum (FBS) used in the differentiation media, ranging from 1 to 10%. This inconsistency in FBS concentrations may contribute to the inconsistent differentiation of cells. To improve the utility of the SH-SY5Y cell line as a model for neuronal cell culture, we investigated the impact of FBS concentrations in the differentiation media using Dulbecco's modified eagle medium and 10 μM all-trans-retinoic acid (ATRA). The aim of this study was to optimise the concentrations of FBS in the differentiation media of SH-SY5Y cells. Our study focused on assessing the length of dendrites in neuronal cells and the expression of β-III tubulin, a marker indicative of mature neurons. SH-SY5Y cells were differentiated with 10 µM of ATRA for 7 days. Four treatment groups with different FBS concentrations (1%, 3%, 5%, and 10%) were examined to assess the alteration of cellular morphology and dendritic length. The expression of the mature neuron marker β-III tubulin was evaluated using immunocytochemistry technique.ResultsSH-SY5Y cells' dendrite length was significantly longer (p < 0.05) when there was a higher concentration of FBS in the differentiation medium. The result was confirmed with the significant increase of β-III tubulin expression (p < 0.001) of the differentiated SH-SY5Y cells that have been incubated with higher concentrations of FBS in the differentiation medium.ConclusionsWe concluded that optimised concentrations of FBS in the differentiation media display longer length of the dendrites and express higher production of β-III tubulin in the differentiated SH-SY5Y cells. The consistency of serum concentration used in the differentiation media is important to produce a sustainable in vitro neuronal model of SH-SY5Y cells for neurodegenerative studies.

Optimal isolation, culture, and invitro propagation of spermatogonial stem cells in Huaixiang chicken.

Spermatogonial stem cells (SSCs) comprise the foundation of spermatogenesis and hence have great potential for fertility preservation of rare or endangered species and the development of transgenic animals and birds. Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs invitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8-79 days of age) of Huaixiang chicken. We found that the testes of 21-day-old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02 g/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic-activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30 ng/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20 ng/mL), bFGF (30 ng/mL), or LIF (5 ng/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21-day-old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.

PCR-Based DNA Detection of Bovine on Medium of Cultured Meat for Consumption: Religious Perspectives

Food technology advancements have led to the development of cultured or artificial meat produced in a laboratory setting. Recently, Israel and Singapore have built factories specifically for cultured meat production. Malaysia is also informed about a cultured meat processing facility, which is expected to be completed in 2024. This alternative is a viable solution to meet the boosting needs and demand for meat-based food products while minimising the environmental impact of traditional livestock farming. Nevertheless, despite its potential benefits, cultured meat faces diverse challenges, including religious concerns for faiths with specific dietary requirements. Therefore, this research intended to determine the detection of DNA of bovine on the cultured medium by using polymerase chain reaction (PCR) analysis. The polymerase chain reaction analysis was conducted by targeting the mitochondrial DNA of the cytochrome oxidase II (COII) gene sequence and produced an amplicon size of 165 bp. The PCR was obtained by using the sample of medium mix with different concentrations (10-20%) of Foetal Bovine Serum (FBS) (Capricorn Scientific, Ebsdorfergrund, Germany), and the cell was harvested on different days. For DNA extraction, GENEAID Blood/Cell DNA mini kit (Taipei, Taiwan) was used. The findings demonstrate that DNA concentration in foetal bovine serum content was detected on the cultured medium. The presence of DNA contradicts religions such as Islam and Judaism that have strict standard dietary practices known as halal and kosher respectively. This study serves as a reference for the consumption of cultured meat for the consumer, particularly for Muslim and Jewish communities.

Comparison of NK cell count between 1% and 10% FBS culture media concentration by FACS analysis

Recently, there has been a growing research interest in the application of immunotherapy for cancer treatment. Various approaches have been developed, and one of these methods involves utilizing Natural Killer (NK) cells of the immune system to specifically target cancer cells. Research on this method necessitates the mass and stable production of NK cells through culturing. For NK cell culture, Fetal Bovine Serum (FBS) is widely used in the industry at concentrations ranging from 10% to 20%. In an effort to explore a more efficient culturing technique, this study aims to investigate the effect of 1% FBS on cell viability of NK cells in cell culture. Because NK cells are usually defined by cell surface molecules, the efficacy of using a lower FBS concentration was quantitatively measured using Fluorescence-Activated Cell Sorting (FACS). The cell counts of cells cultured in 1% FBS was compared to those of cells cultured in 10% FBS. The results showed that no significant difference was found between cell count of cells cultured in 1% and 10% FBS. Thus, 1% FBS concentration culturing method was not found to be inferior. Further research is needed on the effect of replicating cancer microenvironment on NK cell viability.

