Fetal Bovine Serum Research Articles

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R’s (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

Establishment of COS-BK cells persistently infected with archetype BK polyomavirus.

BK polyomavirus (BKPyV) was the first human polyomavirus to be isolated from an immunosuppressed kidney transplant recipient in 1971. BKPyV reactivation causes BKPyV-associated nephropathy and hemorrhagic cystitis. However, the mechanisms underlying BKPyV replication remain unclear. In the present study, we performed the long-term cultivation of COS-7 cells transfected with archetype KOM-5 DNA, which were designated as COS-BK cells. BKPyV derived from COS-BK cells was characterized by analyzing the amount of the virus based on hemagglutination, viral replication, and the production of viral protein 1 (VP1). Immunostaining showed that VP1-positive cells accounted for a small percentage of COS-BK cells. The nucleotide sequences encompassing the origin of the DNA replication of BKPyV derived from COS-BK cells were generated from KOM-5 by the deletion of an 8-bp sequence, which did not involve T antigen binding sites. BKPyV replicated most efficiently in COS-BK cells in DMEM containing 2% fetal bovine serum. These results indicate that COS-BK cells are a suitable culture system for studying the persistent infection of archetype BKPyV.

Thermoresponsive block-copolymer brush-modified interfaces for effective fabrication of hepatocyte sheets

Hepatic tissue engineering has been investigated for the treatment of liver disease. One promising tissue is the hepatocyte sheet, cultured in temperature-responsive cell culture dishes. However, the preparation of hepatocyte sheets requires precoating of the dish with fetal bovine serum (FBS), which is not desirable for clinical applications. To overcome this issue, we developed a thermoresponsive polymer-modified glass with an affinity for hepatocytes. Poly(N-p-vinylbenzyl-O-β-d-galactopyranosyl-(1→4)-d-guliconamide)-b-poly(N-isopropylacrylamide) (PVLA-b-PNIPAAm) was prepared through two steps of atom transfer radical polymerization (ATRP). The prepared copolymer brushes were characterized using X-ray photoelectron spectroscopy, atomic force microscopy, gel permeation chromatography, contact angle measurements, and fluorescence labeled-fibronectin and -lectin adsorption assays. These characterizations showed that the smooth block copolymer layer was successfully modified by ATRP. The feasibility of using the prepared polymer brush-grafted glass as a hepatocyte sheet fabrication dish was investigated using primary rat hepatocytes. The PVLA-b-PNIPAAm brushes exhibited hepatocyte adhesion and proliferation without FBS precoating. By reducing the temperature from 37 °C to 20 °C, the hepatocyte sheet was recovered from the copolymer brush. These results indicated that the prepared PVLA-b-PNIPAAm brushes can be utilized for effective hepatocyte sheet fabrication without the need for FBS precoating.

Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer

ObjectiveThe effects of arsenic trioxide (As2O3) on hepatocellular carcinoma have been documented widely. Autophagy plays dual roles in the survival and death of cancer cells. Therefore, we investigated the exact role of autophagy in As2O3-induced apoptosis in liver cancer cells. MethodsThe viability of hepatoma cells was determined using the MTT assay with or without fetal bovine serum. The rate of apoptosis in liver cancer cells treated with As2O3 was evaluated using flow cytometry, Hoechst 33258 staining, and TUNEL assays. The rate of autophagy among liver cancer cells treated with As2O3 was detected using immunofluorescence, Western blot assay and transmission electron microscopy. ResultsUpon treatment with As2O3, the viability of HepG2 and SMMC-7721 cells was decreased in a time- and dose-dependent manner. The apoptosis rates of both liver cancer cell lines increased with the concentration of As2O3, as shown by flow cytometry. Apoptosis in liver cancer cells treated with As2O3 was also shown by the activation of the caspase cascade and the regulation of Bcl-2/Bax expression. Furthermore, As2O3 treatment induced autophagy in liver cancer cells; this finding was supported by Western blot, immunofluorescence of LC3-II and beclin 1, and transmission electron microscopy. In liver cancer cells, As2O3 inhibited the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway that plays a vital role in both apoptosis and autophagy. The PI3K activator SC-79 partially reversed As2O3-induced autophagy and apoptosis. Furthermore, inhibiting autophagy with 3-methyladenine partially reversed the negative effects of As2O3 on cell viability. Serum starvation increased autophagy and amplified the effect of As2O3 on cell death. ConclusionAs2O3 induces apoptosis and autophagy in liver cancer cells. Autophagy induced by As2O3 may have a proapoptotic effect that helps to reduce the viability of liver cancer cells. This study provides novel insights into the effects of As2O3 against liver cancer.Please cite this article as: Deng ZT, Liang SF, Huang GK, Wang YQ, Tu XY, Zhang YN, Li S, Liu T, Cheng BB. Autophagy plays a pro-apoptotic role in arsenic trioxide-induced cell death of liver cancer. J Integr Med. 2024; Epub ahead of print.

