Protocol to identify S-acylated proteins in hippocampal neurons using ω-alkynyl fatty acid analogs and click chemistry.

S-acylation, commonly palmitoylation, is the addition of fatty acids to cysteines to regulate protein localization and function. S-acylation detection has been hampered by limited sensitivity and selectivity in low-protein, costly samples like cultured neurons. Here, we present a protocol for sensitive and selective bioorthogonal labeling and click-chemistry-based detection of S-acylated proteins in primary hippocampal neurons. We describe steps for metabolically labeling neurons with alkynyl fatty acid, click chemistry, NeutrAvidin-based capture, and elution with hydroxylamine.

Analysis of current technology status for the industrialization of cultured meat.

Cultured meat is expected to become an important material for future food production; however, contrary to initial expectations, the full-scale industrialization of cultured meat is slow and the actual level and opened technology amount is very limited. This study reviews the publicly available technologies of cultured meat and suggests future developmental directions and research agenda. As a result of analyzing papers, patents, and press releases published over the past 10 years, it was found that cultured meat production technology is still at the prototype production level. This is because most papers published are about culture medium and scaffold development, culture conditions, and there is almost no research on finished cultured meat products. Worldwide, most of the filed patents are for producing cultured meat principles; most of them do not use food-grade materials and are not economically feasible for industrialization. Therefore, future research on the industrialization of cultured meat should focus on effective acquisition technologies for satellite cells; cell lineage and undifferentiated state maintenance technologies; the development of serum-free media and culture devices; the prevention of genetic modification, safety verification, and mass production. Furthermore, basic research on mechanisms and influencing factors related to cultured meat production is warranted.

Effect of Heat Treatment on the Material Property and Cell Viability of Wire Arc Additively Manufactured Ti6Al4 V.

Wire arc additive manufacturing (WAAM) holds promise for producing medium to large industrial components. Application of WAAM in the manufacturing of biomedical materials has not yet been evaluated. The current study addresses two key research questions: first, the suitability of the WAAMed Ti6Al4V alloy for biomedical applications, and second, the effect of Ti6Al4V's constituents (α and ß phases) on the cell viability. The WAAMed Ti6Al4V alloy was fabricated (as-deposited: AD) using a metal inert gas (MIG)-based wire arc system using an in-house designed shielding chamber filled with argon. Subsequently, samples were subjected to solution treatment (950 °C for 1 h), followed by aging at 480 °C (T1), 530 °C (T2), and 580 °C (T3) for 8 h and subsequent normalization to ambient conditions. Microstructural analysis revealed ∼45.45% of α'-Ti colonies in the as-deposited samples, reducing to 23.26% postaging at 580 °C (T3). The α-lath thickness and interstitial oxygen content in the sample were observed to be proportional to the aging temperature, peaking at 580 °C (T3). Remarkably, during tribocorrosion analysis in simulated body fluid, the 580 °C-aged T3 sample displayed the lowest corrosion rate (7.9 µm/year) and the highest coefficient of friction (CoF) at 0.58, showing the effect of increasing oxygen content in the alloy matrix. Cell studies showed significant growth at 530 and 580 °C by day 7, correlated with higher oxygen content, while other samples had declining cell density. Additionally, optimal metallurgical property ranges were identified to enhance the Ti6Al4V alloy's biocompatibility, providing crucial insights for biomedical implant development.

Gel microspheres enhance the stemness of ADSCs by regulating cell-ECM interaction.

The gel microsphere culture system (GMCS) showed various advantages for mesenchymal stem cell (MSC) expansion and delivery, such as high specific surface area, small and regular shape, extensive adjustability, and biomimetic properties. Although various technologies and materials have been developed to promote the development of gel microspheres, the differences in the biological status of MSCs between the GMCS and the traditional Petri dish culture system (PDCS) are still unknown, hindering gel microspheres from becoming a culture system as widely used as petri dishes. In the previous study, an excellent "all-in-one" GMCS has been established for the expansion of human adipose-derived MSCs (hADSCs), which showed convenient cell culture operation. Here, we performed transcriptome and proteome sequencing on hADSCs cultured on the "all-in-one" GMCS and the PDCS. We found that hADSCs cultured in the GMCS kept in an undifferentiation status with a high stemness index, whose transcriptome profile is closer to the adipose progenitor cells (APCs) in vivo than those cultured in the PDCS. Further, the high stemness status of hADSCs in the GMCS was maintained through regulating cell-ECM interaction. For application, bilayer scaffolds were constructed by osteo- and chondro-differentiation of hADSCs cultured in the GMCS and the PDCS. The effect of osteochondral regeneration of the bilayer scaffolds in the GMCS group was better than that in the PDCS group. This study revealed the high stemness and excellent functionality of MSCs cultured in the GMCS, which promoted the application of gel microspheres in cell culture and tissue regeneration.