Establishment and characterization of a golden pompano (Trachinotus blochii) fin cell line for applications in marine fish pathogen immunology

Pompano fishes have been widely farmed worldwide. As a representative commercial marine species of the Carangidae family, the golden pompano (Trachinotus blochii) has gained significant popularity in China and worldwide. However, because of rapid growth and high-density aquaculture, the golden pompano has become seriously threatened by various diseases. Cell lines are the most cost-effective resource for in vitro studies and are widely used for physiological and pathological research owing to their accessibility and convenience. In this study, we established a novel immortal cell line, GPF (Golden pompano fin cells). GPF has been passaged over 69 generations for 10 months. The morphology, adhesion and extension processes of GPF were evaluated using light and electron microscopy. GPF cells were passaged every 3 days with L-15 containing 20 % fetal bovine serum (FBS) at 1:3. The optimum conditions for GPF growth were 28 °C and a 20 % FBS concentration. DNA sequencing of 18S rRNA and mitochondrial 16S rRNA confirmed that GPF was derived from the golden pompano. Chromosomal analysis revealed that the number pattern of GPF was 48 chromosomes. Transfection experiments demonstrated that GPF could be utilized to express foreign genes. Furthermore, heavy metals (Cd, Cu, and Fe) exhibited dose-dependent cytotoxicity against GPF. After polyinosinic-polycytidylic acid (poly I:C) treatment, transcription of the retinoic acid-inducible gene I-like receptor (RLR) pathway genes, including mda5, mita, tbk1, irf3, and irf7 increased, inducing the expression of interferon (IFN) and anti-viral proteins in GPF cells. In addition, lipopolysaccharide (LPS) stimulation up-regulated the expression of inflammation-related factors, including myd88, irak1, nfκb, il1β, il6, and cxcl10 expression. To the best of our knowledge, this is the first study on the immune response signaling pathways of the golden pompano using an established fin cell line. In this study, we describe a preliminary investigation of the GPF cell line immune response to poly I:C and LPS, and provide a more rapid and efficient experimental material for research on marine fish immunology.

Black soldier fly bioconversion to cultivated meat media components using blue catfish gut microbiome

This study aimed to develop low-cost media for cell-cultivated seafood using gut-microbial community-assisted fermentation. Black soldier fly larvae (Hermetia illucens) were used as the substrate and exposed to gut microbial communities isolated from blue catfish (Ictalurus furcatus). Fermentation led to a decrease in species richness, with the loss of important chitinase and protease-producing genera such as Pseudomonas and Clostridiaceae. However, there was an increase in Paraclostridium and members of the Enterobacteriaceae. In addition, the effect of fermented hydrolysates from BSFL on the proliferation of zebrafish embryo fibroblasts was tested in comparison to fetal bovine serum (FBS) in in vitro cell cultivation. Lower concentrations of FBS resulted in decreased cell density and altered cell morphology. The supplementation of hydrolysate B at high peptide concentrations had cytotoxic effects on the cells, while at lower peptide concentrations, it improved cell proliferation only in cultures with 2.5 % FBS.

Unraveling the neuroprotective mechanisms of naltrexone against aluminum-induced neurotoxicity

