Fetal Bovine Serum Research Articles

Study on the feasibility of using livestock blood as a fetal bovine serum substitute for cultured meat.

Fetal bovine serum (FBS) accounts for the largest portion of the cost of cultured meat production or cell culture experiments and is highly controversial in terms of animal welfare because it is taken from the fetus of a pregnant cow during slaughtering. Nevertheless, FBS is the most important supplement in the cell culture manufacturing process. This study aimed to develop an FBS substitute from slaughterhouse waste blood to reduce the cost of FBS in cultured meat production through various experiments. Our study successfully demonstrated that adult livestock blood obtained from slaughterhouses can effectively replace FBS. Our substitute, when cultured with bovine myosatellite cells, demonstrated cell growth that was either equivalent to or superior to that of commercial FBS. In the process of muscle generation through differentiation, the substitutes from bovine and chicken formed 70%-75% more bovine muscle compared to the control group using FBS. Furthermore, using the FBS substitute can reduce cell culture costs by approximately 61% compared to using commercial FBS. Therefore, the groundbreaking FBS substitute will not only contribute to the development of technology to mass-produce cultured meat using livestock byproducts but will also lower the production cost of media for experimental cell culture or vaccine production.

AIE pyrene-based luminescent zinc MOF for selective and sensitive ATP and ADP sensing in water by analyte-induced structure decomposition

Adenosine triphosphate (ATP) is a natural and universal energy carrier that produces adenosine diphosphate (ADP) by releasing chemical energy through hydrolysis, which plays a key role in various biological processes. Hence, the design of probes for the selective ATP and ADP sensing among various nucleosides is significant. In this work, 4,4′-(pyrene-1,6-diyl)dibenzoic acid (H2PDBA) has been synthesized and shows an aggregation-induced emission (AIE) effect in a THF-H2O mixture at 10 % water fraction. Based on H2PDBA, a new zinc metal–organic framework (MOF), namely {[Zn(PDBA)]·2(CH3)2NH}n (Zn-MOF, PDBA = deprotonation of H2PDBA), has been constructed under hydrothermal conditions with a coordination-induced emission (CIE) effect. Significantly, Zn-MOF has been utilized to detect ATP and ADP in water with high selectivity and quick response time (within 1 min) by fluorescence quenching. The Ksv values and detection limits have been measured to be 7.83/8.78 × 103 M−1 and 0.82/1.71 μM for ATP and ADP, respectively. The sensing mechanism investigation indicates that the interactions between the ATP or ADP and Zn2+ nodes of the framework lead to the partial collapse of the framework, followed by the release of the ligand H2PDBA with weak luminescence in pure water. Moreover, Zn-MOF has successfully been applied for the determination of ATP and ADP with the recovery method by using fetal bovine serum samples.

Ultrastable and Redispersible Zwitterionic Bottlebrush Micelles for Drug Delivery.

Bottlebrush copolymers are increasingly used for drug delivery and biological imaging applications in part due to the enhanced thermodynamic stability of their self-assemblies. Herein, we discuss the effect of hydrophilic block chemistry on the stability of bottlebrush micelles. Amphiphilic bottlebrushes with zwitterionic poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and nonionic polyethylene glycol (PEG) hydrophilic blocks were synthesized by "grafting from" polymerization and self-assembled into well-defined spherical micelles. Colloidal stability and stability against disassembly were challenged under high concentrations of NaCl, MgSO4, sodium dodecyl sulfate, fetal bovine serum, and elevated temperature. While both types of micelles appeared to be stable in many of these conditions, those with a PMPC shell consistently surpassed their PEG analogs. Moreover, when repeatedly subjected to lyophilization/resuspension cycles, PMPC micelles redispersed with no apparent variation in size or dispersity even in the absence of a cryoprotectant; PEG micelles readily aggregated. The observed excellent stability of PMPC micelles is attributed to the low critical micelle concentration of the bottlebrushes as well as to the strong hydration shell caused by ionic solvation of the phosphorylcholine moieties. Zwitterionic micelles were loaded with doxorubicin, and higher loading capacity/efficiency, as well as delayed release, was observed with increasing side-chain length. Finally, hemocompatibility studies of PMPC micelles demonstrated no disruption to the red blood cell membranes. The growing concern regarding the immunogenicity of PEG-based systems propels the search for alternative hydrophilic polymers; in this respect and for their outstanding stability, zwitterionic bottlebrush micelles represent excellent candidates for drug delivery and bioimaging applications.