Topical Delivery of microRNA-125b by Framework Nucleic Acids for Psoriasis Treatment.

Psoriasis is a chronic and recurrent inflammatory dermatitis characterized by T cell imbalance and abnormal keratinocyte proliferation. MicroRNAs (miRNAs) hold promise as therapeutic agents for this disease; however, their clinical application is hindered by poor stability and limited skin penetration. This study demonstrates the utilization of Framework Nucleic Acid (FNA) for the topical delivery of miRNAs in psoriasis treatment. By utilizing miRNA-125b as the model drug, FNA-miR-125b was synthesized via self-assembly. The successful synthesis and stability of FNA-miR-125b in bovine fetal serum (FBS) were verified through gel electrophoresis. Subsequently, flow cytometry was employed to investigate the cell internalization on HaCaT cells, while qPCR determined the effects of FNA-miR-125b on cellular functions. Additionally, the skin penetration ability of FNA-miR-125b was assessed. Finally, a topical administration study involving FNA-miR-125b cream on imiquimod (IMQ)-induced psoriasis mice was conducted to evaluate its therapeutic efficacy. The FNA-miR-125b exhibited excellent stability, efficient cellular internalization, and potent inhibition of keratinocyte proliferation. In the psoriasis mouse model, FNA-miR-125b effectively penetrated the skin tissue, resulting in reduced epidermal thickness and PASI score, as well as decreased levels of inflammatory cytokines.

Human platelet lysate enhances in vivo activity of CAR-Vδ2 T cells by reducing cellular senescence and apoptosis

Background aimsVγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. MethodsSpecifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. ResultsWe found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity. ConclusionsThis study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement.

Long-Term Porcine Retina Explants as an Alternative to InVivo Experimentation.

The porcine retina represents an optimal model system to study treatment approaches for inherited retinal dystrophies owing to close anatomical similarities to the human retina, including a cone enriched visual streak. The aim of this work was to establish a protocol to keep explants in culture for up to 28days with good morphological preservation. Two to four retina explants per eye were obtained from the central part of the retina and transferred onto a membrane insert with the photoreceptors facing down. Different medium compositions using Neurobasal-A medium containing 100 or 450mg/dL glucose and combinations of fetal calf serum, B-27 with or without insulin and N-2 were tested. We developed a tissue quality score with robust markers for different retinal cell types (protein kinase C alpha, peanut agglutinin and 4',6-diamidino-2-phenylindol). Retinae were kept until 28days with only little degradation. The best results were attained using Neurobasal-A medium containing 100mg/dL glucose supplemented with B-27 containing insulin and N-2. For an easy preparation process, it is necessary to minimize transport time and keep the eyes on ice until dissected. Heat-mediated decontamination by the butcher has to be avoided. Using a standardized protocol, porcine retina explants represent an easy to handle intermediate model between in vitro and in vivo experimentation. This model system is robustly reproducible and contributes to the implementation of the 3R principle to minimize animal experimentation. This model can be used to test future therapeutic approaches for inherited retinal dystrophies.

Turn-On Fluorescent Probe for BSA Detection Constructed by Supramolecular Assembly.