Aluminum (Al) is a known neurotoxic trace element linked to Alzheimer’s disease (AD). Naltrexone, an opioid antagonist, has shown promising effects in reducing neuroinflammation at lower doses than those prescribed for addiction. This study aimed to determine the neuroprotective effects of naltrexone on Al-induced neurotoxicity in an in vitro AD model. The SH-SY5Y cells were first cultivated in a standard growth medium. Subsequently, the cells were induced to differentiate by decreasing the concentration of fetal bovine serum and introducing retinoic acid (RA) into the culture media. Subsequently, the inclusion of brain-derived neurotrophic factor (BDNF) was implemented in conjunction with RA. The process of differentiation was concluded on the seventh day. Study groups (n = 3) were designed as the control group, naltrexone group, Al group, Al-Nal group, Alzheimer’ model (AD) group, Alzheimer model + Al-exposed group (AD-Al), Alzheimer model + Nal applied group (AD-Nal) and Alzheimer model + Al-exposed + Nal applied group (AD-Al-Nal). Hyperphosphorylated Tau protein as the specific marker of AD was measured in all groups. Glycogen synthase kinase-3 (GSK-3)β, Protein phosphatase 2A (PP2A), Akt and Wnt signaling pathways were analyzed comparatively. In addition, oxidative stress parameters (total antioxidant capacity, lipid peroxidase, protein carbonyl and reactive oxygen species) were measured comparatively in the study groups. The results showed that naltrexone reduced hyperphosphorylated tau protein levels by regulating GSK-3β, PP2A, Akt and Wnt signaling. Also, exposure to naltrexone decreased oxidative stress parameters. Based on these results, naltrexone shows promise as a potential therapy for AD, subject to additional clinical assessments.

Bovine mammary epithelial cells can grow and express milk protein synthesis genes at reduced fetal bovine serum concentration.

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions.

Development of a spotted sea bass (Lateolabrax maculatus) bulbus arteriosus cell line and its application to fish virology and immunology

The bulbus arteriosus tissue of teleosts, which is located at the forefront of the heart, is used to reduce the pulse pressure. In this study, we constructed a permanent cell line (LmAB) for the first time using bulbus arteriosus tissue from spotted sea bass (Lateolabrax maculatus). This cell line has been passaged more than 80 times. Currently, it can be subcultured in L-15 medium with 8 % fetal bovine serum added. The optimal fetal bovine serum concentration and culture temperature for LmAB cells at 62 passages are 20 % and 28 °C, respectively. This cell line consists predominantly of epithelial-like cells. We used 18S rRNA gene sequencing to confirm that LmAB cells originated from spotted sea bass. Karyotype analysis revealed that 43 % of LmAB cells in passage 63 had 48 chromosomes. Exogenous plasmid transfection revealed that LmAB cells can express the green fluorescent protein gene with a transfection efficiency of up to 40 %, indicating that these cells can be used for in vitro genetic research. LmAB cells showed susceptibility to nervous necrosis virus, largemouth bass ulcer syndrome virus, and infectious spleen and kidney necrosis virus, which results in severe cytopathic effects. PCR analysis verified that these viruses can replicate in LmAB cells, and analysis of cytoskeletal F-actin patterns verified that infected cells exhibit serious changes in their actin cytoskeleton. LmAB cells infected with these three viruses showed increased expressions of interferon signaling pathway genes (IFNd, IFNγ-rel, and ISG15), indicating that the host interferon signaling pathway participates in the antiviral immune response. These findings indicate that our newly developed LmAB cell line is a valuable resource for future research in genetics, virology, and immunology.

Development of artificial bone graft via in vitro endochondral ossification (ECO) strategy for bone repair

Endochondral ossification (ECO) is a form of bone formation whereby the newly deposited bone replaces the cartilage template. A decellularized artificial cartilage graft (dLhCG), which is composed of hyaline cartilage matrixes, has been developed in our previous study. Herein, the osteogenesis of bone marrow-derived MSCs in the dLhCG through chondrogenic differentiation, chondrocyte hypertrophy, and subsequent transdifferentiation induction has been investigated by simulating the physiological processes of ECO for repairing critical-sized bone defects. The MSCs were recellularized into dLhCGs and subsequently allowed to undergo a 14-day proliferation period (mrLhCG). Following this, the mrLhCG constructs were subjected to two distinct differentiation induction protocols to achieve osteogenic differentiation: chondrogenic medium followed by chondrocytes culture medium with a high concentration of fetal bovine serum (CGCC group) and canonical osteogenesis inducing medium (OI group). The formation of a newly developed artificial bone graft, ossified dLhCG (OsLhCG), as well as its capability of aiding bone defect reconstruction were characterized by in vitro and in vivo trials, such as mRNA sequencing, quantitative real-time PCR (qPCR), immunohistochemistry, the greater omentum implantation in nude mice, and repair for the critical-sized femoral defects in rats. The results reveal that the differentiation induction of MSCs in the CGCC group can realize in vitro ECO through chondrogenic differentiation, hypertrophy, and transdifferentiation, while the MSCs in the OI group, as expected, realize ossification through direct osteogenic differentiation. The angiogenesis and osteogenesis of OsLhCG were proved by being implanted into the greater omentum of nude mice. Besides, the OsLhCG exhibits the capability to achieve the repair of critical-size femoral defects.