Co nanoparticles confined in N-doped hollow carbon nanospheres as multifunctional oxidase mimic for colorimetric detection of L-Cysteine and Hg2+

In contrast to peroxidase-mimicking nanozymes, oxidase mimics directly employ molecular oxygen (O2) as oxidant to generate reactive oxygen species (ROS). Generally, transition metal-based nanozymes exhibit poor oxidase-like activity. We herein constructed a colorimetric sensor based on Co-based oxidase mimic, where the Co nanoparticles were encapsulated in hollow N-doped carbon nanospheres (Co-N/C). Co nanoparticles confined catalyst effectively anchored the active part, which greatly influenced the catalytic activity of the oxidase-mimic nanozymes. Due to the antioxidative property of L-Cysteine (L-Cys) and the specific complexation between L-Cys and Hg2+, the rationally designed Co-N/C could be utilized to detect L-Cys and Hg2+. The proposed sensing platform exhibited wide linear ranges of 1–30 μM for L-Cys and 0.6–30 μM for Hg2+, respectively. The excellent selectivity of the constructed sensor showed the reliability and practicality for the assay of L-Cys and Hg2+ in fetal bovine serum. The spatial confinement can prevent the aggregation of Co nanoparticles, boosting the stability and catalytic performance in colorimetric sensing.

7260 Nuclear Corepressor 1 (NCoR1) plays important role in the regulation of insulin gene transcription in mouse Min6 pancreatic beta cells

Abstract Disclosure: H. Shimizu: None. Y. Horibata: None. I. Amano: None. M.J. Ritter: None. M. Ohyama: None. M. Domae: None. H. Sugimoto: None. A.N. Hollenberg: None. Background: Nuclear Corepressor 1 (NCoR1) is a transcriptional corepressor which has been recognized as an important player in the regulation of hepatic lipogenesis and hematopoiesis in mouse embryo. NCoR1 is also widely expressed in mouse insulin sensitive organs, for instance adipocyte, skeletal muscle, and hepatocytes. Mouse genetic deletion strategies of either NCoR1 or SMRT(NCoR2; an ortholog corepressor of NCoR1) demonstrated that each corepressor effects the insulin sensitivity in the tissues. However, the role of each corepressor in pancreatic beta cell is not still elucidated. Methods: To clarify this, we took the gene-targeting strategy for NCoR1 using CRISPR Cas-9 and made the pancreatic beta Min6 clone which lacks NCoR1 protein (Min6-NCoR1KO; NKO cells). For this study, wild type Min6 cells (WT) and NKO cells were cultured in the Dulbecco's DMEM medium with 10% fetal bovine serum for 4-5 days, and the expression analyses compared between two cell types were performed. Results: Both comprehensive proteomics and following expression analyses compared between WT and NKO cells demonstrated that NKO cells have significantly lower expression level of insulin (INS-1 and INS-2), accompanied with high expression of histone deacetylase 6 (HDAC6). For further analyses, we also made the Min6 clone which lacks SMRT(Min6-SMRTKO; SKO cells). Interestingly, SKO cell had higher insulin and lower HDAC6 levels, accompanied with significant up-regulation of NCoR1. Conclusion: Taken together, these data indicate that NCoR1 has dominant effects in the regulation of both histone deacetylation and insulin gene transcription in Min6 cells. Further investigations for the function of NCoR1 in the insulin gene transcription observed in Min6 cells will be needed. Presentation: 6/2/2024