Knockout cancer by nano-delivered immunotherapy using perfusion-aided scaffold-based tumor-on-a-chip.

Cancer is a multifactorial disease produced by mutations in the oncogenes and tumor suppressor genes, which result in uncontrolled cell proliferation and resistance to cell death. Cancer progresses due to the escape of altered cells from immune monitoring, which is facilitated by the tumor's mutual interaction with its microenvironment. Understanding the mechanisms involved in immune surveillance evasion and the significance of the tumor microenvironment might thus aid in developing improved therapies. Although in vivo models are commonly utilized, they could be better for time, cost, and ethical concerns. As a result, it is critical to replicate an in vivo model and recreate the cellular and tissue-level functionalities. A 3D cell culture, which gives a 3D architecture similar to that found in vivo, is an appropriate model. Furthermore, numerous cell types can be cocultured, establishing cellular interactions between TME and tumor cells. Moreover, microfluidics perfusion can provide precision flow rates, thus simulating tissue/organ function. Immunotherapy can be used with the perfused 3D cell culture technique to help develop successful therapeutics. Immunotherapy employing nano delivery can target the spot and silence the responsible genes, ensuring treatment effectiveness while minimizing adverse effects. This study focuses on the importance of 3D cell culture in understanding the pathophysiology of 3D tumors and TME, the function of TME in drug resistance, tumor progression, and the development of advanced anticancer therapies for high-throughput drug screening.

Protocol for mapping physiological DSBs using in-suspension break labeling in situ and sequencing.

Physiological double-stranded breaks (DSBs) are a major source of genomic instability. Here, we present a protocol for mapping physiological DSBs by in-suspension break labeling in situ and sequencing (sBLISS) in a single-nucleotide resolution. We describe steps for cell fixation, labeling of DSBs, DNA isolation followed by in vitro transcription (IVT), reverse transcription, and library preparation. sBLISS provides a map of DSBs over the genome and can be used to study the role of different factors in DSB formation. For complete details on the use and execution of this protocol, please refer to Hidmi et al.1.

Protocol to develop human alveolar macrophage-like cells from mononuclear cells or purified monocytes for use in respiratory biology research.

Human alveolar macrophages are a unique myeloid subset critical for understanding pulmonary diseases and are difficult to access. Here, we present a protocol to generate human alveolar macrophage-like (AML) cells from fresh peripheral blood mononuclear cells or purified monocytes. We describe steps for cell isolation, incubation in a defined cocktail of pulmonary surfactant and lung-associated cytokines, phenotype analysis, and validation with human alveolar macrophages. We then detail procedures for quality control and technical readouts for monitoring microbial response. For complete details on the use and execution of this protocol, please refer to Pahari et al.1 and Neehus et al.2.

Cell Culture Models for Hepatitis B and D Viruses Infection: Old Challenges, New Developments and Future Strategies.

Chronic Hepatitis B and D Virus (HBV and HDV) co-infection is responsible for the most severe form of viral Hepatitis, the Hepatitis Delta. Despite an efficient vaccine against HBV, the HBV/HDV infection remains a global health burden. Notably, no efficient curative treatment exists against any of these viruses. While physiologically distinct, HBV and HDV life cycles are closely linked. HDV is a deficient virus that relies on HBV to fulfil is viral cycle. As a result, the cellular response to HDV also influences HBV replication. In vitro studying of HBV and HDV infection and co-infection rely on various cell culture models that differ greatly in terms of biological relevance and amenability to classical virology experiments. Here, we review the various cell culture models available to scientists to decipher HBV and HDV virology and host-pathogen interactions. We discuss their relevance and how they may help address the remaining questions, with one objective in mind: the development of new therapeutic approaches allowing viral clearance in patients.

Mass Production of Rg1-Loaded Small Extracellular Vesicles Using a 3D Bioreactor System for Enhanced Cardioprotective Efficacy of Doxorubicin-Induced Cardiotoxicity.