Research on Different Compound Combinations of Realgar-Indigo Naturalis Formula to Reverse APL Arsenic Resistance By Regulating Autophagy through mTOR Pathway

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia, and 90% of APL patients carry positive fusion gene of the promyelocytic leukemia protein (PML)- retinoic acid receptor α (RARα), which drives the development of APL. All-trans retinoic acid and arsenic trioxide is able to cure 90% of APL patients by targeting PML-RARα. However, there are still 5%-10% of patients who will relapse due to drug resistance. Compound realgar-indigo naturalis formula (RIF) was developed based on the characteristics of APL pathogenesis. It contains four Chinese herbs: realgar, natural indigo, danshen root and heterophylly falsestarwort root. Clinical studies have confirmed that RIF has a certain efficacy on APL, which shown non-inferiority compared with arsenic trioxide. In addition, RIF combined with all-trans retinoic acid has become the first-line treatment regimen in APL guidelines in China. This study further clarified the efficacy of RIF on arsenic-resistant APL, explored the mechanism, and laid the foundation for further improving the efficacy of APL. O bjective: To investigate the mechanism of different combinations of the compound of RIF in reversing arsenic resistance in APL. M ethods: The arsenic-resistant cell line HL60-PML A216V-RARα, which could stably express PML A216V-RARα, was established by lentiviral transfection. Among compounds of RIF, the active compound of realgar is tetra-arsenic tetra-sulfide (As 4S 4, A), for natural indigo is indirubin (I), for danshen root is tanshinone IIa (T), and the for heterophylly falsestarwort root is total saponins of Radix Pseudostellariae (S). The four compounds were applied on the HL60-PML A216V-RARα cell line. Cell viability was detected by CCK-8 method. Hoechst staining was used to observe cell morphology, Annexin V/PI double staining was applied to detect cell apoptosis, JC-1 detection was used to detect mitochondrial membrane potential, flow cytometry was used to detect CD33 expression to evaluate cell differentiation, and Western blotting was used to observe the changes in the levels of PML A216V-RARα, apoptosis-related factors, PI3K/AKT/mTOR and autophagy in the control, A, ITS and AITS groups. The PI3K inhibitor LY294002 and the autophagy inhibitor Baf A1 were combined and applied to observe the changes in the levels of PML A216V-RARα, mTOR and p62 after blocking the PI3K or autophagy pathway. Results: The arsenic-resistant cell line HL60-PML A216V-RARα was successfully established. Compared with the control group, A, ITS and AITS groups all inhibited the growth of HL60-PML A216V-RARα cells in a time-dependent manner ( P &amp;lt; 0.0001). The IC50 value of AITS was significantly lower than the other groups ( P &amp;lt; 0.0001), and AITS group exhibited the strongest inhibitory effect, followed by ITS and A. Hoechst staining and JC-1 detection both showed that AITS had the strongest effect in inducing apoptosis. Annxin V/PI detection showed that the apoptosis rate of the control group was 10.04%, 15.19% for A group, 12.46% for ITS group, and 28.05% for AITS group. The differences between the groups were statistically significant ( P &amp;lt; 0.0001). Detection of CD33 expression showed: control group (79.72%), ITS group (79.19%), A group (28.60%), AITS group (24.46%), and there were significant differences between groups ( P &amp;lt; 0.001 or P &amp;lt; 0.0001). AITS could reduce PML A216V-RARα protein, caspase3, Bcl-2 levels more than ITS and A ( P &amp;lt; 0.05 or P &amp;lt; 0.0001), and ITS was better than AITS and A in increasing cleaved PARP-1 levels ( P &amp;lt; 0.0001). AITS decreased the levels of PI3K, mTOR, P-mTOR, AKT, p-AKT and p62 and increased the level of LC3B ( P &amp;lt; 0.001 or P &amp;lt; 0.0001). Electron microscopic detection of autophagy showed that AITS had the highest level of autophagy, AITS combined with LY294002 increased the level of p62 and decreased the level of mTOR ( P &amp;lt; 0.0001) compared to the AITS group; while when AITS was combined with Baf A1, the level of p62 increased ( P &amp;lt; 0.0001) and the level of mTOR also increased ( P &amp;lt; 0.001). Autophagy could induced by serum starvation. As the concentration of fetal bovine serum decreased, the effects of degrading PML A216V-RARα and p62, and LC3B level increasing were stronger. C onclusion: Tanshinone IIa, indirubin and total saponins of Radix Pseudostellariae could enhance the effect of tetra-arsenic tetra-sulfide down-regulating PML A216V-RARα, and the mechanism was suggested to be related to inhibiting mTOR pathway to activate autophagy.