Medium/Long Chain Hydrophobic Deep Eutectic Solvent-Based Liquid Phase Microextraction for the Determination of Rhein, Emodin, and Chrysophanol with Detection by High-Performance Liquid Chromatography (HPLC)

Medium/long-chain fatty acids/alcohols as hydrogen bond donors coupled with methyltrioctylammonium chloride were used to prepare hydrophobic deep eutectic solvents (DESs) that were employed for the extraction of the anthraquinones (rhein, emodin, and chrysophanol) from fetal bovine serum. Hydrophobic DES-based liquid-phase microextraction and high-performance liquid chromatography were developed. The most suitable combination was optimized for liquid-phase microextraction. Spiked fetal bovine serum was analyzed with satisfactory extraction recoveries.

4D-printed shape-programmable [H+]-responsive needles for determination of urea

Four-dimensional printing (4DP) technologies are revolutionizing the fabrication, functionality, and applicability of stimuli-responsive analytical devices. More practically, 4DP technologies are effective in fabricating devices with complex geometric designs and functions, and the degree of shape programming of 4D-printed stimuli-responsive devices can be optimized to become a reliable analytical strategy. Although shape-programming modes play a critical role in determining the analytical characteristics of 4D-printed stimuli-responsive sensing devices, the effect of shape-programming modes on the analytical performance of 4D-printed stimuli-responsive devices remains an unexplored subject. We employed digital light processing three-dimensional printing (3DP) with acrylate-based photocurable resins and 2-carboxyethyl acrylate (CEA)-incorporated photocurable resins for 4DP of the bending, helixing, and twisting needles. Upon immersion in samples with pH values above the pKa of CEA, the electrostatic repulsion among the dissociated carboxyl groups of polyCEA caused swelling of the CEA-incorporated part and [H+]-dependent shape programming. When coupling with the derivatization reaction of the urease-mediated hydrolysis of urea, the decline in [H+] induced shape programming of the needles, offering reliable determination of urea based on the shape-programming angles. After optimizing the experimental conditions, the helixing needles provided the best analytical performance, with the method's detection limit of 0.9 μM. The reliability of this analytical method was validated by determining urea in samples of human urine and sweat, fetal bovine serum, and rat plasma with spike analyses and comparing these results with those obtained from a commercial assay kit. Our demonstration and analytical results suggest the importance of optimizing the shape-programming modes to improve the analytical performance of 4D-printed stimuli-responsive shape-programming sensing devices and emphasize the benefits and applicability of 4DP technologies in advancing the development and fabrication of stimuli-responsive sensing devices for chemical sensing and quantitative chemical analyses.

Schistosoma japonicum infection-mediated downregulation of lncRNA Malat1 contributes to schistosomiasis hepatic fibrosis by the Malat1/miR-96/Smad7 pathway