BACKGROUND: Small extracellular vesicles (sEVs) obtained from human umbilical cord mesenchymal stromal cells (MSCs) have shown cardioprotective efficacy in doxorubicin-induced cardiotoxicity (DIC). However, their clinical application is limited due to the low yield and high consumption. This study aims to achieve large-scale production of sEVs using a three-dimensional (3D) bioreactor system. In addition, sEVs were developed to deliver Ginsenoside Rg1 (Rg1), a compound derived from traditional Chinese medicine, Ginseng, that has cardioprotective properties but limited bioavailability, to enhance the treatment of DIC. METHODS: The 3D bioreactor system with spinner flasks was used to expand human umbilical cord MSCs and collect MSC-conditioned medium. Subsequently, sEVs were isolated from the conditioned medium using differential ultra-centrifugation (dUC). The sEVs were loaded with Ginsenoside Rg1 by electroporation and evaluated for cardioprotective efficacy using Cell Counting Kit-8 (CCK-8) analysis, Annexin V/PI staining and live cell count of H9c2 cells under DIC. RESULTS: Using the 3D bioreactor system with spinner flasks, the expansion of MSCs reached ~600 million, and the production of sEVs was up to 2.2 × 1012 particles in five days with significantly reduced bench work compared to traditional 2D flasks. With the optimized protocol, the Ginsenoside Rg1 loading efficiency of sEVs by electroporation was ~21%, higher than sonication or co-incubation. Moreover, Rg1-loaded sEVs had attenuated DOX-induced cardiotoxicity with reduced apoptosis compared to free Ginsenoside Rg1 or sEVs. CONCLUSIONS: The 3D culture system scaled up the production of sEVs, which facilitated the Rg1 delivery and attenuated cardiomyocyte apoptosis, suggesting a potential treatment of DOX-induced cardiotoxicity.

EGFR and PI3K Signalling Pathways as Promising Targets on Circulating Tumour Cells from Patients with Metastatic Gastric Adenocarcinoma.

The prognosis for metastatic gastric adenocarcinoma (mGAC) remains poor. Gene alterations in receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and their downstream effectors including catalytic subunit alpha of the phosphatidylinositol 3-kinase (PIK3CA) are common in mGAC. Targeted RTK and phosphatidylinositol-3-kinase (PI3K) treatments have demonstrated clinical benefits in other solid tumours and are key potential targets for clinical development against mGAC given the presence of recurrent alterations in these pathways. Furthermore, combination RTK/PI3K treatments may overcome compensatory mechanisms that arise using monotherapies, leading to improved patient outcomes. Herein, we investigated RTK/PI3K single and combination drug responses against our unique human mGAC-derived PIK3CA gain-of-function mutant, human epidermal growth factor receptor 2 (HER2)-negative, EGFR-expressing circulating tumour cell line, UWG02CTC, under two- and three-dimensional culture conditions to model different stages of metastasis. UWG02CTCs were highly responsive to the PI3K p110α-subunit targeted drugs PIK-75 (IC50 = 37.0 ± 11.1 nM) or alpelisib (7.05 ± 3.7 µM). Drug sensitivities were significantly increased in 3D conditions. Compensatory MAPK/ERK pathway upregulation by PI3K/Akt suppression was overcome by combination treatment with the EGFR inhibitor gefitinib, which was strongly synergistic. PIK-75 plus gefitinib significantly impaired UWG02CTC invasion in an organotypic assay. In conclusion, UWG02CTCs are a powerful ex vivo mGAC drug responsiveness model revealing EGFR/PI3K-targeted drugs as a promising combination treatment option for HER2-negative, RAS wild-type mGAC patients.

Development and evaluation of a chicken embryo fibroblast cell culture based live attenuated Indian strain duck plague vaccine.