Toward a Transportable Cell Culture Platform for Evaluating Radiotherapy Dose Modifying Factors

The current tools for validating dose delivery and optimizing new radiotherapy technologies in radiation therapy do not account for important dose modifying factors (DMFs), such as variations in cellular repair capability, tumor oxygenation, ultra-high dose rates and the type of ionizing radiation used. These factors play a crucial role in tumor control and normal tissue complications. To address this need, we explored the feasibility of developing a transportable cell culture platform (TCCP) to assess the relative biological effectiveness (RBE) of ionizing radiation. We measured cell recovery, clonogenic viability and metabolic viability of MDA-MB-231 cells over several days at room temperature in a range of concentrations of fetal bovine serum (FBS) in medium-supplemented gelatin, under both normoxic and hypoxic oxygen environments. Additionally, we measured the clonogenic viability of the cells to characterize how the duration of the TCCP at room temperature affected their radiosensitivity at doses up to 16 Gy. We found that (78±2)% of MDA-MB-231 cells were successfully recovered after being kept at room temperature for three days in 50% FBS in medium-supplemented gelatin at hypoxia (0.4±0.1)% pO2, while metabolic and clonogenic viabilities as measured by ATP luminescence and colony formation were found to be (58±5)% and (57±4)%, respectively. Additionally, irradiating a TCCP under normoxic and hypoxic conditions yielded a clonogenic oxygen enhancement ratio (OER) of 1.4±0.6 and a metabolic OER of 1.9±0.4. Our results demonstrate that the TCCP can be used to assess the RBE of a DMF and provides a feasible platform for assessing DMFs in radiation therapy applications.

Establishment and characterization of a muscle cell line from golden pompano (Trachinotus ovatus) and its application in viral susceptibility

A new cell line, golden pompano muscle (GPM), was developed and characterized from the muscle tissue of the golden pompano in this work. GPM cells were mostly fibroblast-like and proliferated well in Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS) (concentration of 10% to 20%) at 28 °C. The cell line has been stably passed over 80 generations and has been successfully cryopreserved. Molecular characterization of cytochrome oxidase subunit I (COI) confirmed that the GPM cells used in this study were from the golden pompano. Chromosome analysis showed that the diploid chromosome number of GPM cells was 2 N = 48. The enhanced green fluorescent protein (EGFP) reporter gene was successfully expressed in the GPM cell line by liposome transfection technique, and the transfection efficiency of GPM cells was about 60%, which suggests that GPM cells are available for research on gene expression in vitro. Virus susceptibility assay showed that GPM cells were susceptible to red-spotted grouper nervous necrosis virus (RGNNV), evidenced by the occurrence of cytopathic effect (CPE) and the increased viral protein synthesis capacity. Furthermore, viral replication significantly increased the expression levels of interferon (IFN) related signaling molecules and pro-inflammatory cytokines in cells. Therefore, our work developed and characterized a new cell line from golden pompano muscle, which allowed for functional studies of genetic manipulation, viral pathogenesis, and the interactions of host and virus.