BackgroundSchistosoma japonicum infection causes hepatic fibrosis, a primary cause of morbidity and mortality associated with the disease, and effective treatments are still lacking. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenic process of various tissue fibroses. However, the role of lncRNAs in schistosomiasis hepatic fibrosis (HF) is poorly understood. Understanding the role of lncRNAs in schistosomiasis HF will enhance knowledge of disease processes and aid in the discovery of therapeutic targets and diagnostic biomarkers.MethodsDifferentially expressed lncRNA profiles in primary hepatic stellate cells (HSCs) of mice infected with S. japonicum were identified using high-throughput lncRNA sequencing. Primary HSCs were isolated from infected mice using collagenase digestion and density-gradient centrifugation, cultured in DMEM with 10% fetal bovine serum. Dual-luciferase reporter assays, nuclear cytoplasm fractionation and RIP assays were employed to assess the relationship between Malat1 and miRNA-96. Malat1 lentivirus and ASO-Malat1 were constructed for forced expression and downregulated expression of Malat1. The Malat1-KO mouse was constructed by CRISPR/Cas9 technology. Pathological features of the liver were evaluated by hematoxylin-eosin (HE), Masson’s trichrome staining and immunohistochemistry (IHC). The expression levels of fibrosis-related genes were determined by quantitative real-time PCR (qRT-PCR) and Western blot.ResultsA total of 1561 differentially expressed lncRNAs were identified between infected and uninfected primary HSCs. Among the top altered lncRNAs, the downregulated Malat1 was observed in infected HSCs and verified by qPCR. Treatment of infected mice with praziquantel (PZQ) significantly increased the Malat1 expression. Elevated Malat1 expression in infected primary HSC reduced the expressions of profibrogenic genes, whereas Malat1 knockdown had the opposite effect. Moreover, Malat1 was found to interact with miR-96, a profibrotic miRNA, by targeting Smad7. Forced Malat1 expression reduced miR-96 levels in infected primary HSCs, attenuating fibrogenesis and showing negative correlation between Malat1 expression and the expression levels of miR-96 and profibrogenic genes α-SMA and Col1α1. Notably, in Malat1-KO mice, knockout of Malat1 aggravates schistosomiasis HF, while restored Malat1 expression in the infected HSCs reduced the expression of profibrogenic genes.ConclusionsWe demonstrate that lncRNA is involved in regulation of schistosomiasis HF. Elevated lncRNA Malat1 expression in infected HSCs reduces fibrosis via the Malat1/miR-96/Smad7 pathway, thus providing a novel therapeutic target for schistosomiasis HF. Furthermore, Malat1 expression is sensitive to PZQ treatment, thus offering a potential biomarker for assessing the response to chemotherapy.Graphical abstract

Comparative Analysis of Serum and Serum-Free Medium Cultured Mesenchymal Stromal Cells for Cartilage Repair.

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair.

Phenotypic characteristics of adipocyte-like cells generated from C2C12 myoblasts cultured with chicken serum

The aim of this study was to clarify the transcriptional and metabolic characteristics of C2C12 myoblasts cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 20% chicken serum (CHS) (C2C12-CHS cells) compared with C2C12 myoblasts cultured in DMEM containing 20% fetal bovine serum (FBS) (C2C12-FBS cells). After 3 days of culture, C2C12-CHS cells showed a marked accumulation of lipid droplets, accompanied by increased expression levels of brown adipocyte-related genes (i.e., Bmp7, Prdm16, Ucp1, Cidea, Pgc1α, Cox7a1, Cox8, and β3-adorenoceptor). Furthermore, stimulation of β3-adorenoceptor by its selective agonist, mirabegron, increased the mRNA expression of Ucp1 and Pgc1α in C2C12-CHS cells. Wide-targeted metabolomic analysis performed by gas chromatography-tandem mass spectrometry revealed that the metabolic profile of C2C12-CHS cells was obviously different to that of C2C12-FBS cells. Additionally, the metabolomic analysis indicated that β3-adrenoceptor stimulation by mirabegron upregulated energy metabolism in C2C12-CHS cells as seen in brown adipocytes. These results suggest that C2C12-CHS cells may differentiate into brown adipocyte-like cells, accompanied by increased functional β3-adrenoceptor.

Cellular uptake of CPX-351 by scavenger receptor class B type 1-mediated nonendocytic pathway

The proper uptake of drugs in liposome formulations into target cells markedly impacts therapeutic efficacy. The protein corona (PC), formed by the adsorption of serum proteins onto the liposome surface, binds to specific surface receptors of target cells, influencing the uptake pathway. We investigated the uptake pathway into leukemia cells based on PC analysis of CPX-351, a liposome containing cytarabine and daunorubicin in a fixed 5:1 synergistic molar ratio. The PC of CPX-351 mixed with fetal bovine serum was analyzed by nanoflow liquid chromatography-tandem mass spectrometry. CPX-351 uptake in HL-60, K562, and THP-1 leukemia cell lines, was measured by flow cytometry using daunorubicin fluorescence. The major components of CPX-351 PC were apolipoproteins A-I and A-II, which bind to scavenger receptor class B type 1 (SR-BI), a nonendocytic pathway that takes up only liposome contents. SR-BI was expressed in each cell, and its expression correlated with CPX-351 uptake. The uptake was significantly decreased by the inhibition of clathrin-mediated endocytosis and macropinocytosis. Additionally, blocks lipid transport-1 (BLT-1), a selective inhibitor of SR-BI, decreased the uptake; however, high-dose BLT-1 addition significantly increased the uptake, which was more strongly inhibited by macropinocytosis suppression compared with clathrin-mediated endocytosis. BLT-1 enhances the binding of SR-BI to liposomes in a dose-dependent manner. These findings indicate that the enhancement of binding between SR-BI and CPX-351 activates different pathways, such as macropinocytosis, distinct from CPX-351 alone. SR-BI may be a biomarker for CPX-351 therapy, and the combination of CPX-351 with high-dose BLT-1 may augment therapeutic efficacy.