Duck plague (DP) is an acute, contagious and fatal disease, caused by duck enteritis virus (DEV), with worldwide distribution causing several outbreaks and posing severe economic losses. The present study was carried out with a goal of development of a live attenuated cell culture based DP vaccine using an Indian strain of DEV and evaluation of its safety, efficacy along with complete genome analysis. The live attenuated DP vaccine (DPvac/IVRI-19) was developed by serial propagation of a virulent isolate of DEV (DEV/India/IVRI-2016) in the chicken embryo fibroblast (CEF) primary cell culture. Adaptation of DEV in CEF cell culture was indicated by more rapid appearance of cytopathic effects (CPE) and gradual increase of virus titre, which reached up to 107.5 TCID50/mL after 41 passages. The safety, immunogenicity and efficacy of the vaccine were determined by immunization trials in ducklings. The DPvac/IVRI-19 was found to be avirulent and completely safe in the ducklings. Further, the vaccine induced both humoral and cell mediated immune responses and afforded 100% protection against the virulent DEV challenge. A comparison of the whole genome of DPvac/IVRI-19 (MZ911871) and DEV/India/IVRI-2016 (MZ824102) revealed significant number of mutations, which might be associated with viral attenuation. Phylogenetic tree of DEV/India/IVRI-2016 revealed its evolutionary relationship with other DEV isolates, but it formed a separate cluster with certain unique mutations. Thus, with the proven safety and 100% efficacy, the DPvac/IVRI-19 is suitable for large scale production with precisely pure form of vaccine and has potential utility at national and global levels.

Activation of the Anaphase Promoting Complex Restores Impaired Mitotic Progression and Chemosensitivity in Multiple Drug-Resistant Human Breast Cancer.

The development of multiple-drug-resistant (MDR) cancer all too often signals the need for toxic alternative therapy or palliative care. Our recent in vivo and in vitro studies using canine MDR lymphoma cancer cells demonstrate that the Anaphase Promoting Complex (APC) is impaired in MDR cells compared to normal canine control and drug-sensitive cancer cells. Here, we sought to establish whether this phenomena is a generalizable mechanism independent of species, malignancy type, or chemotherapy regime. To test the association of blunted APC activity with MDR cancer behavior, we used matched parental and MDR MCF7 human breast cancer cells, and a patient-derived xenograft (PDX) model of human triple-negative breast cancer. We show that APC activating mechanisms, such as APC subunit 1 (APC1) phosphorylation and CDC27/CDC20 protein associations, are reduced in MCF7 MDR cells when compared to chemo-sensitive matched cell lines. Consistent with impaired APC function in MDR cells, APC substrate proteins failed to be effectively degraded. Similar to our previous observations in canine MDR lymphoma cells, chemical activation of the APC using Mad2 Inhibitor-1 (M2I-1) in MCF7 MDR cells enhanced APC substrate degradation and resensitized MDR cells in vitro to the cytotoxic effects of the alkylating chemotherapeutic agent, doxorubicin (DOX). Using cell cycle arrest/release experiments, we show that mitosis is delayed in MDR cells with elevated substrate levels. When pretreated with M2I-1, MDR cells progress through mitosis at a faster rate that coincides with reduced levels of APC substrates. In our PDX model, mice growing a clinically MDR human triple-negative breast cancer tumor show significantly reduced tumor growth when treated with M2I-1, with evidence of increased DNA damage and apoptosis. Thus, our results strongly support the hypothesis that APC impairment is a driver of aggressive tumor development and that targeting the APC for activation has the potential for meaningful clinical benefits in treating recurrent cases of MDR malignancy.

Unraveling the Impact of Extracellular Vesicle-Depleted Serum on Endothelial Cell Characteristics over Time.

Extracellular vesicles (EVs) are produced by all kinds of cells, including endothelial cells. It has been observed that EVs present in fetal bovine serum (FBS), broadly used in cell culture, can be a confounding factor and lead to misinterpretation of results. To investigate this phenomenon, human brain microvascular endothelial cells (HBMECs) were cultured for 2 or 24 h in the presence of EV-depleted FBS (EVdS). Cell death, gene and protein expression, and the presence of EVs isolated from these cells were evaluated. The uptake of EVs, intercellular adhesion molecule 1 (ICAM-1) expression, and monocyte adhesion to endothelial cells exposed to EVs were also evaluated. Our results revealed higher apoptosis rates in cells cultured with EVdS for 2 and 24 h. There was an increase in interleukin 8 (IL8) expression after 2 h and a decrease in interleukin 6 (IL6) and IL8 expression after 24 h of culture. Among the proteins identified in EVs isolated from cells cultured for 2 h (EV2h), several were related to ribosomes and carbon metabolism. EVs from cells cultured for 24 h (EV24h) presented a protein profile associated with cell adhesion and platelet activation. Additionally, HBMECs exhibited increased uptake of EV2h. Treatment of endothelial cells with EV2h resulted in greater ICAM-1 expression and greater adherence to monocytes than did treatment with EV24h. According to our data, HBMEC cultivated with EVdS produce EVs with different physical characteristics and protein levels that vary over time.