The fluorescent probe method has attracted significant research attention due to its high sensitivity and reproducibility in detecting bovine serum albumin (BSA). In this study, we constructed a fluorescent probe for BSA detection by assembling an amphiphilic organic fluorescent molecule, termed 2-(2'-hydroxyphenyl) benzothiazole (HBT-11), with BSA. In an aqueous solution, HBT-11 exhibited a weak fluorescence emission at 501 nm. However, the addition of BSA substantially enhanced the fluorescence emission at 501 nm, indicating that the assembly was driven by electrostatic interactions between HBT-11 and BSA. HBT-11, serving as a fluorescent probe for BSA detection, demonstrated a limit of detection (LOD) as low as 3.92 nmol L-1, excellent photostability, high selectivity, and robust anti-interference capability. Notably, we successfully applied HBT-11 for detecting BSA in fetal bovine serum and selectively imaging BSA in HeLa cells.

Effect of delayed addition of fetal calf serum to the culture medium on in vitro embryo production from creole heifers raised in a high altitude in Ecuador

El estudio tuvo como objetivo evaluar el efecto del suero fetal bovino (FCS) agregado al medio de cultivo los días 5 a 7 después de la fertilización in vitro sobre la producción y posterior criotolerancia de blastocistos de vaquillonas criollas ecuatorianas. Se recolectaron complejos cúmulo-ovocitos (COCs) inmaduros mediante aspiración intravaginal guiada por ultrasonido (OPU) de 10 vaquillonas criollas y de ovarios de matadero en ocho sesiones. Los COCs se sometieron a maduración in vitro (MIV), fecundación (FIV) y cultivo (IVC). El día 1 después de la FIV, los presuntos embriones de cada fuente de ovocitos (OPU [O] y Matadero [A]) se asignaron aleatoriamente en dos grupos según si se añadió FCS al 2.5% (v/v) al medio de cultivo el día 5 después de la FIV: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+ y 4) A-FCS-. El día 7 después de la FIV se vitrificaron embriones de alta calidad. La criotolerancia de los embriones vitrificados-calentados se evaluó según la reexpansión del blastocele a las 2 h y la posterior reexpansión y eclosión a las 24 y 48 h de incubación. La tasa de blastocistos el día 7 no difirió entre la fuente de ovocitos y el grupo FCS. Después del proceso de vitrificación/calentamiento, la adición de FCS solo afectó la tasa de reexpansión a las 2 h, independientemente de la fuente de ovocitos (p<0.05). Asimismo, la tasa de eclosión de los blastocistos a las 48 h de incubación se vio drásticamente afectada únicamente en los ovocitos derivados de OPU (p<0.01). En conclusión, la suplementación de FCS el día 5 después de la FIV no mejoró la producción de blastocistos y afectó negativamente la criotolerancia de embriones bovinos in vitro derivados de vaquillonas criollas criadas en los Andes ecuatorianos.

Surface property and in vitro toxicity effect of insoluble particles given by protein corona: Implication for PM cytotoxicity assessment

In vitro toxicological assessment helps explore key fractions of particulate matter (PM) in association with the toxic mechanism. Previous studies mainly discussed the toxicity effects of the water-soluble and organic-soluble fractions of PM. However, the toxicity of insoluble fractions is relatively poorly understood, and the adsorption of proteins is rarely considered. In this work, the formation of protein corona on the surface of insoluble particles during incubation in a culture medium was investigated. It was found that highly abundant proteins in fetal bovine serum were the main components of the protein corona. The adsorbed proteins increased the dispersion stability of insoluble particles. Meanwhile, the leaching concentrations of some metal elements (e.g., Cu, Zn, and Pb) from PM increased in the presence of proteins. The toxicity effects and potential mechanisms of the PM insoluble particle–protein corona complex on macrophage cells RAW264.7 were discussed. The results revealed that the PM insoluble particle–protein corona complex could influence the phagosome pathway in RAW264.7 cells. Thus, it promoted the intracellular reactive oxygen species generation and induced a greater degree of cell differentiation, significantly altering cell morphology. Consequently, this work sheds new light on the combination of insoluble particles and protein corona in terms of PM cytotoxicity assessment.