Optimizing the Procedure for Manufacturing Clinical-grade Genetically Manipulated NK cells for Adoptive Immunotherapy

BackgroundEx vivo expanded natural killer (NK) cells hold significant potential as antitumor effector cells for adoptive immunotherapy. However, producing clinical-grade genetically modified NK cells in sufficient quantities presents a considerable challenge. MethodsWe tested RPMI 1640, KBM581, SCGM, NK MACS, X-VIVO 15, and AIM-V, each supplemented with fetal bovine serum (FBS), human AB serum, human platelet lysate (HPL), or Immune Cell Serum Replacement (SR) combined with feeder cells, to produce cytotoxic NK cells. Subsequent analyses were conducted to assess cell viability, expansion folds, cytotoxicity, immunophenotype, and transcriptome profile of NK cells under certain conditions. Furthermore, transfer plasmids varying in transgene size, promoter elements, backbones and packaging plasmids with different envelopes were used to transduce NK cells and differences in transduction efficiency were compared. Nucleofection was performed every two days from Day 0 to Day 12 to determine the optimal time window for gene editing. ResultsNK cells cultured in KBM581 medium supplemented with SR exhibited the best expansion, achieving over 5000-fold increase within two weeks and exceeding 25,000-fold expansion within three weeks. Additionally, NK cells cultured in KBM581 medium with human AB serum demonstrated the highest cytolytic activities and exhibited higher expression of NKp30, 2B4, PRF1, Granzyme B, and IL2RG. Baboon envelope (BaEV) pseudotyped lentivirus outperformed BaEV-Vesicular Stomatitis Virus type-G (VSVG) hybrid envelope lentivirus, achieving robust NK cell transduction. Additionally, efficient gene knockout efficiency was achieved in NK cells on day 4 to day 6 post feeder cell activation using LONZA DN-100 program, which can strike a balance between editing efficiency and cell expansion. ConclusionsThis research presents a Good Manufacturing Practice (GMP)-compliant protocol utilizing a feeder cell expansion system for the large-scale production of highly cytotoxic Natural Killer (NK) cells. The protocol facilitates genetic modification of these cells, positioning them as promising candidates for universal therapeutic applications in immunotherapy.

A dual-template molecularly imprinted electrochemical sensor assisted with BO-XGBoost algorithm for simultaneous determination of dopamine and acetaminophen