Shape of scaffold controlling the direction of cell migration.

Cell migration plays an important role in the development and maintenance of multicellular organisms. Factors that induce cell migration and mechanisms controlling their expression are important for determining the mechanisms of factor-induced cell migration. Despite progress in the study of factor-induced cytotaxis, including chemotaxis and haptotaxis, precise control of the direction of cell migration over a wide area has not yet been achieved. Success in this area would update the cell migration assays, superior cell separation technologies, and artificial organs with high biocompatibility. The present study therefore sought to control the direction of cell migration over a wide area by adjusting the three-dimensional shape of the cell scaffold. The direction of cell migration was influenced by the shape of the cell scaffold, thereby optimizing cell adhesion and protrusion. Anisotropic arrangement of these three-dimensional shapes into a periodic structure induced unidirectional cell migration. Three factors were required for unidirectional cell migration: 1) the sizes of the anisotropic periodic structures had to be equal to or lower than the size of the spreading cells, 2) cell migration was restricted to a runway approximately the width of the cell, and 3) cells had to be prone to extension of long protrusions in one direction. Because the first two factors had been identified previously in studies of cell migration in one direction using two-dimensional shaped patterns, these three factors are likely important for the mechanism by which cell scaffold shapes regulate cell migration.

Post-transcriptional regulation of the T-box gene midline via the 3'UTR in Drosophila is complex and cell and tissue-dependent.

The T-box proteins have a 180-230 amino acid DNA-binding domain, first reported in the Brachyury (T) protein. It is highly conserved among metazoans. They regulate a multitude of cellular functions in development and disease. Here, we report post-transcriptional and translational regulation of midline (mid), a Tbx member in Drosophila. We found that the 3'UTR of mid has mRNA degradation elements and AT-rich sequences. In Schneider S2 cells, mid mRNA could be detected only when the transgene was without the 3'UTR. Similarly, the 3'UTR linked to the Renilla Luciferase reporter significantly reduced the activity of the Luciferase. Whereas deleting only the degradation elements from the 3'UTR resulted in reduced activity but not as much. Overexpression of mid in MP2, an embryonic neuroblast, showed no significant difference in the levels of mid mRNA between the two transgenes, with and without the 3'UTR, indicating the absence of post-transcriptional regulation of mid in MP2. Moreover, while elevated mid RNA was detected in MP2 in nearly all hemisegments, only a fifth of those hemisegments had elevated levels of the protein. Over-expression of the two transgenes resulted in MP2-lineage defects about the same frequency. These results indicate a translational/post-translational regulation of mid in MP2. The regulation of ectopically expressed mid in the wing imaginal disc was complex. In the wing disc, where mid is not expressed, the ectopic expression of the transgene lacking the 3'UTR had a higher level of mid RNA and the protein and had a stronger phenotypic effect. These results indicate that the 3'UTR can subject mid-mRNA to degradation in a cell and tissue-specific manner. We further report a balancer-mediated transgenerational modifier effect on the expression and gain of function effects of the two transgenes.

Noncontact elasticity measurement of hydrogels in a culture dish using reverberant optical coherence elastography.

Estimating the elasticity of hydrogel phantoms in a cell culture plane is important for understanding the cell behavior in response to various types of mechanical stimuli. Hence, a noncontact tool for measuring the elastic properties of hydrogel phantoms in such three-dimensional cell cultures is required. A well-known method to determine the mechanical properties of hydrogels is the transient wave method. However, due to the multiple reflections of waves from the boundaries, a bigger cell culture plane or multiple directional filters may be required. In this study, we utilized reverberant shear wave elastography, which is based on the autocorrelation principle, to evaluate the shear wave speed in hydrogel samples within a culture dish. Numerical simulations were performed first to confirm the validity of the reverberant elastography method. Subsequently, we used this method to measure the wave speeds in hydrogel phantoms with different concentrations. Shear rheology tests were also performed, and their results were found to be in good agreement with the measured shear wave speeds. The proposed method could be useful for measuring the elasticity of tissues in tissue engineering applications in an inexpensive and noncontact manner.