Optimizing SH-SY5Y cell culture: exploring the beneficial effects of an alternative media supplement on cell proliferation and viability

In the quest to unravel the mysteries of neurological diseases, comprehending the underlying mechanisms is supreme. The SH-SY5Y human neuroblastoma cell line serves as a crucial tool in this endeavor; however, the cells are known for its sensitivity and slow proliferation rates. Typically, this cell line is cultured with 10% Fetal Bovine Serum (FBS) supplement. Nu-Serum (NuS), a low-protein alternative to FBS, is promising to advance cell culture practices. Herein, we evaluated the substitution of NuS for FBS to test the hypothesis that an alternative serum supplement can aid and promote SH-SY5Y cell proliferation and differentiation. Our findings revealed that the NuS-supplemented group exhibited a notable increase in adhered cells compared to both the FBS and serum-free (SF) groups. Importantly, cell viability remained high in both sera treated groups, with the NuS-supplemented cells displaying significantly larger cell sizes compared to the SF-treated group. Furthermore, cell proliferation rates were higher in the NuS-treated group, and neuroblast-like morphology was observed earlier than FBS group. Notably, both FBS and NuS supported the differentiation of these cells into mature neurons. Our data supports NuS as an alternative for SH-SY5Y cell culture, with the potential to elevate the quality of research in the neuroscience field.

Critical role of the composition of the cell culture medium on cell attachment and viability on PLA biocomposite scaffolds under in vitro assay conditions

In tissue engineering applications 3D scaffolds undertake the function of the native extracellular matrix (ECM) for cell attachment, survival, proliferation, and differentiation. For successful long-term implementation scaffolds need to mimic the ECM in vitro and in vivo. Many studies have focused on protein adsorption as the most critical factor for cell attachment. To improve protein adsorption on polymeric scaffolds various physical and chemical surface modification techniques, such as changing surface porosity, surface energy, and introducing various functional surface chemical groups have been investigated. It is well studied and documented that complex interactions between the biomaterial surface and the cell culture components in in vitro assay conditions define the scaffold performance. In this study influence of in vitro assay components on HepG2 liver cell attachment and proliferation on 3D printed poly(lactic acid) (PLA) scaffolds coated with various polymeric electrospun webs were investigated. MTT assays were performed on the scaffolds by using 3 different cell culture media; (i) basal cell culture medium (DMEM), (ii) DMEM supplemented with 10% albumin, and (iii) DMEM supplemented with 10% fetal bovine serum (FBS). Results obtained have shown dramatic improvement in cell attachment and proliferation in DMEM supplemented with 10% FBS when compared with DMEM and DMEM containing 10% albumin. Our findings clearly demonstrate synergistic effects of all bioactive components present in the FBS medium in significantly improving the HepG2 cell attachment and proliferation on polymeric scaffolds under in vitro conditions, regardless of the substrate structure, composition, and surface properties.

Enzymatic Synthesis of TNA Protects DNA Nanostructures

AbstractXeno‐nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3’ terminus of DNA oligonucleotide, resulting in DNA‐TNA chimera resistant to exonuclease digestion. Moreover, TdT‐catalyzed TNA extension supports one‐pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self‐assembly of DNA origami nanostructures (DONs). Such TNA‐protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

Calcitonin Inhibits Phenotypic Switching of Aortic Smooth Muscle Cells and Neointimal Hyperplasia through the AMP-Activated Protein Kinase/Mechanistic Target of Rapamycin Pathway.

Calcitonin (CT) is a peptide hormone secreted by the parafollicular C cells of the thyroid gland, salmon calcitonin was originally extracted from the hind cheek of salmon. Neointimal hyperplasia refers to the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). In this study, a rat model of restenosis was employed to explore the impact of calcitonin on neointima proliferation. Calcitonin was administered via continuous injections for a duration of 14 days postsurgery, and the expression of proteins associated with proliferation, migration, and phenotypic switching was assessed using the vascular smooth muscle cells. Additionally, metabolomic analyses were conducted to shed light on the mechanisms that underlie the role of calcitonin in the development of cardiovascular disease. In our study, we found that calcitonin possesses the capability to dispute the proliferation, migration, and phenotypic transformation of VSMCs induced by platelet-derived growth factor-BB (PDGF-BB) and 15% fetal bovine serum in vitro. Calcitonin has demonstrated a favorable impact on smooth muscle cells, both in vitro and in vivo. More specifically, it has been observed to mitigate phenotypic switching, proliferation, and migration of these cells. Moreover, calcitonin has been identified as a protective factor against phenotypic switching and the formation of neointima, operating through the AMP-activated protein kinase/mechanistic target of rapamycin (mTOR) pathway.

Enzymatic Synthesis of TNA Protects DNA Nanostructures.