The simultaneous detection of dopamine (DA) and acetaminophen (AP) is crucial for diagnosing and treating related mental disorders. However, accurately determining DA and AP in biological samples remains challenging due to the interference from other biomolecules, such as ascorbic acid (AA), uric acid (UA), epinephrine (EP), etc. Here, we present a dual-template molecularly imprinted electrochemical sensor for the selective recognition of DA and AP. Reduced graphene(rGO)-gold nanoparticles(AuNPs) composite were utilized to enhance the effective area and increase the electron transfer rate of glassy carbon electrode(GCE), thus the electrochemical response signals were amplified. This sensor combined the advantages of nanocomposites and molecularly imprinted polymer (MIP) to achieve highly sensitive and selective detection of DA and AP. However, decreases in the current response of some analytes and variations in relationships of the peak current versus analyte concentration will occur due to adsorption competition on active sites in multianalyte mixtures, rendering the detection range constrained and traditional linear fitting methods inaccurate for predicting analyte concentrations. Therefore, a concentration prediction model based on XGBoost was developed for the sensor to achieve the reliable multi-component determination of DA and AP within the identical measurement range as individual detection. In this model, nine characteristic parameters were extracted from the differential pulse voltammetry (DPV) response curve and Bayesian optimization (BO) was adopted for automatic hyperparameter search, thereby the prediction errors were reduced and the generalization ability was improved. Experiments indicated that the MIP/AuNPs/rGO/GCE exhibited a wide detection range of 2–240 μM and 3–240 μM for DA and AP, with detection limits down to 0.26 μM and 0.33 μM, respectively. In addition, the intelligent MIP/AuNPs/rGO/GCE sensor based on BO-XGBoost model provides more accurate prediction results for untrained concentration combinations obtained from fetal bovine serum samples, indicating that the proposed detection scheme holds potential in future clinical diagnosis.

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.

Establishment of lung cancer cell lines and tumorigenesis in mice from malignant pleural effusion in patients with lung cancer.

Lung cancer was often diagnosed by malignant pleural effusion (MPE). Excessive MPE is generally discarded. The establishment of cell lines and the generation of cancer mouse models have the potential to be directly linked to personalized medicine. This study aimed to establish cell lines and generate mouse models using MPE. Cells derived from 5 mL of MPE were cultured in several conditions, including 100% MPE supernatant and Roswell Park Memorial Institute-1640 supplemented with 10% fetal bovine serum (FBS) or 10% MPE supernatant. When steady cell growth was observed, fewer cells were spread and the colonies were selected to establish the cell line. Cells derived from 10 mL of MPE were inoculated subcutaneously into non-obese diabetic-severe combined immunodeficiency (NOD-scid) and NOD.Cg-Prkdcscid Il2rgtmlWjl /SzJ (NSG) mice to assess tumorigenic potential. MPEs were obtained from 28 lung cancer patients, 23 of whom had adenocarcinoma. Cell lines were established from 5 patients (18%). Tumorigenesis was observed in 6 of 28 cases (21%). However, in 7 cases, the mice (7 NSG and 1 NOD-scid mice) became progressively weaker, lost their hair, and died within 12 weeks without tumorigenesis. The appearance and pathological findings were consistent with graft-versus-host disease. Cell line establishment and tumorigenesis in mice were associated with a lower response to first-line therapy and poorer prognosis of patients. When MPEs were simply utilized, the cell line establishment rate was 18% and the engraftment rate in mice was 21%. The prognosis of patients who underwent cell line establishment and engraftment in mice was poor.

Primary isolation and cultivation of retinal Müller cells in Sprague-Dawley rats

Objective: Müller cells in the retina are one of the most important supporting cells in the retina, belonging to the category of glial cells. This study used gradient low concentration enzyme repeated digestion method to isolate Müller cells in the retina of Sprague-Dawley (SD) rats. Methods: Retinal Müller cells were harvested in retina from newborn SD rats. Initially, the retinal tissue was carefully dissected, digested with trypsin at 37°C for 15 minutes, and filtered. Following the centrifugation and resuspension in DMEM/F12 medium with 10% fetal bovine serum, cells were cultured in a gelatin-coated plate. After reaching confluence, cells were digested with low-concentration trypsin to improve purity. Immunofluorescence staining was performed using GFAP antibody staining and DAPI counterstain to confirm cell identity. Results: Retinal Müller cells began adhering and forming clusters within 24 hours, with confluence achieved by days 3-6. After subculturing, the cells exhibited uniform morphology, elongated shapes, and round/oval nuclei. Immunofluorescence on day 7 showed over 80% GFAP-positive reaction, confirming their glial nature and the successful isolation of Müller cells. Conclusion: This method is effective for obtaining a stable population of Müller cells, which can be used for further research on retina.