The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Potential pro-inflammatory impact of scleral lens midday fogging on human corneal epithelial cells: An in vitro study.

PURPOSE: Midday fogging (MDF) occurs when particulate material accumulates in the fluid reservoir (FR) beneath scleral lenses (SL), and its impact on epithelial cells is unknown. This study examines the in vitro pro-inflammatory effect of the FR on human corneal epithelial cells in varying degrees of MDF. METHODS: Normal SL neophytes were recruited to wear SL 8 h daily for 4 days. Following 8 h on days 1 and 4, optical coherence tomography (OCT) images were acquired for MDF quantification using ImageJ, and the FR was collected. FR samples from the same eye were later pooled, diluted 2-fold and applied on human telomerase-immortalized corneal epithelial (hTCEpi) cells cultured on Terasaki microwell plates. Tumor necrosis factor (TNF)-α and culture media were used as positive and negative controls, respectively. After a 30-minute treatment, the nuclear factor-kappa B (NF-κB) pathway was measured by NF-κB-p65 immunofluorescence and images were analyzed with ImageJ. Pearson's correlation was conducted to determine the association between median nuclear fluorescence and MDF. RESULTS: Fourteen FR samples with a mean volume of 22 ± 16 µl were tested. Mean MDF severity following 8 h of SL wear was 25 ± 17 units (range 7 - 64). The median nuclear fluorescence (NF-κB-p65 translocation) in cultured hTCEpi cells ranged from 31.43 to 45.16 while the negative and positive controls were 44.71 ± 1.72 and 108.77 ± 68.38, respectively. Although a potential positive trend between MDF and median nuclear fluorescence was observed, Pearson's correlation analysis revealed no significant association (r = +0.48, P = 0.09). CONCLUSIONS: The results suggest that the FR can trigger NF-κB-p65 translocation in hTCEpi cells, which may be associated with MDF severity. This study introduces the use of Terasaki microwell plates for immunofluorescence studies of the FR. The technique is simple, minimizes sample usage, and does not require expensive instrumentation.

Establishment of a new equine embryo brain primary cell culture with long-term expansion.

Primary cell cultures derived from human embryo lung play a crucial role in virology by aiding virus propagation and vaccine development. These cultures exhibit a notable ability to undergo multiple subcultures, often reaching up to 70 passages. However, finding alternative primary cell cultures with similar longevity and usefulness is challenging. In this study, we introduce a novel primary culture cells derived from equine embryo brain (FEB), which cells exhibited remarkable long-term cultivation potential. The FEB was established and maintained using Sumitomo Nerve-Cell Culture System Comparison studies were conducted with fetal equine kidney cell line (FEK-Tc13) to assess growth rates and subculture longevity. Immunological characterization was performed using neuronal markers to confirm the neural nature of FEB cells. Viral growth assessments were conducted using equine herpesviruses (EHV-1 and EHV-4) to evaluate infectivity and cytopathic effects in FEB cells. PCR analysis and real-time PCR assays were employed to detect viral genomic DNA and transcription activity of EHVs in infected FEB cells. FEB cells demonstrated faster growth rates compared to fetal equine kidney cell line (FEK-Tc13 cells) and exhibited sustained subculture capability exceeding 50 passages. Immunostaining confirmed the glial identity of FEB cells. Both equine herpesviruses 1 and 4 EHV-1 and EHV-4 viruses efficiently replicated in FEB cells, resulting in clear cytopathic effects. PCR analysis detected genomic DNA of EHVs in infected FEB cells, indicating successful viral infection. The establishment of FEB cells with extended subculture capability highlights their potential utility as a model system for studying neural cell biology and viral infections.

Protocol to culture enteric glial cells from the submucosal and myenteric plexi of neonatal and juvenile pig colons.

Here, we present our protocol to culture enteric glial cells from the submucosal and myenteric plexus of neonatal and juvenile pig colons. We describe steps for colon isolation, microdissection, and enzymatic and mechanical dissociation. We include procedures for passaging and analyzing cell yield, freeze/thaw efficiency, and purity. This protocol allows for the generation of primary cultures of enteric glial cells from single-cell suspensions of microdissected layers of the colon wall and can be used to culture enteric glia from human colon specimens. For complete details on the use and execution of this protocol, please refer to Ziegler et al.1.