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with improved biological stabilities and offer powerful molecular tools such as aptamers and catalysts. However, XNA application has been hindered by a very limited repertoire of tool enzymes, particularly those that enable de novo XNA synthesis. Here we report that terminal deoxynucleotide transferase (TdT) catalyzes untemplated threose nucleic acid (TNA) synthesis at the 3' terminus of DNA oligonucleotide, resulting in DNA-TNA chimera resistant to exonuclease digestion. Moreover, TdT-catalyzed TNA extension supports one-pot batch preparation of biostable chimeric oligonucleotides, which can be used directly as staple strands during self-assembly of DNA origami nanostructures (DONs). Such TNA-protected DONs show enhanced biological stability in the presence of exonuclease I, DNase I and fetal bovine serum. This work not only expands the available enzyme toolbox for XNA synthesis and manipulation, but also provides a promising approach to fabricate DONs with improved stability under the physiological condition.

Multifunctional chitosan-based hydrogels: characterization and evaluation of biocompatibility and biodegradability in vitro

Creation of novel remedies efficient in supporting wound healing remains an actual task in pharmacology. Hydrogels showed high efficiency in wound healing and tissue regeneration due to viscosity, elasticity and fluidity that provide them with functional characteristics similar to that in extracellular matrix. The aim of the study was to create chitosan-based hydrogels functionalized with different components (chondroitin-6-sulfate, hyaluronic acid, N-stearoylethanolamine) and to estimate their biocompatibility and biodegradabili­ty in vitro. For the first time, a lipid substance N-stearoylethanolamine (NSE) known as suppressor of pro-inflammatory cytokines expression was used as hydrogel component (1.95 mg/g). FTIR analysis confirmed the complexation of chitosan molecule with hyaluronate, chondroitin-6-sulfate, NSE. MTT-test and Trypan blue exclusion test were used to study hydrogels cytotoxicity towards human cells of different tissue origin. Biodegradability of hydrogels was evaluated using direct hydrogel contact with cells and cell-independent degradation. It was shown that chondroitin-6-sulfate (<2 mg/ml), hyaluronic acid (<2 mg/ml) and NSE (26 µg/ml) did not demonstrate significant toxic effects towards pseudonormal human cells of the MCF10A, HaCat, HEK293 lines and mouse cells of the Balb/3T3 line. The studied hydrogels were stable in saline solution, while in a complete culture medium containing 10% fetal bovine blood serum they underwent degradation in >24 h. The identified biodegradability of the chitosan-based hydrogels is important for the release of noncovalently immobilized NSE into biological medium. Further studies on laboratory animals with experimental wounds are expected to explore the potential of created hydrogels as anti-inflammatory and wound-healing agents. Keywords: biodegradability, chitosan hydrogels, chondroitin-6-sulfate, FTIR analysis, human pseudonormal cells, hyaluronic acid, N-stearoylethanolamine, toxicity

Potential Effect of Bovine Colostrum on Mesenchymal Stem Cells for Regenerative Therapy

Bovine colostrum is the first mammary secretion after parturition; it is rich in Igs and bioactive compounds and could play a role in the development of naturally based products with positive effects on human health. In this discussion, we critically examine the effect of bovine colostrum on the properties of mesenchymal stem cells. Multipotent mesenchymal stem cells (MSCs) are a heterogeneous subset of fibroblast-like cells that can be isolated from various biological tissues, such as bone marrow, umbilical cord, and adipose tissues. They are characterized by their ability to self-renew and differentiate into cells of the mesodermal lineage, including adipocytes, osteocytes, and chondrocytes. Additionally, MSCs display an immunomodulatory capacity due to their ability to interact with effector cells typical of both innate and adaptive immune responses. Considering these important properties, MSCs have gained increasing attention in the field of regenerative medicine in recent decades. To date, most experimental protocols are based on cell culture media supplemented with fetal bovine serum (FBS) to promote the ex vivo expansion of MSCs while preserving their differentiative and immunomodulatory capacities. Future trends could involve the application of bovine colostrum in regenerative medicine.