Grifola frondosa extract as a fetal bovine serum supplement for the culture of bovine muscle satellite cells under low serum conditions

Expensive fetal bovine serum (FBS) is a major obstacle to the production of cultivated meat. However, because FBS substitutes do not sufficiently induce cell proliferation, a good alternative is to reduce the amount of FBS and use ingestible additives to promote cell proliferation. In this study, Grifola frondosa extract (GFE) was used to investigate its potential as an additive to promote myogenesis of bovine muscle satellite cells from Hanwoo cattle under low serum conditions (10 % FBS). GFE treated with 10 % FBS only during the proliferation period not only increased cell proliferation and related biomarkers in a concentration-dependent manner (0.78–12.5 μg/mL), but also increased cell differentiation. Additionally, differentiation was promoted when cells were with GFE treated only during the differentiation period. Especially GFE at 12.5 µg/mL induced significantly higher proliferation and differentiation rates than 20 % FBS medium. In particular, compared to treatment alone in the proliferation or differentiation periods, GFE treatment in both periods contributed to an increase in the differentiation rate and significantly enhanced total protein production. The integration of GFE into cultivated meat production presents a promising approach to reducing FBS dependence, lowering costs, and enhancing scalability, aligning with sustainability and consumer acceptance goals.

Integration of MUTZ-Langerhans cells into a 3D full-thickness skin equivalent and influences of serum reduction and undefined medium supplements on differentiation

The MUTZ-3 cell line is a surrogate for Langerhans cells (LCs) employed in New Approach Methodologies for assessing the skin sensitizing potential of chemicals. However, MUTZ-3 cells must first be differentiated to achieve the LC-typical phenotype. As all protocols use high fetal calf serum (FCS) concentrations, we aimed at reducing, or even replacing FCS, while maintaining MUTZ-LC characteristics. Additionally, we assessed the impact of the poorly defined 5637-conditioned medium (5637CM) on MUTZ-LC differentiation.With reducing the FCS content by 75 %, the desired differentiation status was achieved after 7 instead of 14 days, identified by elevated CD207 and CD1a expression. Culture with Ultroser G, a synthetic surrogate for FCS, resulted in an insufficient number of MUTZ-LCs. 5 % FCS-differentiated MUTZ-LCs could be activated with DNCB, an extreme sensitizer, as demonstrated by increased CD83 expression. 5637CM did not affect MUTZ-LC differentiation and is therefore not needed as a supplement.For their intended role in an immunocompetent skin model to assess the sensitizing potential of chemicals, MUTZ-LCs were successfully integrated into the Phenion® Full-Thickness skin model, as demonstrated by CD1a expression. These results are important steps towards medium standardization and the generation of an immunocompetent skin model according to the 3R principles.

Assessing the biocompatibility and stability of CeO2 nanoparticle conjugates with azacrowns for use as radiopharmaceuticals.

The application of nanoparticles is promising for the purposes of nuclear medicine due to the possibilities of using them as vectors and transporters of radionuclides. In this study, we have successfully synthesised conjugates of CeO2 nanoparticles and azacrown ligands. Then, the radiolabelling conditions with radionuclides 65Zn, 44Sc and 207Bi were selected and the kinetic stability of the complexes in biologically significant media was evaluated. Optimum conditions for CeO2-APTES-L and CeO2-APTES-DOTA labelling were found: 0.1 g l-1 conjugate and 10-9 M metal cations at 90 °C for complexes with [65Zn]Zn2+, [44Sc]Sc3+ and [207Bi]Bi3+. CeO2-APTES-L-44Sc (radiochemical purity more than 90%) was stable in fetal bovine serum. The obtained results enabled us to choose the most promising complex for biomedical applications for carrying out in vitro and in vivo biodistribution research. Nanoceria and its derivative showed no obvious toxicity to human endothelial cells EA.hy926. Then, the in vivo stability of the studied scandium complex was demonstrated. Taken together, our studies show that functionalised cerium oxide nanoparticles lead to stable radiolabelled nanosystems that may be used for targeted drug delivery, diagnosis and treatment of oncological diseases.

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.