Preparation and Characterization of Gellan Gum Hydrogel as Therapeutic Protein Delivery for Wound Healing

Therapeutic proteins like platelet-rich plasma have been used as adjunct therapies for wound healing. The delivery of these proteins may require a special carrier as a controlled release to prolong and optimize the healing effects on the affected tissues. The present study focuses on preparing and characterizing a hydrogel made from gellan gum to act as a scaffold to carry therapeutic proteins intended for wound healing. Fetal bovine serum (FBS) was used as a representative for therapeutic proteins due to its ability to stimulate cell proliferation in vitro. FBS, gellan gum (GG) hydrogel, and FBS-loaded gellan gum hydrogel (GF) were prepared and characterized by the detection of its functional groups through FTIR and elemental analysis through CHNS analyzer, confirming the entrapment of biomolecules of FBS into GG. The protein release study showed a burst release of protein from all GF variants with subsequent gradual slow release over 72 hours period. Cell viability (MTT) assay showed an increasing trend of cell viability percentage with the increasing concentration of FBS loaded into GG hydrogel. The results of this study support the potential use of GG hydrogel as a carrier of therapeutic proteins for wound regeneration.

Quantitation of tizoxanide in multiple matrices to support cell culture, animal and human research

Currently nitazoxanide is being assessed as a candidate therapeutic for SARS-CoV-2. Nitazoxanide is rapidly broken down to its active metabolite tizoxanide upon administration. Unlike many other candidates being investigated, tizoxanide plasma concentrations achieve antiviral levels after administration of the approved dose, although higher doses are expected to be needed to maintain these concentrations across the dosing interval in the majority of patients. Here an LC-MS/MS assay is described that has been validated in accordance with Food and Drug Administration (FDA) guidelines. Fundamental parameters have been evaluated, and these included accuracy, precision and sensitivity. The assay was validated for human plasma, mouse plasma and Dulbecco’s Modified Eagles Medium (DMEM) containing varying concentrations of Foetal Bovine Serum (FBS). Matrix effects are a well-documented source of concern for chromatographic analysis, with the potential to impact various stages of the analytical process, including suppression or enhancement of ionisation. Herein a validated LC-MS/MS analytical method is presented capable of quantifying tizoxanide in multiple matrices with minimal impact of matrix effects. The validated assay presented here was linear from 15.6 ng/mL to 1000 ng/mL. The presented assay here has applications in both pre-clinical and clinical research and may be used to facilitate further investigations into the application of nitazoxanide against SARS-CoV-2.

Comparison of Single-step and Sequential Embryo Culture Systems: Replacement of Serum with Platelet Lysate

Background: More understanding of the physiological needs of the embryo during its growth from the zygote to the blastocyst stage, as well as the composition of the uterine and fallopian tubal secretions, has led to the development of single-step or sequential culture media. Objectives: The present study aimed to investigate the utilization of mCR2aa culture medium as a sequential culture medium for embryo development, supplemented with platelet lysate (PL) to decrease the concentration of fetal bovine serum (FBS) in the embryo culture medium. Methods: After maturation and fertilization of sheep oocytes obtained from a slaughterhouse, potential zygotes were cultured in mCR2aa medium without FBS for two days (C1, 1 - 3 days of culture). In the second two days of culture (C2, 3 - 5 days of culture), 2.5% or 5% of FBS and 5% or 10% of PL were used. In the remaining days of culture (C3, 5 - 9 days of culture), 2.5%, 5%, or 10% of FBS, and 0%, 2.5%, 5%, or 10% of PL were used. These percentages were compared to the commercial BO-IVC™ medium and mCR2aa medium, both of which contained 10% of FBS. Results: No significant difference was observed in the number of produced blastocysts between different treatments and the control. However, the number of hatched blastocysts in BO-IVC™, as a single-step culture medium, significantly differed from that of the other treatments. The number of hatched blastocysts in the sequential culture medium was higher in media supplemented with 2.5% FBS and 10% PL in the C2 mCR2aa medium, and 2.5% FBS without PL in the C3 mCR2aa medium, but no significant difference was observed in other treatments. However, increasing the concentration of FBS and PL in the C3 mCR2aa medium significantly decreased the number of hatched blastocysts. Conclusions: An increase in the percentage of PL to 10% necessitated a decrease in FBS to 2.5% in the C2 mCR2aa medium, while PL in the C3 mCR2aa medium was not required in the presence of 2.5% FBS.