Ultrasensitive detecting of dopamine in complex components by field effect transistor sensor based on the synergistic enhancement effect and overcoming debye length limitations

Field effect transistor (FET) has attracted high attention in biomolecules detection. However, the sensitivity of FET-based biosensors is often limited by the charge screening effect. In addition, the facile and ultra-sensitive detection for small biomolecules, which possess weak charge or electroneutral, remains to be studied. Here, the self-assembled monolayer AuNPs/three-dimensional (3D) crumpled graphene FET is structured for a biosensor by shrinking flexible polystyrene (PS) films through heat treatment method and realized label-free and ultra-sensitive detection of dopamine (DA) using DA aptamer as probes immobilized on the AuNPs/3D crumpled graphene surface. The nanoscale deformation caused by thermal expansion effect of flexible graphene/PS can effectively reduce charge screening and the synergistic application of crumpled graphene and AuNPs can promote electron transfer and the graphene carrier concentration, leading to conductivity increase and hydrophilicity enhancement of 3D graphene. In addition, the binding of DA aptamer to DA will cause conformational changes of the aptamer molecule, which affects the charge transport properties of the sensor, thus improving its selectivity and stability. The biosensor can also easily distinguish interfering substances for DA detection in complex components (PBS, human urine, and fetal calf serum) with the detection limits as low as 60, 240 and 316 zM (10−19–10−11 M), respectively. DA released from exocytosis induced by K+ stimulation was selectively detected. The results show that the biosensor can be used as an excellent tool for ultrasensitive molecular recognition and detecting the effects of exogenous reagents on living cells, which is promising for clinical diagnosis and early disease prevention.

Highly selective colorimetric determination of glutathione based on sandwich-structured nanoenzymes composed of gold nanoparticle-coated molecular imprinted metal-organic frameworks.

Asandwich-structured composite nanoenzyme (NH2-MIL-101(Fe)@Au@MIP) was prepared using molecularly imprinted polymers, metal-organic frameworks, and gold nanoparticles and a highly selective glutathione (GSH) colorimetric sensor was constructed. The inner part of the composite nanoenzymes is a metal-organic framework loaded with gold nanoparticles (AuNPs), NH2-MIL-101(Fe)@Au, which has superior peroxidase-like activity compared with NH2-MIL-101(Fe). This is due to the surface plasmon resonance effect of AuNPs. GSH can form strong Au-S bonds with AuNPs, which can significantly reduce the enzymatic activity of NH2-MIL-101(Fe)@Au, thereby changing the absorbance at 450nm of the sensing system. The degree of change in absorbance is correlated with the concentration of GSH. In the outer part, the molecularly imprinted polymer with oxidized glutathione (GSSG) as a dummy template provided specific pores, which significantly improved the selectivity of the sensing system. The sensor showed good GSH sensing performance in the range 1 ~ 50μM with a lower limit of detection (LOD) of 0.231μM and good sensing performance in fetal bovine serum, indicating its highpotential for clinical diagnostic applications.

Characterization of Myosatellite Stem Cells from Black Bone Chicken and Effect of Different Animal Serums on Growth Performance

Isolation of muscle stem cells (myosatellite stem cells) of black bone chickens using enzymatic digestion, the pronase, and screening by low-speed centrifugation process combined with 40 μm cell strainer filtration showed that this method was able to separate chicken muscle stem cells efficiently. The results proved that the isolated cells were myosatellite stem cells expressing marker genes, c-met, Pax7, MyoD, and myogenin, by RT-PCR assay. Additionally, immunofluorescence staining with monoclonal antibodies confirmed the isolated cells were MSCs, with positive staining for canonical markers proteins: Pax7, Desmin, and actin. Evaluating the influence of fetal bovine serum (FBS), horse serum (HS), and chicken serum (CKS) supplements on MSC proliferation revealed that FBS emerged as the most effective despite its escalating cost and emerging need for alternatives. Notably, Chicken Serum (CKS) demonstrated potential as an alternative, closely following the cell proliferation rate achieved by FBS. Our findings affirm the efficacy of the devised isolation method and highlight the potential of using chicken serum in cell culture protocols. HIGHLIGHTS This research established a technique for isolating myosatellite stem cells from black bone chickens through enzymatic processes combined with gradient methods. Centrifugation aids in obtaining a purer isolation of these stem cells and allows for the evaluation of various animal serums’ effects. In cell cultures, the performance of chicken serum showed no significant difference compared to bovine serum. GRAPHICAL ABSTRACT